Cyclic compounds and their use in assays for detecting antibodies - Patents.com

Abstract

Kind Code: A1 Peptide-based cyclic compounds containing epitopes derived from amyloidogenic proteins involved in systemic amyloidosis are provided, which are useful for immunoassays and efficacy assays in antibody drug research and development.

Description

[Technical Field]

[0001] The present disclosure relates generally to cyclic compounds comprising peptides containing epitopes of systemic amyloidogenic proteins, and their use for detecting, quantifying, and validating therapeutically useful antibodies and equivalent binding molecules. The 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, and are particularly useful in conducting clinical trials, for marketing authorization applications, and for quality control of approved pharmaceutical products. [Background technology]

[0002] Over the past 30 years, monoclonal antibody drugs have matured from research subjects to improved technologies, from clinical studies to commercialization. In recent years, the number of monoclonal antibody drugs approved for sale has increased rapidly, with the approval of the 100th monoclonal antibody product by the U.S. Food and Drug Administration (FDA) in 2021 marking a milestone. In 2019, 9 of the 20 best-selling drugs were monoclonal antibody drugs (Mullard, Nature Reviews Drug Discovery 20, 491-495 (2021), DOI: 10.1038 / d41573-021-00079-7).

[0003] One promising application of therapeutic antibodies is the treatment of amyloidosis, which is caused by toxic amyloid aggregation. Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, exhibit highly prevalent and fatal forms of localized amyloidosis, in which amyloid deposits form within the nervous system, triggering the death of specific types of nerve cells. In systemic amyloidosis, such as immunoglobulin light chain, transthyretin, and dialysis-associated amyloidosis, multiple organs are affected because amyloid-forming proteins are distributed to various parts of the body as they migrate from their synthesis site. Antibodies and antibody fragments have already proven to be effective anti-amyloid molecules. For example, aducanumab has shown dose-dependent clearance of amyloid deposits in Alzheimer's disease patients and was recently approved by the FDA for use in the treatment of Alzheimer's disease.

[0004] The discovery and development of antibody drugs typically rely on immunoassays, for example, to detect and quantify antibodies, and the sensitivity of the assay depends on the affinity of the antibody to its target protein. Similarly, the sensitivity and reliability of potency assays depend on the affinity of the antibody to its target, especially in cellular assays. Therefore, due to the importance of immunoassays in various stages of antibody screening and drug discovery, there is a constant need for improvements in assays, including the optimization of various parameters and the adoption of various strategies. The goal is to increase the sensitivity, specificity, accuracy, and reproducibility of the assay. Important parameters and measures that are commonly subject to improvement include, to name a few, the optimization of antigen coating, the use of effective blocking agents to prevent nonspecific binding, antibody dilution, incubation conditions, detection systems, and signal amplification.

[0005] Thus, the problem underlying the present disclosure is the provision of immunoassay systems and methods for detecting, isolating, and characterizing antibodies. Summary of the Invention

[0006] The present disclosure generally relates to cyclic compounds comprising peptide or protein fragments, also referred to as cyclic peptide compounds, that contain an epitope of an antibody or equivalent binding molecule, and the use of such cyclic peptide compounds in methods for determining the efficacy of antibodies or binding molecules, and generally in the fields of drug discovery and diagnostics. The disclosure further relates to the use of cyclic peptide compounds in efficacy assays, e.g., for determining or comparing the efficacy of binding molecules such as anti-TTR antibodies. The assay methods of the present disclosure are useful, for example, in evaluating drug candidates for clinical trials, in testing batch releases of pharmaceutical compositions containing antibodies or other binding molecules, in applying for marketing approval, and in quality control of approved pharmaceutical products. More specifically, the cyclic peptide compounds contain an epitope of an amyloidogenic protein and can be used in assays to detect amyloid-specific antibodies and corresponding binding fragments thereof. In this regard, the cyclic compounds of the present disclosure contain an epitope of an antibody, and thus the cyclic compounds are designed to be bound by an antibody. As explained further below, preferably, the epitope of the peptide is selected from epitopes that are accessible to antibody binding only in misfolded and / or aggregated forms of the protein, e.g., the epitope is exposed only in abnormal protein aggregates. Also, 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 in an ELISA assay than amyloid-forming proteins or protein aggregates, and preferably higher than the corresponding linear peptides.

[0007] As shown in the accompanying examples, cyclic peptides containing TTR epitopes (cyclic TTR peptides) have been used as target antigens in immunological and biological assays for the detection of antibodies and for measuring their efficacy, respectively.

[0008] In particular, as exemplified in Example 1, cyclic peptides as target antigens provide higher sensitivity of ELISA assays for antibodies than native antigens, i.e., aggregated TTR, also referred to as misfolded TTR. The same was observed in reporter gene assays, as exemplified in Examples 2 and 5, demonstrating that cyclic peptides provide improved ADCP assays. Surprisingly, when cyclic peptides were used as antigens instead of TTR aggregates, the assays showed significant improvements in sensitivity and reliability. Without intending to be bound by theory, the exceptional performance of assays using cyclic peptides may be due to the highly stable conformation adopted by cyclic peptides, which is believed to be due to the fact that cyclic peptides are constrained by connecting both ends to each other, thus mimicking the stability of protein aggregates. However, as shown in Examples 2 and 5, even taking such theoretical considerations into account, 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 intending to be bound by theory, this may be due to the smaller size of peptides compared to proteins and the resulting increased epitope density, leading to both improved binding capacity and enhanced avidity effects. Furthermore, epitopes in cyclic peptides may be more accessible than in full-length proteins. Nevertheless, the cyclic TTR peptide containing linker amino acids, totaling 31 amino acids, is only one-quarter the size of the full-length TTR protein, and the size of the cyclic peptide cannot a priori explain the observed effect. A further reason may be better conformational control of synthetic peptides compared to recombinant proteins. In particular, although over 95% of the peptides are cyclized, it is unclear what proportion of misfolded aggregated TTR protein adopts an 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, using the experimental results described in the accompanying Examples, it is now possible to retrospectively develop superior explanatory approaches and theories for envisioning additional cyclic peptides with suitable epitopes for the detection and identification of potent antibodies against amyloidogenic proteins, particularly systemic amyloidogenic proteins, which would not have been foreseen without the knowledge of the present results and the teachings of the present disclosure. In this regard, and again without wishing to be bound by any theory, it is noteworthy that cryo-electron microscopy studies have recently shown that the amyloid structures of systemic amyloidogenic proteins, such as ATTR and AL amyloidosis caused by misfolding of immunoglobulin light chains (LC), are similar on the one hand, but substantially different on the other hand from the amyloid structures of localized amyloidogenic proteins, such as tau; see Figure 5 in Schmidt et al., Nat. Commun. 10 (2019), 5008, https: / / doi.org / 10.1038 / s41467-019-13038. It is therefore reasonable to assume that the present results for cyclic peptides derived from TTR may also apply to other systemic amyloidogenic proteins.

[0009] Thus, the present disclosure provides cyclic compounds comprising peptides containing epitopes of systemic amyloidogenic proteins, where the epitope is preferably accessible to antibody binding only in the misfolded and / or aggregated form of the protein, as in the case of neoepitopes, and / or where the epitope is at least absent in the physiologically active form of the protein, e.g., in the case of epitopes accessible in the monomeric form of TTR protein that are hidden in the physiologically active tetramer and no longer accessible to antibody binding. As illustrated in the accompanying examples, the cyclic peptide compounds of the present disclosure are particularly useful in immunological assays, such as ELISA assays that may be used for drug discovery or diagnostic methods, as well as in potency assays for characterizing therapeutically useful antibodies and equivalent binding molecules in which antibody Fc-mediated activity plays a key role in their mechanism of action.

[0010] In particular, the remarkable performance of the cyclic peptide compounds as target antigens was first demonstrated in the ELISA assay described in Example 1. The EC 50 The EC value is the EC of an antibody that binds to a protein aggregate and a linear peptide containing the same epitope as the cyclic peptide. 50 The cyclic peptides had the highest binding affinity to the antibody, i.e., the lowest EC 50 As mentioned above, the higher binding affinity between antibodies and cyclic peptides compared to protein aggregates can be attributed to a higher epitope density, which results in an apparent improved binding affinity due to a higher binding capacity and a higher avidity effect, due to better accessibility of epitopes in cyclic peptides compared to full-length proteins, and / or due to better control of the conformation of synthetic peptides compared to recombinant proteins. Thus, in one embodiment, the cyclic peptide compounds and cyclic peptides of the present disclosure each provide a higher binding affinity with an antibody than with a target protein, which is higher than the corresponding linear peptide derived from the corresponding linear peptide in an ELISA assay such as that described in the accompanying Example 1, and is preferably at least 2-fold, more preferably at least 3-fold, 4-fold, or 5-fold, 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 utility of antibodies, particularly as effective drugs for treating amyloidosis, depends not only on their ability to bind to aggregates but also on antibody Fc-mediated activity, which plays a key role in their mechanism of action. Antibody binding to Fc receptors on cell surfaces triggers a number of important and diverse biological responses, including the phagocytosis and destruction of antibody-coated particles (called antibody-dependent cellular phagocytosis, or ADCP). When manufacturing pharmaceutical compositions, it is not enough to simply formulate a drug substance into a dosage form; the resulting formulation must also be approved by the regulatory authorities of the country in which the pharmaceutical composition will be used. In the United States, the responsible regulatory authority is the FDA (http: / / www.fda.gov / ), and in Europe, for example, the European Medicines Agency (EMEA) (http: / / www.emea.eu.int / ).

[0012] The approval process is highly regulated, and drug developers must submit a significant amount of information about their drug candidates to regulatory authorities in order to obtain approval. This may include information about the drug candidate's potency and the corresponding assays for determining potency. Such potency assays help characterize the drug product, monitor lot-to-lot consistency, and ensure the stability of the product. The potency of antibodies in which Fc binding to Fc receptors plays a key role in their mechanism of action is traditionally measured through the use of biological assays in which the effect being assessed depends on Fc-Fc receptor binding.

[0013] Therefore, assays to characterize drug products, monitor lot-to-lot consistency, and ensure drug product stability are clinically important and must be sensitive enough to detect differences that may affect the drug product's mechanism of action and function.

[0014] As shown in Example 2, the use of cyclic peptides of the present disclosure provided an improved ADCP assay. Unexpectedly, when cyclic peptides were used as antigens instead of TTR aggregates, the assay demonstrated significant improvements in sensitivity and reliability. Furthermore, the initially developed highly sensitive ADCP assay as described in Example 2 has been confirmed and validated; see Example 5. In particular, the assay outlined in Example 5 has been shown to have the ability to detect potency changes associated with Fc domain mutations in the range of 40% to 180%, i.e., up to a 60% loss of potency and up to an 80% increase in potency.

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

[0016] In one embodiment, such a potency assay comprises the following steps: (a) contacting a cyclic peptide compound of the present disclosure as a target antigen with a binding molecule under conditions that allow the 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; (b) contacting the binding molecule-antigen complex with a population of effector cells, preferably Jurkat cells, that express an Fc receptor, preferably the human Fc receptor FcγRI (CD64), and that have been engineered to carry a reporter gene that preferably encodes a bioluminescent protein, preferably luciferase, under the control of a response element that responds to activation by the Fc receptor, preferably an NFAT (nuclear factor of activated T cells) response element, under conditions that allow 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 reporter gene activity, 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 indicates efficacy of the binding molecule, preferably, the mechanism of action of the Fc domain is to induce antibody-dependent cell-mediated phagocytosis (ADCP).

[0017] Detailed embodiments for potency assays are described and claimed in WO 2023 / 099788 A1, which is incorporated herein by reference.

[0018] Thus, the cyclic peptide compounds of the present disclosure containing epitopes of amyloidogenic proteins can be used as target antigens in place of the amyloidogenic proteins themselves, resulting in highly sensitive and reliable efficacy assays as described above. The cyclic peptides of the present disclosure are particularly suitable for assays to determine the efficacy of antibodies and Fc domain-containing binding molecules that bind to amyloidogenic TTR or other amyloidogenic proteins involved in systemic amyloidosis.

[0019] Without wishing to be bound by theory, it is believed that the advantage of cyclic peptides can be explained by the fact that, in contrast to linear peptides, which are extremely flexible and can adopt a virtually infinite number of conformations, cyclic peptides are constrained by the attachment of their ends to each other, resulting in much less flexibility and therefore more stable conformations. In other words, cyclic peptides have lower entropy than the same amino acid sequence in linear form.

[0020] Thus, the provision of cyclic peptide compounds according to the present disclosure represents an important contribution to the art, given their remarkable utility as suitable targets for studying binding between target antigens and corresponding target antigen-binding molecules, for example, in assays requiring high sensitivity. Nevertheless, the present disclosure also relates to linear forms of cyclic peptide compounds and cyclic peptides, for example, for use as precursors or controls for preparing cyclic peptide compounds in experiments.

[0021] The above-mentioned assay, i.e., the potency assay using the cyclic peptide compound of the present disclosure, can be applied to the method of producing pharmaceutical compositions containing target antigen-binding molecules, and after production, the potency of the binding molecules is first analyzed. Based on this result, it is evaluated whether the binding molecules can be used in pharmaceutical compositions. In particular, only binding molecules that are deemed to be potent according to the assay are selected for further use and formulated into pharmaceutical compositions containing pharmaceutically acceptable carriers.

[0022] The potency assay can also be used in a method for analyzing and selecting batches of pharmaceutical compositions of target antigen-binding molecules, in which a sample of the batch to be analyzed and a control sample are subjected to the potency assay, and the reporter gene activity of the sample is compared with that of the control. Batches in which the sample exhibits reporter gene activity greater than, equal to, or not substantially less than that of the control are ultimately selected for further use. Thus, this assay can be used to verify lot-to-lot consistency.

[0023] The present disclosure further relates to kits preferably designed for carrying out potency assays as disclosed herein, in particular for assaying the potency of binding molecules comprising an Fc domain to induce ADCP, which kits comprise at least the cyclic peptide compounds of the present disclosure or the corresponding linear precursors, and which may also serve as controls similar to those shown for the TTR peptide in the Examples. (i) a population of effector cells that express an Fc receptor, preferably the human Fc receptor FcγR, and that have been engineered to carry a reporter gene under the control of a response element that responds to activation by the Fc receptor, preferably wherein the population of effector cells is a population of Jurkat cells, and the reporter gene encodes a photoprotein, preferably luciferase under the control of an NFAT response element; (ii) a corresponding substrate of the reporter; and preferably the kit comprises: (iii) a solid support, preferably a microtiter plate, preferably a 96-well plate including a lid; (iv) washing, blocking and assay / sample dilution buffers; and / or (v) a monomeric control of the target antigen and / or a positive control anti-target antigen antibody.

[0024] Immunological and biological assays such as those described herein are exemplified using cyclic peptides of the present disclosure comprising a TTR epitope as a target antigen and an anti-TTR antibody (e.g., NI-301.37F1, etc.), which is disclosed in International Publication No. WO 2015 / 092077(A1) and has been shown to be capable of activating the immune system to clear TTR fibrils in animal models; see International Publication No. WO 2020 / 094883(A1). The physiological form of TTR is a tetrameric protein that exhibits amyloidogenic properties when it dissociates into monomers to form transthyretin amyloidosis (ATTR), a systemic amyloidosis. Systemic amyloidosis is a protein misfolding disease caused by extracellular deposition of amyloid, resulting in organ dysfunction. Localized amyloidosis, on the other hand, refers to intracellular and / or extracellular amyloid deposition occurring only in organs or tissues where precursor proteins are synthesized, such as intracellular tau protein fibrils and extracellular amyloid-β fibrils and plaques in Alzheimer's disease. In principle, the cyclic peptide compounds of the present disclosure can include any peptide or protein fragment capable of forming a cyclic peptide compound, particularly a peptide or protein fragment containing the neoepitope described above. In a particularly preferred embodiment, the (neo)epitope is hidden in the natively folded conformation of the target antigen but accessible to antibody binding after unfolding and aggregation, such as the linear epitope WEPFA of the antibody NI-301.37F1, located at positions 41-45 of the mature TTR protein. Similarly, the methods of the present disclosure are applicable to any cyclic peptide that contains and displays epitope(s) of the target antigen-binding molecule being tested, for example, epitopes exposed only in misfolded variants, structural epitopes on aggregates, fibrils, and / or oligomers, epitopes on extracellular variants of otherwise intracellular physiological proteins, or epitopes specific to exogenous pathogens such as fungi, bacteria, and viruses.

[0025] Nevertheless, according to this example, the cyclic peptide compounds of the present disclosure preferably comprise a peptide or protein fragment comprising an epitope derived from an amyloidogenic protein involved in systemic amyloidosis, preferably an epitope exposed in a misfolded, non-physiological form of the protein, such as transthyretin, and are therefore particularly suitable for, and therefore preferably used in, methods for detecting antibodies specific for an amyloidogenic protein involved in systemic amyloidosis, particularly antibodies that bind to the misfolded, non-physiological form of the protein, and for determining the efficacy of such antibodies.

[0026] The cyclic peptide compounds of the present disclosure are particularly useful for determining the efficacy of antibodies to activate ADCPs.

[0027] Furthermore, the cyclic peptide compounds of the present disclosure are particularly useful in methods for identifying and optionally obtaining antibodies that bind to amyloidogenic proteins involved in systemic amyloidosis, which methods typically comprise: (a) providing and optionally producing one or more potential amyloidogenic protein-binding antibodies or a source thereof; (b) subjecting one or more potential amyloidogenic protein-binding antibodies or sources thereof to a binding assay comprising a cyclic peptide compound of the present disclosure; and (c) identifying and optionally obtaining an antibody (an antibody of the invention) that has been determined to bind to the cyclic peptide compound.

[0028] This method can be combined with each of the potency assays of the present disclosure and those described in WO 2023 / 099788 A1, and / or any other suitable method to further determine the diagnostic or preferably therapeutic utility of the antibodies of the invention.

[0029] Thus, a further embodiment of the present disclosure is a method for producing a pharmaceutical composition comprising an antibody that binds to an amyloidogenic protein, comprising at least: (a) providing and optionally producing one or more potential amyloidogenic protein-binding antibodies or a source thereof; (b) subjecting the one or more potentially amyloidogenic protein-binding antibodies or a source thereof to a binding assay comprising a cyclic peptide compound of the present disclosure; (c) identifying and optionally obtaining an antibody that binds to the cyclic peptide compound (an antibody of the invention); and (d) formulating the antibody or derivative thereof identified and optionally obtained in step (c) with a pharmaceutically acceptable carrier.

[0030] Sources of antibodies are not limited and include, for example, natural and synthetic antibodies obtained from immunized laboratory animals such as rodents, preferably mice, and 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 lines such as CHO and HEK; see also the "Detailed Description of the Disclosure" for additional sources of antibodies and other target binding molecules.

[0031] Preferably, the binding assay used in the above method comprises an ELISA such as that performed in Example 1.

[0032] In a preferred embodiment of the disclosed method for identifying and obtaining the antibodies of the present invention, and their further use when formulated into a pharmaceutical composition and in drug discovery, respectively, the antibody identified and optionally obtained in step (c) competes with the reference antibody for binding to the amyloidogenic protein, and preferably the antibody of the present invention has a lower EC2 for the amyloidogenic protein than the reference antibody. 50 It has.

[0033] Unless otherwise defined in this application, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of 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. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0034] Further embodiments of the present disclosure will become apparent from the following description, examples, and claims. Those skilled in the art will understand that any characterization of a general feature of the following general embodiments can be, and preferably is intended to be, combined with one or more characterizations of other features of such general embodiments. Additionally, unless otherwise specifically indicated, the antibody embodiments described herein, including the examples, although preferred, are intended as examples, and any target-binding molecule may be extrapolated. [Brief explanation of the drawings]

[0035] [Figure 1] Figure 1 shows the improved sensitivity of the ELISA assay. Comparison of the binding specificity of antibody NI-301.37F1 to the peptides TTR34-54cyc, TTR40-49, biotin.TTR40-49, and mis-WT-TTR (A) with the binding specificity of antibody NI-301.37F1 to the peptides TTR34-54cyc, biotin.TTR34-54cyc, TTR40-49, biotin.TTR40-49, and mis-WT-TTR (B) using an ELISA assay demonstrates specific binding of NI-301.37F1 to mis-WT-TTR, and binding of NI-301.37F1 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 the curve for biotin.TTR40-49. [Figure 2]1 shows the improvement of the ADCP assay by using a cyclic peptide compound as a target antigen. Measuring the potency of antibody NI-301.37F1 in an ADCP assay using a cyclic TTR peptide (TTR34-54cyc) demonstrates the ability of antibody NI-301.37F1 RS to activate phagocytosis in a dose-responsive, i.e., dose-dependent, manner, characterized by an EC50 of 19.8 ng / mL. [Figure 3-1] FIG. 1 shows that a comparison of the ADCP assay using TTR34-54cyc as the target antigen to detect changes in antibody activity with low concentration samples (NI-301.37F1 50% (A) and 70% (B)) and high concentration samples (NI-301.37F1 130% (C) and 150% (D)) of the NI-301.37F1 reference sample (NI-301.37F1 RS) demonstrated that the assay was capable of detecting a 50% loss of antibody activity and a 50% increase in antibody activity, respectively. [Figure 3-2] FIG. 1 shows that a comparison of the ADCP assay using TTR34-54cyc as the target antigen to detect changes in antibody activity with low concentration samples (NI-301.37F1 50% (A) and 70% (B)) and high concentration samples (NI-301.37F1 130% (C) and 150% (D)) of the NI-301.37F1 reference sample (NI-301.37F1 RS) demonstrated that the assay was capable of detecting a 50% loss of antibody activity and a 50% increase in antibody activity, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present disclosure relates to cyclic compounds comprising peptides or protein fragments containing epitopes derived from amyloidogenic proteins involved in systemic amyloidosis. As described herein, typically, the epitopes of the peptides and protein fragments in the cyclic compounds of the present disclosure are epitopes of antibodies, i.e., amyloid / aggregate-specific antibodies, which bind to the cyclic compounds. Accordingly, the present disclosure also relates to the use of such cyclic peptide compounds in the fields of drug discovery and diagnostics, and in methods for determining the efficacy of target antigen-binding molecules containing Fc domains, particularly their efficacy in activating antibody-dependent cell-mediated phagocytosis (ADCP). Preferably, the target antigen-binding molecule is an antibody that binds to an amyloidogenic protein. The present disclosure further relates to the use of cyclic peptide compounds in corresponding efficacy assays, which are particularly useful for batch release of pharmaceutical compositions containing antibodies or similar binding molecules, particularly when conducting clinical trials, applying for marketing authorization, and for quality control of approved pharmaceuticals.

[0037] Unless otherwise stated, terms used herein are given the definitions set out in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000, expanded 2003, ISBN 0 19 850673 2; 2nd Edition, 2006, ISBN 0-19-852917-1 978-0-19852917-0.

[0038] The term "protein" as used throughout this specification includes fragments and peptides of (full-length) proteins that contain and expose the epitope of the target antigen-binding molecule, e.g., antibody, being tested.

[0039] Reference to a "cyclic peptide" herein can refer to a fully proteinaceous compound, for example, where the linker is 2, 3, 4, 5, 6, 7, or 8 amino acids, or where no linker is present. For example, a stretch of amino acids comprising a natural protein sequence, i.e., an antibody epitope, allows for cyclization, for example, due to the presence of two cysteines at an appropriate distance, without the addition of extra amino acids. It is understood that the properties described for the cyclic peptides determined in the examples can be introduced into other compounds, for example, cyclic peptide compounds containing non-amino acid linker molecules. When a cyclic compound is composed of amino acids, the terms "cyclic peptide" and "cyclic compound" can be used interchangeably. Furthermore, the term "cyclic compound" can be used interchangeably with the term "cyclic peptide compound."

[0040] The term "linker" as used herein refers to a chemical moiety that can be covalently attached directly or indirectly to a protein fragment or peptide as defined herein. Linker termini can be linked, for example, to generate a cyclic peptide compound. Linkers can be located at the N- and C-terminal positions. Alternatively, linkers can be located at internal positions "some distance" from the termini. Linkers can contain one or more functional moieties, such as one or more cysteine ​​(C) residues. Linkers can also be linked to other proteins or components via functional moieties. Cyclic peptide compounds containing linkers are longer than the peptide or protein fragment itself.

[0041] As used herein, the term "functional 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 reacts 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 another 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. As used herein, a strong non-covalent bond refers to 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.

[0042] During the course of experiments conducted in accordance with the present disclosure, it was unexpectedly discovered that a cyclic peptide containing an epitope of TTR, specifically the epitope recognized by the anti-TTR antibody NI-301.37F1 (WEPFA SEQ ID NO: 1), which selectively binds with high affinity to TTR aggregates of either wild-type or mutant TTR as described in WO 2015 / 092077(A1), is an excellent target antigen in ELISA and ADCP assays. As described in Example 1 and illustrated in Figure 1, the antibody exhibited highly specific binding to the cyclic peptide compound, with the binding affinity of the antibody to the cyclic peptide being an order of magnitude higher than that of its natural target antigen, i.e., misfolded TTR, for which the antibody was initially screened and identified. This remarkable effect was both unexpected and advantageous. This is because the above-described cyclic peptide compounds can not only replace the preparation of full-length amyloidogenic proteins and aggregates / fibrils of full-length amyloidogenic proteins, which tend to be more variable and time-consuming than the preparation of cyclic peptides, but also because, as exemplified in Examples 1, 2, and 5 and shown in Figures 1-3, the cyclic peptide compounds represent excellent target antigens in binding assays such as ELISA and functional assays such as ADCP, which require high sensitivity and reproducibility.

[0043] The cyclic TTR peptide was originally designed to solve the crystal structure of the Fab fragment of antibody NI-301.37F1 in complex with its TTR antigen in order to obtain information about the three-dimensional structure of the antibody-antigen complex and understand its mechanism of action. It was fortuitous that the cyclic TTR peptide was used to replace full-length recombinant TTR protein in an ELISA assay to determine antibody NI-301.37F1, i.e., an IgG antibody. Surprisingly, the ELISA assay proved to be much more sensitive and reliable than the use of recombinant TTR protein; see Example 1 and Figure 1 . Subsequent experiments have even more surprisingly demonstrated that the use of the cyclic TTR peptide substantially improves the sensitivity and reliability of the potency assay.

[0044] As described below, structural analysis by cryo-electron microscopy (cryo-EM) revealed that both ends of the unresolved loop are in contact with each other, suggesting that the epitopes and peptide sequences of the cyclic peptides and cyclic peptide compounds, respectively, could be similarly selected based on this. Thus, in addition to or instead of peptide design on the crystallographic structure of the Fab peptide antigen, the cryo-EM structure can be used to select suitable epitopes and amino acid sequences containing them for designing cyclic peptides of the present disclosure that exhibit advantageous properties similar to those of the cyclic TTTR peptide, which has experimentally proven to be a highly effective and reliable tool in ELISA and ADCP assays. As previously described herein, it is noteworthy that cryo-EM studies have shown that the amyloid structures of systemic amyloidogenic proteins, such as ATTR and AL amyloidosis caused by immunoglobulin light chain (LC) misfolding, are similar to, but substantially different from, those of localized amyloidogenic proteins, such as tau; see Figure 5 in Schmidt et al., Nat. Commun. 10 (2019), 5008, https: / / doi.org / 10.1038 / s41467-019-13038. Therefore, it is reasonable to expect that the present results for TTR-derived cyclic peptides may also apply to at least other systemic amyloidogenic proteins.

[0045] Methods for generating crystal structures of antibodies and their Fab fragments against peptides and peptide antigen complexes, 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.

[0046] This knowledge now provides an opportunity to create additional 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 selected, additional cyclic peptides, such as those described herein, that mimic, for example, antibody-binding epitopes of amyloidogenic proteins can be designed using the program PEP-FOLD, which can predict peptide structure from amino acid sequences and which, when applied to TTR cyclic peptides, will reasonably predict epitope presentation.

[0047] Thus, the present disclosure relates to cyclic peptide compounds and their linear precursors, including peptides containing epitopes of systemic amyloidogenic proteins, where the epitope is preferably accessible to antibody binding only in the misfolded and / or aggregated form of the protein, as in the case of neoepitopes, and / or where the epitope is absent, at least in the physiologically active form of the protein, as in the case of epitopes accessible in the monomeric form of the TTR protein that are hidden in the physiologically active tetramer and no longer accessible to antibody binding. Such peptides are preferably antigenic peptides and antigenic cyclic peptide compounds, respectively. As illustrated 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 reporter gene assays used as efficacy assays, as disclosed herein.

[0048] The cyclic peptide compounds of the present disclosure can either consist of an epitope recognized by a target antigen-binding molecule, such as an antibody or antigen-binding molecule specific to an amyloidogenic protein, or can comprise or consist of a protein fragment or peptide containing an epitope recognized by a target antigen-binding molecule, which means that, for example, additional amino acids or other chemical entities used to cyclize the peptide or protein fragment, as further described below, can be present in the protein fragment or peptide that forms the cyclic peptide compound.

[0049] The additional amino acids may be amino acids naturally located adjacent to the epitope sequence, i.e., amino acids flanking the epitope sequence, or amino acids present in the protein sequence from which the protein fragment or peptide is derived. That is, the protein fragment or peptide forming the cyclic peptide compound comprises an epitope of the target antigen-binding molecule and additional amino acids adjacent to and flanking the epitope. The number of these adjacent / flanking amino acids may vary, for example, from 1, 2, or 3 to 50 amino acids, preferably from 1, 2, or 3 to 40 amino acids, more preferably from 1, 2, or 3 to 30 amino acids, more preferably from 1, 2, or 3 to 20 amino acids, and more preferably from 10 to 20 amino acids, where the amino acids are either evenly distributed between the N-terminus and C-terminus of the epitope sequence, or unevenly distributed, e.g., 7 amino acids added to the N-terminus and 9 amino acids added to the C-terminus of the epitope.

[0050] Additionally or alternatively, in one embodiment, the protein fragment or peptide comprises a linker, i.e., the protein fragment or peptide may comprise an epitope recognized by a target antigen-binding molecule without any adjacent amino acids and a linker, or may comprise an epitope as defined above, adjacent amino acids, 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 a target antigen-binding molecule, amino acids adjacent to the epitope, and a linker. Preferably, the linker is directly or indirectly covalently bound to the N-terminal residue of the protein fragment or peptide and the C-terminal residue of the protein fragment or peptide.

[0051] Methods for cyclizing peptides are generally known in the art. For example, cyclization can be achieved by chemical crosslinking, especially using chemical scaffolds. Crosslinking requires functional groups, and only a few protein chemical targets account for the majority of crosslinking techniques, such as primary amines (-NH2) (this group is present at the N-terminus of each polypeptide chain and in the side chains of lysine residues); carboxyls (-COOH) (this group is present at the C-terminus of each polypeptide chain and in the side chains of aspartic acid and glutamic acid); and sulfhydryls (-SH) (this group is present in the side chain of cysteine).

[0052] Scaffold-based cyclization is one of the most frequently used methods because it can be applied to chemically or biologically synthesized peptides. Generally, scaffold compounds such as organic halides (most frequently organic bromides) selectively react with the sulfhydryl groups of cysteines. Non-sulfhydryl groups, such as primary amines of lysines or the N-terminal amino group in peptides, can also be used for cyclization using, for example, N-hydroxysuccinimide (NHS)-containing chemistries. Specifically designed unnatural amino acids can also be used to cyclize peptides via bioorthogonal reactions. For example, if an azide-containing amino acid, such as azidohomoalanine or azidophenylalanine, is present in a peptide, copper-mediated click reaction with an alkyne-bearing scaffold can result in cyclization.

[0053] Additionally, cysteines can be linked to each other between their side chains via disulfide bonds (-SS-), or amide cyclization can occur without a scaffold (head-to-tail or backbone cyclization).

[0054] For example, a peptide having "C" residues at its N-terminus and C-terminus, such as the cyclic TTR compound used in the Examples, GCGGGRKAADDTWEPFASGKTSESGEGGGCG (SEQ ID NO: 17), can be reacted by SS-cyclization to produce a cyclic peptide. Cyclic peptide compounds can be synthesized as linear molecules with linkers covalently attached to or near the N-terminus or C-terminus of a TTR peptide or a peptide containing a relevant epitope as described herein, and then cyclized, providing a precursor that is also the subject of this disclosure. Alternatively, some of the linkers are covalently attached to or near the N-terminus and some are covalently attached to or near the C-terminus prior to cyclization. In either case, the linear compound is cyclized, for example, by SS-bond cyclization. Thus, the compounds can be cyclized by covalently bonding 1) at or near the N- or C-terminus of the peptide plus linker, forming a peptide bond (e.g., cyclizing the backbone), 2) at or near the N- or C-terminus with a side chain in the peptide plus linker, or 3) two side chains in the peptide plus linker. In this context, "near" is defined as within 1, 2, or 3 amino acid residues of the N- or C-terminus. Preferably, the linker is attached at the N- or C-terminus.

[0055] As described above, peptides can be cyclized by oxidation of thiol- or mercaptan-containing residues at or near the N- or C-terminus, or internally within the peptide, including, for example, cysteine ​​and homocysteine. For example, two cysteine ​​residues on the sides of the peptide can be oxidized to form a disulfide bond. Oxidizing reagents that can be used include, for example, oxygen (air), dimethyl sulfoxide, oxidized glutathione, cystine, copper(II) chloride, potassium ferricyanide, thallium(III) trifluoroacetate, or other oxidizing reagents known to those skilled in the art and can be used in conjunction with methods known to those skilled in the art. Crosslinkers are also known in the art and can be selected, for example, based on the functional groups used for crosslinking; see, for example, the crosslinker selection tool provided by Thermo Fisher Scientific.

[0056] Thus, in one embodiment, the linker comprises a functional moiety, e.g., an amino acid bearing one of the above-mentioned functional groups, such as lysine, aspartic acid, glutamic acid or cysteine, a non-naturally occurring amino acid, e.g., azidohomoalanine or azidophenylalanine, or an equivalently functional molecule, e.g., polyethylene glycol (PEG).

[0057] When the functional moiety is a naturally occurring amino acid, such as lysine, aspartic acid, glutamic acid, serine, threonine, or cysteine, the functional moiety does not necessarily have to be present in the linker, but can also be present in the epitope or in adjacent amino acids present in the protein fragment or peptide that form the cyclic peptide. Thus, the cyclization of the peptide and protein fragment, respectively, can also be performed without a linker. Thus, in one embodiment, the protein fragment or peptide forms the cyclic peptide compound of the present disclosure without a linker. The bond can occur through 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.

[0058] In preferred embodiments, at least one functional moiety is present in the linker, i.e., the linker comprises one or more functional moieties. The linker can comprise or consist of any amino acid, including unnatural amino acids, but preferably comprises at least one of the above-mentioned functional moieties, i.e., unnatural amino acids such as lysine, aspartic acid, glutamic acid, or cysteine, azidohomoalanine, or azidophenylalanine, or equivalently functional molecules such as polyethylene glycol (PEG). In preferred embodiments, the linker comprises cysteine ​​as the functional moiety.

[0059] Thus, in preferred embodiments, linkers of any length and sequence can be described using the following sequence: X-nX-1FX1-Xn, where F is any functional moiety, preferably C (cysteine), and X is any amino acid, including unnatural amino acids. In further preferred embodiments, 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).

[0060] 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 functional moiety is cysteine ​​(C). Thus, preferably, cyclization is carried out using a scaffolding compound such as an organic halide, preferably an organic bromide, which selectively reacts with the sulfhydryl group of cysteine, or via a disulfide bridge. Most preferably, cyclization is carried out via a disulfide bridge.

[0061] In a preferred embodiment, the linker contains 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, particularly 1, 2, 3, 4, 5, 6, 7, or 8 amino acids, and / or equivalent functional molecules, and / or combinations thereof. When the linker contains only amino acids, it is preferred that at least one amino acid, preferably cysteine, having any of the above-mentioned functional groups is present among the amino acids. 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).

[0062] As mentioned above, the length of the linker can vary, for example, it can be 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 GCGGGG (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: 15) or GGGCG (SEQ ID NO: 16), 4 amino acids, such as GCGG (SEQ ID NO: 159) or GGCG (SEQ ID NO: 160), or 3 amino acids, such as GCG.

[0063] Most preferably, the linker in the cyclic peptide compound comprises or consists of GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16).

[0064] As illustrated in the Examples, cyclic peptide compounds containing the epitope WEPFA of antibody NI-301.37F1 disclosed in WO 2015 / 092077(A1) (which is a neoepitope in the sense that it is located at positions 41-45 of the mature TTR protein and is hidden in the natively folded conformation of the TTR protein but is accessible to antibody binding after unfolding and aggregation) have been shown to be highly suitable target antigens for immunological and biological assays, for example, reporter gene assays used as potency assays.

[0065] Thus, in a preferred embodiment, the cyclic peptide compound preferably comprises a (neo)epitope derived from any protein whose aggregation leads to a disease phenotype.

[0066] The physiological function of proteins is highly dependent on their correct three-dimensional conformation. Disruptions in the proper folding of newly synthesized or pre-existing proteins, as well as in the pathways involved in the refolding (molecular chaperones) or degradation (ubiquitin-proteasome and autophagy) of misfolded proteins, can lead to intracellular and / or extracellular protein aggregation. These precipitates of misfolded proteins form either ordered (e.g., amyloid fibrils) or disordered (e.g., inclusion bodies) protein aggregates that dissociate only in the presence of high concentrations of detergents or denaturing buffers (Schroder, Acta Neuropathol 125 (2013), 1-2).

[0067] Amyloid diseases are characterized by the deposition of crossed-β-sheet amyloid fibrils composed of misfolded and / or misassembled proteins. The pathological hallmark of these disorders, amyloid fibrils, can be deposited systemically or localized in specific organs. The development of amyloidosis is often age-related and associated with a decline in quality of life and substantial suffering for both patients and their families. Alzheimer's disease is an example of localized cerebral amyloidosis, and type 2 diabetes is an example of localized extracerebral amyloidosis, both of which are age-related. Systemic amyloid diseases, often associated with age, are less common and include TTR amyloidosis. Amyloidosis can be sporadic, i.e., derived from a normal protein sequence, or hereditary (familial), i.e., derived from a protein with one or more point mutations. Furthermore, there are infectious amyloidoses such as transmissible spongiform encephalopathies caused by the aggregation of prion proteins (Ankarcrona et al., J Intern Med. 280 (2016), 177-202).

[0068] As illustrated in the accompanying examples, the present disclosure provides cyclic peptide compounds that preferably comprise epitopes that are normally exposed in abnormal protein aggregates of amyloidogenic proteins, making the cyclic compounds particularly useful in contributing to reliable methods for determining the efficacy of antibodies and antibody-based drugs in terms of their ability to activate FC domain / receptor-mediated effector functions such as antibody-dependent cell-mediated phagocytosis (ADCP); The antibody preferably targets an epitope on the abnormal protein aggregate; see also Examples 5-8 of WO 2023 / 099788 A1.

[0069] In a preferred embodiment, the (neo)epitope is derived from an amyloidogenic protein or aggregate thereof involved in systemic amyloidosis, such as transthyretin (TTR), in particular wild-type and mutant TTR, preferably wild-type TTR, immunoglobulin light chain (LC), immunoglobulin heavy chain (LH), serum amyloid A (SAA), leukocyte chemoattractant protein 2 (LECT2), gelsolin, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, β2-microglobulin, in particular wild-type and mutant β2-microglobulin, cystatin C, ABriPP, prion protein, and lysozyme; see, for example, Benson et al., Amyloid 25 (2018), 215-219 and Muchtar et al. See, e.g., et al., Journal of Internal Medicine 289 (2021), 268-292. Thus, the cyclic peptide compound includes a peptide derived from any one of the listed proteins, and preferably, the peptide includes at least four amino acids derived from the protein.

[0070] In a preferred embodiment, the amyloidogenic protein is TTR, and therefore the cyclic peptide compound comprises a protein fragment of TTR or a peptide derived from TTR.

[0071] The protein fragment or peptide in the cyclic peptide compound of the present disclosure is at least 4 amino acid residues of an amyloidogenic protein, preferably at least 5 amino acid residues, more preferably at least 10 amino acid residues, more preferably at least 15 amino acid residues, and most preferably at least 20, 21, 22, 23, 24, or 25 amino acid residues. More specifically, at least the epitope of the target antigen-binding molecule can consist of only 4 amino acids, as known to those skilled in the art, which may be supplemented with an appropriate number of amino acids sufficient and necessary for cyclization and / or other linker moieties.

[0072] However, in principle, there is no limit to the length of the peptide as long as it can be cyclized and recognized by the target-binding molecule. Thus, the cyclic peptide compounds of the present disclosure and used herein may include proteins, fragments thereof, or peptides thereof containing from four amino acids to all amino acids of an amyloidogenic protein. Preferably, the protein fragment or peptide in the cyclic peptide compound contains 4 to 100 amino acids, more preferably 4 to 90 amino acids, more preferably 4 to 80 amino acids, more preferably 4 to 70 amino acids, more preferably 4 to 60 amino acids, more preferably 4 to 50 amino acids, more preferably 4 to 45 amino acids, more preferably 4 to 40 amino acids, more preferably 4 to 35 amino acids, more preferably 4 to 30 amino acids, more preferably 4 to 25 amino acids, or 4 to 24 amino acids, or 4 to 23 amino acids, or 4 to 22 amino acids, or 4 to 21 amino acids, or 4 to 20 amino acids, preferably 5 to 25 amino acids, or 5 to 24 amino acids, or 5 to 23 amino acids, or 5 to 22 amino acids, or 5 to 21 amino acids, or 5 to 20 amino acids.

[0073] The amino acids represent either 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, and these amino acid residues include the epitope and adjacent amino acids.

[0074] 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, including the epitope WEPFA (SEQ ID NO: 1), from the amyloidogenic protein TTR, and five 10-amino acid linker sequences at the N- and C-termini of the 21-amino acid stretch derived from TTR. Thus, in a preferred embodiment, the cyclic peptide compound consists of a total of 20 to 40, more preferably 25 to 35, and most preferably 30 plus or minus 1, 2, 3, or 4 amino acids, or is configured such that its structure resembles the corresponding peptide when non-amino acid residues are incorporated, for example as linkers. In this embodiment, the amino acid sequence derived from an amyloidogenic protein present in the cyclic peptide compound may consist of 10 to 40, preferably 15 to 25, most preferably 20±1, 2, 3, or 4 amino acids, optionally supplemented with a linker sequence preferably 5 to 20, more preferably 5 to 15, most preferably 10±1, 2, 3, or 4 amino acids in length, either distributed at both the N-terminus and C-terminus or at only one terminus. Linker or "filler" sequences may also be located within the amino acid sequence derived from an amyloidogenic protein, for example, when the epitope of the target-binding molecule is a structural or discontinuous epitope.

[0075] As mentioned above, the cyclic peptide compound preferably comprises a peptide derived from an amyloidogenic protein or aggregate thereof involved in systemic amyloidosis, and preferably the peptide comprises at least four amino acids derived from the protein, and most preferably the amyloidogenic protein is TTR, and therefore the cyclic peptide compound comprises a protein fragment of TTR or a peptide derived from TTR.

[0076] The sequence of the cyclic TTR peptide set forth in SEQ ID NO: 17 further reveals that this peptide contains seven amino acids (RKAADDT (SEQ ID NO: 162)) added to the N-terminus of the epitope WEPFA (SEQ ID NO: 1) and nine amino acids SGKTSESGE (SEQ ID NO: 163) added to the C-terminus of the epitope.

[0077] Thus, in one embodiment, a cyclic peptide of the present disclosure comprises at least four 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), and the cyclic peptide further comprises at least seven amino acids added to the N-terminus of SEQ ID NO: 1 and / or at least nine amino acids added to the C-terminus of SEQ ID NO: 1, or a variant thereof. In a preferred embodiment, the cyclic peptide comprises the sequence WEPFASG (SEQ ID NO: 4). Preferably, the N- and / or C-terminal amino acids of the cyclic peptide compound of the present disclosure are attached 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) attached 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) attached to the epitope / peptide sequence WEPFA (SEQ ID NO: 1) at the C-terminus. Preferably, the C-terminal amino acid threonine (T) of the first peptide sequence RKAADDT (SEQ ID NO: 162) is linked 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 linked 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 include a linker comprising or consisting of GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16). More particularly, in a preferred embodiment, a cyclic peptide of the present disclosure comprises a first linker (L1) preferably comprising the sequence GCGGG (SEQ ID NO: 15) linked directly or indirectly 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) linked directly or indirectly 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 with the N-terminus of L1 attached to the C-terminus of L2 via a peptide bond.

[0078] Generally, a TTR protein fragment or peptide 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 four amino acids derived from the TTR protein, and the four amino acids can be, for example, any one of the amino acids listed in Table 1 below.

[0079] [Table 1-1]

[0080] [Table 1-2]

[0081] In a preferred embodiment, the TTR peptide comprises at least four amino acid residues, preferably all amino acids, of the amino acid sequence exposed in the misfolded mutant and on the aggregates, fibrils, and / or oligomers, respectively, and is, for example, WEPFA (SEQ ID NO: 1), which is the peptide recognized by the antibody NI-301.37F1 or NI-301.28B3 disclosed in WO 2015 / 092077(A1); EEFXEGIY (SEQ ID NO: 2), which is the peptide recognized by the antibody NI-301.59F1 disclosed in WO 2015 / 092077(A1); or EEFXEGIY (SEQ ID NO: 3), which is the peptide recognized by the antibody NI-301.35G11 disclosed in WO 2015 / 092077(A1). ELXGLTXE (SEQ ID NO: 3), a peptide recognized by the antibody NI-301.12D3 disclosed in WO 2015 / 092077(A1) (for example, WEPFASG (SEQ ID NO: 4)), a peptide recognized by the antibody NI-301.12D3 disclosed in WO 2015 / 092077(A1) (for example, TTAVVTNPKE (SEQ ID NO: 5)), a peptide recognized by the antibody NI-301.18C4 ... KCPLMVK and VFRK (SEQ ID NOs: 6 and 7), which represent peptides comprising a structural epitope requiring at least C of the first sequence and V and F of the second sequence, and which are epitopes recognized by the antibody NI-301.44E4 of WO 2015 / 092077(A1) (for example, Higaki et al., among others).EHAEVVFTA (SEQ ID NO: 8), a peptide recognized by the antibody 14G8 / PRX004 disclosed in Amyloid 23 (2016), 86-97; GPRYTIAA (SEQ ID NO: 9), a peptide recognized by the antibody 18C5 described in, for example, WO 2019 / 071205; VHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVE (SEQ ID NO: 10), a peptide recognized by the antibody described in, for example, WO 2014 / 124334 (A2), which binds to TTR30-66; ALLSPYSYSTTAV (SEQ ID NO: 11), which is a peptide recognized by antibody 371M described in, for example, WKALGISPFHE (SEQ ID NO: 12), which is a peptide recognized by antibody 371M described in, for example, WO 2015 / 115332 (A1); SYSTTAVVTN (SEQ ID NO: 13), which is a peptide recognized by antibody 313M (RT24) described in, for example, WO 2015 / 115331 (A1); or LLSPYSYSTTAVVTNPKE (SEQ ID NO: 14), which is a peptide recognized by an antibody described in, for example, WO 2014 / 124334 (A2), which binds to TTR100-127.

[0082] Most preferably, the TTR peptide of the present disclosure comprises the amino acid sequence WEPFA (SEQ ID NO: 1).

[0083] All of the above peptides are derived from the TTR protein, with the peptide or fragment VFRK (SEQ ID NO:7) located at the N-terminus of the peptide WEPFA (SEQ ID NO:1), and the peptide or fragment ELXGLTXE (SEQ ID NO:3) located at the C-terminus of the peptide WEPFA (SEQ ID NO:1). Thus, in one embodiment, a cyclic peptide of the present disclosure comprises part or all of a first motif VFRK (SEQ ID NO:7) at its N-terminus and / or part or all of a second motif ELXGLTXE (SEQ ID NO:3) at its C-terminus, wherein X in SEQ ID NO:3 is any naturally occurring amino acid, preferably X at position 3 of SEQ ID NO:3 is histidine (H) and X at position 7 of SEQ ID NO:3 is threonine (T).

[0084] In one embodiment, a cyclic peptide of the present disclosure comprises a portion of a first motif VFRK (SEQ ID NO: 7) comprising at least two amino acids at its N-terminus and a portion of a second motif ELXGLTXE (SEQ ID NO: 3) comprising at least one amino acid at its C-terminus, preferably wherein the amino acids comprising part of the first motif comprise the dipeptide RK of SEQ ID NO: 7 and the amino acids comprising part of the second motif comprise the N-terminal E of SEQ ID NO: 3.

[0085] 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, most preferably comprising the amino acid sequence WEPFA (SEQ ID NO: 1) and adjacent amino acids and linkers at the N- and C-termini of the peptide, which linkers may in principle comprise any of the linker sequences described above, preferably 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 is shown as a suitable target antigen in Examples 1, 2, and 5.

[0086] The present disclosure also relates to a variant of a cyclic peptide compound of the present disclosure comprising the sequence WEPFA (SEQ ID NO: 1) and further comprising a variation of 5% to 20% in terms of amino acid sequence identity, wherein the variation is in an amino acid added to the N-terminus and / or 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 in the N-terminal sequence RKAADDT (SEQ ID NO: 162) and / or an amino acid substitution, addition, or deletion in the C-terminal sequence SGKTSESGE (SEQ ID NO: 163), and particularly preferably the variation is due to an amino acid substitution.

[0087] Preferably, the cyclic peptide compounds or variants thereof of the present disclosure, characterized by comprising the sequence WEPFA (SEQ ID NO: 1) and comprising the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 17), optionally comprising the above-mentioned flanking amino acids, respectively, also include cyclic peptide compounds comprising additional amino acids and peptide / protein fragments, respectively. Thus, in one embodiment, the cyclic peptide compound or variants thereof of the present disclosure further comprises 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 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),

[0088] Alternatively, the cyclic peptide compound or variant thereof of the present disclosure does not contain a second immunogenic sequence selected from the group consisting of EEFXEGIY (SEQ ID NO:2) (wherein X in SEQ ID NO:2 is any amino acid, preferably, 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).

[0089] Additionally or alternatively, the cyclic peptide compounds or variants thereof of the present disclosure do not contain any of the immunogenic sequences of (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.

[0090] When referring to cyclic peptide compounds, the above-mentioned variants are also included.

[0091] In one embodiment, the cyclic peptide compounds of the present disclosure and their precursors, or protein fragments or peptides within the cyclic peptide compounds, respectively, are further derivatized or modified. For example, proteins and / or other agents can be coupled to the cyclic peptide compounds, which can function, for example, as probes in in vitro studies. For this purpose, any functional moiety capable of reacting (e.g., forming a covalent or non-covalent but strong bond) can be used. These proteins and / or other agents can be, for example, carrier proteins such as bovine serum albumin (BSA) used in immunoblot or immunohistochemical assays. Furthermore, the other agent can be a dye.

[0092] The present disclosure further relates to compositions comprising the cyclic peptide compounds disclosed herein or their linear precursors. The compositions can comprise additional excipients, such as buffers, stabilizers, and / or diluents. In one embodiment, the compositions comprise cyclic peptide compounds that are further derivatized as described above, e.g., the compositions comprise cyclic peptide compounds further comprising a conjugate such as a dye.

[0093] Also provided herein are peptide microarrays containing the cyclic peptide compounds of the present disclosure. Peptide arrays are known to those skilled in the art and comprise peptides displayed on a solid surface, usually a glass or plastic chip. Peptide arrays are commonly used to study the binding properties, functionality, and kinetics of protein-protein interactions. 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.

[0094] As shown in Example 1, antigen-binding molecules (here, anti-TTR antibodies) showed strong binding affinity to cyclic peptides in ELISA assays. Therefore, cyclic peptide compounds are suitable target antigens in assays used to detect and quantify antigen-binding molecules such as antibodies.

[0095] Therefore, the present disclosure further relates to the use of the cyclic peptide compounds disclosed herein or the compositions disclosed herein in any type of assay for the analysis of the interaction between a target antigen-binding molecule and a target antigen, for example, for the detection of a target antigen-binding molecule, which may also include quantification. In a preferred embodiment, such an assay is an ELISA assay.

[0096] Cyclic peptides have further been shown to be particularly suitable target antigens in the efficacy assays described herein. Thus, in a further preferred embodiment, the present disclosure relates to the use of the cyclic peptide compounds disclosed herein or the compositions disclosed herein for determining the efficacy of antigen-binding molecules, such as antibodies or any other binding molecules comprising an Fc domain, preferably the efficacy of antibodies as defined herein. The determination of efficacy is preferably carried out using the assays disclosed herein, i.e., the assays described in Examples 2 and 5 and WO 2023 / 099788(A1) (the contents of which are incorporated herein by reference).

[0097] Potency testing is performed as part of product suitability, comparability, 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, potency, and stability of products used throughout clinical trials. Similarly, potency measurements are used to ensure that only product lots, i.e., batches that meet defined specifications or acceptance criteria, are administered throughout all phases of clinical studies and after market approval. Potency is defined as "the specific performance or ability of a product to produce a given result as demonstrated by appropriate laboratory tests or by well-controlled clinical data obtained by administering the product in the intended manner." Ideally, potency assays represent the product's mechanism of action (i.e., relevant therapeutic activity or intended biological effect); see "Guidance for Industry - Potency Tests for Cellular and Gene Therapy Products," U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research, January 2011. In the context of the assay of the present invention, the "potency" of a target antigen-binding molecule, specifically an antibody as a pharmaceutical, is therefore a measure of its activity in the ADCP assay relative to the activity of a reference standard (of the pharmaceutical) whose activity in the ADCP assay and activity level, respectively, have been assessed or are known. Thus, a higher potency of an antibody / preparation compared to a reference means that the antibody / preparation has a higher binding activity in the ADCP assay, i.e., a lower EC 50 A lower potency of an antibody / formulation compared to a reference means that the antibody / formulation has a 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 has an EC value 0.7 times that of the reference sample, NI-301.37F1 RS (100%). 50 In contrast, antibody NI-301.37F1 50% mimics an antibody with lower potency (loss of activity) and exhibits an EC2 value twice that of the reference sample, NI-301.37F1 RS. 50values ​​are shown (100%); see Example 2. As shown in Example 5, even potency changes of 60% to 80% could be observed. Thus, a target antigen-binding molecule (e.g., an antibody) exhibiting increased potency has, e.g., at least 1%, e.g., at least 5%, e.g., at least 10%, or more (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) lower EC relative to a reference sample, as determined, e.g., by an ADCP assay described herein. 50 Alternatively, a target antigen-binding molecule (e.g., an antibody) exhibiting reduced potency may have, e.g., at least a 1%, e.g., at least a 5%, e.g., at least a 10% or more (e.g., at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) higher EC value relative to a reference sample, e.g., when measured in an ADCP assay described herein. 50 However, according to GLP and GMP, the allowable variation (i.e., imprecision) in potency measurements is ±20%, and therefore this variation is the preferred limit for the target antigen-binding molecule tested.

[0098] As described above, the cyclic peptide compounds and corresponding compositions of the present disclosure are preferably used to determine the potency of antigen-binding molecules, and the determination of potency is preferably carried out using the assays disclosed herein, i.e., Examples 2 and 5 and WO 2023 / 099788(A1) (the contents of which are incorporated herein by reference).

[0099] Such a potency assay preferably comprises the following steps: (a) contacting a cyclic compound of the present disclosure as a target antigen with a binding molecule under conditions that allow for the formation of a binding molecule-antigen complex; (b) contacting a population of effector cells engineered to express an Fc receptor and harbor a reporter gene with the binding molecule-antigen complex under conditions that allow binding of the Fc domain to the Fc receptor under the control of a response element that responds to activation by the Fc receptor, wherein binding of the Fc domain to the Fc receptor results in intracellular signaling that mediates quantifiable reporter gene activity; and c) detecting reporter gene activity, 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 indicates efficacy of the binding molecule.

[0100] More particularly, such an assay preferably comprises at least the following steps: i) spotting the target antigen, i.e., the cyclic peptide compound of the present disclosure, into the wells of a microplate, i.e., coating a microplate (96-well plate) with the target antigen overnight (or 18 hours ± 2 hours) at 4°C, preferably diluting the cyclic compound to 3 μg / mL in PBS buffer at pH 7.4; ii) contacting the target antigen with the target antigen-binding molecule under conditions that allow the formation of a binding molecule-target antigen complex, preferably at 37°C for 30 minutes; iii) contacting the complex comprising the binding molecule and the target antigen with effector cells, i.e., adding effector cells, also called reporter cells, to the complex, which express an Fc receptor and a reporter gene under the control of a response element responsive to activation by the Fc receptor, preferably the effector cells are genetically engineered cells, more preferably Jurkat cells expressing the FcγRI receptor and the luciferase gene under the control of an NFAT transcription factor, and the complex is preferably incubated at 37° C. for 6 hours; iv) adding a substrate solution, preferably a luminescent substrate solution; and v) detecting a signal, preferably a luminescent signal, using a luminometer.

[0101] A detailed description of the steps of such a potency assay is provided in WO 2023 / 099788 A1, in particular pages 23-32 and 38-46, the contents of which are incorporated herein by reference.

[0102] The binding molecule whose potency, particularly its potency to induce ADCP, is determined using the method described herein, i.e., the potency assay using the cyclic peptides of the present disclosure, can be any binding molecule that binds to a 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 a method to determine the potency, preferably the potency to induce ADCP, of any binding molecule that binds to the cyclic peptide compound.

[0103] Preferably, the binding molecule is an antibody or any other binding molecule comprising an Fc domain. In a preferred embodiment, the cyclic peptide compounds are used to determine the potency of anti-TTR antibodies, particularly their potency in inducing ADCP, most preferably the potency of the anti-TTR antibody NI-301.37F1 (which comprises in its variable region or binding domain the amino acid sequences of the VH and VL chains of SEQ ID NO: 19 and SEQ ID NO: 21 or SEQ ID NO: 23 and SEQ ID NO: 21).

[0104] [Table 2]

[0105] The present disclosure further relates to methods for preparing pharmaceutical compositions comprising said binding molecules as defined above, i.e., preferably antibodies that bind to amyloidogenic proteins involved in systemic amyloidosis, most preferably anti-TTR antibodies.

[0106] In a first step, the binding molecule and the formulation are provided and preferably manufactured, respectively. Systems and methods for the recombinant production of antibodies, their corresponding binding molecules, fragments, derivatives and mimetics are known in the art. In particular, their recombinant production in host cells, purification, modification, formulation into pharmaceutical compositions and therapeutic use, as well as terms and characteristics common in the art, may be relied upon by a person skilled in the art when carrying out the present disclosure as claimed (see, for example, Antibodies A Laboratory Manual, 2nd Edition, 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA; Frenzel et al., Front Immunol. 4 (2013), 217, doi:10.3389 / fimmu.2013.00217; Lalonde and Durocher, Journal of Biotechnology 251 (2017), 128-140, doi:10.1016 / j.jbiotec.2017.04.028; Tripathi and (See Shrivastava, Front. Bioeng. Biotechnol. 7 (2019), 420, DOI: 10.3389 / fbioe.2019.00420), which also describes antibody purification and storage; antibody engineering, including the use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis, immunoblotting protocols, and state-of-the-art screening and labeling techniques. The production of DARPins is described, for example, in Stumpp et al., Drug Discovery Today 13 (2008), 695-701, and references cited therein, and Hanenberg et al., J Biol Chem 289 (2014), 27080-27089, DOI: 10.1074 / jbc.M114.564013. Furthermore, preparation of formulations can be carried out in any manner desirable and / or suitable for the formulation.

[0107] In the next step, the binding molecule is subjected to the method described herein.In particular, the binding molecule is subjected to the method for determining the efficacy of the binding molecule, particularly the efficacy of inducing ADCP, thereby using the cyclic peptide compound of the present disclosure as the target antigen.The information obtained from the assay is used as part of the evaluation of whether the binding molecule can be used as a pharmaceutical composition, that is, whether the formulation containing the binding molecule meets the criteria for injection into patients agreed with the regulatory authorities of the country where the formulation can be injected.Furthermore, this information is used to identify the binding molecule for use in the pharmaceutical composition.

[0108] In a further preferred embodiment, the binding molecules are formulated as pharmaceutical compositions together with a pharmaceutically acceptable carrier, particularly those binding molecules that have been found to be useful by the methods described herein, i.e., potency assays using the cyclic peptide compounds of the present invention. Useful binding molecules have, for example, an EC50 activity in the (sub)nanomolar range when assessed by the methods of the present disclosure. 50The binding molecule exhibits a value similar to that of a reference standard, for example, a potency of at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, and more preferably 100% as compared to the potency of a positive control. Pharmaceutically acceptable carriers and administration routes can be obtained from 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 the Sciences, Philadelphia, ISBN 0-683-306472; Vaccine Protocols, 2nd Edition, edited by Robinson et al., Humana Press, Totowa, NJ, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 2nd Edition, 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, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject at an appropriate dosage. Administration of a suitable composition can be achieved in various ways. Examples include administering a composition containing a pharmaceutically acceptable carrier orally, intranasally, rectally, topically, intraperitoneally, intravenously, intramuscularly, subcutaneously, transdermally, intrathecally, and intracranially.

[0109] The present disclosure also provides a process for preparing a pharmaceutical or diagnostic agent 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% of the potency of a positive control to activate an ADCP. The process includes producing the binding molecule as described above to obtain a batch of the binding molecule. The potency of the batch, particularly the potency of the batch to activate an ADCP described herein, is then analyzed. The process further includes preparing a pharmaceutical or diagnostic agent from the batch, provided that the batch is determined to have at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, and more preferably 100% of the potency of the positive control, particularly the potency of the ADCP.

[0110] The control is either a reference standard, an antibody known to have the efficacy of activating ADCP, e.g., an antibody approved by a regulatory agency, and / or the batch being analyzed has been stored and / or exposed to stress conditions, and the control is the value of reporter gene activity of a sample taken from the batch or a corresponding batch before storage and / or exposure to the stress conditions.

[0111] The present disclosure also provides a method for determining the efficacy of such an antibody, which method is part of an application for marketing approval to sell the formulation as a pharmaceutical composition. The present disclosure also provides a method for applying for marketing approval of a formulation comprising a binding molecule, the method comprising a method described herein for determining the efficacy of the binding molecule in the formulation.

[0112] As mentioned above, the methods described herein are used as batch release potency assays, i.e., the methods are useful for analyzing various batches from the manufacture of, for example, a given target antigen-binding molecule.

[0113] Continuous manufacturing of pharmaceutical formulations results in the production of various batches of a product for pharmaceutical release. A key feature of manufacturing is ensuring that the various batches meet the same standards. These standards are usually set in conjunction with regulatory agencies. Typically, each batch is tested and inspected using a number of different assays to ensure that the batch is of sufficient quality to be approved for market. This can be accomplished using the potency assays described herein using the cyclic peptide compounds of the present disclosure.

[0114] 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, which comprises, in a first step, evaluating the potency of a sample of the batch, particularly its potency in activating ADCP, using a method using the cyclic peptide compounds described herein. As described above, reporter gene activity is a measure of the potency of a binding molecule. Therefore, the reporter gene activity of the sample is compared with that of a control, and batches are selected in which the sample exhibits greater, equal, or not substantially less reporter gene activity than the control. In one embodiment, batches are selected in which the sample exhibits 80% or more, preferably 90%, preferably 95%, preferably 98%, preferably 99%, and more preferably 100% reporter gene activity compared to the control. The selected batches can be further packaged, for example, in kits and distributed to customers.

[0115] Thus, the present disclosure relates to a process for validating a batch of target binding molecules, i.e., a process for determining the quality of target antigen (e.g., aggregation protein) binding molecules, in which a sample of the batch is tested for its potency to activate ADCP using a potency assay described herein using cyclic peptide compounds of the present disclosure, and the batch is validated for distribution only if the potency of the sample of the batch to activate ADCP is at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, and more preferably 100% compared to the potency of the positive control to activate ADCP.

[0116] In preferred embodiments, the method is particularly useful for analyzing and selecting batches of pharmaceutical compositions comprising anti-TTR antibodies and for validating batches of anti-TTR antibodies for distribution, respectively. The control can be a reference standard and / or, if the batch being analyzed has been stored and / or exposed to stress conditions, the control can be the value of reporter gene activity of a sample taken from the batch or a corresponding batch before storage and / or exposure to the stress conditions.

[0117] The binding of the binding molecule of the formulation to the Fc receptor is compared to the binding of the reference standard to the Fc receptor, and the therapeutic efficacy of the binding molecule of the formulation is assessed by whether its ability to bind to the Fc receptor is the same as, or substantially the same as, that of the reference standard.

[0118] As noted above, the potency of a batch of samples 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% of that of the reference standard. However, the specific extent to which the FcR binding profile of a binding molecule of a drug product and the FcR binding profile of the reference standard may differ can be established on a case-by-case basis, e.g., determined in cooperation with appropriate regulatory authorities.

[0119] To be able to determine FcR binding in a reliable and consistent manner, the FcR binding of the binding molecules of the drug product and the reference standard must be performed using the same assay, preferably using the assay described herein. Determining the binding of the reference standard is typically performed first to establish a standard against which any subsequent batches of binding molecules can be compared. However, determining the binding of the reference standard may be performed simultaneously with or after determining the FcR binding of the binding molecules of the drug product.

[0120] Accordingly, the present disclosure further relates to the use of the cyclic peptide compounds of the present disclosure in the above-mentioned methods and processes, i.e., in a method for manufacturing a pharmaceutical composition, a process for preparing a medicament or diagnostic agent, a process for applying for marketing approval to sell the formulation as a pharmaceutical composition, a method for applying for marketing approval of the formulation, a method for analyzing and selecting at least one batch of a pharmaceutical composition, a process for validating a batch of a target binding molecule, i.e., a process for determining the quality of a target antigen-binding molecule for distribution, and particularly in a method for analyzing and selecting a batch of a pharmaceutical composition comprising an anti-TTR antibody and a method for validating a batch of an anti-TTR antibody.

[0121] The present disclosure further 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.

[0122] To verify that the analyzed binding molecule actually induces phagocytosis and results in the engulfment of a target antigen, e.g., a cyclic peptide compound, a phagocytosis assay can be performed, for example, via the in vivo phagocytosis assay described in Prakash et al., Chem Sci. 12 (2021), 10901-10918 for monitoring the phagocytic uptake of amyloid beta in real time, or preferably, via the in vitro phagocytosis assay described in Examples 1 and 2 of WO 2023 / 099788 (A1), the contents of which are incorporated herein by reference. These assays demonstrated that antibody NI-301.37F1_W1 indeed induces phagocytosis of TTR aggregates. Thus, the disclosed methods for assaying the efficacy of binding molecules can be combined with phagocytosis assays, particularly in vitro phagocytosis assays. Furthermore, verification that the analyzed binding molecules indeed induce phagocytosis, resulting in the engulfment of target antigens such as aggregated TTR, can be performed using the patient-derived amyloid xenograft animal model disclosed in WO 2020 / 094883(A1).

[0123] Furthermore, the binding of the analyzed binding molecule to its corresponding antigen can be verified by methods known in the art, for example, via ELISA or BLI as shown in Example 1. Thus, the disclosed methods for assaying the potency of a binding molecule can be combined with methods for determining the binding of a binding molecule to its antigen.

[0124] As mentioned above, determining the efficacy of a drug is an important step in the development, including the evaluation of new therapeutic agents for treating diseases. In the context of the present disclosure, the above-mentioned method is used for the development, evaluation, and batch release of antibody drugs and other target antigen-binding molecules that utilize the effector function of the Fc domain for the treatment of target protein-related diseases, particularly protein aggregation diseases, such as systemic and localized amyloidosis.

[0125] Thus, the cyclic peptide compounds of the present invention are particularly useful as target antigens in assays for measuring the efficacy of antibodies that normally target protein aggregates, i.e., amyloidogenic proteins, and are therefore useful in the treatment of protein aggregation diseases, such as systemic and localized amyloidosis, particularly diseases associated with TTR aggregation.

[0126] Furthermore, the cyclic peptide compounds of the present disclosure or compositions containing them 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, for example, to identify antibodies equivalent to NI-301.37F1. Similarly, the cyclic peptides can be used, for example, by phage display, to screen for antibodies against amyloidogenic proteins, particularly for anti-TTR antibodies in general.

[0127] Furthermore, cyclic peptides can be used to study the pharmacokinetic profile, i.e., half-life of an antibody in plasma in in vivo non-human animal studies as well as in human clinical trials, using, for example, antibody NI-307.37F11 (NI006) or antibody NNC6019-0001 (PRX004). Furthermore, cyclic peptide compounds can be used, for example, during the course of antibody treatment, to measure the plasma concentration of the antibody and assist in dosing to maintain sustained levels of the antibody.

[0128] The cyclic peptides can also be used to identify antibodies equivalent to known antibodies, particularly antibodies equivalent to the mentioned anti-TTR antibodies, particularly antibody NI-307.37F11, for example, by competitive assays commonly known in the art, and all such uses are therefore also part of this disclosure.

[0129] Further disclosed herein is a kit comprising at least the cyclic peptide compound of the present disclosure or its linear precursor, optionally together 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., for detection, which may also include quantification of the target antigen-binding molecule, most preferably for determining the potency of an antigen-binding molecule comprising an Fc domain, such as an antibody. In a preferred embodiment, the potency is determined using an assay disclosed herein, particularly the assay described in the accompanying Examples and WO 2023 / 099788(A1), respectively; therefore, the kit preferably includes means for performing a corresponding potency assay. In a further preferred embodiment, the antigen-binding molecule is an antigen-binding molecule as defined herein above, preferably an antigen-binding molecule comprising an Fc domain, such as an antibody, most preferably an anti-TTR antibody. Thus, the kit can be used for the purposes listed above.

[0130] In one embodiment, the kit comprising the cyclic peptide compound comprises: (i) a population of effector cells engineered to express the human Fc receptor FcγR and to carry a reporter gene under the control of a response element that responds to activation by the Fc receptor; (ii) a substrate corresponding to the reporter; and optionally, (iii) a solid support, preferably a microtiter plate, preferably a 96-well plate including a lid; (iv) washing, blocking and assay / sample dilution buffers, and / or (v) a monomeric control of the target antigen and / or a positive control anti-target antigen antibody.

[0131] In a preferred embodiment, the effector cell population is a Jurkat cell population expressing a gene encoding FcγR, preferably FcγRI, and a luminescent protein, preferably luciferase, under the control of an NFAT transcription factor, and the kit includes a luminescent substrate solution. Furthermore, the cyclic peptide compound preferably contains an epitope of an anti-TTR antibody and an epitope of TTR, respectively, and the binding molecule is an anti-TTR antibody.

[0132] Preferably, the kit is adapted to assay the efficacy of a binding molecule comprising an Fc domain to induce ADCP.

[0133] Furthermore, the cyclic peptide compounds of the present disclosure and their linear precursors are particularly useful in methods for identifying, and optionally obtaining, antibodies and equivalent binding molecules (e.g., of the type described hereinabove) that bind to amyloidogenic proteins involved in systemic amyloidosis, which methods typically comprise the following steps: (a) providing and optionally producing one or more potential amyloidogenic protein-binding antibodies or a source thereof; (b) subjecting one or more potential amyloidogenic protein-binding antibodies or sources thereof to a binding assay comprising a cyclic peptide compound of the present disclosure; and (c) identifying and optionally obtaining an antibody (an antibody of the invention) that has been determined to bind to the cyclic peptide compound.

[0134] This method can be combined with the potency assays of the present disclosure and / or those described in WO 2023 / 099788 A1, and / or any other suitable method for further determining the diagnostic or preferably therapeutic utility of the antibodies of the invention. As mentioned above, the antibodies of the present invention may be various types of antigen-binding molecules.

[0135] Accordingly, there is provided a method for producing a pharmaceutical composition comprising an antibody that binds to a systemic amyloidogenic protein, the method comprising at least (a) providing and optionally producing one or more potential amyloidogenic protein-binding antibodies or a source thereof; (b) subjecting the one or more potentially amyloidogenic protein-binding antibodies or a source thereof to a binding assay comprising a cyclic peptide compound of the present disclosure; (c) identifying and optionally obtaining antibodies that bind to the cyclic peptide compounds (antibodies of the invention); and (d) formulating the antibody or derivative thereof identified and optionally obtained in step (c) with a pharmaceutically acceptable carrier.

[0136] Sources of antibodies are not limited and include, for example, natural and synthetic antibodies obtained from immunized laboratory animals, such as rodents, preferably mice, and most preferably Ig-humanized mice; human blood or its fractions, preferably containing memory B cells; recombinant antibody libraries, such as phage, yeast, and ribosomal systems; and mammalian cell lines, such as CHO and HEK. In one embodiment, nanobodies (also known as VHHs) derived from serum of animals of the Camelidae family can be screened using the cyclic peptide compounds of the present disclosure; see, for example, Lyu et al., Anal. Chem. 94 (2022), 7970-7980; Muyldermans, The FEBS Journal 288 (2021) 2084-210. In this regard, IgG antibody binding fragments known to bind to amyloidogenic proteins can be used as reference antibodies or sources for the identification and preparation of nanobodies, respectively. Similarly, synthetic alternatives to antibodies can be designed with computational modeling, e.g., modular peptide binders such as designed armadillo repeat proteins (dArmRPs), can be screened; see, e.g., Gisdon et al., Biological Chemistry 403 (2022), 535-543.

[0137] Preferably, the binding assay used in the above method comprises an ELISA such as that performed in Example 1.

[0138] In a preferred embodiment of the disclosed method for identifying and obtaining the antibodies of the present invention, and their further use when formulated into a pharmaceutical composition and in drug discovery, respectively, the antibody identified and optionally obtained in step (c) competes with the reference antibody for binding to the amyloidogenic protein, and preferably the antibody of the present invention has a lower EC2 for the amyloidogenic protein than the reference antibody. 50 The preparation and formulation of the antibodies and similar target-binding molecules of the present invention obtained by the methods of the present invention can be carried out as described above for the target antigen-binding molecules.

[0139] The present disclosure also relates to antigen-binding molecules, particularly antibodies, that have been screened for the cyclic peptide compounds of the present disclosure 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 described in the accompanying Examples for screening.

[0140] Several documents are cited throughout the text of this specification. The contents of all cited references (including literature, issued patents, published patent applications, etc., cited throughout this application, including background sections and manufacturer's specifications, instructions, etc.) are expressly incorporated herein by reference; however, no admission is made that the cited documents are in fact prior art with respect to this invention.

[0141] 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. [Example]

[0142] Example 1: Cyclic peptides as target antigens provide higher sensitivity of ELISA assays for antigen-binding molecules The ability of antigen-binding molecules to bind to cyclic peptides was evaluated by ELISA using a cyclic peptide containing amino acid residues 34 to 54 of wild-type TTR (biotinylated and non-biotinylated forms of TTR34-54cyc) as the target antigen and the anti-TTR antibody NI-301.37F1 as the antigen-binding molecule. Furthermore, the TTR peptide TTR40-49, biotinylated TTR peptide TTR40-49, and misfolded wild-type TTR (mis.WT-TTR) were used as antigen controls.

[0143] The cyclic peptide TTR34-54cyc (1.36 mg / mL) was manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. In particular, a peptide containing 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. The TTR peptide (TTR40-49, 1.25 mg / mL) containing 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 contain an aminohexanoic acid (Ahx) spacer between their N-termini and the biotin residue, 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), respectively. Misfolded wild-type TTR was prepared as follows: Purified wild-type TTR protein from human plasma was obtained from Bio-Rad Laboratories (California, USA; 7600-0604) and subjected to protein A / G chromatography, followed by custom purification on a lectin column to remove residual immunoglobulins. Plasma-purified WT-TTR was prepared as a 1 mg / mL solution 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 for 4 hours with shaking at 1000 rpm. 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).

[0144] Two ELISA assays were performed: the first (ELISA-1) analyzed antibody binding to the peptides TTR34-54cyc, TTR40-49, biotin.TTR40-49, and mis-WT-TTR; the second (ELISA-2) analyzed antibody binding to the peptides TTR34-54cyc, biotin.TTR34-54cyc, TTR40-49, biotin.TTR40-49, and mis-WT-TTR.

[0145] Specifically, 96-well microplates 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 h at 37°C. Each target antigen was diluted to a concentration of 10 μg / mL in PBS buffer (pH 7.4). Nonspecific binding sites were blocked for 1 h at room temperature (RT) with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% Tween 20 in PBS buffer. The NI-301.37F1 antibody (Neurimmune AG, Zurich, Switzerland; NI-301.37F1) was diluted in blocking buffer in duplicate 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 with horseradish peroxidase (HRP), followed by measurement of HRP activity with a standard colorimetric assay (Thermo Fisher Scientific, Waltham, MA, USA). Data were analyzed using GraphPad Prism software. EC 50 Values ​​were estimated using nonlinear regression of individual data points using a log(agonist) vs. response model with variable slope. Data fitting was performed by least squares regression.

[0146] The ELISA results confirmed the binding of NI-301.37F1 to mis.WT-TTR. Furthermore, the ELISA assays showed that NI-301.37F1 binding to the cyclic TTR34-54cyc and biotin.TTR34-54cyc peptides was much stronger than that to mis.WT-TTR, i.e., approximately 10-fold stronger. Notably, in ELISA-1, the EC of NI-301.37F1 binding to the cyclic TTR34-54cyc peptide was significantly higher than that to mis.WT-TTR. 50 The EC of NI-301.37F1 binding to mis.WT-TTR was 27 pM. 50 The EC was 338 pM; see Figure 1A. In ELISA-2,50 Although the measured values ​​were higher, the approximately 10-fold difference between the binding of cyclic NI-301.37F1 to the TTR34-54cyc peptide and that to mis.WT-TTR remained. In particular, the EC 50 The EC of NI-301.37F1 binding to mis.WT-TTR was 0.66 nM. 50 was 8.3 nM; see Figure 1B. Binding of NI-301.37F1 to TTR40-49 and biotin.TTR40-49 was not observed in either ELISA assay.

[0147] Example 2: Cyclic peptides as target antigens provide an improved ADCP assay An ADCP assay for determining the potency of antigen-binding molecules has been developed. The assay has been evaluated, for example, for its ability to measure the potency of the antibody NI-301.37F1 in activating phagocytosis of the cyclic TTR peptide (TTR34-54cyc) in vitro using a reporter cell line expressing human Fcγ receptor 1 (FcγR1) and the cyclic peptide as the antigen.

[0148] ADCP reporter assay To measure the potency of the NI-301.37F1 reference sample (NI-301.37F1 RS, 100%) and test samples with 50% lower (NI-301.37F1 50%), 30% lower (NI-301.37F1 70%), 30% higher (NI-301.37F1 130%), and 50% higher (NI-301.37F1 150%) concentrations in activating phagocytosis of TTR34-54cyc, a commercially available FcγR1 ADCP reporter bioassay (Promega, Madison, WI, USA, catalog numbers GA1341 and GA1345) was applied. This bioluminescent cell-based assay relies on a genetically engineered Jurkat T cell line expressing human FcγR1 with a luciferase reporter driven by an NFAT response element. FcγR1 activation by the antibody-target complex leads to activation of NFAT pathway signaling and luciferase expression, which is detected using a bioluminescent luciferase substrate. Briefly, 96-well plates were 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) using a blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% Tween 20 in PBS buffer. A dilution plate for the assay was prepared by diluting the NI-301.37F1 antibody to the indicated concentrations (500 ng / mL to 0.4 ng / mL) in ADCP buffer (96% RPMI 1640 medium, 4% low IgG serum). Assays were performed by adding 1 unit volume of antibody diluent and incubation was carried out for 30 minutes at 37°C and 5% CO2, followed by the addition of 1 unit volume of FcγR1 reporter cells in ADCP buffer at a density of approximately 1.65 x 10^5 cells / well. Assays were incubated for 6 hours at 37°C and 5% CO2, after which luminescent substrate (Bio-Glo™ Luciferase Assay Reagent) was added. Luminescence measurements (integration time: 1000 ms, settling time: 0 ms) were taken after 15 minutes of incubation at room temperature.

[0149] NI-301.37F1 RS vs. NI-301.37F1 50%, NI-301.37F1 70%, NI-301.37F1 130%, and NI-301.37F1 150% In the first experiment, antibody NI-301.37F1 RS showed an EC 50 It was shown that the antibody exhibited a dose-response characterized by a β-associated β-association. Specifically, plates were coated with 3 μg / mL of the cyclic peptide TTR34-54cyc, and antibody dilutions ranging from 500 to 0.4 ng / mL were tested. These conditions yielded a reasonable response curve with a stable slope and lower and upper asymptote; see Figure 2.

[0150] Further experiments were performed in which the response of this assay was tested at NI-301.37F1 concentrations of 50%, 70%, 130%, and 150%. As shown in Figures 3A-D and Table 3 below, NI-301.37F1 50% had an EC of 39 ng / mL. 50 The dose response was characterized by EC 50 The 1.97-fold increase in NI-301.37F was almost perfectly matched by half the concentration of the NI-301.37F 50% sample. NI-301.37F1 70% had an EC of 30.4 ng / mL. 50 The dose response was characterized by EC 50 The 1.43-fold increase in NI-301.37F1 was in excellent agreement with the expected difference of 1.54-fold. NI-301.37F1 130% had an EC of 13.6 ng / mL. 50 The dose response was characterized by EC 50 The 0.77-fold increase in NI-301.37F1 was in perfect agreement with the expected difference of 0.77-fold. NI-301.37F1 150% had an EC of 13.5 ng / mL. 50 The dose response was characterized by EC 50 The 0.67x increase in was almost perfectly matched by the expected difference of 0.70x.

[0151] Thus, assay variation was minimal, and the results demonstrated that the assay was responsive to changes in antibody concentration. In particular, the FcγR1 ADCP assay was shown to have the ability to detect up to a 50% loss of antibody activity and up to a 50% increase in antibody activity with excellent precision.

[0152] [Table 3]

[0153] Example 3: Evaluation of additional cyclic peptides as target antigens for antigen-binding molecules As shown in Example 1, the cyclic peptide TTR34-54cyc was successfully used as the target antigen for 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 either the TTR epitope EHAEVVFTA (SEQ ID NO: 8) or the TTR epitope GPRRYTIAA (SEQ ID NO: 9), namely, TTR89-97cyc and TTR101-109cyc, as described above, was assessed in 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, supra. For the ELISA assay, 96-well microplates are coated with the two cyclic peptides TTR89-97cyc and TTR101-109cy and an antigen control, and the assay is performed as described in Example 5 supra.

[0154] Example 4: Evaluation of additional cyclic peptides as target antigens in the ADCP assay As shown in Example 2, the efficacy of anti-TTR antibody NI-301.37F1 in activating the phagocytosis of the cyclic TTR peptide (TTR34-54 cyc) in vitro was successfully determined using an ADCP assay. Therefore, the efficacy of anti-TTR antibodies in activating the phagocytosis of two cyclic peptides, TTR89-97 cyc and TTR101-109 cyc, will be evaluated in further ADCP assays.

[0155] The commercially available FcγR1 ADCP reporter bioassay (Promega, Madison, WI, USA, catalog numbers GA1341 and GA1345) described in Example 2 was applied to measure the phagocytosis-activating potency of the two cyclic peptides in the anti-TTR antibody reference sample (anti-TTR antibody RS, 100%) and test samples at 50% lower (anti-TTR antibody 50%), 30% lower (anti-TTR antibody 70%), 30% higher (anti-TTR antibody 130%), and 50% higher (anti-TTR antibody 150%) concentrations.

[0156] Anti-TTR antibodies showed a dose-response, with anti-TTR 50% antibody showing approximately two-fold increased EC compared with anti-TTR RS antibody. 50 The anti-TTR antibody 70% showed an approximately 1.5-fold increased EC value compared to the anti-TTR antibody RS. 50 The EC values ​​for anti-TTR antibody 130% were approximately 0.77-fold reduced compared to anti-TTR antibody RS. 50 The EC values ​​for anti-TTR antibody 150% were approximately 0.70-fold reduced compared to anti-TTR antibody RS. 50 It is expected that the value will be

[0157] Example 5: Validation of the ADCP assay An ADCP assay for determining the potency of antigen-binding molecules having 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 the antibody NI-301.37F1 is illustratively evaluated for its ability to determine the potency of activating phagocytosis of the cyclic TTR peptide (TTR34-54cyc) in vitro.

[0158] The reliability and accuracy of the cyclic peptide-based ADCP assay has been confirmed under validated experimental conditions to determine antibody potency ranging from 40% to 180% of theoretical relative activity.

[0159] ADCP reporter assay with luminescent readout for NI-301.37F1 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 incubating for 18±2 hours at 5° C. On day 2, the coating solution was removed, and after adding 200 μL of blocking buffer (2% BSA and 0.1% Tween 20 in DPBS), the plates were incubated for 60±5 minutes at room temperature on a plate shaker at 300 rpm.

[0160] Standard solutions of the reference standard and test items were prepared in parallel; see Figure 4. The reference standard (RS) corresponded to an antibody concentration of 4000 ng / mL in ADCP buffer (4% low IgG serum (v / v) in RPMI 1640 medium). Test items were generated from a reference standard stock solution with an antibody concentration of 49.9 mg / mL.

[0161] [Table 4]

[0162] Additionally, FcγR1 effector cell suspension was prepared using the instructions of a commercially available FcγR1 ADCP reporter bioassay kit (Promega, USA, #GA1345). Approximately 3 × 10 6 cells / mL were obtained.

[0163] One assay plate was prepared to analyze one test item and compare it with the reference standard in triplicate per dose. The assay plate was incubated with blocking buffer, after which the blocking buffer was removed. After washing the assay plate with 200 μL of DPBS, 55 μL of antibody standard solution was transferred to the assay plate and incubated at 37°C and 5% CO2 for 30 ± 5 minutes. After the assay plate incubation period, 55 μL of effector cell suspension was transferred to the assay plate and incubated at 37°C and 5% CO2 for 6 hours ± 15 minutes. The plate layout is shown in Table 5.

[0164] [Table 5]

[0165] The assay plate was removed from the incubator and incubated at room temperature for 10 ± 5 minutes, after which 110 μL of Bio-Glo™ Luciferase Assay Reagent (Promega, USA, #GA1345) was added. The assay plate was then covered with a black lid and incubated at room temperature for 15 ± 5 minutes. Finally, the luminescence of the assay plate was measured using a multiplate reader with glow-type luminescence reading capabilities. The results are shown in Table 6.

[0166] [Table 6]

[0167] Conclusion: These data demonstrate the utility of the disclosed cyclic TTR peptides (e.g., TTR34-54cyc) in screening anti-TTR antibody candidates that can potently activate phagocytosis of TTR amyloid plaques. This example also provides a validated ADCP reporter assay method to easily and reliably determine the efficacy of anti-TTR antibodies as agents for treating and / or managing TTR amyloidosis.

Claims

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

2. 2. The cyclic compound according to claim 1, comprising a linker, wherein the linker is preferably an amino acid linker or a non-amino acid linker.

3. 3. The cyclic compound of claim 2, wherein the linker is covalently attached to or near the N-terminal residue of the peptide and the C-terminal residue of the peptide.

4. 4. The cyclic compound according to claim 1, wherein the peptide in the cyclic compound comprises at least 5, preferably at least 10, more preferably at least 15, and most preferably at least 20, 21, 22, 23, 24, or 25 amino acid residues of the amyloidogenic protein.

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

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

7. 7. The cyclic compound of claim 6, wherein the TTR peptide comprises at least four amino acid residues of any one of amino acid sequences selected from WEPFA (SEQ ID NO: 1), EEFXEGIY (SEQ ID NO: 2), ELXGLTXE (SEQ ID NO: 3), WEPFASG (SEQ ID NO: 4), TTAVVTNPKE (SEQ ID NO: 5), KCPLMVK and VFRK (SEQ ID NOs: 6 and 7), EHAEVVFTA (SEQ ID NO: 8), GPRRYTIAA (SEQ ID NO: 9), VHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVE (SEQ ID NO: 10), ALLSPYSYSTTAV (SEQ ID NO: 11), WKALGISPFHE (SEQ ID NO: 12), SYSTTAVVTN (SEQ ID NO: 13), and LLSPYSYSTTAVVTNPKE (SEQ ID NO: 14), and wherein X can be any naturally occurring amino acid.

8. The cyclic compound of claim 6 or 7, wherein the cyclic compound comprises at least four consecutive amino acid residues of the peptide sequence WEPFA (SEQ ID NO: 1), and further comprises at least seven amino acids added to the N-terminus of SEQ ID NO: 1 and / or at least nine amino acids added to the C-terminus of SEQ ID NO: 1, or variants thereof.

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

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

11. The cyclic compound according to any one of claims 8 to 10, wherein the amino acid at the N-terminus and / or 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) added to the N-terminus of the peptide sequence WEPFA (SEQ ID NO: 1), and / or a second peptide sequence SGKTSESGE (SEQ ID NO: 163) added to the C-terminus of the peptide sequence WEPFA (SEQ ID NO: 1).

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

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

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

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

17. 17. The cyclic compound according to any one of claims 1 to 16, comprising 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).

18. 18. The cyclic compound of claim 17, wherein L1 is directly or indirectly linked to the N-terminus of the peptide sequence WEPFA (SEQ ID NO: 1), and L2 is directly or indirectly linked to the C-terminus of the peptide sequence WEPFA (SEQ ID NO: 1).

19. 19. The cyclic compound of claim 18, wherein L1 and L2 are linked to form a cyclic peptide, preferably wherein the N-terminus of L1 is linked to the C-terminus of L2 via a peptide bond.

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

21. 21. The cyclic compound of claim 20, wherein the epitope is accessible for binding by the antibody only in misfolded and / or aggregated forms of the protein, preferably wherein the epitope is exposed in abnormal protein aggregates.

22. 22. The cyclic compound of claim 20 or 21, which provides a higher binding affinity with the antibody than the amyloid-forming protein or protein aggregate, and preferably also higher than the corresponding linear peptide in an ELISA assay.

23. 23. The cyclic compound according to any one of claims 8 to 22, comprising part or all of a first motif VFRK (SEQ ID NO: 7) at the N-terminus and / or part or all of a second motif ELXGLTXE (SEQ ID NO: 3) at the C-terminus, wherein X in SEQ ID NO: 3 is any naturally occurring amino acid, preferably, 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. 23. The cyclic compound according to any one of claims 8 to 22, comprising a portion of the first motif VFRK (SEQ ID NO: 7) comprising at least two amino acids at the N-terminus and a portion of the second motif ELXGLTXE (SEQ ID NO: 3) comprising at least one amino acid at the C-terminus, wherein preferably the amino acids constituting the portion of the first motif comprise the dipeptide RK of SEQ ID NO: 7 and the amino acids constituting the portion of the second motif comprise the N-terminal E of SEQ ID NO:

3.

25. 25. The cyclic compound of any one of claims 1 to 24, comprising or consisting 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 variation of 5% to 20% in terms of amino acid sequence identity, wherein the variation is in an amino acid added to the N-terminus and / or 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 in the N-terminal sequence RKAADDT (SEQ ID NO: X) and / or an amino acid substitution, addition or deletion in the C-terminal sequence SGKTSESGE (SEQ ID NO: Y), and particularly preferably the variation is due to an amino acid substitution.

27. 27. The cyclic compound of 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. 27. The cyclic compound of any one of claims 1 to 26, wherein the cyclic compound 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. 27. The cyclic compound of any one of claims 1 to 26, which does not contain any of the immunogenic sequences: (a) the N-terminus of VFRK (SEQ ID NO: 7) in the human FTP sequence of SEQ ID NO: T; and / or (b) the C-terminus of ELHGLTTE (SEQ ID NO: 3) in the human FTP sequence of SEQ ID NO:

164.

30. 30. The cyclic compound of 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. A composition according to claim 32, comprising 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 a cyclic compound according to any one of claims 1 to 30 or a precursor according to claim 31, optionally together with reagents and / or instructions for use.

36. 36. The kit of claim 35, (i) a population of effector cells that express an Fc receptor, preferably the human Fc receptor FcγR, and that have been engineered to carry a reporter gene under the control of a response element that is responsive to activation by said Fc receptor, preferably said population of effector cells is a population of Jurkat cells, and said reporter gene encodes a photoprotein, preferably luciferase under the control of an NFAT response element; (ii) a substrate corresponding to said reporter, and preferably (iii) a solid support, preferably a microtiter plate, preferably a 96-well plate including a lid; (iv) washing, blocking and assay / sample dilution buffers; and / or (v) a monomer control of the target antigen 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 use of the composition according to claim 32 or 33, use of the array according to claim 34, or use of the kit according to claim 35 or 36, for detecting or quantifying an antigen-binding molecule.

38. 38. Use according to claim 37, wherein the detection or quantification is carried out by an immunological assay, preferably an 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, or the array according to claim 34, or the kit according to claim 35 or 36, for determining the efficacy of an antigen-binding molecule comprising an Fc domain.

40. 40. The use according to claim 39, wherein determining the efficacy of the antigen-binding molecule comprises the following steps: (a) contacting a cyclic compound of any one of claims 1 to 30 with a binding molecule under conditions that allow the formation of a binding molecule-antigen complex; (b) contacting a population of effector cells engineered to express an Fc receptor and harbor a reporter gene with the binding molecule-antigen complex under conditions that allow binding of the Fc domain to the Fc receptor, under the control of a response element responsive to activation by the Fc receptor, wherein binding of the Fc domain to the Fc receptor results in intracellular signaling that mediates quantifiable reporter gene activity; and (c) detecting the reporter gene activity, The use, 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 activity of the reporter gene indicates efficacy of the binding molecule.

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

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

43. The use according to any one of claims 40 to 42, wherein the cells do not overexpress FcγRIIa (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. The use according to any one of claims 40 to 45, wherein the response element is the NFAT (nuclear factor of activated T cells) response element.

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

48. 48. The 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. 49. The use of claim 48, wherein the antibody is an IgG1 antibody, such as an IgG1, lambda antibody or an IgG1, kappa antibody.

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

51. 51. The use of any one of claims 37 to 50, wherein the binding molecule is an anti-FTP antibody that is NI-301.37F1 and comprises in its variable region or binding domain 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.

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

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

54. A method for identifying and optionally obtaining an antibody or its corresponding antigen-binding molecule that binds to an amyloidogenic protein involved in systemic amyloidosis, comprising: (a) providing, and optionally producing, one or more potential amyloidogenic protein-binding antibodies or amyloidogenic protein-binding molecules thereof, or a source 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 (an antibody of the invention) or binding molecule that has been determined to bind to said cyclic compound.

55. A method for producing a pharmaceutical composition comprising an antibody that binds to an amyloidogenic protein or an antigen-binding molecule thereof, comprising: (a) providing, and optionally producing, one or more potential amyloidogenic protein-binding antibodies or amyloidogenic protein-binding molecules thereof, or a source 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 (antibody of the invention) or binding molecule that binds to the cyclic compound; and (d) formulating the antibody or binding molecule or derivative thereof identified and optionally obtained in step (c) with a pharmaceutically acceptable carrier.

56. 56. The method of claim 54 or 55, wherein the source of the antibody is selected from the group consisting of: an immunized laboratory animal, such as a rodent, preferably a mouse, most preferably an Ig-humanized mouse; human blood or a fraction thereof, preferably containing memory B cells; recombinant antibody libraries such as phage, yeast and ribosomal systems, or mammalian cell lines such as CHO and HEK.

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

58. The antibody identified and optionally obtained in step (c) competes with a reference antibody for binding to said amyloidogenic protein, and preferably said antibody of the invention has a lower EC2 for said amyloidogenic protein than said reference antibody. ５０ 58. The method of any one of claims 54 to 57, comprising:

59. 1. A method for analyzing and selecting at least one candidate target antigen-binding molecule, comprising: (a) subjecting at least two target antigen-binding molecules to an assay for determining the efficacy by using the cyclic compound according to any one of claims 1 to 30, preferably the assay according to any one of claims 39 to 53; (b) comparing the reporter gene activities 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 at least one batch of pharmaceutical compositions of target antigen-binding molecules, 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 said assay being an assay according to any one of claims 39 to 53; (b) comparing the reporter gene activity of the sample with the reporter gene activity of a control; and (c) selecting batches in which the samples exhibit greater than, equal to, or not substantially less reporter gene activity than the control, preferably batches in which the samples exhibit greater than, equal to, or 80% or more reporter gene activity than the control, preferably wherein the control is a reference standard and / or the batch to be analysed has been stored and / or exposed to stress conditions, and the control is the value of reporter gene activity of a sample taken from the batch or a corresponding batch before storage and / or exposure to the stress conditions.

61. 61. The method of any one of claims 54 to 60, wherein in a further step, the antigen-binding molecule is assayed for its ability to induce phagocytosis of and / or bind to the misfolded / aggregated amyloidogenic protein using a full-length amyloidogenic protein, preferably a misfolded / aggregated amyloidogenic protein.

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

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