Novel potency assay for antibody-based drugs and useful tools therefor

Abstract

Provided are novel methods for measuring antibody efficacy. Furthermore, methods and kits for the production, quality control, and batch release of pharmaceutical compositions containing antibody-based agents are provided. Additionally, peptide-based cyclic compounds containing epitopes from amyloidogenic proteins involved in systemic amyloidosis are described, which are generally useful in antibody efficacy and antibody binding assays, as well as in the screening and acquisition of antibodies of interest.

Description

[Technical field]

[0001] The present invention relates generally to a novel method for characterizing therapeutically useful antibodies and equivalent binding molecules, in which antibody Fc-mediated activity plays a crucial role in the mechanism of action, and in particular to a method suitable as a potency assay when performing clinical trials and applying to market approval and quality control of approved drugs, and which is particularly useful for batch release of pharmaceutical compositions comprising antibodies or similar binding molecules. In a further aspect, the present invention relates to cyclic compounds comprising peptides containing epitopes of systemic amyloidogenic proteins, which can be used in such potency assays. [Background technology]

[0002] Monoclonal antibody drugs have matured over the past 30 years, from research targets to improved technologies, from clinical studies to commercialization. In recent years, the number of monoclonal antibody drugs approved for marketing has increased rapidly, with the groundbreaking 100th monoclonal antibody product approved by the United States Food and Drug Administration (FDA) in 2021. In 2019, 9 out 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 caused by toxic amyloid aggregates. Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, represent a highly prevalent class of fatal localized amyloidosis that forms in the nervous system when amyloid deposits induce the death of certain neuronal cell types. In systemic amyloidosis, such as immunoglobulin light chain, transthyretin, and dialysis-associated amyloidosis, several organs are affected when amyloidogenic proteins are distributed to different parts of the body as they migrate from the site of synthesis. 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 has recently been approved by the FDA for use in the treatment of Alzheimer's disease.

[0004] The therapeutic utility of antibodies, especially as effective drugs for the treatment of amyloidosis, depends not only on their ability to bind aggregates, but also on antibody Fc-mediated activities, which play a crucial role in the mechanism of action. Binding of antibodies to Fc receptors on cell surfaces induces several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles (called antibody-dependent cellular phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cellular cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0005] One important mechanism of action (MoA) for antibodies targeting aggregating proteins such as amyloid beta (Aβ) is ADCP. Antibodies binding to target proteins result in the presentation of multivalent Fc domains that can bind to high- and low-affinity Fcγ receptors on patrolling immune cells such as macrophages, recruiting them to specific regions. Clustering of Fc receptors results in membrane deformation around the target antigen, activation of intracellular signaling, and changes in actin cytoskeleton dynamics that ultimately cause phagocytosis of the target antigen by phagocytes.

[0006] Therefore, antibodies and corresponding binding molecules are promising agents for the prevention and treatment of protein aggregation diseases. However, when making a pharmaceutical composition, it is not enough to formulate the drug substance into a preparation, it is also essential that the obtained preparation is approved by the national regulatory agency in which the pharmaceutical composition will be used. In the United States, the responsible regulatory agency is the FDA (http: / / www.fda.gov / ), and in Europe, it is, for example, the European Agency for the Evaluation of Medical Products (EMEA) (http: / / www.emea.eu.int / ).

[0007] The approval process is thoroughly regulated and requires drug developers to submit a great deal of information about their formulation candidates to regulatory agencies for approval. This may include information about the potency of the formulation candidate and the corresponding assays to measure potency. Such potency assays help characterize the product, monitor lot-to-lot consistency, and ensure product stability.

[0008] The potency of antibodies, where binding of Fc to Fc receptors plays a crucial role for the mechanism of action, is traditionally measured by the use of bioassays where the effect being evaluated depends on Fc-Fc receptor binding. Such assays may include ADCC, ADCP, or induction or inhibition of T cell activation, which requires antibody cross-linking. However, the assays developed so far are often highly burdensome, require expensive equipment (e.g., flow cytometers), and are quite complicated. For example, ADCP assays are usually a two-step process in which an antibody must bind to a target, and macrophages must recognize, bind, and phagocytose the antibody bound to the target. International application WO 2017 / 157961 A1 describes such a method for assaying ADCP by measuring the uptake of aggregated proteins, exemplified by Aβ. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Mullard,Nature Reviews Drug Discovery 20,491-495(2021),DOI:10.1038 / d41573-021-00079-7 Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, assays to characterize products, monitor lot-to-lot consistency, and ensure product stability must be clinically relevant, relatively easy to perform, and sensitive enough to detect differences that may affect the product's mechanism of action and function. [Means for solving the problem]

[0011] The present invention relates generally to a novel method for characterizing therapeutically useful antibodies and equivalent binding molecules, where antibody Fc-mediated activity plays a crucial role in the mechanism of action, in particular a method suitable as a potency assay, particularly useful for batch release of pharmaceutical compositions comprising antibodies or similar binding molecules, when performing clinical trials and applying to market approval and quality control of approved drugs. The present invention further relates to cyclic compounds comprising peptides or protein fragments comprising an epitope of an antibody or equivalent binding molecule, and the use of such cyclic compounds generally in the drug discovery and diagnostic fields, together with a method for measuring the potency of the antibody or binding molecule.

[0012] More specifically, the present invention relates to a novel method for measuring the phagocytosis-related potency of target antigen-binding molecules that contain Fc domains, and the use of the method in the production and quality control of pharmaceutical compositions that contain such molecules, where in a preferred embodiment, the target antigen is preferably an amyloidogenic protein in aggregated, misfolded, and non-physiological form. As illustrated in Examples 3 and 4 and the corresponding figures, a stable and highly sensitive reporter gene assay has been developed that is suitable for measuring antibody potency and has the ability to detect the reduced potency associated with Fc domain modification. As further illustrated in Example 6 and the corresponding figures, the performance of the reporter gene assay has been further improved by using a cyclic peptide as the target antigen, which contains an epitope of an amyloidogenic protein.

[0013] In accordance with the present invention, several experiments have been performed to apply the ADCP assay of WO 2017 / 157961 A1, exemplified with Aβ, to the systemic amyloidogenic protein transthyretin (TTR). In a first set of experiments, an in vitro assay was developed that included human-derived macrophages, fluorescently labeled L55P-TTR protein, and aggregated TTR-selective antibodies; see Example 1. However, some variability was observed in the phagocytic activity between macrophages obtained from different blood donors. Therefore, to eliminate this source of variability, an in vitro phagocytosis assay was developed again using the human monocytic THP1 cell line instead of fresh PBMCs (see Example 2), but again some variability was observed between replicates.

[0014] The present invention provides an improved assay that is particularly suitable for determining the potency of antibodies and binding molecules containing Fc domains that bind to amyloidogenic TTR or other amyloidogenic proteins involved in systemic amyloidosis.Accordingly, in another set of experiments carried out within the scope of the present invention, different types of cell assays have been evaluated with different success.Finally, as illustrated in Examples 3 and 4, it has been found that an assay that utilizes mammalian cells, in particular Jurkat cells, genetically modified to express human Fc receptors, in particular Fc receptor FcγRI (CD64), as effector cells, provides very reliable results, especially for systemic amyloidogenic proteins such as TTR as target antigen, and this setup is particularly suitable for potency assays against target antigens that exist as aggregates.

[0015] In another set of experiments, a cyclic peptide containing a TTR epitope (cyclic TTR peptide) is used as the target antigen instead of TTR aggregates. Unexpectedly, this assay showed a significant improvement in sensitivity and reliability. Without intending to be bound by theory, the extraordinary performance of the assay with cyclic peptides may be due to the very stable conformation adopted by the cyclic peptide, since it is constrained by having two ends connected together, thus reproducing the stability of protein aggregates. However, as shown in Example 6, even taking these theoretical considerations into account, the assay with cyclic peptides as the target antigen is an order of magnitude more accurate and sensitive than the assay with target antigens present as protein aggregates. Furthermore, without intending to be bound by theory, this may be due to the smaller size of the peptides compared to proteins and the higher epitope density obtained, which accounts for both the higher binding capacity and the higher avidity effect. Furthermore, epitopes in cyclic peptides may be more accessible compared to full-length proteins. Nevertheless, the size of the cyclic peptide cannot a priori explain the observed effect, since the cyclic TTR peptide including the linker amino acids with a total of 31 amino acids is only 4 times smaller than the full-length TTR protein. A further reason may be the better control of the conformation of the synthetic peptide compared to the recombinant protein. In particular, while more than 95% of the peptide is cyclized, it is unknown that a fraction of the misfolded aggregated TTR protein adopts an amyloid conformation. Since mis.WT-TTR is a heterogeneous mixture of conformations, a significant fraction of the protein may form amorphous aggregates instead of amyloid. In summary, together with the results of the experiments described in the attached Examples 5-8, we have now laid down a good explanatory approach and theory, and can predict further cyclic peptides with epitopes suitable for the detection and identification of potent antibodies against amyloidogenic, especially systemic amyloidogenic, proteins, which could not have been foreseen without knowledge of these results and teachings of the present invention.In this context, and again without wishing to be bound by any theory, it is noteworthy that cryo-EM studies have recently shown that the amyloid structure of systemic amyloidogenic proteins such as ATTR and AL amyloidosis, which result from misfolding of the immunoglobulin light chain (LC), is on the one hand similar, but on the other hand substantially different, from that of local amyloidogenic proteins such as tau; see Figure 5 in Schmidt et al., Nat. Commun. 10 (2019), 5008, https: / / doi.org / 10.1038 / s41467-019-13038. It is therefore reasonable to assume that this result for the TTR-derived cyclic peptide can also be applied to other systemic amyloidogenic proteins.

[0016] Thus, in a further aspect, the present invention specifically relates to the presentation of cyclic compounds comprising peptides containing epitopes of systemic amyloidogenic proteins, the epitopes being accessible for binding by antibodies preferably only in misfolded and / or aggregated forms of the protein, such as in the case of neoepitopes, and / or the epitopes being hidden in the bioactive tetramer and no longer accessible for antibody binding, e.g. in the case of epitopes accessible in the monomeric TTR protein, at least not present in the bioactive form of the protein. As illustrated in Example 6, the cyclic compounds of the present invention are particularly useful in potency assays of the present invention.

[0017] The remarkable performance of the cyclic compounds as target antigens is demonstrated in the ELISA assay described in Example 5. EC for antibodies binding to cyclic peptides 50 The EC value is for an antibody that binds to a protein aggregate and a linear peptide that contains the same epitope as the cyclic peptide. 50 Again, the cyclic peptides had the highest binding affinity to the antibody, i.e., the lowest EC 50As mentioned above, the highest binding affinity between the antibody and the cyclic peptides compared to the protein aggregates may be due to a higher epitope density resulting in a better apparent binding affinity, due to a higher binding capacity and a higher avidity effect, due to a better accessibility of the epitopes in the cyclic peptides to the full-length protein, and / or due to a better control of the conformation of the synthetic peptide compared to the recombinant protein. Thus, in one embodiment, the cyclic compounds and cyclic peptides of the present invention each provide a higher binding affinity between the antibody and the target protein (derived from and higher than the corresponding linear peptide), 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 binding affinity compared to the full-length target protein and / or linear peptide, for example in an ELISA assay as described in the attached Example 5.

[0018] Without being bound by theory, the advantage of cyclic peptides over linear peptides of similar sequence can be explained by the extreme flexibility of linear peptides, which can adopt a virtually infinite number of conformations, in contrast to cyclic peptides, which are constrained by having two ends connected together and therefore have much less flexibility and adopt more stable conformations. In other words, cyclic peptides have lower entropy than the same amino acid sequence in linear form.

[0019] Thus, the presentation of the cyclic compounds according to the present invention represents an important contribution to the art, given their remarkable usefulness as suitable targets for testing for binding between target antigens and corresponding target antigen-binding molecules in assays requiring high sensitivity. Nevertheless, the present invention also relates to linear forms of the cyclic compounds and cyclic peptides, respectively, for use, for example, as precursors for preparing cyclic compounds or as controls in experiments.

[0020] As further described in Example 3, the effector cells used in the assays of the present invention contain a reporter gene under the control of a response element that is responsive to activation by an Fc receptor. Because reporter gene activity can be measured via a standard photometer, no expensive and complicated equipment is required.

[0021] Briefly, a method for measuring the potency of a molecule that binds to a target antigen and comprises an Fc domain, comprising: (a) contacting a target antigen with a binding molecule under conditions that allow for the formation of a binding molecule-target antigen complex; (b) contacting the binding molecule-target antigen complex with a population of effector cells that express an Fc receptor and harbor a reporter gene under the control of a response element that is responsive to activation by the Fc receptor, under conditions that allow binding of the Fc receptor to the Fc domain of the binding molecule, resulting in intracellular signaling and mediating quantifiable reporter gene activity; (c) detecting a signal induced by reporter gene activity; wherein at least one MoA of an Fc domain of a binding molecule is mediated through binding of the Fc domain to an Fc receptor, and reporter gene activity is indicative of potency of the binding molecule.

[0022] Such assay can be applied in the method of preparing pharmaceutical compositions containing target antigen-binding molecules, and the efficacy of said binding molecules is first analyzed after preparation.Based on the result, it is evaluated whether the binding molecules can be used in pharmaceutical compositions.In particular, only the binding molecules that are considered to be strong based on the assay are selected for further use and formulated as pharmaceutical compositions with pharmaceutically acceptable carriers.

[0023] The potency assay of the present invention can also be used in a method for analyzing and selecting batches of pharmaceutical compositions of target antigen-binding molecules, where samples of the batch to be analyzed and control samples are subjected to said potency assay, and the reporter gene activity of the samples is compared with that of the control. Batches whose samples show greater, equal or insubstantial lower reporter gene activity compared with the control are finally selected for further use. Thus, the method of the present invention can be used to verify the consistency between lots.

[0024] The present invention preferably relates to a kit designed to carry out the method of the present invention and in particular to assay the efficacy of a binding molecule comprising an Fc domain for inducing ADCP, comprising at least: (i) a population of effector cells that express an Fc receptor and have been genetically modified to harbor a gene encoding a reporter under the control of a response element that is responsive to activation by the Fc receptor; (ii) a corresponding substrate of the reporter; and optionally (iii) target antigen; (iv) a microtiter plate, preferably a 96 or 384 well plate including a lid; (v) recommendations for buffers, diluents, substrates and / or solutions, and instructions for use, particularly instructions on how to perform the assays of the invention; (vi) washing, blocking and assay / sample dilution buffers; and / or (vii) a positive control target antigen-binding molecule, preferably an antibody The present invention further relates to a kit comprising:

[0025] In one embodiment, the kit of the present invention comprises at least (i) a population of effector cells that have been genetically modified to express an Fc receptor and harbor a gene encoding a reporter under the control of a response element that is responsive to activation by the Fc receptor; (ii) the corresponding substrate of the reporter; and (iii) a target antigen, the kit optionally comprising: (iv) a microtiter plate, preferably a 96 or 384 well plate including a lid; (v) recommendations for, and instructions for, buffers, diluents, substrates and / or solutions, particularly instructions on how to carry out the assays of the invention; (vi) washing, blocking and assay / sample dilution buffers; and / or (vii) a positive control target antigen-binding molecule, preferably an antibody Further includes:

[0026] In a preferred embodiment, the kit of the invention comprises, instead of or in addition to a target antigen, a cyclic compound comprising a peptide comprising an epitope from an amyloidogenic protein involved in systemic amyloidosis, and / or comprises a precursor of the cyclic compound, wherein the compound is in linear form, which may then also serve as a control similar to that shown for the TTR peptide in the examples.

[0027] The method, i.e., the potency assay of the present invention, uses the amyloidogenic protein TTR and its aggregates as target antigens, and cyclic peptides containing TTR epitopes, respectively, and anti-TTR antibodies, such as NI-301.37F1, which are disclosed in International Application Publication No. WO 2015 / 092077 A1 and described as capable of activating the immune system for the removal of TTR fibrils in animal models; see International Application Publication No. WO 2020 / 094883 A1. In its physiological form, TTR is a tetrameric protein that expresses amyloidogenic properties when it dissociates into monomers and forms the systemic amyloidosis transthyretin amyloidosis (ATTR). Systemic amyloidosis is a protein misfolding disorder resulting from extracellular deposition of amyloid leading to organ failure, whereas local amyloidosis refers to intracellular and / or extracellular amyloid deposits occurring exclusively in organs or tissues of precursor protein synthesis, such as intracellular tau protein fibrils and extracellular amyloid-β fibrils and plaques in Alzheimer's disease. In principle, the method of the invention is applicable to any target antigen, in particular any protein that in its pathogenic variants forms a neo-epitope, e.g. an epitope that is exclusively exposed in the misfolded variant, a conformational epitope on aggregates, fibrils and / or oligomers, an epitope on an extracellular variant of an otherwise intracellularly located physiological protein, or an epitope specific for exogenous pathogens such as fungi, bacteria and viruses. Furthermore, the method of the present invention can in principle be carried out with any type of antigen, including aggregates, fibrils, oligomers, (misfolded) monomers, as well as protein fragments and peptides that contain and display epitopes of the target antigen-binding molecule to be tested, where the peptides are preferably provided in cyclic form. Similarly, the cyclic compounds of the present invention can in principle include any peptide or protein fragment capable of forming a cyclic compound, in particular a peptide or protein fragment that contains the neo-epitope described above.In a particularly preferred embodiment, the (neo)epitope is hidden in the natively folded conformation of the target antigen, but is accessible for antibody binding after unfolding and aggregation, such as, for example, the linear epitope WEPFA of antibody NI-301.37F1, located at positions 41-45 of the mature TTR protein.

[0028] Nevertheless, in accordance with this example, the method of the invention is preferred as it is particularly suited to measuring the potency of antibodies targeted against amyloidogenic proteins, preferably against aggregates of misfolded, non-physiological forms of proteins such as transthyretin and their amyloidogenic forms, and preferably against fragments and peptides of proteins in cyclic form, comprising epitopes from amyloidogenic proteins, preferably from epitopes exposed in misfolded, non-physiological forms of proteins such as transthyretin.

[0029] The methods of the invention are particularly useful for determining the efficacy of antibodies to activate ADCP.

[0030] Furthermore, the cyclic compounds of the invention, and their linear precursors, are useful in a method for identifying, and optionally obtaining, antibodies that bind to amyloidogenic proteins involved in systemic amyloidosis, typically comprising the steps of: (a) providing, and optionally producing, an antibody or a source thereof that binds to one or more potentially amyloidogenic proteins; (b) subjecting an antibody or a source thereof that binds to one or more potentially amyloidogenic proteins to a binding assay comprising a cyclic compound of the invention; (c) identifying, and optionally obtaining, an antibody that has been determined to bind to the cyclic compound (a target antibody); The method is particularly useful in methods comprising:

[0031] This method may be combined with a potency assay of the invention, and / or any other suitable method for further determining the diagnostic or preferably therapeutic utility of a subject antibody.

[0032] Therefore, a further embodiment of the present invention is a method of making a pharmaceutical composition comprising an antibody that binds to an amyloidogenic protein, comprising at least (a) providing, and optionally producing, an antibody or a source thereof that binds to one or more potentially amyloidogenic proteins; (b) subjecting the antibody or a source thereof that binds to said one or more potentially amyloidogenic proteins to a binding assay comprising a cyclic compound of the invention; (c) identifying, and optionally obtaining, an antibody that binds to the cyclic compound (a target antibody); (d) formulating the antibody or derivative thereof identified and optionally obtained in step (c) with a pharma- ceutically acceptable carrier; The method comprises:

[0033] Sources of antibodies include, but are not limited to, immunized laboratory animals such as rodents, preferably mice, most preferably Ig-humanized mice; human blood or fractions thereof, preferably containing memory B cells; natural and synthetic antibodies obtained from recombinant antibody libraries such as phage, yeast, and ribosomal-based or mammalian cell systems, e.g., CHO and HEK; see also the Detailed Description of the Invention for additional sources of antibodies and other target binding molecules.

[0034] The binding assay used in the above described methods preferably comprises an ELISA, for example as performed in Examples 5 and 7.

[0035] In a preferred embodiment of the method of the invention for identifying and obtaining an antibody of interest and its further use when formulated in pharmaceutical compositions and drug discovery, respectively, the antibody identified in step (c) and optionally obtained competes with a reference antibody for binding to an amyloidogenic protein, wherein preferably the antibody of interest has a lower EC for the amyloidogenic protein than the reference antibody. 50 has.

[0036] Unless otherwise defined in this application, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, 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 are not intended to be limiting.

[0037] Further embodiments of the present invention will be 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 combined, and preferably is intended to be combined, with one or more characterizations of other features of such general embodiments. Furthermore, unless otherwise specifically indicated, the embodiments described herein for antibodies, including the examples, are intended to be exemplary, even if not preferred, and may be extrapolated herein to any target binding molecule. [Brief description of the drawings]

[0038] [Figure 1-1]NI-301.37F1_3 in vitro phagocytosis assay using human-derived macrophages. NI-301.37F1_3 induces TTR phagocytosis in a concentration-dependent and FcR-dependent manner. Throughout the description and figures, "NI-301.37F1" can also be referred to as "37F1". Antibody concentration-dependent TTR uptake was specifically mediated by NI-301.37F1_3, requiring binding to Fc receptors and low antibody concentrations as measured by a standard fluorescent plate reader (A). Quantification by FACS of double-positive cells (cells positive for both TTR and NI-301.37F1_3) showed that the frequency of double-positive cells increased from a background level of 3% to 6% in the presence of 10 nM NI-301.37F1_3, and further increased to 16% in the presence of 80 nM NI-301.37F1_3 (B). Quantification of cells double positive for TTR and antibody internalized into acidic vesicles showed that the frequency of double positive cells increased from a basal level of 3.5% to 5.5% in the presence of 10 nM NI-301.37F1_3 and further increased to 8.8% in the presence of 80 nM NI-301.37F1_3, where antibody-dependent phagocytosis of TTR was specifically induced by NI-301.37F1_3 and not by the isotype control antibody, which did not induce phagocytosis above background levels at 10 nM and 80 nM (C). [Figure 1-2] Same as above. [Diagram 2]In vitro phagocytosis assay using THP1 cells. NI-301.37F1_3 induced phagocytosis of mis.TTR488 by THP1 cells in a concentration-dependent manner. Quantification of intracellular mis.TTR-488 fluorescence in THP1 cells incubated with 1× or 0.7×_NI-301.37F1_3 dilution series (mean ± SD of triplicates) (A). Both NI-301.37F1_W1 non-GMP DP and NI-301.37F1_W1 GMP DS induced phagocytosis of mis.TTR-488 by THP1 cells within the same concentration range. Quantification of intracellular mis.TTR-488 fluorescence in THP1 cells incubated with NI-301.37F1_W1 non-GMP DP and NI-301.37F1_W1 GMP DS dilution series (mean ± SD of triplicates) (B). [Diagram 3] Comparison of the binding of antibodies NI-301.37F1 batch 3 (37F1_3) (A) and NI-301.37F1 batch W1 (37F1_W1) (B) to mis.WT-TTR batch 5 and mis.WT-TTR batch 6 using ELISA showed that the binding of 37F1_3 and 37F1_W1 to mis.WT-TTR_b6 was virtually identical to that of mis.WT-TTR_b5. [Figure 4] Evaluation of the ADCP assay of the present invention using mis.WT-TTR as target antigen for its ability to detect changes in antibody activity by comparison with the NI-301.37F1 batch W1 reference sample (NI-301.37F1_W1 RS) and a half-concentrated test sample (NI-301.37F1_W1 50%) showed that the assay has the ability to detect a 50% decrease in antibody activity. Mean ± SD of triplicates. [Diagram 5]Evaluation of the ADCP assay of the present invention using mis.WT-TTR as the target antigen for its ability to detect changes in antibody activity by comparison of the NI-301.37F1_W1 reference sample (NI-301.37F1_W1 RS) with a sample having a lower concentration (NI-301.37F1_W1 65%, plate 1) (A) and a sample having a higher concentration (NI-301.37F1_W1 135%, plate 2) (B) showed that the assay using the horizontal plate layout has the ability to detect a 35% decrease in antibody activity and a 35% increase in antibody activity, respectively. [Figure 6] Evaluation of the ADCP assay of the present invention using mis.WT-TTR as the target antigen for its ability to detect changes in antibody activity by comparison with NI-301.37F1_W1 RS, 65% and 135% using a perpendicular assay layout showed that the assay using the perpendicular format has the ability to detect ±35% changes in antibody activity. Mean ±SD of triplicates. [Figure 7] Binding of stressed NI-301.37F1_W1 samples ((A) reference sample, PBCA pH 3.4, Tris pH 10, H2O2; (B) reference sample, Form° buffer pH 5.8, PBS pH 7.4) to mis.WT-TTR was analyzed by ELISA, and the results showed that the stressed NI-301.37F1_W1 samples exhibited binding affinities to mis.WT-TTR that were highly comparable to the reference NI-301.37F1_W1 sample and characterized by an EC50 in the sub-nanomolar range. (C) Tabular summary of the results. [Figure 8] Binding of stressed NI-301.37F1_W1 samples to mis.WT-TTR was analyzed by BLI and the tabular summary showed that stressed NI-301.37F1_W1 samples exhibited binding affinity to mis.WT-TTR that was comparable to the reference NI-301.37F1_W1 sample and characterized by a KD in the low nanomolar range. [Figure 9]Evaluation of the ADCP assay of the present invention for its ability to detect reduced potency by comparison of NI-301.37F1_W1 RS samples stressed with PBCA and Tris buffers (A) and with formulation and H2O2 buffers (B) demonstrated that the assay has the ability to detect reduced potency associated with Fc domain alterations. [Figure 10] Improved sensitivity of ELISA assay. Comparison of the binding specificity of antibody NI-301.37F1 to (A) peptides TTR34-54cyc, TTR40-49, biotin.TTR40-49, and mis-WT-TTR, and (B) peptides TTR34-54cyc, biotin.TTR34-54cyc, TTR40-49, biotin.TTR40-49, and mis-WT-TTR using ELISA assays showed specific binding of NI-301.37F1 to mis.WT-TTR, and binding of NI-301.37F1 to the cyclic TTR34-54cyc peptide was approximately 10 times stronger than binding to mis.WT-TTR. The curve for peptide TTR40-49 matches the one of biotin.TTR40-49. [Figure 11] Improved ADCP assay by using cyclic peptide compounds as target antigens. Measurement of the potency of antibody NI-301.37F1 in an ADCP assay using cyclic TTR peptide (TTR34-54cyc) shows that the ability of antibody NI-301.37F1 RS to activate phagocytosis in a dose-response, i.e., dose-dependent manner, is characterized by an EC50 of 19.8ng / ml. [Figure 12-1]An ADCP assay using TTR34-54cyc as the target antigen to detect changes in antibody activity by comparison of the NI-301.37F1 reference sample (NI-301.37F1 RS) with samples having lower concentrations (NI-301.37F1 50% (A) and 70% (B)) and with higher concentrations (NI-301.37F1 130% (C) and 150% (D)) showed that the assay has the ability to detect a 50% decrease in antibody activity and a 50% increase in antibody activity, respectively. [Figure 12-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention relates to a novel method for measuring the potency of target antigen-binding molecules comprising an Fc domain, in particular the potency of activating antibody-dependent cell-mediated phagocytosis (ADCP), and the use of this method in the production and quality control of pharmaceutical compositions comprising such molecules and in the validation of batches of said compositions.Furthermore, the present invention relates to a kit, preferably designed for and usable in the method of the present invention.In a further aspect, the present invention relates to a cyclic compound comprising an epitope of a protein recognized by an antibody or an equivalent binding molecule, and which can be used as a target antigen in the method according to the present invention.

[0040] Unless otherwise specified, the terms used herein are given the definitions provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0-19-850673-2; Second edition published 2006, ISBN 0-19-852917-1 978-0-19852917-0.

[0041] The term "protein" as used throughout the description includes fragments and peptides of (full length) proteins that contain and expose the epitope of the target antigen binding molecule to be tested, e.g., an antibody.

[0042] "Cyclic peptide" herein can refer to a compound that is entirely proteinaceous, but for example, the linker is 2, 3, 4, 5, 6, 7 or 8 amino acids, or the linker is absent. For example, it is conceivable that a natural protein sequence, i.e., an amino acid stretch, that contains an epitope of an antibody, allows for cyclization without adding extra amino acids, for example, due to the presence of two cysteines within a suitable distance. It is understood that the properties described for the cyclic peptides determined in the examples can be incorporated into other compounds, for example, cyclic compounds that contain non-amino acid linker molecules. "Cyclic peptide" and "cyclic compound" can be used interchangeably when the cyclic compound is composed of amino acids.

[0043] The term "linker" as used herein means a chemical moiety that can be directly or indirectly covalently attached to a protein fragment or peptide as defined herein. The linker termini can be linked, for example, to generate a cyclic compound. The linker can be at the N-terminus and C-terminus. Alternatively, the linker can be at an internal position "some distance" from the termini. The linker can include one or more functionalizable moieties, such as one or more cysteine ​​(C) residues. The linker can also be linked to other proteins or components via functionalizable moieties. The cyclic compound containing the linker is longer in length than the peptide or protein fragment itself.

[0044] The term "functionalizable moiety" as used herein refers to a chemical entity having a "functional group", which as used herein refers to a group or single atom of an atom that will react with another group or single atom of an atom (so-called "complementary functional group") to form a chemical interaction between the two groups or atoms. In the case of cysteine ​​(C), the functional group can be -SH, which can undergo reaction to form a disulfide bond. The reaction with another group of the atom can be a covalent bond or a strong non-covalent bond, such as a biotin-streptavidin bond, which can have a dissociation constant (Kd) of about 1e-14. A strong non-covalent bond, as used herein, means an interaction with a Kd of at least 1e-9, at least 1e-10, at least 1e-11, at least 1e-12, at least 1e-13, or at least 1e-14.

[0045] Potency testing is performed as part of product suitability testing, comparability testing, and stability testing. These tests are used to measure product attributes related to product quality and manufacturing control and are performed to ensure the identity, purity, strength (potency), and stability of the product used during all phases of clinical trials. Similarly, potency measurements are used to demonstrate that only product lots, i.e., batches, that meet defined specifications or acceptance criteria are administered during all phases of clinical studies and after market approval. Potency is defined as "the specific performance or ability of a product, as demonstrated by appropriate laboratory tests or well-controlled clinical data obtained through administration of the product in the intended manner to obtain a given result." Ideally, potency assays will represent the mechanism of action of the product (i.e., the relevant therapeutic activity or intended biological effect); see Guidance for Industry-Potency Tests for Cellular and Gene Therapy Products, USDepartment of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research, January 2011. Therefore, in the context of the assay of the present invention, the "potency" of a target antigen-binding molecule, specifically an antibody as a formulation, is a measure of its activity in the ADCP assay relative to the activity of a reference standard (of the formulation), where the activity and level of activity in the ADCP assay, respectively, have been assessed or are known. Thus, a higher potency compared to the reference of an antibody / formulation means that the antibody / formulation has a higher binding activity, i.e. a lower EC 50 A lower potency compared to the antibody / formulation reference indicates 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% reproduces an antibody with higher potency, with an EC value 0.7-fold higher than the reference sample NI-301.37F1 RS (100%). 50In contrast, antibody NI-301.37F1 50% reproduces an antibody with lower potency (reduced activity) and has an EC value two times higher than the reference sample NI-301.37F1 RS (100%). 50 values ​​are shown; see Example 6. Thus, a target antigen binding molecule (e.g., an antibody) exhibiting increased potency will have, e.g., at least a 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, e.g., as determined in an ADCP assay described herein. 50 Alternatively, a target antigen binding molecule (e.g., an antibody) exhibiting reduced potency has been determined to have an EC value that is 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) higher, e.g., relative to a reference sample, e.g., as determined in an ADCP assay described herein. 50 However, according to GLP and GMP, the variability (i.e., imprecision) allowed for potency measurements is a preferred limit of + / - 20% for the target antigen-binding molecule tested.

[0046] As mentioned above, measuring the efficacy of a drug is an important step in the development, including evaluation of new therapeutic agents for the treatment of disease. In the context of the present invention, such methods are used in the development, evaluation and batch release of antibody-based drugs and other targeted antigen-binding molecules that utilize the effector functions of the Fc domain for the treatment of diseases associated with target proteins, in particular protein aggregation disorders such as systemic and localized amyloidosis.

[0047] The physiological functions of proteins are highly dependent on their precise three-dimensional structure. Disturbances in the proper folding and refolding of newly synthesized or existing proteins (molecular chaperones) or in pathways involved in the degradation of misfolded proteins (ubiquitin-proteasome and autophagy systems) 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 (Schroeder,Acta Neuropathol 125(2013),1-2).

[0048] Amyloid diseases are characterized by the deposition of crossed-β-sheet amyloid fibrils composed of misfolded and / or misassembled proteins. The amyloid fibrils that are the pathological hallmark of these disorders can be either systemically deposited or localized to specific organs. The development of amyloidosis is often associated with aging and is associated with a reduced quality of life and considerable 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 diseases are associated with aging. Systemic forms of amyloid diseases, which are also often associated with aging, are less common and include TTR amyloidosis. The origin of amyloidosis is either sporadic (i.e., from a normal protein sequence) or hereditary (familial) (i.e., from a protein harboring one or more point mutations). In addition, there are infectious forms of amyloidosis, such as transmissible spongiform encephalopathies, which result from the aggregation of prion proteins (Ankarcrona et al., J Intern Med. 280 (2016), 177-202).

[0049] The present invention provides a reliable method 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), where the antibodies preferably target epitopes on pathological protein aggregates or epitopes of cyclic peptides, preferably epitopes that are normally exposed in pathological protein aggregates.

[0050] However, as mentioned above, in principle the method of the invention is applicable to any protein that forms a neoepitope in any kind of target antigen, in particular in its pathogenic mutants, and to any protein fragment or peptide, respectively, preferably in cyclic form, that contains such a neoepitope; see above.

[0051] Thus, in its broadest aspect, the present invention provides a method for measuring the potency of a target antigen-binding molecule comprising an Fc domain, comprising the steps of: (a) contacting a target antigen with a binding molecule under conditions that allow for the formation of a binding molecule-target antigen complex; (b) contacting the binding molecule-target antigen complex with a population of effector cells that express an Fc receptor and that have been engineered to harbor a reporter gene under the control of a response element that is responsive to activation by the Fc receptor, under conditions that allow binding of the Fc domain to the Fc receptor, resulting in intracellular signaling and mediating quantifiable reporter gene activity; (c) detecting a signal induced by reporter gene activity; wherein at least one mechanism of action of the Fc domain of the binding molecule is mediated through binding of the Fc domain to an Fc receptor, and reporter gene activity is indicative of efficacy of the binding molecule.

[0052] As mentioned above, the method of the invention is applicable to any target antigen, in particular any protein that forms a neo-epitope in its pathogenic variants, e.g. epitopes that are only exposed in misfolded variants, conformational epitopes on aggregates, fibrils and / or oligomers, epitopes on extracellular variants of otherwise physiological proteins located intracellularly, or epitopes specific for exogenous pathogens such as fungi, bacteria and viruses. Moreover, the method of the invention can in principle be carried out with any kind of antigen, including aggregates, fibrils, oligomers, (misfolded) monomers, as well as protein fragments and peptides that contain and display epitopes of the target antigen-binding molecule to be tested. Therefore, according to the method of the present invention, the target antigen is preferably a protein, more preferably an extracellular protein, even more preferably a protein aggregate and fibril, respectively, or (misfolded) oligomer, protofibril, or (misfolded) monomer, even more preferably an amyloidogenic protein, preferably an amyloidogenic protein in systemic amyloidosis, and most preferably TTR and its aggregates. As mentioned above, the protein also includes the corresponding fragments and peptides that contain the (neo)epitope of the target antigen-binding molecule. Thus, in another preferred embodiment of the method of the present invention, the target antigen is a protein fragment or peptide that contains the epitope recognized by the target antigen-binding molecule. In other words, the target antigen used according to the method of the present invention is derived from a protein, more preferably from an extracellular protein, even more preferably from a protein capable of forming aggregates and fibrils, respectively, or from (misfolded) oligomers, protofibrils, or (misfolded) monomers, even more preferably from an amyloidogenic protein, preferably from an amyloidogenic protein in systemic amyloidosis, and most preferably from a protein fragment or peptide derived from TTR and its aggregates, wherein the protein fragment or peptide comprises an epitope of the protein as recognized by the target antigen-binding molecule.

[0053] In one embodiment of the method of the present invention, the target antigen comprises or consists of a protein fragment or peptide containing a (neo) epitope of the target antigen-binding molecule. As illustrated in Example 6 and Figures 11 and 12, the potency assay of the present invention, i.e., the ADCP assay herein, can be substantially improved by using a cyclic compound comprising a peptide containing a (neo) epitope of the target antigen-binding molecule of the anti-TTR antibody herein. Thus, in a preferred embodiment of the method of the present invention, the protein fragment or peptide, respectively, is cyclized to form a cyclic compound; see also above. The cyclic compound is characterized as described in the previous preceding section "Summary of the invention" and in the further sections referring to essentially cyclic compounds.

[0054] The cyclic compounds provided herein and used in accordance with the present invention may either be composed of an epitope recognized by a target antigen-binding molecule, or may comprise or consist of a protein fragment or peptide that comprises an epitope recognized by a target antigen-binding molecule, which means that, for example, additional amino acids or other chemical entities used in cyclization of the peptide or protein fragment, as further described below, may be present in the protein fragment or peptide that forms the cyclic compound.

[0055] The additional amino acids may be amino acids naturally located adjacent to the epitope sequence, i.e., amino acids flanking the epitope sequence and present in the protein sequence from which the protein fragment or peptide is derived, i.e., the protein fragment or peptide forming the cyclic compound comprises the epitope of the target antigen-binding molecule and further amino acids adjacent to and flanking the epitope, respectively. The number of those adjacent / flanking amino acids may vary, for example, from 1, 2, or 3 amino acids to 50 amino acids, preferably from 1, 2, or 3 amino acids 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, more preferably from 10 to 20 amino acids, where the amino acids are distributed either evenly or unevenly on the N- and C-terminal sides of the epitope sequence, for example, an additional 7 amino acids on the N-terminal side and 9 amino acids on the C-terminal side of the epitope.

[0056] Additionally or alternatively, the protein fragment or peptide may, in one embodiment, comprise 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 and adjacent amino acids as defined above, and a linker. In a preferred embodiment, the protein fragment or peptide used according to the present invention forming a cyclic compound 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 coupled to the N-terminal residue of the protein fragment or peptide and the C-terminal residue of the protein fragment or peptide.

[0057] Methods for cyclization of peptides are generally known in the art. For example, cyclization can be carried out by chemical cross-linking, especially using chemical scaffolds. Cross-linking requires functional groups, and only a few protein chemical targets account for the majority of cross-linking techniques, such as primary amines (-NH2), which are present at the N-terminus of each polypeptide chain and in the side chains of lysine residues; carboxyls (-COOH), which are present at the C-terminus of each polypeptide chain and in the side chains of aspartic acid and glutamic acid; and sulfhydryls (-SH), which are present in the side chains of cysteine.

[0058] Scaffold-based cyclization is one of the most frequently used methods because it is applicable to chemically or biologically synthesized peptides. In general, scaffold compounds such as organic halides (most often organobromides) selectively react with the hydrosulfur groups of cysteines. Also, non-hydrosulfur groups, such as the primary amines of lysines or the N-terminal amino group in peptides, can be used for cyclization with chemicals containing, for example, N-hydroxysuccinimide (NHS). Also, specially designed unnatural amino acids can be used for cyclization in peptides via bioorthogonal reactions. For example, if an azide-containing amino acid, such as azidohomoalanine or azidophenylalanine, is present in a peptide, a copper-mediated click reaction with an alkyne-bearing scaffold can trigger cyclization.

[0059] Additionally, the cysteines can be linked together via disulfide bonds (-SS-) between their side chains, or amide cyclization can be performed without a scaffold at all (head-to-tail, or backbone cyclization).

[0060] For example, peptides having "C" residues at the N-terminus and C-terminus, such as the cyclic TTR compound GCGGGGRKAADDTWEPFASGKTSESGEGGGCG (SEQ ID NO: 17) used in Examples 5-8, can be reacted by SS-cyclization to produce cyclic peptides. Cyclic compounds can be synthesized as linear molecules with linkers covalently attached at or near the N-terminus or C-terminus of the peptide, including the TTR peptide, or related epitopes provided as precursors prior to cyclization, as referred to herein and also subject of the present invention. Alternatively, prior to cyclization, some of the linkers are covalently attached at or near the N-terminus and some are covalently attached at or near the C-terminus. In either case, the linear compound is cyclized, for example, by SS bond cyclization. Thus, the compounds may be covalently cyclized at 1) the N-terminus and / or C-terminus of the peptide plus linker to form a peptide bond (e.g., cyclize the backbone), 2) at or near the N-terminus or C-terminus with a side chain on the peptide plus linker, or 3) at two side chains on the peptide plus linker. In this context, "near" is defined as within 1, 2, or 3 amino acid residues of the N-terminus or C-terminus. Preferably, the linker is coupled to the N-terminus or C-terminus.

[0061] As mentioned above, peptides can be cyclized by oxidation of thiol or mercaptan-containing residues at or near the N-terminus or C-terminus, or in the interior of the peptide, including, for example, cysteine ​​and homocysteine. For example, two cysteine ​​residues flanking the peptide can be oxidized to form a disulfide bond. Oxidation reagents that can be used include, for example, oxygen (air), dimethylsulfoxide, oxidized glutathione, cystine, copper(II) chloride, potassium ferricyanide, thallium(III) trifluroacetate, or other oxidation reagents that can be known to those skilled in the art and can be used in a manner that is 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 to be used for crosslinking, see, for example, the crosslinker selection tool provided by Thermo Fisher Scientific.

[0062] Thus, in one embodiment, the linker comprises a functionalizable moiety, e.g., an amino acid bearing one of the above functional groups, such as lysine, aspartic acid, glutamic acid, or cysteine, a non-naturally occurring amino acid, such as azidohomoalanine or azidophenylalanine, or an equivalently functionalizable molecule, such as polyethylene glycol (PEG).

[0063] When the functionalizable moiety is a naturally occurring amino acid such as lysine, aspartic acid, glutamic acid, serine, threonine, or cysteine, the functionalizable moiety does not necessarily have to be present in the linker, but can also be present in the epitope or adjacent amino acids present in the protein fragment or peptide that forms 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 a cyclic compound when used according to the present invention without a linker. The linkage 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.

[0064] In a preferred embodiment, at least one functionalizable moiety is present in the linker, i.e., the linker comprises one or more functionalizable moieties. The linker may comprise or consist of any amino acid, including unnatural amino acids, but preferably comprises at least one of the above-mentioned functionalizable moieties, i.e., lysine, aspartic acid, glutamic acid, or cysteine, unnaturally occurring amino acids such as azidohomoalanine or azidophenylalanine, or equivalently functionalizing molecules such as polyethylene glycol (PEG). In a preferred embodiment, the linker comprises cysteine ​​as a functionalizable moiety.

[0065] Thus, in a preferred embodiment, a linker of any length and sequence can be described by the following sequence -nX-1FX1-Xn, where F is any functionalizable moiety, preferably C (cysteine), and X is any amino acid, including unnatural amino acids. In a further preferred embodiment, the linker amino acid is selected from alanine (A), or glycine (G), or serine (S), or from alanine (A) and glycine (G), or from glycine (G) and serine (S), but preferably is glycine (G).

[0066] 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), but preferably glycine (G), and the functionalizable moiety is cysteine ​​(C). Thus, preferably, the cyclization is carried out by a scaffold compound, such as an organic halide, preferably an organic bromide, which selectively reacts with the hydrosulfur group of cysteine, or via a disulfide bridge. Most preferably, the cyclization is carried out via a disulfide bridge.

[0067] In a preferred embodiment, the linker comprises 1 to 40 amino acids, preferably 1 to 35 amino acids, more preferably 1 to 30 amino acids, more preferably 1 to 25 amino acids, more preferably 1 to 20 amino acids, more preferably 1 to 10 amino acids, more preferably 1 to 9 amino acids, and most preferably 1 to 8 amino acids, in particular 1, 2, 3, 4, 5, 6, 7, or 8 amino acids and / or molecules that function equivalently, and / or combinations thereof, where if the linker comprises only amino acids, preferably at least one amino acid having any of the above functional groups, preferably cysteine, is present among the amino acids. The other amino acids contained in the linker can be selected from any known amino acid, including unnatural amino acids, but are preferably alanine (A) and / or glycine (G), preferably glycine (G).

[0068] As mentioned above, the length of the linker can vary and can be, for example, 9 amino acids, such as GGGGCGGGG (SEQ ID NO:148), or 8 amino acids, such as GGGCGGGG (SEQ ID NO:149), GGCGGGGG (SEQ ID NO:150) or GCGGGGGG (SEQ ID NO:151), or 7 amino acids, such as GGGGCGG (SEQ ID NO:152), GGGCGGG (SEQ ID NO:153), GGCGGGG (SEQ ID NO:154) or GCGGGGG (SEQ ID NO:155), 6 amino acids, such as GGGCGG (SEQ ID NO:156), GGCGGG (SEQ ID NO:157) or GCGGGG (SEQ ID NO:158), 5 amino acids, such as GCGGG (SEQ ID NO: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.

[0069] Most preferably, the linker in the cyclic compound comprises or consists of GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16).

[0070] In the first step of the method of the present invention, the target antigen is prepared and contacted with the binding molecule under conditions that allow the formation of the binding molecule-target antigen complex. Different incubation times can be selected as long as the binding of the binding molecule to the target antigen occurs. Thus, the incubation conditions can be varied and the optimal conditions can be tested. For example, any incubation condition that allows the binding of the binding molecule to its corresponding antigen can be tested by methods known in the art, for example, via ELISA or BLI. Preferably, the incubation time is 30 minutes and is preferably carried out at 37°C.

[0071] Contacting the target antigen with the binding molecule can be carried out in solution or by immobilizing the target antigen on a solid support, such as a microplate, to which the binding molecule is added.

[0072] In one embodiment of the method of the invention, target antigens, e.g. protein aggregates, oligomers, protofibrils, fibrils, misfolded monomers or alternatively protein fragments or peptides presenting (neo)epitopes of the subject antibodies and antibody-based drugs, preferably cyclic compounds of the invention, are contacted with binding molecules in solution.

[0073] In a preferred embodiment of the invention, the target antigen, e.g., protein aggregates, oligomers, protofibrils, fibrils, misfolded monomers, or alternatively, protein fragments or peptides presenting the (neo)epitopes of the subject antibodies and antibody-based drugs, are immobilized on a solid support, preferably on a microtiter plate, preferably in the form of a cyclic compound. In this context, it is understood that the target antigen may be modified, e.g., at or near its C-terminus or N-terminus, for the purpose of immobilizing the target antigen on the solid support. Additionally or alternatively, modifications may be provided to stabilize the target antigen, e.g., to prevent oxidation or otherwise degradation that is not critical for binding.

[0074] The target antigen can be immobilized on a solid support by common means known in the art, for example, directly coated by hydrophobic interactions without the need for heterologous functional groups, such as the biotin-streptavidin system, which can also be used for immobilization.

[0075] After incubation of the binding molecule with the target antigen, a population of modified effector cells is added, which express Fc receptors and harbor a reporter gene under the control of a response element responsive to activation by Fc receptors. The binding molecule-target antigen complex is contacted with the effector cells under conditions that allow the Fc domain of the target antigen binding molecule to bind to the Fc receptor of the effector cells. As mentioned above, different incubation times can be selected, as long as the binding of the binding molecule bound to the target antigen to the effector cells is ensured. In a preferred embodiment, the effector cells and the binding molecule-target antigen complex are incubated at 37° C. for about 6 hours.

[0076] Alternatively, all components, i.e., target antigen, binding molecule and effector cells, can be added simultaneously and co-culture results in binding of the binding molecule to the target antigen and to Fc receptors on the surface of the effector cells.

[0077] The binding of the binding molecule to the Fc receptor results in intracellular signal transduction that mediates the expression of a reporter gene that results in a quantifiable signal when an appropriate substrate is added. The reporter gene activity indicates the potency of the binding molecule, which means that a high reporter gene activity resulting in a strong signal indicates a high potency of the binding molecule, and a low reporter gene activity resulting in a weak signal indicates a low potency of the binding molecule.

[0078] Thus, there is a strong correlation between the ability of an antibody-based drug product, which relies on its mechanism of action on the recruitment of cells expressing Fc receptors, to bind to Fc receptors and the therapeutic efficacy of the drug product when administered to a patient in need thereof.

[0079] The potency of a formulation is a measure of the activity in a particular assay relative to the activity of a reference standard of the formulation against which the therapeutic effect may be evaluated. In particular, in the case of binding molecules, such as antibodies, that act by binding to Fc receptors, the method according to the present invention is suitable for use in determining the potency of a formulation, since the binding of the binding molecule to the Fc receptor is a direct indicator of the mechanism of action of the binding molecule.

[0080] In principle, any reporter gene can be used as long as it gives a detectable signal. For example, any reporter gene capable of catalyzing the conversion of a chromogenic, fluorogenic, or chemiluminescent substrate can be used. Such enzymes are known to those skilled in the art and include, for example, β-galactosidase, chloramphenicol acetyltransferase, and luciferase enzymes. In a preferred embodiment of the present invention, a gene encoding a bioluminescent protein, preferably luciferase, is used.

[0081] Binding of the Fc domain of a binding molecule to an Fc receptor of an effector cell mediates at least one effector function, i.e., one mechanism of action (MoA) of the Fc domain, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0082] In one embodiment of the present invention, MoA is ADCP. It is defined as a highly regulated process in which an antibody removes a target via connecting its Fc domain to a specific receptor on a phagocyte and inducing phagocytosis. In the context of the present invention, ADCP refers to the mechanism by which the Fc receptor of a phagocyte binds to a binding molecule, for example, an antibody that is bound to a target antigen, such as an aggregated protein or a cyclic compound that contains an epitope of the protein of interest, stimulating the phagocyte to internalize the protein and the cyclic compound, respectively. However, for the assay of the present invention, it is sufficient that a signal that induces ADCP is induced and causes reporter gene expression.

[0083] The reporter gene used in the method of the present invention is under the control of a response element that is responsive to activation by Fc receptors. The control of gene transcription and translation in response to stimuli is required to trigger the majority of biological responses, such as cell proliferation, differentiation, survival and immune response. These non-coding regions of DNA, called response elements, contain specific sequences that are recognition elements for transcription factors that regulate the efficiency of gene transcription and thus the amount and type of protein produced by cells in response to stimuli. In reporter assays, expression of the reporter gene is driven using standard molecular biology methods by modifying the response element that is responsive to the stimuli. The DNA is then transfected or transduced into cells that contain all the machinery that specifically responds to the stimuli, and the level of transcription, translation or activity of the reporter gene is measured as a surrogate measure of the biological response.

[0084] In one embodiment, the response element used in the methods of the invention comprises an NFAT (nuclear factor of activated T cells) response element, an AP-1 (Fos / Jun) response element, an NF AT / API response element, an NFKB response element, a FOXO response element, a STAT3 response element, a STAT5 response element, or an IRF response element. In some embodiments, the Fc receptor activation response element is arranged as a tandem repeat (such as about any of 2, 3, 4, 5, 6, 7, 8, or more tandem repeats). The Fc receptor activation response element may be located 5' or 3' to the reporter coding sequence.

[0085] Preferably, the assay of the present invention uses the same ADCP signaling pathway as that which naturally occurs during phagocytosis.In particular, like macrophages, when the binding molecule that is bound to target antigen binds to Fc receptor, the same signaling as ADCP is activated, which means that the assay of the present invention reflects the in vivo molecular pathway for Fc receptor-mediated phagocytosis through macrophages.Therefore, in a preferred embodiment of the present invention, the reporter gene is under the control of nuclear factor of activated T cells (NFAT) transcription factor.

[0086] Effector cells express Fc receptors. Fc receptors belong to a family of receptors specific for certain amino acids in the constant region of immunoglobulins. Their expression on individual cells depends on the type of receptor. Receptors for almost all immunoglobulin classes have been described. They are called FcγR (for IgG class), FcαR (for IgA class) and FcεR (for IgE class). Thus, in one embodiment of the invention, the FcR is an FcγR, FcαR or FcεR family member. Preferably, the effector cells used in the present invention express FcγR.

[0087] FcγRs have been identified that differ in their affinity to bind IgG and the relative affinity to bind IgG isotypes. The Fc receptors used in the present invention may be full-length Fc receptors or fragments thereof, where the fragments retain the ability to bind to Fc domains, such as the extracellular domain. The Fc receptors used in the present invention may also be wild-type Fc receptors of any allotype or mutants thereof, the function of which correlates with the function of the Fc receptor to which the FcR binding molecule binds in vivo. The Fc receptors used in the present invention may also be peptides that are not naturally occurring Fc receptors (or fragments or derivatives thereof), where the peptides are capable of binding to the FcR binding region of the Fc portion of an antibody, and the binding of the FcR binding molecule to the Fc binding peptide correlates with the function of the Fc receptor to which the FcR binding molecule binds in vivo.

[0088] Any Fc receptor can be selected that is suitable for mediating ADCP, including, for example, FcγRIIa (CD32a), FcγRI (CD64), and FcγRIIIa (CD16a). In a preferred embodiment, the Fc receptor is FcγR, more preferably FcγRI. Optionally, the effector cells do not express or overexpress FcγRIIa (CD32a) and / or FcγRIIIa (CD16a).

[0089] In one embodiment, the effector cell endogenously expresses an Fc receptor, i.e., the cell contains an endogenous sequence encoding an Fc receptor, where the cell is, for example, a macrophage, mast cell, monocyte, neutrophil, or dendritic cell.

[0090] In another preferred embodiment, the effector cells are modified to express Fc receptors, i.e. the cells are modified to contain a heterologous sequence encoding an Fc receptor. In principle, any cell suitable for expressing Fc receptors can be used. For example, the cells can be 8V-2, THP-1, CHO, 293-T, 3T3, 4Tl, 721, 9L, A2780, A172, A20, A253, A431, A-549, ALC, 816, 835, 8CP-1, 8EAS-28, bEnd.3, 8HK-21, 8R293, 8xPC3, C3H-10Tl / 2, C6, Cal-27, COR-L23, COS-7, CML The cell may be selected from the group consisting of Tl, CMT, CT26, 017, OH82, OU145, OuCaP, EL4, EM2, EM3, EMT6 / AR1, FM3, H1299, H69, H854, H855, HCA2, HEK-293, Hela, Hepalele7, HL-60, HMEC, HT-29, HUVEC, Jurkat, J558L, JY, K562, Ku812, KCL22, KGl, KYOl, MCF-7, R8L, Saos-2, SK8R3, SKOV-3, T2, T-470, T84, U373, U937, Vero, and J774. In a preferred embodiment, the cell is a Jurkat cell.

[0091] In the methods of the invention, the potency of a target antigen-binding molecule that contains an Fc domain is determined, which binds to any protein that forms a neoepitope in the pathogenic variant; see above.

[0092] The molecule whose efficacy is evaluated by the method of the present invention can be any molecule capable of binding to a target antigen. In one embodiment, such a molecule comprises an Fc domain. Preferably, the target antigen binding molecule is an antibody or any fragment, derivative or mimetic thereof that comprises an Fc domain. The target antigen comprises a full-length Fc domain or an FcR binding fragment of an Fc domain, so long as it maintains functionality.

[0093] As used herein, an "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain: CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains comprise binding domains that interact with target antigens, e.g., target proteins. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" also includes antibody formats that do not contain the entire binding domain of, for example, an IgG antibody, but still bind to a target antigen. Such antibody fragments include, for example, single variable domain antibodies, such as nanobodies linked to an Fc domain, in particular nanobodies that are about half the size of conventional antibodies and chimeric nanobody-heavy chain antibodies that combine the advantageous features of an Fc domain (see, for example, Bannas et al., Front. Immunol. (2017), DOI: 10.3389 / fimmu.2017.01603). In general, the term "antibody" encompasses any antibody fragment that contains an Fc domain.

[0094] The antibody may be a monoclonal or polyclonal antibody. A "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition and / or obtained from a population of substantially homogeneous antibodies. A monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. A "polyclonal antibody" refers to a heterogeneous pool of antibodies produced by several different B lymphocytes. Different antibodies in the pool recognize and specifically bind to different epitopes. An "epitope" refers to a polypeptide sequence that binds to antibodies produced in response to the sequence, either by itself or as part of a larger sequence. A target protein, e.g., TTR, may contain linear, discontinuous, and / or conformational epitopes.

[0095] The antibody may be a humanized antibody. "Humanized antibody" refers to an antibody that retains only the protein-binding CDRs from a parent antibody in the context of a human framework. In some embodiments, the antibody is a human antibody. "Human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences or from a human subject. A human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). The term "human antibody" as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., a mouse, are grafted onto human framework sequences (referred to herein as "humanized antibodies"). Human antibodies can be obtained, for example, as described in WO 2008 / 081008 A1. Humanized mice, which have been a prominent source of human antibodies against diverse targets, do not elicit or only poorly elicit immune and memory B cell responses. Several transgenic animal platforms are available, such as OmniAb® from Ligand, USA, Alloy ATX-GK™ mice from USA, and CAMouse™ from CAMAB, China. For example, RenMab™ mice with fully human variable region segments of heavy and kappa chains have been recently developed. For a review of the most prominent antibody engineering techniques used in the development of therapeutic antibody drugs, such as monoclonal antibody humanization, phage display, human antibody mice, single B cell antibody technology, and affinity maturation, see, for example, Lu et al., J. Biomed. Sci. 27 (2020), doi.org / 10.1186 / s12929-019-0592-z, and references cited therein.

[0096] The antibody can be a chimeric antibody, eg, a mouse-human, murine, bispecific or multispecific antibody, or an IgG.

[0097] The antibody may be a recombinant antibody. "Recombinant antibody" generally refers to an antibody that has been prepared, expressed, created, and / or isolated by recombinant means. A review of current antibody production systems is provided in Frenzel et al., Front Immunol. 4 (2013), 217, DOI: 10.3389 / fimmu.2013.00217, and transient expression of human antibodies in mammalian cells is described by Vazquez-Lombardi et al., Nature protocols 13 (2018), 99-117; and Hunter et al., Current Protocols in Protein Science 95 (2019), e77. DOI: 10.1002 / cpps.77. The antibody may be of a particular isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM, which refers to the immunoglobulin class encoded by the heavy chain constant region genes. Each isotype has a unique amino acid sequence and possesses a unique set of isotype epitopes that distinguish it from each other. However, preferably, the potency of IgG, particularly IgG1 antibodies, such as IgG1, lambda antibodies or IgG1, kappa antibodies, is assessed by the methods of the present invention.

[0098] As noted above, the term antibody herein, unless otherwise specified or clearly contradicted by context, includes antibody fragments, derivatives, variants (including deletion variants), and antibody mimetics that retain the ability to specifically bind to an antigen or to an Fc receptor.

[0099] Further molecules fused to an Fc domain, such as antibody mimetics, can be analyzed by the methods of the invention, including, for example, designed ankyrin repeat proteins (DARPins) fused to an Fc domain. Further included are modified Fc-based antibody domains and fragments, e.g., dimeric Fc, mFc, CH2 and mCH3 scaffolds (Ying et al., Biochim Biophys Acta. 1844 (2014), 1977-1982, DOI: 10.1016 / j.bbapap.2014.04.018 Wozniak-Knopp et al. Protein Eng Des Sel. 23 (2010), 289-297, DOI: 10.1093 / protein / gzq005), in which, for example, CDRs have been grafted and / or loop regions have been engineered to form new antigen-binding sites, as well as Fc fusion proteins composed of an immunoglobulin (Ig) Fc domain directly linked to another peptide, protein, or protein domain. For therapeutic purposes, the first description of a CD4-Fc fusion protein during HIV-1 infection in 1989 showed inhibitory activity against syncytia formation, providing proof-of-concept for the use of therapeutic Fc fusion proteins for the treatment of HIV-1 infection (Yang et al. Front Immunol 8(2018), 1860, DOI:10.3389 / fimmu.2017.01860).

[0100] As can be derived from Examples 3 and 4, the assay is exemplified by the amyloidogenic protein TTR and its aggregates, respectively, as target antigen and by the anti-TTR antibody NI-301.37F1 disclosed in International Application WO 2015 / 092077 A1, but in principle the method of the invention can be used for the analysis of any target antigen, in particular any protein that forms a neo-epitope in its pathogenic variants, e.g. an epitope that is only exposed in misfolded variants, a conformational epitope on aggregates, fibrils and / or oligomers, an epitope on an extracellular variant of an otherwise physiological protein located intracellularly, or an epitope specific for exogenous pathogens such as fungi, bacteria and viruses.

[0101] Such a protein can in principle be any protein, preferably any protein whose aggregation causes a disease phenotype.Typically, the protein includes, but is not limited to, transthyretin (TTR), where TTR is wild-type or mutated TTR, preferably wild-type TTR, α-synuclein (α-syn), tau, prion protein (PrP), amyloid beta (Aβ), β2-microglobulin (β2-m), immunoglobulin light chain (LC), immunoglobulin heavy chain (HC), serum amyloid A (SAA), amylin (IAPP), chromosome 9 open reading frame 72 (C9orf72), TAR These include DNA-binding protein 43 (TDP-43), superoxide dismutase 1 (SOD1), RNA-binding protein fused in sarcoma (FUS), huntingtin (htt), optineurin (OPTN), neuroserpin, ABri, Adan, ubiquilin, optineurin, leukocyte chemoattractant protein 2 (LECT2), gelsolin, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, cystatin C, and lysozyme. Additionally, the amyloid fibril-forming protein can be obtained from AmyPro, an open access database that provides a collection of amyloid fibril-forming proteins (Varadi et al., Nucleic Acids Research 46(2018), D387-D392, DOI:10.1093 / nar / gkx950), and / or can be those listed in Table 1 of Benson et al., Amyloid 25(2018), 215-219.

[0102] In a preferred embodiment, the amyloidogenic protein is involved in systemic amyloidosis and is more preferably selected from the following list: 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 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., Journal of Internal Medicine. Please refer to 289(2021), 268-292.

[0103] As illustrated in Example 6, the assay has been successfully performed using a cyclic peptide compound comprising the epitope WEPFA of the antibody NI-301.37F1 disclosed in International Application WO 2015 / 092077 A1, which is a neoepitope in the sense that it is located at positions 41-45 of the mature TTR protein, which is hidden in the natively folded conformation of the TTR protein, but accessible for antibody binding after unfolding and aggregation as the target antigen and the anti-TTR antibody NI-301.37F1 as the target antigen binding molecule.

[0104] Thus, in one preferred embodiment, the target antigen, i.e. a protein fragment or peptide, preferably in the form of a cyclic compound, preferably comprises a (neo)epitope from any protein whose aggregation causes a disease phenotype. Preferably, the (neo)epitope is derived from an amyloidogenic protein or aggregates thereof involved in systemic amyloidosis.

[0105] The protein fragments or peptides used in the potency assays of the cyclic compounds of the present invention contain at least 4, preferably at least 5, more 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 an amyloidogenic protein. More specifically, as known to those skilled in the art, at least the epitope of the target antigen-binding molecule, which may consist of as few as four amino acids that need to be present, may be added with an appropriate number of amino acids and / or other linker moieties that are sufficient and necessary for cyclization.

[0106] However, in principle, there is no limit to the length of the peptide, as long as it can be cyclized and is recognized by the target binding molecule. Thus, the cyclic compounds of the present invention as used herein may include proteins or fragments or peptides thereof that contain from 4 amino acids to all amino acids of an amyloidogenic protein. Preferably, the protein fragment or peptide in the cyclic 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.

[0107] 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, which amino acid residues comprise the epitope and adjacent amino acids.

[0108] The cyclic TTR peptide used in Examples 5 and 6 consists of the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (TTR34-54cyc; SEQ ID NO: 17) having a total of 31 amino acids, including the 5 amino acid epitope WEPFA, and 10 amino acid linker sequences of 5 amino acids each at the N-terminus and C-terminus of the 21 amino acid stretch from TTR. Thus, in a preferred embodiment, the cyclic compound is composed of a total of 20-40 amino acids, more preferably 25-35 amino acids, most preferably 30±1, 2, 3 or 4 amino acids, or is designed 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 the amyloidogenic protein present in the cyclic compound may be comprised of 10-40 amino acids, preferably 15-25 amino acids, most preferably 20±1, 2, 3 or 4 amino acids, optionally with the addition of a linker, distributed either at both termini, the N-terminus and the C-terminus or at only one terminus, preferably 5-20 amino acids in length, more preferably 5-15 amino acids, most preferably 10±1, 2, 3 or 4 amino acids. For example, when the epitope of the target binding molecule is a conformational or discontinuous epitope, a linker sequence or "filler" sequence may be considered to be located within the amino acid sequence derived from the amyloidogenic protein.

[0109] As mentioned above, the method is generally applicable to any target antigen, but preferably the protein fragment or peptide is derived from an amyloidogenic protein and comprises a (neo)pitope of the target antigen-binding molecule. The amyloidogenic protein can in principle be any amyloidogenic protein, for example listed in Table 1 of Benson et al., Amyloid 25 (2018), 215-219 and described above. In a preferred embodiment, the amyloidogenic protein is involved in systemic amyloidosis, and more preferably is selected from the following list: transthyretin (TTR), particularly wild-type and mutant TTR, immunoglobulin light chain (LC), immunoglobulin heavy chain (LH), serum amyloid A (SAA), leukocyte chemoattractant 2 (LECT2), gelsolin, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, β2 microglobulin, particularly wild-type and mutant β2 microglobulin, cystatin C, ABriPP, prion protein, and lysozyme, and thus the target antigen comprises a peptide derived from any one of the listed proteins, and preferably the peptide comprises at least 4 amino acids from the protein.

[0110] In a preferred embodiment, the amyloidogenic protein is TTR, and therefore the target antigen comprises a protein fragment of TTR or a peptide derived from TTR.

[0111] In general, the TTR protein fragment or peptide may be any fragment or peptide derived from the TTR protein. In a preferred embodiment, the TTR fragment or peptide in the cyclic compound used according to the method of the present invention comprises at least four amino acids from the TTR protein, where the four amino acids may be, for example, any one of those listed in Table 1 below.

[0112] [Table 1-1]

[0113]

Table 1-2

[0114]

Table 1-3

[0115] In a preferred embodiment, the TTR peptide comprises at least 4 amino acid residues, and preferably all amino acids of the amino acid sequence that are exposed in misfolded variants and to aggregates, fibrils and / or oligomers, respectively, such as WEPFA (SEQ ID NO: 1), which is a 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 a peptide recognized by the antibody NI-301.59F1 disclosed in WO 2015 / 092077 A1; EEFXEGIY (SEQ ID NO: 3), which is a peptide recognized by the antibody NI-301.35G11 disclosed in WO 2015 / 092077 A1; ELXGLTXE (SEQ ID NO: 3), where X can be any amino acid; WEPFASG (SEQ ID NO: 4), for example, which is the peptide recognized by the antibody NI-301.12D3 disclosed in WO 2015 / 092077 A1; TTAVVTNPKE (SEQ ID NO: 5), for example, which is the peptide recognized by the antibody NI-301.18C4 disclosed in WO 2015 / 092077 A1; KCPLMVK and VFRK (SEQ ID NOs: 6 and 7), which represent peptides comprising a conformational 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;EHAEVVFTA (SEQ ID NO: 8), a peptide recognized by the antibody 14G8 / PRX004 disclosed in Amyloid 23 (2016), 86-97; GPRRYTIAA (SEQ ID NO: 9), a peptide recognized by the antibody 18C5 described, for example, in WO 2019 / 071205 A1; VHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVE (SEQ ID NO: 10), a peptide recognized by the antibody described, for example, in WO 2014 / 124334 A2, which binds to TTR30-66; ALLSPYSYSTTAV (SEQ ID NO: 11), a peptide recognized by the antibody described in WO 2015 / 115332 A1; WKALGISPFHE (SEQ ID NO: 12), a peptide recognized by the antibody 371M described in WO 2015 / 115332 A1; SYSTTAVVTN (SEQ ID NO: 13), a peptide recognized by the antibody 313M (RT24) described in WO 2015 / 115331 A1; or LLSPYSYSTTAVVTNPKE (SEQ ID NO: 14), a peptide recognized by the antibody described in WO 2014 / 124334 A2, which binds to TTR100-127.

[0116] Most preferably, the TTR peptide according to the methods of the present invention comprises the amino acid sequence WEPFA (SEQ ID NO:1).

[0117] As mentioned above, the cyclic compound used according to the method of the present invention preferably comprises a protein fragment or peptide comprising an epitope of an amyloidogenic protein, preferably a TTR epitope, and most preferably an epitope comprising the amino acid sequence WEPFA (SEQ ID NO: 1), and a linker at the N-terminus and C-terminus of the adjacent amino acids and peptides, where the linker may in principle comprise any of the linker sequences described above, and preferably comprises the amino acid sequence GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16). Thus, as a preferred embodiment of the method of the present invention, the cyclic compound comprises 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 5 and 6.

[0118] Thus, the binding molecule whose potency, in particular its potency to induce ADCP, is measured by the method of the invention may be any binding molecule that binds to said target antigen, preferably any protein that induces the disease phenotype in its pathogenic variants, and the corresponding protein fragment or peptide thereof. Exemplary antibodies include, but are not limited to, anti-TTR antibodies, anti-α-syn antibodies, anti-tau antibodies, anti-PrP antibodies, anti-Aβ antibodies, anti-β2-m antibodies, anti-LC antibodies, anti-HC antibodies, anti-SAA antibodies, anti-IAPP antibodies, anti-C9orf72 antibodies, anti-TDP-43 antibodies, anti-SOD1 antibodies, anti-FUS antibodies, anti-htt antibodies, anti-OPTN antibodies, anti-neuroserpin antibodies, anti-ABri antibodies, anti-ADan antibodies, anti-ubiquilin antibodies, anti-optineurin antibodies, anti-LECT2 antibodies, anti-gelsolin antibodies, anti-ApoAI antibodies, anti-ApoAII antibodies, anti-ApoAVI antibodies, anti-ApoCII antibodies, anti-ApoCIII antibodies, anti-fibrinogen antibodies, anti-cystatin C antibodies, anti-ABriPP antibodies, anti-prion antibodies, and anti-lysozyme antibodies.

[0119] In a preferred embodiment, the binding molecule is a binding molecule that binds to a target involved in systemic amyloidosis, and therefore the antibody is preferably selected from the group consisting of an anti-TTR antibody, an anti-LC antibody, an anti-HC antibody, an anti-SAA antibody, an anti-LECT2 antibody, an anti-gelsolin antibody, an anti-ApoAI antibody, an anti-ApoAII antibody, an anti-ApoAVI antibody, an anti-ApoCII antibody, an anti-ApoCIII antibody, an anti-fibrinogen antibody, an anti-β2 microglobulin antibody, an anti-cystatin C antibody, an anti-ABriPP antibody, an anti-prion antibody, and an anti-lysozyme antibody.

[0120] Thus, the assay of the present invention can be used to measure the activity / potency of any suitable binding molecule. Suitable antibodies are known in the art, however, exemplarily, antibodies are listed below.

[0121] Anti-TTR antibodies are preferred to be analyzed by the method of the present invention and are those disclosed in WO 2015 / 092077 A1, in particular those having the amino acid sequence TTR 41-45 (SEQ ID NO: 51 of WO 2015 / 092077A1), in particular NI-301.37F1, NI-301.28B3, and NI-301.12D3. In addition, PRX004, which is currently in Phase 1 trials (ClinicalTrials.gov Identifier: NCT03336580) in patients with ATTR, may be a suitable antibody. The antibody PRX004 corresponds to and corresponds to a humanized version of the murine monoclonal antibody 14G8 described in Higaki et al., Amyloid 23 (2016) 86-97 (see Table 4 on page 91 of WO 2019 / 071206 A1) and described in WO 2016 / 120810 A1 and WO 2018 / 007922 A2, more particularly in WO 2019 / 108689 A1. Further suitable antibodies bind to the same epitope as the antibody PRX004, namely the amino acid TTR. 89-97, or the amino acid TTR 101-109 and are humanized versions of the originally cloned mouse monoclonal antibodies 14G8, 9D5, 5A1, 6C1 described in WO 2016 / 120810A1, WO 2018 / 007924A2, WO 2018 / 007924A2 and WO 2018 / 007923A1. Further suitable antibodies are the humanized versions of the antibody 18C5 described in WO 2019 / 071205A1, the antibody 371M having an epitope at positions 79-89 of human TTR described in WO 2015 / 115332A1, and the antibody 313M (RT24) having an epitope at positions 115-124 of human TTR described in WO 2015 / 115331A1. These antibodies can also be used as control antibodies in the methods of the present invention.

[0122] In a preferred embodiment, the binding molecule comprises a variable heavy (VH) domain having, in its variable region, i.e., in the binding domain, the amino acid sequence depicted in FIG. 1C of WO 2015 / 092077 A1 and set forth in Table 2 herein. H ) chain and variable light (V L1C of WO 2015 / 092077A1 and Table 2 herein, or one or more of the CDRs, in the case of CDR2 and CDR3, may differ in their amino acid sequence by one, two, three or even more amino acids from those set out in FIG. 1C of WO 2015 / 092077A1 and Table 2 herein, wherein the antibody exhibits substantially the same or identical characteristics of the anti-TTR antibody NI-301.37F1 exemplified in the Examples of WO 2015 / 092077A1. The positions of the CDRs are shown in FIG. 1C, explained in the legend to FIG. 1 in WO 2015 / 092077A1 and underlined in Table 2 herein. Additionally or alternatively, the framework regions or the complete VH and / or VL chain are 80% identical to the framework regions depicted in FIG. 1C or FIG. 1M of WO 2015 / 092077 A1 and shown in Table 2 herein, and preferably are 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the framework regions and the VH and / or VL chain, respectively, depicted in FIG. 1C or FIG. 1M of WO 2015 / 092077 A1 and shown in Table 2 herein. Furthermore, cloning and expression of antibody NI-301.37F1 have been performed as described in Examples 1 and 2, pages 110-112 of WO 2015 / 092077 A1, the methods of which are incorporated herein by reference.

[0123] Thus, according to one embodiment of the methods of the invention, the anti-TTR antibody is selected from the group consisting of V, VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, VL16, VL17, VL18, VL19, VL20, VL21, VL2 H and V L The CDRs of the V H and V L Thus, the antibody preferably comprises: (i) a variable heavy (VH) chain comprising VH complementarity determining regions (CDRs) 1, 2, and 3 as follows: and / or a variable light (VL) chain comprising VL CDRs 1, 2, and 3 as follows: (a) VH-CDR1: positions 31 to 35 of SEQ ID NO: 19 (corresponding to SEQ ID NO: 10 of WO 2015 / 092077 A1) or a variant thereof containing one or two amino acid substitutions; (b) VH-CDR2: positions 52 to 67 of SEQ ID NO: 19 (corresponding to SEQ ID NO: 10 of WO 2015 / 092077A1) or a variant thereof containing one or two amino acid substitutions; (c) VH-CDR3: positions 100 to 109 of SEQ ID NO: 19 (corresponding to SEQ ID NO: 10 of WO 2015 / 092077A1) or a variant thereof containing one or two amino acid substitutions; (d) VL-CDR1: positions 24 to 34 of SEQ ID NO: 21 (corresponding to SEQ ID NO: 12 of WO 2015 / 092077 A1) or a variant thereof containing one or two amino acid substitutions; (e) VL-CDR2: positions 50 to 56 of SEQ ID NO: 21 (corresponding to SEQ ID NO: 12 of WO 2015 / 092077 A1) or a variant thereof containing one or two amino acid substitutions, and (f) VL-CDR3: positions 89 to 97 of SEQ ID NO: 21 (corresponding to SEQ ID NO: 12 of WO 2015 / 092077 A1) or a variant thereof containing one or two amino acid substitutions; and / or (ii)(a) the VH chain comprises an amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 23 (corresponding to SEQ ID NO: 10 and SEQ ID NO: 53 in WO 2015 / 092077 A1), or a variant thereof comprising one or more amino acid substitutions; and (b) the VL chain comprises an amino acid sequence represented by SEQ ID NO: 21 (corresponding to SEQ ID NO: 12 of WO 2015 / 092077 A1), or a variant thereof comprising one or more amino acid substitutions; Preferably, the VH and VL chain amino acid sequences are at least 90% identical to SEQ ID NO: 19 or 23 (corresponding to SEQ ID NO: 10 and SEQ ID NO: 53 of WO 2015 / 092077 A1), and SEQ ID NO: 21 (corresponding to SEQ ID NO: 12 of WO 2015 / 092077 A1), respectively.

[0124] According to a preferred embodiment of the method of the present invention, the anti-TTR is NI-301.37F1 and comprises within 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.

[0125] [Table 2]

[0126] Anti-Aβ antibodies include aducanumab (Sevigny et al. Nature 537 (2016), 50-56), bapineuzumab (see review by Kerchner and Boxer, Expert Opin Biol Ther. 10 (2010), 1121-1130, DOI: 10.1517 / 14712598.2010.493872 (including key references cited therein)), gantenerumab (Bohrmann et al., Journal of Alzheimer's Disease 28 (2012), 49-69), crenezumab (Guthrie et al., J Alzheimers Dis. 76 (2020), 967-979, DOI: 10.3233 / JAD-200134), and BAN2401 (Lannfelt et al., Alzheimers Res Ther 6 (2014), 16, DOI: 10.1186 / alzrt246; WO 2007 / 108756 A1), ponezumab (Burstein et al., Clin Neuropharmacol. 36 (2013), 8-13), and solanezumab (Honing et al., N Engl J Med 378 (2018), 321-330, DOI: 10.1056 / NEJMoa1705971).

[0127] Anti-tau antibodies may include those described in Yanamandra et al., Ann Clin Transl Neurol 2 (2015), 278-288, DOI: 10.1002 / acn3.176, WO 2012 / 049570 A1, and WO 2014 / 100600 A1), and in particular antibodies BIIB076 (6C5), BIIB092 (goslanemab), bepraneumab (UCB0107), C2N-8E12, and RG6100 (also described in Medina, Int J Mol Sci. 19 (2018), 1160).

[0128] Anti-alpha-syn antibodies may include those described in WO 2012 / 177972 A1 and WO 2010 / 069603 A1, as well as, in particular, prasinezumab (PRX002), simpanemab (BIIB054), ABBV-0805 and MEDI1341.

[0129] Anti-TDP-43 antibodies, anti-SOD1 antibodies, and anti-IAPP antibodies may include those described in WO 2013 / 061163A2, WO 2012 / 080518A1, in particular the antibody NI-204.12G7, and those described in WO 2014 / 041069A1, in particular the antibodies NI-203.26C11 and NI-203.11B12.

[0130] Anti-C9orf72 antibodies may include those described in WO 2016 / 050822 A2 and WO 2019 / 210054 A1, anti-LC antibodies may include antibodies 11-1F4 and NEOD00 (Muchtar and Gertz, Expert Opinion on Orphan Drugs 5 (2017), 655-663, and anti-PrP antibodies may include antibody PRN100.

[0131] Anti-SAA antibodies may include desamizumab (GSK2398852) and anti-HTT antibodies may include those disclosed in WO 2016 / 016278 A2, in particular the antibodies NI-302.35C1 and NI-302.31F11.

[0132] Further exemplary antibodies and equivalent binding molecules that bind to target antigens such as the aggregation proteins described above are known in the art or can be identified using standard techniques. The assays of the present invention allow for rapid and accurate testing of such antibodies to confirm their ability to induce ADCP.

[0133] In this context, amyloidogenic proteins, particularly systemic amyloidogenic proteins, especially transthyretin (TTR), which are preferably present as protein aggregates, oligomers, fibrils or protofibrils, protein monomers, especially in misfolded structures, and proteins prone to aggregation, such as fragments and synthetic peptides derived therefrom, can be used as target antigens, which contain an epitope of the target antigen-binding molecule-containing antibody or similar Fc domain, preferably a cyclic compound as defined above. In a preferred embodiment, the protein fragment or peptide contains an epitope of any one of the antibodies described herein above, most preferably an epitope of any one of the anti-TTR antibodies referred to herein above.

[0134] In a preferred embodiment of the invention, anti-TTR antibodies are evaluated for their potency in inducing ADCP against aggregated protein TTR as the preferred target antigen and against cyclic compounds containing epitopes of TTR.

[0135] In one embodiment, the method of the present invention comprises at least i) spotting target antigens such as aggregated proteins or cyclic compounds into wells of a microplate (i.e., a microplate (96-well plate) is coated with the target antigens, preferably for 1 hour at 37° C. (protein aggregates) or overnight at 4° C. (cyclic compounds), preferably protein aggregates diluted to a concentration of 10 μg / ml in PBS buffer pH 7.4 and cyclic compounds diluted to 3 μg / ml in PBS buffer 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 for 30 minutes at 37°C; iii) contacting the complex comprising the binding molecule and the target antigen with an effector cell (i.e., effector cells, also called reporter cells, are added to the complex, where the effector cell expresses an Fc receptor and a reporter gene under the control of a response element that is responsive to activation by the Fc receptor, preferably the effector cell is a modified cell, more preferably a Jurkat cell expressing an FcγRI receptor and a luciferase gene under the control of an NFAT transcription factor, and the complex is incubated at 37° C. for 6 hours); iv) adding a substrate solution, preferably a luminescent substrate solution; v) detecting a signal, preferably a luminescent signal, with a luminometer; Includes.

[0136] Coating of the plate with the cyclic compound is preferably performed by immobilization on a plastic surface mainly by hydrophobic interaction, but can also be performed by using a biotin-streptavidin system. However, as described above, instead of spotting the target antigen into the well of a microplate, the contact between the target antigen and the target antigen-binding molecule can also be performed in solution without the target antigen being spotted into the well of a solid support such as a microplate.

[0137] In a preferred embodiment, a step of blocking non-specific binding sites is performed prior to step (ii), preferably blocking is performed with a blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% Tween®-20 in PBS buffer for 1 hour at room temperature.

[0138] In one embodiment, the method of the invention further comprises a step of preparing the target antigen before spotting it on a microplate or other solid support. For protein aggregates, methods for preparing protein aggregates are well known in the art, and may use, for example, the aggregation buffer described in Example 3. Preparation of Ab fibrils is described, for example, in WO 2017 / 157961 A1. Preparation of protein aggregates may further comprise purification of each protein before subjecting it to conditions that allow aggregation. Purification can be performed via protein chromatography followed by a lectin column to remove residual immunoglobulins. Methods for preparing cyclic compounds are also known in the art, as explained above. In a preferred embodiment, the peptide is cyclized via a disulfide bridge between cysteine ​​residues in the linker; see above. Cyclic compounds are prepared in solution and are not subjected to any specific procedure before use, and are therefore in their native monomeric form.

[0139] The method of the invention may further comprise a step of controlling / verifying the quality of the protein aggregates or cyclic compounds. This can be done by various methods, for example by conventional ELISA and / or Biolayer Interferometry (BLI) using antibodies known to bind to aggregated proteins or cyclic compounds. Such methods are described in the attached Examples 3 and 5.

[0140] Different assay setups have been tested for variability in terms of plate layout. In principle, the dilution series of the target antigen-binding molecule, e.g., antibody, can be arranged either horizontally (e.g., wells A1-A12) or vertically (e.g., wells A1-H1). The number of dilution points (e.g., 8 points, 12 points, 16 points, etc.) and the orientation (i.e., the first well can have either the lowest or the highest ligand concentration) can be freely selected. In single-dose assays, the positions of the reference and positive control can be freely selected by the user. However, in the course of experiments carried out according to the present invention implementing it, it was surprisingly found that while both orientations work well enough, a vertical plate layout that allows three samples to be measured simultaneously and in triplicate on the same plate gives the most reliable results. Thus, in one preferred embodiment, the method of the present invention is carried out using a vertical plate layout. Furthermore, in the case of a 96-well microplate, the 24 outer wells exhibit 24% greater variability than that observed with the 60 inner wells, and therefore, preferably, the inner wells are used when carrying out the methods of the present invention.

[0141] When anti-TTR antibodies are analyzed by the methods of the invention, the antibody NI-301.37F1, characterized above, can be used as a control, either as a quality control for aggregated TTR batches or as a positive control for potency assays.

[0142] As can be derived from Examples 3, 4 and 6, the method of the present invention has the ability to detect changes in antibody activity and to detect the loss of antibody potency with respect to Fc domain modification. The latter is tested by subjecting the antibody to stress conditions that reproduce the loss of antibody potency. Thus, the method of the present invention has the ability to detect approximately at least ±35% to ±50% changes in binding molecule activity.

[0143] The present invention further relates to a method for producing a pharmaceutical composition of a target antigen binding molecule as defined above, i.e. a binding molecule which comprises an Fc domain and is preferably an antibody or any fragment or derivative thereof or an antibody mimetic.

[0144] In a first step, the binding molecule and the formulation are provided and preferably produced, respectively. Means and methods for the recombinant production of antibodies, corresponding binding molecules, fragments, derivatives and mimetics thereof are known in the art. In particular, their recombinant production in host cells, purification, modification, formulation in pharmaceutical compositions and therapeutic use, as well as terms and characteristics common in the art, can be relied upon by the skilled artisan when carrying out the invention as claimed (see, for example, Antibodies A Laboratory Manual 2nd edition, 2014 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA; Frenzel et al., Front Immunol. 4 (2013), 217, doi: 10.3389 / fimmu.2013.00217; Lalonde and Durocher, Journal of Biotechnology 251 (2017), 128-140, DOI: 10.1016 / j.jbiotec.2017.04.028; Tripathi and Shrivastava, Front.Bioeng.Biotechnol.7(2019),420,DOI:10.3389 / fbioe.2019.00420), where purification and storage of antibodies; modification of antibodies, such as the use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis, immunoblotting protocols and modern screening and labeling techniques are also described. The production of DARPins is described, for example, in Stumpp et al., Drug Discovery Today 13(2008),695-701 and references cited therein and in Hanenberg et al., J Biol Chem 289(2014),27080-27089,DOI:10.1074 / jbc.M114.564013. Furthermore, the production of the formulations can be carried out in any manner as desired and / or suitable for the formulation in question.

[0145] In the next step, the binding molecule is subjected to the method of the present invention. In particular, the binding molecule is subjected to a method for measuring the potency of the binding molecule, in particular its potency for inducing ADCP. The information obtained from the assay is used as part of the evaluation of whether the binding molecule may be used as a pharmaceutical composition, i.e., whether a formulation containing the binding molecule meets the criteria to be injected into patients as agreed with the national regulatory authority, if the formulation may be injected. Furthermore, the information is used to identify the binding molecule for use in the pharmaceutical composition.

[0146] In a further preferred embodiment of any of the above embodiments of the method of the invention, the target antigen-binding molecule, in particular a target antigen-binding molecule found to be useful by the method of the invention, is formulated as a pharmaceutical composition with a pharma- ceutical acceptable carrier. Useful binding molecules have, for example, an EC20 activity in the (sub)-nanomolar range when assessed by the method of the invention. 50 The binding molecule is one that exhibits a value or exhibits a potency similar to that of a reference standard, for example, at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100%, as compared to the potency of a positive control. Pharmaceutically acceptable carriers and routes of administration can be obtained from the corresponding literature known to those skilled in the art. Pharmaceutical compositions can be formulated according to methods well known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN0-683-306472, Vaccine Protocols 2 nd Edition by Robinson et al.,Humana Press,Totowa,New Jersey,USA,2003;Banga,Therapeutic Peptides and Proteins:Formulation,Processing,and Delivery Systems.2 ndSee, for example, The American Journal of Clinical Chemistry, Vol. 13, No. 1, pp. 1111-1115, 1999. Edition by Taylor and Francis. (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solution, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to subjects in suitable doses. Administration of suitable compositions may be performed in different ways. Examples include administering compositions containing pharma- ceutically acceptable carriers via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.

[0147] The present invention also provides a method for preparing a pharmaceutical or diagnostic agent comprising a target antigen-binding molecule, wherein the potency of the binding molecule for activating ADCP is at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100%, compared to the potency of a positive control. The method includes the preparation of a binding molecule as described above, and a batch of said binding molecule is obtained. The potency of the batch, in particular the potency of the batch for activating ADCP, is then analyzed by the method of the present invention. The method further includes the preparation of a pharmaceutical or diagnostic agent from the batch, but only if it is determined that the batch has an potency of at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100%, in particular for activating ADCP, compared to the potency of a positive control.

[0148] 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 a batch of the analyte that has been stored and / or subjected to stress conditions, and the control is the value of reporter gene activity of a sample taken from that batch or a corresponding batch before storage and / or before being subjected to said stress conditions.

[0149] The invention also provides a method as described above, said method being part of a marketing approval application for selling said formulation as a pharmaceutical composition. The invention also provides a method for applying for marketing approval of a formulation comprising a binding molecule, comprising describing the method of the invention for measuring the potency of the binding molecule in the formulation.

[0150] As mentioned above, the method of the present invention can be used as a potency assay for batch release, i.e., the method of the present invention is useful for analyzing different batches, for example, from the production of a given target antigen-binding molecule.

[0151] Any continuous production of a drug product will result in the production of different batches of product to be released as a pharmaceutical product. A key feature in production is to ensure that the different batches comply with the same standard. This standard is typically set in conjunction with a regulatory body. Typically, each batch will be inspected and tested by several different assays to ensure that the batch is of sufficient quality to be approved in the market. This can be achieved by the method of the present invention.

[0152] Therefore, the present invention also relates to a method for analyzing and selecting at least one batch of pharmaceutical compositions of target antigen-binding molecules as defined above, comprising in a first step the evaluation of the potency of a sample of the batch, in particular its potency for activating ADCP, by the method of the present invention. As mentioned above, reporter gene activity is a measure for the potency of a binding molecule, and therefore the reporter gene activity of the sample is compared to the reporter gene activity of a control, and a batch is selected, in which the sample shows a reporter gene activity greater than, equal to, or not substantially lower than the control. In one embodiment, a batch is selected, in which the sample shows a reporter gene activity greater than, equal to, or more than 80%, preferably 90%, preferably 95%, preferably 98%, preferably 99%, more preferably 100%, in comparison to the control. The selected batch can further be, for example, packed into a kit and distributed to a consumer (costumer).

[0153] Thus, the present invention relates to a method for validating a batch of target binding molecules, i.e. for determining the quality of target antigen (e.g. aggregating protein) binding molecules for distribution, wherein a sample of the batch is tested by the method of the present invention for its potency to activate an ADCP, and the batch is validated for distribution only if the potency of the sample of the batch to activate an ADCP is at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100%, compared to the potency of a positive control to activate an ADCP.

[0154] In a preferred embodiment, the method is particularly useful for analyzing and selecting batches of pharmaceutical compositions comprising anti-TTR antibodies and validating batches of anti-TTR antibodies for distribution, respectively. The control can be a reference standard and / or, if the batch to be analyzed has been stored and / or subjected to stress conditions, the control can be the value of reporter gene activity of a batch or a sample taken from the corresponding batch before storage and / or before being subjected to said stress conditions.

[0155] The binding of the binding molecule of the formulation to the Fc receptor is compared to the binding of a reference standard to the Fc receptor, and the therapeutic effect of the binding molecule of the formulation is assessed from its ability to bind to the Fc receptor to the same or substantially the same extent as the reference standard.

[0156] As mentioned above, the potency of the samples of the batch should preferably be at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100% compared to the potency of the reference standard. However, the particular extent to which the FcR binding profile of the binding molecule of the formulation and the FcR binding profile of the reference standard may differ can be established on a case-by-case basis, e.g., determined in conjunction with the appropriate regulatory body.

[0157] In order to be able to determine FcR binding in a reliable and consistent manner, the FcR binding of the binding molecule of the drug product and the reference standard must be performed using the same assay, preferably using the assay of the present invention.Typically, the determination of the binding of the reference standard is performed first to establish a standard that any subsequent batches of binding molecules can be compared to.However, the determination of the binding of the reference standard may also be performed simultaneously with or after the determination of the FcR binding of the binding molecule of the drug product.

[0158] The present invention further relates to the use of a target antigen binding molecule as defined above, a cyclic compound as defined above, and / or an effector cell as defined above in a method according to the present invention. In particular, the effector cell is engineered to express the human Fc receptor FcγRI (CD64) and harbor a reporter gene under the control of a response element responsive to activation by the Fc receptor.

[0159] Furthermore, the present invention provides at least (i) a population of effector cells that express an Fc receptor and have been genetically modified to harbor a gene encoding a reporter under the control of a response element that is responsive to activation by the Fc receptor; (ii) a corresponding substrate of the reporter; and optionally (iii) target antigen; (iv) a microtiter plate, preferably a 96 or 384 well plate including a lid; (v) Recommendations for, and instructions for use of, buffers, diluents, substrates and / or solutions; (vi) washing, blocking and assay / sample dilution buffers; and / or (vii) a positive control target antigen-binding molecule, preferably an antibody The present invention relates to a kit comprising:

[0160] Preferably, the kit is adapted to carry out the method of the present invention, in particular to assay the efficacy of a binding molecule comprising an Fc domain to induce ADCP. Thus, in a preferred embodiment, the instructions relate to instructions for use of the kit in a method for measuring the efficacy of a target antigen-binding molecule comprising an Fc domain, and preferably to instructions for use in a method for carrying out the assay of the present invention.

[0161] In a preferred embodiment, the population of effector cells is a population of Jurkat cells expressing a gene encoding a luminescent protein, preferably luciferase, under the control of FcγR, preferably FcγRI and NFAT transcription factors, and the kit comprises a luminescent substrate solution.Furthermore, the target antigen is preferably an aggregated protein, more preferably aggregated TTR, or a cyclic compound, each of which contains an epitope of a target binding molecule, more preferably an epitope of an anti-TTR antibody and an epitope of TTR, and the binding molecule is an anti-TTR antibody.

[0162] The method does not necessarily have to be performed on a microtiter plate, but any solid support onto which the target antigen can be spotted or any vial into which the assay components can be incubated would be suitable.

[0163] The present invention further relates to a composition comprising a target antigen-binding molecule of the present invention that has been analyzed, validated and selected according to the present invention, the composition further comprising a pharma- ceutically acceptable carrier.

[0164] To verify that the analyzed binding molecules indeed induce phagocytosis and cause the engulfment of target antigens, e.g., protein aggregates or cyclic compounds, in vitro phagocytosis assays have been performed as described in Examples 1 and 2. These assays show that antibody NI-301.37F1_W1 indeed induces phagocytosis of TTR aggregates. Thus, the methods of the invention for assaying the potency of binding molecules may be combined with in vitro phagocytosis assays.

[0165] Furthermore, the binding of the analyzed binding molecule to its corresponding antigen may be verified by methods known in the art, for example via ELISA or BLI as shown in Examples 3 and 5. Thus, the methods of the invention for assaying the potency of a binding molecule may be combined with methods for determining the binding of a binding molecule to its antigen.

[0166] In certain preferred embodiments of any one of the methods and kits of the invention described above and / or characterized in the claims, (1) the efficacy, i.e. the effector function to be determined, is antibody-dependent cellular phagocytosis (ADCP); (2) the target antigen is an amyloidogenic protein, most preferably an amyloidogenic protein aggregate involved in systemic amyloidosis, preferably in cyclic form, containing an epitope of TTR, most preferably TTR, or a protein fragment or peptide derived therefrom; (3) the Fc receptor is the human Fc receptor FcγRI (CD64); the effector cell is a Jurkat cell, preferably which does not overexpress FcγRIIa (CD32a) and FcγRIII (CD16); the response element is an NFAT (nuclear factor of activated T cells) response element; the reporter gene codes for luciferase; and the target binding molecule is an IgG1 antibody, e.g., an IgG1, lambda antibody or an IgG1, kappa antibody.

[0167] As mentioned in the Summary of the Invention and described above, in a further aspect, the present invention relates to cyclic compounds comprising peptides that include epitopes from amyloidogenic proteins involved in systemic amyloidosis.

[0168] In particular, in the course of experiments performed according to the present invention, it was unexpectedly found that a cyclic peptide comprising an epitope of TTR, in particular the epitope recognized by the anti-TTR antibody NI-301.37F1 (WEPFA (SEQ ID NO: 1) described in WO 2015 / 092077 A1), which selectively binds with high affinity to TTR aggregates of either wild-type or mutant TTR, is an excellent target antigen in ELISA and ADCP assays. As described in Example 5 and illustrated in FIG. 10, the antibody exhibits highly specific binding to the cyclic compound, where the antibody binds to the cyclic peptide. The binding affinity to the cyclic peptides is an order of magnitude higher than that of their native target antigen, i.e., misfolded TTR against which the antibodies were initially screened and identified. This remarkable effect was unexpected and advantageous, not only because such cyclic peptides can replace the preparation of full-length amyloidogenic proteins and their aggregates / fibrils, which are prone to variability and are more time-consuming than the preparation of cyclic peptides, but also because the cyclic compounds represent excellent target antigens in binding assays such as ELISA and functional assays such as ADCP, which require high sensitivity and reproducibility, as exemplified in Examples 5 and 6 and shown in Figures 10-12.

[0169] First, a cyclic TTR peptide was designed to solve the crystal structure of the Fab fragment of the antibody NI-301.37F1 in complex with its TTR antigen, to obtain information about the three-dimensional structure of the antibody-antigen complex and understand its mechanism of action. It is coincidental that the cyclic TTR peptide is used to replace the full-length recombinant TTR protein in the ELISA assay for the determination of the antibody NI-301.37F1, i.e., IgG antibody, and it was unexpectedly shown that the ELISA assay is much more sensitive and reliable than the use of recombinant TTR protein; see Example 5 and Figure 10. Subsequent experiments have demonstrated that, even more unexpectedly, the use of the cyclic TTR peptide substantially improves the sensitivity and reliability of the potency assay of the present invention.

[0170] In what follows, analysis of the cryo-electron microscopy (cryo-EM) structure shows that the two ends of the unsolved loop are in contact with each other, suggesting that the epitopes and peptide sequences in the cyclic peptide and cyclic compound, respectively, may have been similarly selected. Thus, the use of the cryo-EM structure may be used in addition to or as an alternative to peptide design in the crystallographic structure of the Fab-peptide antigen to select suitable epitopes and amino acid sequences containing the same as in the design of the cyclic peptides of the present invention that exhibit the same advantageous properties as the cyclic TTTR peptides that have been experimentally found to be such effective and reliable tools in ELISA and ADCP assays. As mentioned above, it is noteworthy that cryo-EM studies have shown that the amyloid structures of systemic amyloidogenic proteins such as ATTR and AL amyloidosis, which result from misfolding of the immunoglobulin light chain (LC), are on the one hand similar to those of local amyloidogenic proteins such as tau, but on the other hand substantially different; see Figure 5 in Schmidt et al., Nat. Commun. 10 (2019), 5008, https: / / doi.org / 10.1038 / s41467-019-13038. It is therefore prudent to expect that the present results for the TTR-derived cyclic peptides can also be applied to at least other systemic amyloidogenic proteins.

[0171] Methods for generating crystal structures of antibodies and their Fab fragments for their complexes with peptides and peptide antigens, respectively, are well known to those skilled in the art; see, e.g., Amit et al., Science 233 (1986), 747-753. Similarly, applied to cryo-electron microscopy; see, e.g., Schmidt et al. (2019), supra.

[0172] This finding now expands the opportunity to create additional cyclic compounds that contain epitopes of other amyloidogenic proteins. For example, once an epitope has been selected, and a fragment or peptide sequence of an amyloidogenic protein has been selected, the program PEP-FOLD can be used to predict peptide structures from amino acid sequences, and when applied to TTR cyclic peptides appears to reasonably predict the presentation of epitopes, allowing the design of additional cyclic peptides, e.g., as described herein, that reproduce antibody binding epitopes of amyloidogenic proteins.

[0173] Thus, the findings obtained in the experiments carried out within the scope of the present invention allow the creation of cyclic compounds of epitopes from any type of amyloidogenic protein, e.g., as described herein, particularly when derived from systemic amyloidogenic proteins.

[0174] Thus, in a further aspect, the invention relates to cyclic compounds as described above and their linear precursors, comprising peptides containing epitopes of a systemic amyloidogenic protein, preferably epitopes that are accessible for binding by antibodies exclusively in misfolded and / or aggregated forms of the protein, such as in the case of neoepitopes, and / or epitopes that are not present at least in the bioactive form of the protein, for example in the case of epitopes accessible in the monomer of the TTR protein that are hidden in the bioactive tetramer and are no longer accessible to binding antibodies. As illustrated in Example 6, the cyclic compounds of the invention are particularly useful in the potency assays of the invention.

[0175] Most preferably, the cyclic compound of the present invention comprises the amino acid sequence WEPFA (SEQ ID NO:1).

[0176] In one embodiment, the cyclic compounds and their precursors, respectively, or protein fragments or peptides within the cyclic compounds of the invention, are further derivatized or modified. For example, proteins and / or other agents can be coupled to the cyclic compounds, which can act, for example, as probes in in vitro tests. For this purpose, any functionalizable moiety capable of reacting (for example, covalently or non-covalently but making strong bonds) can be used. Those proteins and / or other agents can be carrier proteins, such as bovine serum albumin (BSA), used, for example, in immunoblot or immunohistochemical assays.

[0177] The present invention further relates to compositions comprising the cyclic compounds of the invention or their linear precursors. The compositions may comprise further excipients such as buffers, stabilizers and / or diluents.

[0178] As shown in Example 5, antigen-binding molecules, here anti-TTR antibodies, showed strong binding affinity to the cyclic peptides in an ELISA assay, and therefore the cyclic compounds are suitable target antigens in assays used to detect and quantify antigen-binding molecules such as antibodies.

[0179] Therefore, the present invention further relates to the use of the cyclic compound of the present invention or the composition of the present invention in any kind of assay related to the analysis, e.g. detection, of the interaction between a target antigen-binding molecule and a target antigen, which may also include the quantification of the target antigen-binding molecule. In a preferred embodiment, such an assay is an ELISA assay. In a further preferred embodiment, the present invention relates to the use of the cyclic compound of the present invention or the composition of the present invention for measuring the potency of an antigen-binding molecule, e.g. an antibody or any other binding molecule comprising an Fc domain, preferably an antibody as defined above. The determination of potency is preferably carried out by the assay of the present invention.

[0180] Furthermore, the cyclic compound of the present invention or the composition comprising it can be used for detecting autoantibodies against amyloidogenic proteins or their fragments, oligomers or aggregates.The cyclic compound of the present invention is particularly suitable for detecting autoantibodies against TTR and identifying antibodies equivalent to, for example, NI-301.37F1.Similarly, the cyclic peptide of the present invention can be used for screening antibodies against amyloidogenic proteins, particularly anti-TTR antibodies, in general, for example by phage display.

[0181] Furthermore, the cyclic peptides of the present invention can be used to study the pharmacokinetic profile, i.e., half-life of the antibody, for example, in the case of antibody NI-307.37F11 (NI006), or NNC6019-0001 (PRX004), in plasma in in vivo non-human animal studies and in human clinical trials. Furthermore, the cyclic compounds can be used, for example, in the course of antibody therapy, to measure the plasma concentration of the antibody and to support dosing to maintain a sustained level of the antibody. The cyclic peptides can also be used to identify antibodies that are equivalent to known antibodies, in particular those that are equivalent to the above-mentioned anti-TTR antibodies, in particular antibody NI-307.37F11, for example, by competitive assays that are generally known in the art. Thus, all uses are also part of the present invention.

[0182] Furthermore, the present invention relates to a kit comprising at least the cyclic compound of the present invention 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, for example, the detection may also include the quantification of the target antigen-binding molecule, most preferably for measuring the potency of an antigen-binding molecule comprising an Fc domain, such as an antibody. In a preferred embodiment, the determination of potency is preferably carried out by the assay of the present invention. In a further preferred embodiment, the antigen-binding molecule is an antigen-binding molecule as defined 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.

[0183] In one embodiment, the kit of the invention comprising a cyclic compound comprises: (i) a population of effector cells that express the human Fc receptor FcγR and have been engineered to harbor a reporter gene under the control of a response element that is responsive to activation by the Fc receptor; (ii) a corresponding substrate of 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 Further includes:

[0184] In a preferred embodiment, the population of effector cells is a population of Jurkat cells expressing a gene encoding a luminescent protein, preferably luciferase, under the control of FcγR, preferably FcγRI and an NFAT transcription factor, and wherein the kit comprises a luminescent substrate solution.

[0185] Furthermore, the cyclic compounds of the invention, and their linear precursors, are useful in a method for identifying and, optionally, obtaining antibodies and equivalent binding molecules, e.g. of the type described above, that bind to amyloidogenic proteins involved in systemic amyloidosis, typically comprising the steps of: (a) providing, optionally producing, an antibody or a source thereof that binds to one or more potentially amyloidogenic proteins; (b) subjecting an antibody or a source thereof that binds to one or more potentially amyloidogenic proteins to a binding assay comprising a cyclic compound of the invention; (c) identifying, and optionally obtaining, an antibody that has been determined to bind to the cyclic compound (a target antibody); The method is particularly useful in methods comprising:

[0186] This method can be combined with a potency assay of the invention and / or any other suitable method for further determining the diagnostic or preferably therapeutic utility of the subject antibodies. As mentioned, the subject antibodies can also be different types of antigen-binding molecules.

[0187] Therefore, a further embodiment of the invention is a method of making a pharmaceutical composition comprising an antibody that binds to a systemic amyloidogenic protein, comprising at least (a) providing, optionally producing, an antibody or a source thereof that binds to one or more potentially amyloidogenic proteins; (b) subjecting the antibody or a source thereof that binds to said one or more potentially amyloidogenic proteins to a binding assay comprising a cyclic compound of the invention; (c) identifying, and optionally obtaining, an antibody that binds to the cyclic compound (a target antibody); (d) formulating the antibody or derivative thereof identified and optionally obtained in step (c) with a pharma- ceutically acceptable carrier; The method comprises the steps of:

[0188] Sources of antibodies include, but are not limited to, immune laboratory animals, such as rodents, preferably mice, most preferably Ig-humanized mice; human blood or fractions thereof, preferably containing memory B cells; natural and synthetic antibodies obtained from recombinant antibody libraries, such as phage, yeast, and ribosomal systems or mammalian cell systems such as CHO and HEK; for further sources of antibodies and other target binding molecules, see also the "Detailed Description of the Invention". In one embodiment, nanobodies, also known as VHHs, originating from serum of Camelidae, can be screened with the cyclic compounds of the invention; see, for example, Lyu et al., Anal. Chem. 94 (2022), 7970-7980; Muyldermans, The FEBS Journal 288 (2021) 2084-210. In this regard, binding fragments of IgG antibodies known to bind to amyloidogenic proteins can be used as reference antibodies or sources for identifying and preparing nanobodies (a), respectively. Similarly, synthetic alternatives to antibodies that can be designed by computational modeling can be screened, such as modular peptide binders such as designed armadillo repeat proteins (dArmRPs); see, e.g., Gisdon et al., Biological Chemistry 403 (2022), 535-543.

[0189] The binding assay used in the above described methods preferably comprises an ELISA, for example as performed in Examples 5 and 7.

[0190] In a preferred embodiment of the method of the invention for identifying and obtaining antibodies of interest and their further use when formulated in pharmaceutical compositions and drug discovery, respectively, the antibody identified in step (c) and optionally obtained competes with a reference antibody for binding to an amyloidogenic protein, and preferably the antibody of interest has a lower EC for the amyloidogenic protein than the reference antibody. 50The preparation and formulation of the subject antibodies and similar target binding molecules obtained by the methods of the invention can be carried out as described for the target antigen binding molecules above.

[0191] Several documents are cited throughout the text of this specification. The contents of all cited references (including references cited throughout this application, including the background section and manufacturer's specifications, instructions, etc., issued patents, published patent applications) are expressly incorporated herein by reference; however, no admission is made that any document cited is indeed prior art with respect to this invention.

[0192] 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. EXAMPLES

[0193] Example 1: In vitro phagocytosis assay using human-derived macrophages Phagocytosis of misfolded TTR induced by antibody NI-301.37F1_3 was determined in an in vitro assay involving human-derived macrophages, fluorescently labeled L55P-TTR protein, and the NI-301.37F1_3 antibody selective for ATTR.

[0194] Human-derived macrophages were differentiated in vitro from fresh human monocytes. Briefly, fresh blood donations were received, PBMCs were prepared, and monocytes were extracted by negative depletion on a magnetic column (Miltenyi, Monocyte Isolation Kit II). Monocytes were then differentiated into M2 macrophages by culturing for a minimum of 10 days in macrophage serum-free medium (M-SFM, Life technologies) supplemented with 100 ng / ml macrophage colony-stimulating factor (M-CSF, Miltenyi). Between 10 and 15 days after the start of differentiation, macrophages were detached with trypsin and distributed at a density of 500,000 cells / ml in 96- or 24-well plates. Phagocytosis experiments were performed the following day.

[0195] For in vitro phagocytosis experiments, the L55P-TTR mutant (Wako, Osaka, Japan) was selected because this mutation strongly destabilizes the TTR tetramer, leading to the generation of misfolded TTR protein under physiological conditions. The L55P-TTR protein was coupled to a fluorescent dye to allow direct detection of TTR in macrophages (Atto 488 protein labeling kit from Sigma, or pHrodo Green labeling kit from ThermoFischer).

[0196] Antibody NI-301.37F1_3 and isotype control were coupled to fluorescent dyes (Atto 550 protein labeling kit from Sigma or pHrodo Red labeling kit from ThermoFischer) according to standard procedures, allowing direct detection in macrophages.

[0197] For phagocytosis assays, macrophages were preincubated with fucoidan (Sigma) for 30 min to block nonspecific phagocytosis mediated by scavenger receptors and an Fc receptor inhibitor (Miltenyi) as a negative control condition. L55P-TTR-488 and NI-301.37F1_3-550 or isotype-550 were co-incubated for at least 15 min at room temperature before addition to macrophages. Phagocytosis was performed in triplicate by incubation at 37°C for 2 h in the presence of fucoidan at 0.5 mg / ml, L55P-TTR-488 at 7 μg / ml, NI-301.37F1_3-550 or isotype-550 antibodies at concentrations between 0 and 80 nM, and blocking of FcR at a dilution of 1:100. The reaction was stopped by washing the cells twice with PBS and keeping the plates on ice until measurement. For FACS analysis, macrophages were washed with PBS / EDTA, trypsinized, detached and stored on ice until quantification.

[0198] A standard fluorescent plate reader was used to quantitate the total levels of L55P-TTR-488 incorporated by macrophages. Similar experiments were quantified by FACS to count the number of macrophages incorporating both L55P-TTR and NI-301.37F1_3. The experiment was also repeated with macrophages coated onto cover slips that were washed, fixed, and mounted for confocal microscopy.

[0199] Antibody-mediated TTR uptake was concentration-dependent and required low antibody concentrations (Figure 1A). Uptake was strongly increased by NI-301.37F1_3 already at a concentration of 1 nM, reaching saturation at 10 nM under the assay conditions used. Phagocytosis was mediated by Fc receptors, as shown by complete inhibition in the presence of 1% FcR blocking, and required specific antibody-target interactions, as shown by the absence of TTR uptake in the presence of an isotype control antibody. In parallel experiments, cells specifically positive for both TTR and NI-301.37F1_3 were quantified by analysis by FACS. The frequency of double-positive cells increased from background levels of 3%-6% in the presence of 10 nM NI-301.37F1_3 and further increased to 16% in the presence of 80 nM NI-301.37F1_3 (Figure 1B).

[0200] To further probe the assay, the experiment was repeated using L55P-TTR and NI-301.37F1_3 proteins labeled with the pH-sensitive fluorescent dyes pHrodo Green and pHrodo Red, respectively. These dyes exhibit a strong increase in fluorescence at acidic pH, allowing the assay to target cells internalizing TTR and NI-301.37F1_3 in phagolysosomal vesicles. In agreement with previous experiments, the frequency of double-positive cells increased from a basal level of 3.5%-5.5% in the presence of 10 nM NI-301.37F1_3 and further increased to 8.8% in the presence of 80 nM NI-301.37F1_3 (Figure 1C). This antibody-dependent phagocytosis of TTR was specifically induced by NI-301.37F1_3 and not by an isotype control antibody, which did not induce phagocytosis above background levels at 10 nM and 80 nM. Examination of macrophages by confocal microscopy supported these results, with activated macrophages presenting numerous vesicles positive for both TTR and NI-301.37F1_3. In summary, the results show that NI-301.37F1_3 induces phagocytosis of L55P-TTR by human macrophages in a concentration-dependent manner. Phagocytosis was mediated by Fc receptors and required specific interaction of the antibody with its target protein. Upon phagocytosis, the antibody-target complex was targeted to acidic compartments, likely the phagolysosomal system for target degradation.

[0201] Example 2: In vitro phagocytosis assay using THP1 cells The phagocytosis assay shown in Example 1 showed that, in principle, NI-301.37F1_3 has the ability to activate ATTR phagocytosis, but this approach suffers from variability in phagocytic activity between macrophages obtained from different blood donors. To eliminate this source of variability, the in vitro phagocytosis assay was redeveloped using human monocytic THP1 cell line instead of fresh PBMC. Once established, the ATTR phagocytosis assay using THP1 cells was evaluated for its ability to detect a decrease in antibody potency, which was reproduced by a 30% decrease in antibody concentration.

[0202] THP1 cells (Sigma; 88081201) were cultured in spinner flasks using cell culture medium RPMI1640 (ATCC, Manassas, Virginia, USA; ATCC1640 30-2001) supplemented with 20% fetal bovine serum (FBS), 1x penicillin / streptavidin, and 0.05 mM 2-mercaptoethanol during cell expansion. Cells were cultured at 10 5 ~10 6 For phagocytosis assays, THP1 cells were distributed in 96-well plates at a density of 200,000–400,000 cells / ml and differentiated with phorbol 12-myristate 13-acetate (PMA; Sigma) at 25 ng / ml for 48 h, followed by PMA plus human interferon gamma (IFNγ; Sigma) at 20 ng / ml for an additional 48 h.

[0203] Monomeric F87M / L110M-TTR mutant (AlexoTech AB, Umea, Sweden; T-509-10) was selected because this double mutation prevents the formation of TTR dimers and tetramers, and therefore promotes protein labeling and formation of ATTR aggregates. Following the kit instructions (Sigma, 38371), F87M / L110M-TTR protein was conjugated with Atto-488 fluorescent dye and then aggregated at 1 mg / ml in aggregation buffer (50 mM acetic acid-HCl, 100 mM KCl, 1 mM EDTA, pH 3.0) for 4 h at 37°C, resulting in the generation of fluorescently labeled misfolded TTR aggregates (mis.TTR-488).

[0204] For the phagocytosis assay, antibody dilution series was prepared in Life Cell Imaging Solution (LCIS, ThermoFischer A14291DJ) supplemented with fluorescently labeled misfolded TTR at 150 μg / ml and preincubated for 2 h at RT. In parallel, cell medium was replaced with LCIS supplemented with fucoidan at a final concentration of 0.1 mg / ml and preincubated for 30 min at 37 °C. Phagocytosis assay was initiated by adding 100 μL of mis.TTR-488 / antibody solution to THP1 cells and then incubated for 90 min at 37 °C. The assay was stopped by washing the cells with ice-cold PBS and in a final washing step, wells were filled with LCIS supplemented with background suppressor. Fluorescence of intracellular mis.TTR-488 was measured using a plate reader with excitation set at 498 ± 5 nm, emission at 520 ± 5 nm, duration of 100 ms, bottom reading with all reading sites / well. Phagocytosis assays were performed using NI-301.37F1_3 at concentrations ranging from 0.02 to 5 nM (1x NI301A) as a reference, and a similar dilution series was prepared with NI-301.37F1_3 at 0.7 times the reference concentration (0.7x NI-301.37F1_3). This second condition was used to assess whether the assay had the ability to detect a potential decrease in antibody activity, which was reproduced in this experiment by a 30% decrease in NI-301.37F1_3 concentration.

[0205] The results showed that both 1× and 0.7×_NI-301.37F1_3 induced phagocytosis of mis.TTR-488 by THP1 cells in a concentration-dependent manner. The dose response of 1×_NI-301.37F1_3 was EC 50 The dose response of 0.7× NI-301.37F1_3 was 1.5 nM EC 50 (FIG. 2A, mean ± SD of triplicates). The 25% lower potency observed with 0.7×_NI-301.37F1_3 was in good agreement with the 30% lower antibody concentration in this sample. Mean ± SD of triplicates.

[0206] In vitro phagocytosis assays were further evaluated using NI-301.37F1_W1 non-GMP formulation and NI-301.37F1_W1 GMP bulk drug. NI-301.37F1_3 and NI-301.37F1_W1 antibodies have the same sequence and differ only in their production and purification methods. Both compounds were prepared as a dilution series ranging from 0.09 to 20 nM. Results showed that both NI-301.37F1_W1 non-GMP DP and NI-301.37F1_W1 GMP DS induced phagocytosis of mis.TTR-488 by THP1 cells in a concentration-dependent manner. The NI-301.37F1_W1 non-GMP DP dose response showed an EC of 0.92 nM. 50 The NI-301.37F1_W1 GMP DS dose response was characterized by an EC 50 (Figure 2B). However, this assay showed a high degree of variability in triplicates, with EC 50 This prevented the calculation of confidence intervals.

[0207] Example 3: ADCP assay using FcγR1 reporter cell line to measure the efficacy of antigen-binding molecules to activate phagocytosis of target proteins An ADCP assay was developed to measure the potency of protein aggregate binding molecules. The assay uses a reporter cell line expressing human Fcγ receptor 1 (FcγR1) and exemplifies the antibody NI-301.37F1_W1 for its ability to measure the potency of activating the phagocytosis of misfolded wild-type TTR (mis.WT-TTR) in vitro. Both antibodies NI-301.37F1_W1 and NI-301.37F1_3 refer to the antibody NI-301.37F1 described in the international application WO 2015 / 092077A1, and only differ in their recombinant production and purification methods.

[0208] Preparation and characterization of mis.WT-TTR Wild-type TTR protein purified from human plasma was obtained from Bio-Rad Laboratories, Inc. (California, USA; 7600-0604) and custom purified through protein A / G chromatography followed by lectin column to remove residual immunoglobulin. Plasma-purified WT-TTR was prepared as a solution at a concentration of 1 mg / ml in PBS buffer. Misfolded WT-TTR aggregates (mis.WT-TTR) were prepared in vitro by diluting WT-TTR stock solution to a concentration of 200 μg / ml in aggregation buffer (50 mM acetic acid-HCl, 100 mM KCl, 1 mM EDTA, pH 3.0) and then incubating at 37°C for 4 hours with shaking at 1000 rpm. mis.WT-TTR was aliquoted and stored at -20°C until use. The quality of mis.WT-TTR was confirmed by ELISA and biolayer interferometry (BLI).

[0209] For ELISA, 96-well microplates were coated with mis.WT-TTR diluted to a concentration of 10 μg / ml in PBS buffer, pH 7.4, for 1 h at 37°C. Nonspecific binding sites were blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween-20 in PBS buffer for 1 h at room temperature (RT). NI-301.37F1_3 antibody (Neurimmune AG, Zurich, Switzerland; NI-301.37F1) was diluted in duplicate to the indicated concentrations in PBS and incubated overnight at 4°C. Binding was determined using an anti-human IgG antibody conjugated with horseradish peroxidase (HRP), and HRP activity was then measured by standard colorimetric assay (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA). Data were analyzed with Prism software from GraphPad. EC values ​​were calculated using nonlinear regression of individual data points using a log(agonist) versus log(agonist) response model with variable slope. 50 Values ​​were evaluated. Data fitting was performed by least squares regression.

[0210] BLI was performed on an Octet RED96 instrument (Molecular Devices, LLC, San Jose, California, USA) equipped with an anti-human capture sensor. Binding kinetics were measured at 25°C in 1x kinetic buffer (assay buffer). NI-301.37F1_3 or NI-301.37F1_W1 antibodies were diluted to 5 μg / ml in assay buffer and loaded onto the sensor for 300 s. Mis.WT-TTR aggregates were diluted in assay buffer at six different concentrations and buffer-only conditions were run simultaneously on the seventh and eighth sensors, the latter of which was used as a reference. Association and dissociation were measured for 600 s, respectively. Data were analyzed using reference subtraction (buffer-only conditions) in Data Analysis 8.2. For kinetic analysis, a simple 1:1 binding model was used.

[0211] Quality control was performed on Mis.WT-TTR batch 6 (mis.WT-TTR_b6) by comparing it to the previous batch of mis.WT-TTR (mis.WT-TTR_b5). Analysis was performed by measuring the binding of NI-301.37F1_3 and NI-301.37F1_W1 using ELISA and BLI. ELISA results showed that the binding of NI-301.37F1_3 (Figure 3A) and NI-301.37F1_W1 (Figure 3B) to mis.WT-TTR_b6 was substantially identical to that to mis.WT-TTR_b5. EC of NI-301.37F1_3 binding to mis.WT-TTR_b5 and b6 50 The EC of binding of NI-301.37F1_W1 to mis.WT-TTR_b5 and b6 was 1.3 nM and 2 nM, respectively. 50were 1.0 nM and 1.4 nM, respectively. Mis.WT-TTR_b6 was also compared to b5 using BLI. NI-301.37F1_3 and NI-301.37F1_W1 bound to mis.WT-TTR_b6 with dissociation constants (KD) of 0.93 nM and 0.66 nM, respectively. These values ​​were very similar to the binding affinity of NI-301.37F1_3 for mis.WT-TTR_b5, which was determined in a previous experiment to have a KD of 0.66 nM. Based on this, mis.WT-TTR_b6 was deemed similar to mis.WT-TTR_b5 and suitable for use in ADCP reporter assays.

[0212] ADCP reporter assay To measure the potency of the NI-301.37F1_W1 reference sample (NI-301.37F1_W1 RS) and the half concentrated test sample (NI-301.37F1_W1 50%) to activate phagocytosis of mis.WT-TTR, a commercially available FcγR1 ADCP reporter bioassay (Promega, Madison, Wisconsin, USA; early access (not verified), CS1781C08) is applied. This bioluminescent cell-based assay relies on a genetically modified Jurkat T cell line expressing human FcγR1 together 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 that is detected using a bioluminescent luciferase substrate. Briefly, 96-well plates were coated with mis.WT-TTR diluted to a concentration of 10 μg / ml in PBS buffer, pH 7.4, for 1 h at 37° C. Nonspecific binding sites were blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween-20 in PBS buffer for 1 h at room temperature (RT). NI-301.37F1_W1 antibody was diluted in triplicate to the indicated concentrations in PBS and incubated for 30 min at 37° C. before addition of FcγR1 reporter cells at a density of 77,000 cells / well. Assays were incubated at 37° C. for 6 h before addition of luminescent substrate.

[0213] NI-301.37F1_W1 RS vs. NI-301.37F1_W1 50% In the first experiment, NI-301.37F1_W1 RS was tested using a 10-point concentration range from 2 to 10,000 ng / ml in triplicate. NI-301.37F1_W1 50% was prepared using the same dilution series but starting at a lower concentration (i.e., 5,000 ng / ml) for two time points to reproduce the reduced potency.

[0214] NI-301.37F1_W1 RS had an EC 50 The NI-301.37F1_W1 50% showed a dose-response characterized by an EC of 187 ng / ml (95% confidence interval (CI) 79.4-116.7). 50 The dose response was characterized by an EC 50 The increase in EC of NI-301.37F1_W1 RS was well matched with the lower concentration at two time points in sample NI-301.37F1_W1 50%. 50 and NI-301.37F1_W1 50% EC 50 were statistically different (F(1,52):26.60, p<0.0001). The results showed that the FcγR1 ADCP assay has the ability to detect a 50% decrease in antibody activity. The data also showed that the NI-301.37F1_W1 RS dose response showed four data points in the plateau phase, which was unnecessary and triggered adjustment of the antibody concentration range for subsequent experiments.

[0215] A second set of experiments was performed to 1) adjust the NI-301.37F1_W1 RS concentration range, 2) compare NI-301.37F1_W1 RS with samples having concentrations lower and higher than 35% (NI-301.37F1_W1 65% and 135%, respectively), 3) compare horizontal and vertical plate layouts, and 4) test plate uniformity.

[0216] NI-301.37F1_W1 RS vs. NI-301.37F1_W1 65% and 135% using horizontal layout The NI-301.37F1_W1 RS dose response was adjusted to an 8-point concentration range from 4 to 2000 ng / ml (i.e., 2000, 500, 250, 125, 63, 31, 16, 4 ng / ml) and tested in duplicate using a horizontal plate layout. The NI-301.37F1_W1 RS in plate 1 had an EC of 93.7 ng / ml. 50 In contrast, NI-301.37F1_W1 65% showed a dose response characterized by an EC of 159 ng / ml (95%CI 67.4-128.5). 50 The EC of NI-301.37F1_W1 RS was characterized by a dose response of 118.3-219.4 (Figure 5A). 50 and NI-301.37F1_W1 65% EC 50 were statistically different (F(1,24):7.14, p=0.013). This result demonstrated that the FcγR1 ADCP assay had the ability to detect a 35% decrease in antibody activity. However, there was a 1.7-fold EC 50 The increase in was slightly different from the expected value of 1.35 for NI-301.37F1_W1 65%.

[0217] NI-301.37F1_W1 RS in plate 2 had an EC 50 In contrast, NI-301.37F1_W1 135% showed a dose response characterized by an EC of 76.7 ng / ml. 50 The EC of NI-301.37F1_W1 RS was characterized by a dose response of 1.0-fold higher than that of NI-301.37F1_W1 RS (68.1-86.2) (Figure 5B). 50 and NI-301.37F1_W1 135% EC 50 were statistically different (F(1,24):20.97, p=0.0001). The results showed that the FcγR1 ADCP assay had the ability to detect a 35% increase in antibody activity. However, the EC 50The decrease in was in good agreement with the expected value of 0.65 for NI-301.37F1_W1 135%.

[0218] Comparison of dose responses for NI-301.37F1_W1 RS on plates 1 and 2 showed certain differences in maximum signal intensity that occurred even though these plates were run in parallel, on the same day, and by the same analyst. This difference illustrated the potential benefit of using a perpendicular plate layout that would allow three samples to be measured in triplicate on the same plate.

[0219] NI-301.37F1_W1 RS vs. 65% and 135% using vertical layout The vertical assay layout was evaluated using the same concentration range as the triplicate samples above. In the vertical assay layout, NI-301.37F1_W1 RS had an EC 50 (95%CI 53.2-91.5), NI-301.37F1_W1 65% had an EC of 99.5ng / ml 50 (82.7-119.1), and NI-301.37F1_W1 135% had an EC of 50.3ng / ml. 50 The dose response was characterized by a 1.4-fold EC for NI-301.37F1_W1 (41.1-60.8) (Figure 6). 50 and 0.7-fold EC for NI-301.37F1_W1 135% 50 The decrease in EC was well consistent with the respective sample concentrations. NI-301.37F1_W1 RS and NI-301.37F1_W1 65% had statistically different EC 50 The EC values ​​were statistically different (F(1,40): 5.199, p=0.028), and NI-301.37F1_W1 RS and NI-301.37F1_W1 135% were statistically different. 50 Values ​​were shown to be consistent with those of the FcγR1 ADCP assay (F(1,40): 4.358, p=0.043), demonstrating that the FcγR1 ADCP assay using the perpendicular format has the ability to detect ±35% changes in antibody activity.

[0220] Plate Uniformity Test Plate uniformity assessment was performed with NI-301.37F1_W1 at 12 ng / ml in all 96 wells of the plate. The 24 outer wells showed, on average, 5% lower signal intensity than that measured in the 60 inner wells. This small difference was statistically significant. Furthermore, although all wells gave sufficiently reliable results, the 24 outer wells showed greater variability than that observed in the 60 inner wells.

[0221] Example 4: Evaluation of the FcγR1 ADCP assay using stressed NI-301.37F1_W1 samples To further evaluate the ADCP assay presented in Example 3, antibody NI-301.37F1_W1 was subjected to stress conditions known to potentially cause a loss of antibody efficacy.

[0222] Stressed NI-301.37F1_W1 samples were prepared by dialyzing 25 mg / ml of NI-301.37F1_W1 into five different buffers listed below. Dialysis was performed overnight at 4° C. and then incubated at 40° C. for 19 hours. The stressed samples were then dialyzed again against formulation buffer overnight at 4° C. before being aliquoted and stored at −20° C. The buffers used to prepare the stressed samples were: - Acidic buffer: 20 mM phosphate buffer-citric acid (PBCA) buffer, pH 3.4 - Preparation buffer: 20 mM histidine-HCl, 7% sucrose, 0.02% PS80, pH 5.8 (Form° buffer) -Physiological buffer: PBS, pH 7.4 - Base buffer: 20 mM Tris-HCl, pH 10.0 -Oxidative buffer: 1% H2O2 in PBS

[0223] Stressed NI-301.37F1_W1 samples were characterized by measuring their binding affinity to mis.WT-TTR using ELISA and BLI as described above. Additionally, stressed NI-301.37F1_W1 samples were characterized using SDS-PAGE under reducing and non-reducing conditions and silver staining according to standard techniques to identify possible aggregation or degradation products. By ELISA, stressed NI-301.37F1_W1 samples were highly comparable to the reference NI-301.37F1_W1 sample, with EC values ​​in the sub-nanomolar range. 50 The mis.WT-TTR binding affinity, characterized by , was shown to be lower than that of the reference sample (Figure 7). Samples stressed with PBS buffer, 1% hydrogen peroxide, and to a lesser extent with formulation buffer, showed lower maximum signal intensities than the reference sample.

[0224] Similar results were obtained using BLI and a summary of the binding results obtained by BLI is presented in Figure 8. Stressed NI-301.37F1_W1 samples showed binding affinities for mis.WT-TTR that were comparable to the reference NI-301.37F1_W1 samples and characterized by a KD in the low nanomolar range. For ELISA, samples stressed in PBS buffer or 1% hydrogen peroxide showed lower maximum signal intensities than the reference samples. Using SDS-PAGE and silver staining, samples stressed in formulation buffer, phosphate buffer and Tris buffer showed patterns similar to the reference samples under reducing and non-reducing conditions. NI-301.37F1_W1 samples stressed in PBCA buffer (pH 3.4) showed cleaved forms visible under reducing and non-reducing conditions, while NI-301.37F1_W1 samples stressed in 1% H2O2 showed a pattern clearly different from the reference samples under non-reducing conditions.

[0225] The FcγR1 ADCP assay was performed as described in Example 3 using stressed NI-301.37F1_W1 samples with the goal of assessing whether the assay has the ability to detect reduced potency. For triplicate samples, a vertical assay layout was used. In assay plate 1, NI-301.37F1_W1 RS had an EC of 95 ng / ml. 50 (95%CI 67-127), and NI-301.37F1_W1 stressed under PBCA buffer had an EC 50 (191-292) and NI-301.37F1_W1 stressed under Tris buffer had an EC 50 In assay plate 2, NI-301.37F1_W1 RS showed an EC 50 (95%CI 52-138), and NI-301.37F1_W1 stressed under formulation buffer had an EC 50 (96-144) and NI-301.37F1_W1 stressed under H2O2 buffer had an EC 50 (127-200) showed a dose response characterized by (Figure 9B).

[0226] Considering that the stressed samples showed similar binding affinities to NI-301.37F1_W1 RS in ELISA and BLI assays, these results demonstrated that the FcγR1 ADCP assay has the ability to detect reduced potency upon Fc domain modification.

[0227] Example 5: Cyclic peptides as target antigens provide a more sensitive ELISA assay for antigen-binding molecules The ability of the antigen-binding molecule to bind to the cyclic peptide is illustratively evaluated by an ELISA assay using a cyclic peptide containing amino acid residues 34-54 of wild-type TTR (TTR34-54cyc in biotinylated and non-biotinylated forms) as the target antigen and the anti-TTR antibody NI-301.37F1 as the antigen-binding molecule. In addition, the TTR peptide TTR40-49, the biotinylated TTR peptide TTR40-49, and the misfolded wild-type TTR (mis.WT-TTR) were used as antigen controls.

[0228] Cyclic peptide TTR34-54cyc (1.36 mg / mL) was prepared 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. TTR peptide (TTR40-49, 1.25 mg / mL) containing the amino acid sequence H-TWEPFASGKT-OH (SEQ ID NO: 161) was also prepared by Schafer-N (Copenhagen, Denmark) and stored at -20°C. The biotinylated peptides biotin.TTR34-54cyc and biotin.TTR40-49 each contain an aminohexanoic acid (Ahx) spacer between their N-terminus 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). Misfolded wild-type TTR was prepared as described in Example 3, above.

[0229] Two ELISA assays were performed, where a first ELISA assay (ELISA-1) analyzed antibodies binding to the peptides TTR34-54cyc, TTR40-49, biotin.TTR40-449, and mis-WT-TTR, and a second ELISA assay (ELISA-2) analyzed antibodies binding to the peptides TTR34-54cyc, biotin.TTR34-54cyc, TTR40-49, biotin.TTR40-449, and mis-WT-TTR.

[0230] In particular, 96-well microplates were coated with TTR34-54cyc, TTR40-49, biotin.TTR40-449, and mis-WT-TTR (ELISA-1), and TTR34-54cyc, biotin.TTR34-54cyc, TTR40-49, biotin.TTR40-449, and mis-WT-TTR (ELISA-2), respectively, for 1 hour at 37° C., where each target antigen was diluted to a concentration of 10 μg / ml in PBS buffer at pH 7.4. Nonspecific binding sites were blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween-20 in PBS buffer at room temperature (RT) for 1 hour. NI-301.37F1 antibody (Neurimmune AG, Zurich, Switzerland; NI-301.37F1) was diluted in duplicate to the indicated concentrations (dilution series from 400 nM to 4 pM and 0) in blocking buffer and incubated overnight at 4 °C. Binding was determined using an anti-human IgG antibody conjugated with horseradish peroxidase (HRP), and HRP activity was then measured by a standard colorimetric assay (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA). Data were analyzed with Prism software from GraphPad. EC was calculated using nonlinear regression of individual data points using a log (agonist) versus logarithm (agonist) versus response model with variable slope. 50 Values ​​were evaluated. Data fitting was performed by least squares regression.

[0231] The ELISA results confirmed the binding of NI-301.37F1 to mis.WT-TTR, as seen in Example 3, supra. Furthermore, the ELISA assay showed that the binding of NI-301.37F1 to cyclic TTR34-54cyc and biotin.TTR34-54cyc peptides was much stronger, i.e., about 10-fold stronger, than that to mis.WT-TTR. Notably, in ELISA-1, the binding EC of NI-301.37F1 to cyclic TTR34-54cyc peptide was significantly higher than that to mis.WT-TTR. 50 The binding EC of NI-301.37F1 to mis.WT-TTR was 27 pM. 50 was 338 pM; see FIG. 10A. In ELISA-2, the measured EC 50 Although the values ​​were higher, the approximately 10-fold difference between the binding of NI-301.37F1 to the cyclic TTR34-54cyc peptide and to mis.WT-TTR was maintained. In particular, the binding EC 50 The binding EC of NI-301.37F1 to mis.WT-TTR was 0.66 nM. 50 was 8.3 nM; see FIG. 10B. No binding of NI-301.37F1 to TTR40-49 and biotin.TTR40-49 was observed in both ELISA assays.

[0232] Example 6: Improved ADCP assay by using cyclic peptides as target antigens An ADCP assay for measuring the potency of antigen-binding molecules was developed, using a reporter cell line expressing human Fcγ receptor 1 (FcγR1), and exemplary antibody NI-301.37F1 was evaluated for its ability to measure the potency of activating phagocytosis of cyclic TTR peptide (TTR34-54cyc) in vitro.

[0233] ADCP reporter assay To measure the potency of the NI-301.37F1 reference sample (NI-301.37F1 RS, 100%) and test samples with concentrations lower than 50% (NI-301.37F1 50%), lower than 30% (NI-301.37F1 70%), higher than 30% (NI-301.37F1 130%) and higher than 50% (NI-301.37F1 150%) to activate the phagocytosis of TTR34-54cyc, a commercially available FcγR1 ADCP reporter bioassay (Promega, Madison, Wisconsin, USA, Cat.#GA1341, GA1345, which is the same as CS1781C08 described in Example 3 as early access) is applied as described in Example 3 with slight variations. Briefly, 96-well plates were coated overnight at 4°C with TTR34-54cyc diluted to a concentration of 3 μg / ml in PBS buffer. Non-specific binding sites were blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween-20 in PBS buffer for 1 h at room temperature (RT). Measurement dilution plates were prepared in which NI-301.37F1 antibody was diluted to the indicated concentrations (500ng / mL to 0.4ng / mL) in ADCP buffer (96% RPMI1640 medium, 4% low IgG serum). The assay was performed by adding 1 unit volume of antibody dilution and incubation for 30 min at 37°C and 5% CO2, followed by injecting 1 unit volume of FcγR1 reporter cells at approximately 1.65×10 in ADCP buffer. 5 Cells were added at a density of 1000 x 1000 cells / well. The assay was incubated at 37° C. and 5% CO2 for 6 hours before adding the luminescent substrate (Bio-Glo™ Luciferase Assay Reagent). After 15 minutes of incubation at room temperature, luminescence measurements were performed (acquisition time: 1000 ms, settling time: 0 ms).

[0234] 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 had an EC 50 It has been shown that the antibody exhibits a dose response characterized by a linear correlation between the cyclic peptide TTR34-54cyc and the antibody dilutions ranging from 500 to 0.4 ng / mL. These conditions yielded a reasonable response curve with a stable slope, lower and upper asymptote; see Figure 11.

[0235] Further experiments were performed in which the assay response was tested at NI-301.37F1 concentrations of 50%, 70%, 130% and 150%. As shown in Figures 12A-D and Table 3 below, NI-301.37F1 50% had an EC 50 The dose response was characterized by an EC 50 The increase in NI-301.37F1 was almost perfectly consistent with the lower concentration at the two time points in 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 an EC 50 The 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 an EC 50 The increase in NI-301.37F1 was perfectly consistent with the expected difference of 0.77 fold. The NI-301.37F1 150% had an EC 50 The dose response was characterized by an EC 50 The increase in was almost perfectly consistent with the expected difference of 0.70 fold.

[0236] Thus, little assay variability was observed 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% decrease in antibody activity and up to a 50% increase in antibody activity with excellent precision.

[0237] [Table 3]

[0238] Example 7: Evaluation of additional cyclic peptides as target antigens for antigen-binding molecules As shown in Example 5, the cyclic peptide TTR34-54cyc has been successfully used as a target antigen for the antibody NI-301.37F1 in an ELISA assay. Therefore, the ability of anti-TTR antibodies to bind to further cyclic peptides is analyzed. In particular, the ability of anti-TTR antibodies to bind to two cyclic peptides, namely TTR89-97cyc and TTR101-109cyc, containing either the TTR epitope EHAEVVFTA (SEQ ID NO: 8) or the TTR epitope GPRRYTIAA (SEQ ID NO: 9), as described above, is evaluated by further ELISA assays using said cyclic peptides as target antigens and 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 are prepared as described in Example 5, above. For the ELISA assay, 96-well microplates are coated with the two cyclic peptides TTR89-97cyc and TTR101-109cy as well as an antigen control, and the assay is performed as described in Example 5, supra.

[0239] Example 8: ADCP assay using FcγR1 reporter cell line to measure the potency of antigen-binding molecules to activate phagocytosis of additional cyclic peptides As shown in Example 6, the potency of anti-TTR antibody NI-301.37F1 to activate the phagocytosis of cyclic TTR peptide (TTR34-54cyc) in vitro has been successfully measured in ADCP assay. Therefore, the potency of anti-TTR antibody to activate the phagocytosis of two cyclic peptides, TTR89-97cyc and TTR101-109cyc, is evaluated in further ADCP assay.

[0240] A commercially available FcγR1 ADCP reporter bioassay (Promega, Madison, Wisconsin, USA, Cat. #GA1341, GA1345) described in Example 6 is applied to measure the phagocytosis activating potency of the two cyclic peptides of the anti-TTR antibody reference sample (anti-TTR antibody RS, 100%) and test samples with concentrations lower than 50% (anti-TTR antibody, 50%), lower than 30% (anti-TTR antibody, 70%), higher than 30% (anti-TTR antibody, 130%), and higher than 50% (anti-TTR antibody, 150%).

[0241] Anti-TTR antibodies are expected to show a dose response, where anti-TTR 50% has an approximately 2-fold increased EC compared to anti-TTR RS. 50 The anti-TTR antibody 70% showed an EC value that was increased by approximately 1.5 times compared to the anti-TTR antibody RS. 50 The EC value for anti-TTR antibody 130% was approximately 0.77-fold lower than that for anti-TTR antibody RS. 50 The EC value was reduced by approximately 0.70-fold for anti-TTR antibody 150% compared to anti-TTR antibody RS. 50 The value will be shown.

Claims

1. A method for measuring the potency of a target antigen-binding molecule comprising an Fc domain, comprising: (a) contacting a target antigen with said binding molecule under conditions that allow for the formation of a binding molecule-antigen complex; (b) contacting the binding molecule-antigen complex with a population of effector cells that express an Fc receptor and that have been engineered to harbor a reporter gene under the control of a response element that is responsive to activation by the Fc receptor, under conditions that allow binding of the Fc domain to the Fc receptor, resulting in intracellular signaling and mediating quantifiable reporter gene activity; (c) detecting the reporter gene activity; 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 wherein the reporter gene activity is indicative of efficacy of the binding molecule.

2. The method of claim 1, further comprising formulating the binding molecule with a pharmaceutically acceptable carrier to produce a pharmaceutical composition comprising the target antigen-binding molecule.

3. 2. The method of claim 1, wherein the target antigen is selected or derived from an amyloidogenic protein or aggregate thereof involved in systemic amyloidosis.

4. The method of claim 1, wherein the mechanism of action of the Fc domain is to induce antibody-dependent cell-mediated phagocytosis (ADCP).

5. 10. The method of claim 1 used as a potency assay for batch release.

6. The method of claim 1, wherein the Fc receptor is human Fc receptor FcγRI (CD64).

7. The method of claim 1, wherein the cells do not overexpress FcγRIIa (CD32a), FcγRIII (CD16), or a combination thereof.

8. The method described in claim 1, wherein the cells do not express FcγRIIa (CD32a).

9. The method described in claim 1, wherein the cells do not express FcγRIII (CD16). (a) the effector cells are Jurkat cells; (b) the response element is a NFAT (nuclear factor of activated T cells) response element; (c) the reporter gene encodes a bioluminescent protein; or (d) a combination thereof, The method of claim 1.

11. The method described in claim 10, wherein the bioluminescent protein is luciferase.

12. The method of claim 1 , wherein the binding molecule is selected or derived from an antibody.

13. The method described in claim 12, wherein the antibody is a monoclonal antibody.

14. The method described in claim 12, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

15. The method of claim 12, wherein the antibody is an IgG1 antibody.

16. 10. The method of claim 1, wherein the target antigen comprises transthyretin (TTR) or an aggregate or derivative thereof.

17. 2. The method of claim 1, wherein the binding molecule is an anti-FTR antibody.

18. The method of claim 1, wherein the target antigen comprises a protein fragment or peptide comprising an epitope of the target antigen-binding molecule.

19. 19. The method of claim 18, wherein the protein fragment or peptide forms a cyclic compound.

20. 20. The method of claim 19, wherein the protein fragment or peptide comprises a linker capable of forming the cyclic compound.

21. 21. The method of claim 20, wherein the linker is covalently coupled at or near the N-terminal residue of the peptide and the C-terminal residue of the peptide fragment.

22. 19. The method of claim 18, wherein the target antigen comprises an epitope or neoepitope of an amyloidogenic protein or aggregate thereof involved in systemic amyloidosis.

23. 20. The method of claim 19, wherein the protein fragment or peptide in the cyclic compound comprises five or more amino acid residues of an amyloidogenic protein.

24. The method of claim 23, wherein the protein fragment or peptide in the cyclic compound comprises 10 or more, 15 or more, or 20 or more amino acid residues of the amyloidogenic protein.

25. 24. The method of claim 23, wherein the amyloidogenic protein is selected from the group consisting of transthyretin (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, cystatin C, ABriPP, prion protein, and lysozyme.

26. 26. The method of claim 25, wherein the amyloidogenic protein is TTR and the target antigen comprises a TTR peptide.

27. The TTR peptides are 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), VHVFRKAADDTWEPFASGKTSESGELHGLTTE 27. The method of claim 26, comprising at least four amino acid residues of any one of the amino acid sequences selected from EEFVE (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), where X can be any naturally occurring amino acid.

28. 28. The method of claim 27, wherein the FAP peptide comprises the amino acid sequence WEPFA (SEQ ID NO: 1).

29. 20. The method of claim 19, wherein the linker comprises 1 to 8 amino acids, one or more functionalizable moieties, and / or combinations thereof.

30. (i) the linker amino acid is selected from the group consisting of alanine (A), glycine (G), and serine (S); (ii) the functionalizable moiety is cysteine ​​(C), lysine (K), arginine (R), aspartic acid (D), or glutamic acid (E); or (iii) a combination thereof, 30. The method of claim 29.

31. 31. The method of claim 30, wherein the functionalizable moiety is cysteine ​​(C) and the compound is cyclized via a disulfide bridge.

32. 20. The method of claim 19, wherein the linker in the cyclic compound comprises or consists of GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16).

33. 20. The method of claim 19, wherein the cyclic compound comprises or consists of the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (TTR34-54cyc; SEQ ID NO: 17).

34. 2. The method of claim 1, wherein the binding molecule is an anti-FAT antibody, and the antibody comprises, within 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.

35. The method of claim 34, wherein the antibody is NI-301.37F1.

36. The method of claim 1 , wherein the target antigen is bound on a solid support.

37. The method of claim 36, wherein the solid support is a microtiter plate.

38. 3. The method of claim 1 or 2, further comprising performing step (b) of claim 1 in a vertical plate layout.

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