Patient-derived amyloid xenograft non-human animal model

Abstract

To provide new systems that appropriately model amyloidoses in order to enable the development of new effective amyloidosis therapies.SOLUTION: Provided are a patient-derived amyloid xenograft (PDAX) non-human animal model, uses and production methods thereof, as well as methods comprising the model to determine / obtain anti-amyloid drugs allowing treatment of an amyloidosis or amyloid-related disease, and methods and processes for characterizing, validating, developing and / or quality-controlling and manufacturing such drugs. More specifically, the animal model is characterized by an implant of amyloid transthyretin (ATTR) fibrils derived from the tissue or organ of a patient suffering from an ATTR amyloidosis or ATTR-related disease.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of in vivo animal models, specifically to a patient-derived amyloid xenograft (PDAX) non-human animal model, uses thereof, and methods of making the PDAX model. The present invention also relates to methods and processes for determining / obtaining, characterizing, validating, developing, and / or quality controlling anti-amyloid drugs capable of treating amyloidosis or amyloid-related diseases, and methods and processes for manufacturing pharmaceutical compositions thereof. [Background technology]

[0002] Amyloidosis is a protein folding disease characterized by the extracellular deposition of specific soluble precursor proteins that aggregate into insoluble fibrils; for review, see Hazenberg, Rheum. Dis. Clin. North Am. 39 (2013), 323-345. These rigid and unbranched fibrils, approximately 10 nm in diameter, are characterized by a molecular β-pleated sheet structure, usually composed of peptides arranged in an antiparallel configuration. This structure of the fibrils is responsible for their insolubility, resistance to proteolysis, and binding affinity for the amyloid-specific Congo Red and Thioflavin S dyes. Three mechanisms appear to operate, either independently or in combination: the precursor protein may have an intrinsic propensity to misfold, which becomes evident with age (wild-type transthyretin) or at high serum levels (serum amyloid A protein and immunoglobulin free light chain); inherited or acquired mutant proteins (transthyretin); and proteolytic remodeling of the precursor protein (β-amyloid precursor protein). Interactions with the extracellular matrix also appear to be important and may be involved in the preferential deposition of amyloid in some organs or tissues. Extracellular deposition of amyloid fibrils in organs and tissues results in tissue infiltration and swelling that leads to progressive loss of function of the affected organs.

[0003] Amyloidosis is broadly divided into localized forms, in which deposition is limited to one organ or site of the body, such as Alzheimer's disease with amyloid β protein plaques in the brain, or type 2 diabetes with amyloid amylin deposition in the pancreatic islets of Langerhans, and systemic forms, in which fibril deposition occurs in various organs and tissues throughout the body.One of the most frequent systemic amyloidoses is amyloid transthyretin (ATTR) amyloidosis, in which ATTR fibrils accumulate in multiple organs and tissues, resulting in multiple organ dysfunction with rapid disease progression and fatal outcome.

[0004] ATTR amyloidosis shows two major types of clinical manifestations, with predominant amyloid fibril accumulation in cardiac tissue resulting in cardiomyopathy (ATTR-CM), and fibril accumulation in nerve fibers resulting in polyneuropathy (ATTR-PN) (Ando et al., Orphanet J. Rare Dis. 8:31 (2013) 1-18). TTR has the innate ability to aggregate into insoluble amyloid fibrils. Amyloid misfolding occurs spontaneously at a low rate in presumably all individuals and increases with aging and senescence. In healthy subjects, it is hypothesized that the immune system has the ability to clear amyloid fibrils; however, for reasons that are not yet fully understood, amyloid fibrils escape immune surveillance and accumulate to toxic levels in ATTR patients. There is no specific treatment yet available for ATTR-CM. Conventional heart failure treatments such as beta-blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers are poorly tolerated in ATTR-CM and should be avoided (Gertz et al., J. Am. Coll. Cardiol. 66 (2015), 2451-2466). In the absence of treatments that can specifically reduce the amount of ATTR amyloid, heart transplantation remains the only available approach to restore cardiac function, but is difficult to apply to elderly and vulnerable patient populations. Liver transplantation was considered an option for mutant ATTR, but both peripheral neuropathy as well as cardiac amyloidosis can progress after liver transplantation. Tafamidis (Vyndaqel®), a transthyretin stabilizer, was approved in Europe in November 2011 for patients with early ATTR-PN. However, in a post-approval trial, one year after tafamidis treatment, neurological disability scores worsened by 55%, suggesting that it failed to halt disease progression (Gertz et al., J. Am. Coll. Cardiol. 66 (2015), 2451-2466). Similar results were recently obtained with tafamidis in patients with ATTR-CM.Tafamidis slowed disease progression in patients with ATTR-CM, resulting in a 35% higher survival and hospitalization rate after 30 months of treatment (Maurer et al., N. Engl. J. Med. 379 (2018), 1007-1016); however, similar to what was observed in ATTR-PN, tafamidis treatment was more beneficial for early stage patients, slowing but not halting disease progression and not bringing patients into symptom remission.

[0005] In the development of new drug treatments, animal models are essential to evaluate the efficacy and safety of drugs. In ATTR, as well as in most, if not all, amyloidosis, amyloidosis drug treatments have so far been studied using transgenic animal models, especially transgenic mouse models, which depend in large part on the mutations of the respective precursor proteins observed in human patients. For example, multiple transgenic mouse models have been presented to the scientific community in an attempt to generate models of ATTR amyloidosis, but they all suffer from significant limitations, including the absence or rarity of ATTR amyloid deposits, high variability depending on age, sex, genetic background, and rearing conditions, and in all cases, the lack of a phenotype that mimics patient symptoms (Takaoka et al., Transgenic Research 6(1997), 261-269; Teng et al., Lab Invest. 81(2001), 385-396; Sousa et al., Am. J. Path. 161(2002), 1935-1948; Noguchi, Exp. Anim. 51(2002), 309-316; Panayiotou, BB reports 8(2016), 48-54).

[0006] Analysis of characterizing transgenic mouse models shows that these transgenic mouse models exhibit non-amyloid TTR deposits, which do not have the characteristic coloring properties of amyloid, are soluble instead of insoluble, and do not induce toxicity in mice.Therefore, available models are not suitable for evaluating the potential of compounds that bind and clear insoluble amyloid fibrils in these models that only exhibit soluble deposits.Therefore, there is a need for new systems that appropriately model amyloidosis to enable the development of new effective amyloidosis treatments. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2015 / 092077 A1 [Patent Document 2] WO2014 / 124334 A2 [Patent Document 3] WO2018 / 007923 A3 [Patent Document 4] WO2016 / 120810 Al [Patent Document 5] US2017 / 0058023 A1 [Patent Document 6] US9,879,080 B2 [Non-patent literature]

[0008] [Non-Patent Document 1] Hazenberg, Rheum.Dis.Clin.North Am. 39(2013), 323~345 [Non-Patent Document 2] Ando et al., Orphanet J. Rare Dis. 8:31(2013)1-18 [Non-Patent Document 3] Gertz et al., J.Am.Coll.Cardiol. 66(2015), 2451~2466 [Non-Patent Document 4] Maurer et al., N. Engl. J. Med. 379 (2018), 1007-1016 [Non-Patent Document 5] Takaoka et al., Transgenic Research 6(1997), 261-269 [Non-Patent Document 6] Teng et al., Lab Invest. 81(2001), 385–396 [Non-Patent Document 7] Sousa et al., Am.J.Path. 161(2002), 1935~1948 [Non-Patent Document 8] Noguchi, Exp.Anim. 51(2002), 309~316 [Non-Patent Document 9] Panayiotou, BB reports 8(2016), 48~54 [Non-Patent Document 10] Poster presentation at the XVIth International Symposium on Amyloidosis; March 26-29, 2018; Kumamoto, Japan. [Non-Patent Document 11] Suhr et al., J. Internal Medicine 281 (2017), 337-347 [Non-Patent Document 12] Higashi et al., Brain Research 1184(2007)284~294 [Non-Patent Document 13] Lee et al., Trends in Neurosciences 27(2004), 129-134 [Non-Patent Document 14] Sakata et al., J.Histochem. Cytochem. 53(2005), 237-242 [Non-Patent Document 15] Higaki et al., Amyloid 23(2016), 86-97 [Non-Patent Document 16] Orphanet Journal of Rare Diseases 10(2015), Suppl.1:P39 [Non-Patent Document 17] Yoshimura et al., Bioconjugate Chem. 27(2016), 1532-1539 [Non-Patent Document 18] Leino et al., Journal of Alzheimer's Disease 59(2017), 43-56 [Non-Patent Document 19] Tennent, Methods Enzymol. 309(1999), 26-47. [Non-Patent Document 20] Remington: The Science and Practice of Pharmacy (2000), University of Sciences in Philadelphia, ISBN 0-683-306472 [Non-Patent Document 21] Vaccine Protocols. 2nd ed., Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003 [Non-Patent Document 22] Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd edition, Taylor and Francis (2006), ISBN:0-8493-1630-8 [Non-Patent Document 23] O'Hagan et al., Nature Reviews, Drug Discovery 2(9)(2003), 727-735 [Non-Patent Document 24] Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985) and corresponding current editions. [Non-Patent Document 25] Langer, Science 249(1990), 1527~1533 [Non-Patent Document 26] Smith et al., Anal. Biochem. 150 (1985), 76-85 [Non-Patent Document 27] Pras et al., J. Clin. Invest. 47(1968), 924-33 Summary of the Invention [Means for solving the problem]

[0009] The present invention relates generally to patient-derived amyloid xenograft (PDAX) non-human animal models, more specifically, the animal models are characterized by implants of amyloid transthyretin (ATTR) fibrils derived from tissues or organs of patients suffering from ATTR amyloidosis or an ATTR-related disease.

[0010] According to the invention, the tissue is cardiac tissue, renal tissue, liver tissue, gastrointestinal tissue, skin tissue, muscle tissue, tongue tissue, adipose tissue, salivary gland tissue, lymph node tissue, brain tissue, pancreatic tissue, or any ATTR amyloidoma, and the amyloid fibrils are implanted subcutaneously, or implanted in the kidney or subcapsule, peritoneum, muscle, brain, ventricle, nerve, eye, tongue, or heart of an animal model.

[0011] In one embodiment of the invention, the animal is a mouse, a rat, ie, a rodent, or a non-human primate, ie, in general, a non-human mammal.

[0012] In a further embodiment of the invention, the animal is non-transgenic for at least the amyloid fibril protein.

[0013] In another aspect, the present invention relates to a method for determining and / or obtaining an anti-amyloid drug capable of treating amyloidosis or amyloid-related disease, comprising administering a drug or its variant to a model of the present invention and determining amyloid fibrils in the model, wherein accelerated clearance or reduction of amyloid fibrils when the drug or its variant is administered compared to a control indicates suitability for the anti-amyloid drug. In one embodiment, the method comprises collecting tissue biopsies at a first and second time point after administration, and analyzing and quantifying amyloid fibrils, including immunohistochemistry. Preferably, the amount of amyloid fibrils is expressed as a percentage of the implant tissue area, and the amyloid stained area of ​​the implant tissue area in the group treated with the drug or its variant is significantly lower than that in the control group. In additional or alternative embodiments, the accelerated clearance or reduction of amyloid fibrils is observed in a dose-dependent manner.

[0014] In one embodiment of the method of the invention, the drug comprises an anti-amyloid fibril protein antibody or an amyloid fibril binding molecule.

[0015] In another embodiment of the invention, the drug is an antibody and the control is a corresponding isotype antibody.

[0016] In a further embodiment of the invention, the antibody is of human origin, preferably an antibody derived from a human memory B cell, and a variant thereof comprises a heterologous constant domain, preferably said variant antibody is a chimeric antibody and said heterologous constant domain is derived from the same species as the animal used in the model.

[0017] In further embodiments of the invention, the drug is administered intravenously, intraperitoneally, subcutaneously, or orally.

[0018] In another aspect, the present invention provides a process for the manufacture of a pharmaceutical composition comprising an anti-amyloid drug and a pharma- ceutically acceptable carrier, comprising the steps of subjecting a drug or a variant thereof to a method according to the present invention for determining and / or obtaining an anti-amyloid drug, and mixing said drug determined as a suitable anti-amyloid drug with a pharma- ceutically acceptable carrier.

[0019] In one embodiment of the present invention, the pharmaceutical composition is designed for the treatment of amyloidosis or an amyloid-related disease.

[0020] A further aspect of the present invention relates to a method for the characterization, validation, development and / or quality control of anti-amyloid drugs capable of treating amyloidosis or amyloid-related diseases, comprising the steps of subjecting a drug or a variant thereof to the method according to the present invention for determining and / or obtaining an anti-amyloid drug, communicating information obtained by said method to a client, contracting party or collaboration partner and / or selecting said drug determined to be a suitable anti-amyloid drug, and optionally using said anti-amyloid drug or a pharmaceutical composition comprising said anti-amyloid drug for the treatment of amyloidosis or amyloid-related diseases. Thus, one aspect of the present invention relates to the use of a model according to the present invention for drug characterization, quality control and / or development, preclinical and / or parallel clinical trials, or drug selection or validation in the manufacture of a medicament for the treatment of amyloidosis or amyloid-related diseases.

[0021] In another aspect, the present invention relates to a method of generating a patient-derived amyloid xenograft (PDAX) non-human animal model of the present invention comprising isolation of ATTR fibrils from a tissue biopsy obtained from a patient suffering from ATTR amyloidosis or an ATTR-related disease, and implantation of said isolated amyloid fibrils in a non-human animal, preferably having a total protein concentration of about 0.5-5 mg / ml, preferably about 1-4 mg / ml, and most preferably about 2±0.5 mg / ml, preferably as determined by a BCA assay. [Brief description of the drawings]

[0022] [Figure 1] Figure 1 shows the presence of large ATTR aggregates detected by NI-301.37F1 in amyloid fibril extracts prepared from human heart tissue. A: Semi-native SDS-PAGE and Western blot analysis with NI-301.37F1 of tetrameric WT-TTR (100 ng, not detected), misfolded WT-TTR (100 ng), and 0.2 μg, 2.0 μg, and 20 μg ATTR fibril extracts per lane. B: Western blot of antibody NI-301.37F1 (10 nM) binding to ATTR fibrils extracted from postmortem heart tissue (samples no. 4, 67, 87, and 70) obtained from four different patients with ATTR amyloidosis, using a 30 second exposure time. Binding to sample 70 is very faint, consistent with the very low amount of ATTR detection by IHC in this tissue. NI-301.37F1 did not bind to the same protein fractions prepared from human heart tissue without ATTR amyloidosis (samples 40, 18, 60, and 48) and from one sample with amyloidosis without TTR (A+TTR- sample 94). [Figure 2A]Characterization of subcutaneous ATTR fibril implants in mice. A: 24 hours after implantation, fibril grafts were recognizable by their characteristic shape and morphology, distinct from adjacent skin tissue. Fibril grafts were Congo red positive and stained strongly by IHC with human TTR antibody Dako A0002, indicating the presence of ATTR fibrils. Fibril grafts were infiltrated by cells that were negative for conventional mouse macrophage markers CD68, F4 / 80, and Iba1. [Figure 2B] Characterization of subcutaneous ATTR fibril implants in mice (B) Fibril implants stained positive by IHC with CD11b antibody, indicating that the cells infiltrating the ATTR implants and clearing the fibrils were neutrophils. [Figure 3A] Figure 1 shows that the ATTR-specific antibody NI-301.37F1 binds patient-derived ATTR fibrils in a dose-dependent manner in vivo. A: 48 hours after subcutaneous fibril implantation and intravenous antibody injection, fluorescently labeled NI-301.37F1 (NI-301.37F1-VT680) accumulated in a dose-dependent manner on ATTR fibril implants as detected by Thioflavin S fluorescence. This was not observed with the isotype antibody (isotype-VT680). Representative images with n=5 mice per dose level. [Figure 3B] Figure 1 shows that the ATTR-specific antibody NI-301.37F1 binds patient-derived ATTR fibrils in a dose-dependent manner in vivo. B: A linear correlation was observed between the antibody concentration in plasma and on the ATTR fibril grafts in vivo. Dose-dependent increases in NI-301.37F1-VT680 concentration in plasma (upper left panel) and NI-301.37F1-VT680 fluorescence intensity on the fibril grafts (upper right panel) over the dose range 0.05-15 mg / kg are shown. Linear correlation between NI-301.37F1-VT680 concentration and fluorescence intensity over the dose range 0.05-15 mg / kg (lower panel). Dotted line: linear approximation ±95%CI, R2=0.846, n=5 per dose level. [Figure 4]FIG. 1: Treatment with mouse chimera NI-301.37F1 accelerates ATTR fibril clearance in vivo. IHC quantification of remaining human TTR in ATTR fibril grafts 6 and 96 hours after implantation and treatment administration. Intravenous administration of ch.NI-301.37F1 at 5 mg / kg accelerated fibril clearance. n=9-10 mice per group, 2-way ANOVA combined with Sidak's multiple comparison test: p<0.0001. [Diagram 5] FIG. 1 shows that treatment with mouse chimera NI-301.37F1 accelerates ATTR fibril clearance in vivo in a dose-dependent manner. ATTR fibril quantification using TTR IHC. N=5 per group, 1-way ANOVA combined with Dunnett's multiple comparison test: ns: not significant (p>0.05), **: p=0.001, ***: p<0.001. [Figure 6A] Characterization of subcutaneous ATTR fibril implants in mice upon treatment with mouse chimera NI-301.37F1. A: IBA1 staining on subcutaneous ATTR fibril grafts 6 and 96 hours after implantation and treatment with 5 mg / kg ch.NI-301.37F1 or isotype intravenously. Representative images from n=5 mice per treatment group and time point using 5 non-consecutive sections per animal. [Figure 6B] Characterization of subcutaneous ATTR fibril implants in mice upon treatment with mouse chimera NI-301.37F1. B: CD11B staining on ATTR fibril grafts 6 and 96 hours after implantation and treatment with 5 mg / kg ch.NI-301.37F1 or isotype intravenously. Representative images from n=5 mice per treatment group and time point using 5 non-consecutive sections per animal. [Figure 6C]Characterization of subcutaneous ATTR fibril implants in mice upon treatment with mouse chimera NI-301.37F1. C: LY6G staining on ATTR fibril grafts 6 and 96 hours after implantation and treatment with 5 mg / kg ch.NI-301.37F1 or isotype intravenously. Representative images from 3 different mice per treatment group and time point. Staining performed on 5 mice per group and with 2 non-consecutive sections per animal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention relates to a patient-derived amyloid xenograft (PDAX) non-human animal model. In the absence of a suitable in vivo animal model for testing anti-amyloid drugs, the inventors have developed a simple but highly effective model for this purpose, which is advantageously not necessarily based on transgenic animals. The PDAX model is characterized by implants of ATTR fibrils isolated from tissues or organs of patients suffering from amyloid transthyretin (ATTR) amyloidosis or ATTR-related diseases. Surprisingly, the amyloid fibril implants are maintained in animals for several days and are also amenable to drug treatment. In particular, as exemplarily shown for mice implanted with ATTR fibrils in Examples 1 and 2, the amyloid fibril implants are tolerated in experimental animals and present characteristic amyloid features in tissue biopsies after histological analysis. Furthermore, as shown in Examples 3 and 4, the PDAX model has been validated with anti-ATTR specific antibodies, which have been shown to actively promote the clearing of ATTR fibrils in another mouse model in vivo. The PDAX model is therefore highly suitable for testing compounds or drugs for their suitability as anti-amyloid drugs.

[0024] Previously, in a poster presentation at the XVIth International Symposium on Amyloidosis; March 26-29, 2018; Kumamoto, Japan, Higaki et al. reported on an in vivo model of target engagement and antibody-dependent cellular phagocytosis (ADCP) by a systemically administered humanized antibody against misfolded TTR (mis-TTR antibody), in which aggregates of His-tagged TTR-V30M suspended in a basement membrane matrix (Matrigel) were implanted subcutaneously in mice.

[0025] However, although in principle the implantation method could be used to generate a PDAX animal model according to the present invention, the ADCP model mouse of Higaki et al. only represents a suitable animal model according to the present invention as such and may not be suitable for the intended purpose.

[0026] For example, despite the fact that human ATTR amyloidosis is a monogenic disease, there is a lot of variation in its phenotype and ATTR fibril composition, e.g., there are different types of amyloid fibrils, type A consisting of a C-terminal ATTR fragment and full-length TTR, whereas the other type B consists only of full-length TTR, which may be related to the phenotypic diversity for the TTR Val30-Met mutation; see Suhr et al., J. Internal Medicine 281 (2017), 337-347.

[0027] Moreover, it is known that in various types of systemic amyloidosis caused by amyloid fibril protein, the deposition of amyloid fibril protein in tissues and organs is colocalized and / or coaggregated with one or more different amyloid fibril proteins.Indeed, as known in the art and published in, for example, Higashi et al., Brain Research 1184 (2007) 284-294, tau- and alpha-synuclein and TAR-DNA binding protein 43 (TDP43) are found to coexist in the brain pathology of Alzheimer's disease and Lewy body dementia; see also Lee et al., Trends in Neurosciences 27 (2004), 129-134.

[0028] Colocalization of apolipoprotein AI (apoAI) with amyloid deposits in ATTR amyloidosis has also been found in amyloid A (AA) amyloidosis (AA amyloidosis), immunoglobulin (Ig) lambda light chain amyloidosis (Alambda amyloidosis), Ig kappa light chain amyloidosis (Akappa amyloidosis), and Aβ2M amyloidosis; see Sakata et al., J. Histochem. Cytochem. 53 (2005), 237-242. Thus, artificial in vitro generated TTR-V30M aggregates may not reflect the actual structure of ATTR fibrils in vivo, i.e., in patients suffering from ATTR amyloidosis.

[0029] In addition, whether ADCP has any significance for the suitability of anti-TTR antibodies for in vivo therapeutic applications may be questionable and remains to be proven, since the antibodies used in the model are humanized mouse monoclonal antibodies raised against artificial antigenic peptides containing cryptotopes within the TTR sequence and, to date, have only been characterized in vitro; see Higaki et al. Amyloid 23 (2016), 86-97.

[0030] In contrast, the PDAX model of the present invention has been validated with the conformation-specific human-derived monoclonal antibody NI-301.37F1 against TTR aggregates using tissues and fibrils from human patients, respectively, and the antibody has been demonstrated to have potential for in vivo diagnostic and therapeutic use in transgenic mice (FAP mice) expressing exclusively human V30M-TTR protein and no mouse TTR protein; see WO2015 / 092077 A1 and Michalon et al., Orphanet Journal of Rare Diseases 10 (2015), Suppl.1:P39.

[0031] Previously, Yoshimura et al., Bioconjugate Chem. 27 (2016), 1532-1539, established a method of orthotopic implantation of amylin (islet amyloid polypeptide (IAPP)) aggregates and amylin imaging probes, in particular 99m A mouse model of islet amyloidosis intended to study the development of Tc-labeled pyridylbenzofuran derivatives has been described. Specifically, orthotopic amylin aggregates were surgically implanted into BALB / c-nu / nu mice (8-10 weeks old, male) by diluting the peptide solution; see Yoshimura et al. (2016), supra, p. 1537, section "Orthotopic Amylin Aggregates for Implantation" in the left column.

[0032] However, although in principle the implantation method can be used to generate a PDAX animal model according to the present invention, the Yoshimura et al. islet amyloid model mouse represents a suitable animal model for the purposes of the present invention, but may not be suitable for the intended purpose. This is because, similar to the above-mentioned Higaki et al. (2018) in vivo model for ATTR, amylin aggregates prepared in vitro cannot be expected to (fully) reflect the in vivo structure of amylin. For example, an association between phosphorylated TDP43 (pTDP43) and IAPP was observed, which was strong and prominent, especially in subjects with DM and high IAPP in the pancreas; see Leino et al., Journal of Alzheimer's Disease 59 (2017), 43-56.

[0033] Thus, one reason why most previous approaches to provide antibodies for the treatment of amyloidosis have failed is that previous antibody candidates have been screened using recombinant amyloid proteins and their fibrils produced in vitro, which, however, do not take into account the actual environment and three-dimensional structure of amyloid fibrils in diseased tissues or organs, which may also contain other amyloid fibril-forming proteins.

[0034] In a preferred embodiment, the ATTR fibrils to be implanted are isolated from cardiac tissue, renal tissue, liver tissue, gastrointestinal tissue, skin tissue, muscle tissue, tongue tissue, adipose tissue, salivary gland tissue, lymph node tissue, brain tissue, pancreatic tissue, or any ATTR amyloidoma. In a preferred embodiment, the amyloid fibrils are isolated from cardiac tissue. Methods for isolating amyloid fibrils from tissue have been previously described, for example, by Tennent, Methods Enzymol. 309 (1999), 26-47. In particular, according to the present invention, the amyloid fibrils are isolated using a detergent-free protocol to preserve the native conformation of the fibrils. The effectiveness of the isolation can be verified by standard techniques known to those skilled in the art, such as semi-native SDS-PAGE followed by Western blot. As described in Example 1 and shown in Figure 1, amyloid transthyretin (ATTR) fibrils can be effectively isolated from post-mortem frozen cardiac tissue.

[0035] According to the present invention, amyloid fibrils are implanted subcutaneously or subcapsularly, and implanted in kidney, peritoneum, muscle, brain, ventricle, nerve, eye, tongue, or heart.As shown in the examples, amyloid fibrils are conveniently injected subcutaneously into the thigh of mice, where they form deposits that are amenable to drug treatment and further analysis in skin biopsy.Specifically, Example 2 shows that after histological analysis, the deposited amyloid fibrils in the skin biopsy of the model according to the present invention show characteristic features of ATTR amyloid in human biopsy, i.e., Congo red positive and strongly stained by IHC with human TTR antibody Dako A0002 (Figure 2A).Therefore, in a preferred embodiment, amyloid fibrils are implanted subcutaneously.

[0036] Commonly used model animals suitable for the present invention are mice, rats, i.e. rodents, or non-human primates, i.e. generally non-human mammals. In a preferred embodiment of the present invention, the animal is a mouse. Mice as model organisms for the purposes of the present invention are advantageously convenient and inexpensive to breed and have a short generation time, i.e. many animals can be produced in a relatively short period of time.

[0037] Although at least the mouse genome is relatively easy to manipulate, the establishment of transgenic mouse lines or other animals always involves costs and a rather large amount of time. Therefore, it is very advantageous that wild-type animals can be used for the PDAX model according to the present invention. Therefore, in one embodiment of the present invention, the animal is non-transgenic at least for amyloid fibril proteins. Of course, transgenic animals can also be used where appropriate, for example to better reflect the actual patient situation. For example, it is conceivable that amyloid fibrils are implanted in immunodeficient mice to create a model of a patient suffering from a disease associated with immunodeficiency in addition to amyloidosis. This allows the analysis of anti-amyloid drugs in a combined patient system with respect to additional diseases, for example immunodeficiency or other diseases for which a transgenic animal model is available.

[0038] In a further aspect, the present invention relates to a method for determining and / or obtaining an anti-amyloid drug capable of treating amyloidosis or amyloid-related disease, comprising administering a drug or its variant to a model of the present invention and determining amyloid fibrils in said model, and accelerating clearance or reduction of amyloid fibrils when said drug is administered compared to a control indicates suitability for the anti-amyloid drug, and preferably, said accelerated clearance or reduction is observed in a dose-dependent manner. Rather than the intended drug, a variant of the drug may be used and tested in the PDAX model of the present invention, particularly in the case of protein drugs such as antibodies or receptor-based drugs that may tend to induce an immune response in animals. For example, as illustrated in the examples, the drug that is ultimately produced is a parent fully human antibody, but a chimeric version of an otherwise fully human antibody may be used for testing.

[0039] Those skilled in the art know various methods of determining amyloid fibrils. As detailed in Examples 2-5, amyloid fibrils can be analyzed in tissue sections by Congo Red staining, Thioflavin S staining, or immunohistochemistry with appropriate antibodies. To determine whether drug treatment accelerates the clearance or reduction of amyloid fibrils compared to control, amyloid fibrils are then quantified using appropriate devices and processes, such as automated microscopy and image analysis processes. Furthermore, as mentioned in Examples 4 and 5, ATTR fibrils undergo spontaneous fibril clearance processes in the model. Thus, the acceleration of fibril clearance or reduction by treatment with the drug being evaluated is the output for suitability as an anti-amyloid drug. Thus, the method preferably includes collecting tissue biopsies at the first and second time points after administration, and analyzing and quantifying amyloid fibrils, including immunohistochemistry. In a further preferred embodiment, the amount of amyloid fibrils is expressed as a percentage of the implant tissue area, and the amyloid stained area of ​​the implant tissue area in the group treated with the drug or its variant is significantly lower than in the control group.

[0040] In a preferred embodiment, the first time point is 3 to 10 hours, more preferably 5 to 8 hours, and most preferably 6 hours, after administration of the drug or its variant, and the second time point is 48 to 156 hours, more preferably 72 to 120 hours, and most preferably 96 hours, after administration of the drug or its variant.

[0041] In principle, all available methods for diagnosing amyloidosis in patient biopsies can be applied to the method of the present invention to determine / analyze amyloid fibril grafts in model organisms.

[0042] As shown in Examples 3-5, ATTR-specific antibodies have been shown to specifically bind implanted amyloid fibrils and accelerate their clearance. Thus, in one embodiment of the method of the invention, the drug comprises an anti-amyloid fibril protein antibody or an amyloid fibril binding molecule. In a further embodiment, the drug is an antibody and the control is a corresponding isotype antibody. Suitable antibody candidates are known from the prior art, for example anti-transthyretin (TTR) antibodies are disclosed in WO2015 / 092077 A1, WO2014 / 124334 A2, WO2018 / 007923 A3, WO2016 / 120810 Al, US2017 / 0058023 A1, and US9,879,080 B2. Of course, it is envisaged within the scope of the invention that particularly novel antibodies or compounds in general may be applied to the method of the invention to be evaluated for their suitability as anti-amyloid drugs. Besides antibodies, all types of drugs, including, for example, small molecules, can be conveniently tested by the methods of the present invention for their suitability as anti-amyloid drugs.

[0043] As mentioned, most previous approaches to provide drugs in the treatment of amyloidosis have used screening of lead candidate compounds with recombinant amyloid proteins and their fibrils produced in vitro, which, however, do not take into account the actual environment and three-dimensional structure of amyloid fibrils in the diseased tissue or organ, which may also contain other amyloid fibril-forming proteins.

[0044] In contrast, the PDAX animal model of the present invention allows for the acquisition and selection of anti-amyloid compounds, particularly antibodies, that can be reasonably expected to be specific enough to selectively bind to target amyloid proteins, e.g., at desired and necessary locations of toxic amyloid deposits, in the patient to be treated.

[0045] In certain preferred embodiments, the candidate antibodies are humanized, human-like or human antibodies, preferably human-derived antibodies, most preferably human-derived antibodies isolated from human memory B cells, and recombinant variants thereof, which typically comprise substantially the variable heavy and light chains of the original human-derived antibody, as well as human constant domains, preferably of IgG1 or IgG4 subtype, but not necessarily identical to those of the original human-derived antibody.

[0046] However, as illustrated in the Examples for testing and validation in the PDAX model of the present invention, preferably a humanized, human-like or human antibody variant is used, which variant comprises a heterologous constant domain, preferably said variant antibody is a chimeric antibody and said heterologous constant domain is derived from the same species as the animal used in the model.

[0047] Those skilled in the art are aware of various routes for administering drugs to model animals.Therefore, in one embodiment of the method of the present invention, drug is administered intravenously, intraperitoneally, subcutaneously or orally.For example, drug is intravenously injected into the tail vein of the mouse receiving the implant of isolated amyloid fibrils, as shown in Examples 3 and 4.

[0048] A further aspect of the present invention relates to a process for the manufacture of a pharmaceutical composition comprising an anti-amyloid drug or a variant thereof and a pharma- ceutically acceptable carrier, in which an anti-amyloid drug determined to be a suitable anti-amyloid drug by the method of the present invention as described above is mixed with a pharma- ceutically acceptable carrier.

[0049] Pharmaceutically acceptable carriers and administration routes can be obtained from the corresponding literature known to those skilled in the art. The pharmaceutical composition of the present invention can be formulated according to methods well known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000), University of Sciences in Philadelphia, ISBN 0-683-306472; Vaccine Protocols 2nd Edition, Robinson et al., eds., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd Edition, Taylor and Francis (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art, and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. The composition containing such carriers can be formulated according to well-known conventional methods. These pharmaceutical compositions can be administered to subjects in appropriate doses. Administration of the appropriate compositions can be accomplished by various modes. Examples include administering compositions containing pharma- ceutically acceptable carriers by oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations, such as nasal spray formulations, contain purified aqueous or other solutions of active substances, together with preservatives and isotonicity agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa. Pharmaceutical compositions for oral administration, such as single domain antibody molecules (e.g., "Nanobodies™"), are also envisioned in the present invention. Such oral formulations can take the form of tablets, capsules, powders, liquids, or semisolids. Tablets can contain solid carriers, such as gelatin or adjuvants.Formulations for rectal or vaginal administration may be provided as a suppository with a suitable carrier; see also O'Hagan et al., Nature Reviews, Drug Discovery 2(9)(2003), 727-735. Further guidelines regarding formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th Edition (1985) and corresponding current editions. For a brief review of methods for drug delivery, see Langer, Science 249(1990), 1527-1533.

[0050] In one embodiment of the process of the present invention, the pharmaceutical composition is designed for the treatment of ATTR or an ATTR-related disease, whereby the amyloidosis is characterized by ATTR deposits in the patient, in particular its respective precursor protein.

[0051] In another aspect, the present invention relates to a method for characterizing, validating, developing, and / or quality control of anti-amyloid drugs that can treat amyloidosis or amyloid-related diseases.For example, anti-amyloid drugs are characterized in dose-response studies, target association experiments (drug binding to target), or characterized for treatment mode of action, such as local activation of specific immune response by drugs at fibril graft site, or PK / PD relationship, such as dose-response as a function of drug exposure level.Thereby, the information about drugs that are subjected to the method for determining and / or obtaining anti-amyloid drugs of the present invention as described above is transmitted to clients, contracting parties, or collaboration partners.Furthermore, the drug that is determined to be a suitable anti-amyloid drug can be selected, and optionally, the anti-amyloid drug or pharmaceutical composition containing the anti-amyloid drug is used to treat amyloidosis or amyloid-related diseases.

[0052] The present invention further relates to the use of PDAX model for drug characterization, quality control, and / or development, preclinical and / or parallel clinical trials, or drug selection or validation in the manufacture of medicines for the treatment of amyloidosis or amyloid-related diseases.The method of using the model of the present invention to characterize drugs, for example, in dose-response studies, or target association experiments (drug binding to target), is well known to those skilled in the art.Furthermore, according to the present invention, the model is used to characterize the treatment mode of action of drugs, for example, the local activation of specific immune response by drugs at the fibril graft site, or PK / PD relationship, for example, dose response as a function of drug exposure level.

[0053] In this context, PDAX model can also be used in the context of personalized medicine, which means that the amyloid fibrils from individual patients are isolated and analyzed individually for their response to anti-amyloid drugs using the model of the present invention.With regard to such use of the model of the present invention, advantageously, it is possible to determine whether treatment is effective in a particular patient even before treating the patient and before exposing the patient to possible adverse effects.Therefore, the use of the model of the present invention allows more targeted amyloidosis treatment.

[0054] In a further aspect, the present invention relates to a method of making the patient-derived amyloid xenograft (PDAX) non-human animal model of the present invention, comprising the isolation of ATTR fibrils from a tissue biopsy obtained from a patient suffering from ATTR amyloidosis or an ATTR-related disease, and implantation of said isolated amyloid fibrils in a non-human animal, preferably wherein said fibril preparation has a total protein concentration of amyloid fibrils of about 0.5-5 mg / ml, preferably about 1-4 mg / ml, most preferably about 2±0.5 mg / ml, preferably as determined by a bicinchoninic acid (BCA) assay, and is highly viscous; see, e.g., Smith et al., Anal. Biochem. 150 (1985), 76-85. An example of how the model of the present invention is made is depicted in Example 1. As mentioned above, the tissue biopsy from which amyloid fibrils are isolated may be cardiac tissue, renal tissue, liver tissue, gastrointestinal tissue, skin tissue, muscle tissue, tongue tissue, adipose tissue, salivary gland tissue, lymph node tissue, brain tissue, pancreatic tissue, or any other tissue with deposited amyloidoma. Those skilled in the art are aware of various methods for isolating amyloid fibrils, for example, isolation methods that preserve the native conformation of the fibrils are described in detail in Tennent, Methods Enzymol. 309 (1999), 26-47. As shown in Example 1, the isolated amyloid fibrils are implanted by subcutaneous injection, although other forms of implantation such as subcapsular implantation, implantation in the kidney, peritoneum, muscle, brain, ventricle, nerve, eye, tongue, or heart are conceivable. EXAMPLES

[0055] The following Examples 1-5 serve to illustrate embodiments of the present invention. It will be appreciated that the present invention is not intended to be limited by the foregoing description, which is intended to serve to illustrate embodiments of the present invention. The patient-derived amyloid xenograft (PDAX) mouse model presented in the following examples includes subcutaneous implantation of patient-derived ATTR fibrils obtained from postmortem cardiac tissue.

[0056] Example 1: Creation of PDAX model Isolation of ATTR fibrils Frozen human heart samples showing extensive TTR amyloid infiltration were processed for biochemical extraction of amyloid TTR fibrils. A detergent-free protocol was used to preserve the fibril conformation as much as possible (Pras et al., J. Clin. Invest. 47 (1968), 924-33; Tennent, Methods Enzymol. 309 (1999), 26-47). Briefly, the procedure consisted of repeated mechanical homogenization in ice-cold TE buffer (10 mM Tris pH 8.0, 140 mM NaCl, 10 mM EDTA, 0.1% (wt / vol) NaN3, protease inhibitor cocktail) to extract and eliminate soluble proteins, followed by repeated mechanical homogenization in ice-cold pure water. This has been described to eliminate calcium-dependent binding of SAP protein on amyloid fibrils, allowing suspension of amyloid fibrils in pure water (Pras et al. (1968), supra). ATTR fibrils were further purified by precipitation with NaCl and EDTA and concentrated by centrifugation. Total protein concentration was determined by BCA assay and adjusted to 2 mg / ml to ensure high viscosity of the fibril preparation. Each fibril preparation was verified by semi-native SDS-PAGE using standard procedures as described below, and by Western blot with ATTR fibril-specific antibody NI-301.37F1 (10 nM). To preserve aggregates during the analysis process, samples were mixed with loading buffer and loaded directly onto SDS-PAGE gels, omitting the usual heat denaturation step. For each sample, 10.0 μg of total protein was loaded onto a 4-12% bis-tris gel in MOPS buffer and run at 200 V for 40 min. Proteins were transferred to nitrocellulose membranes by semi-dry blotting for 60 min at 20 V. The membranes were blocked for 1 h in blocking buffer (2% BSA, 0.1% tween-20 in PBS buffer, pH 7.4) and incubated overnight at 4° C. with NI-301.37F1 antibody diluted to 10 nM in blocking buffer. Detection was performed with an HRP-conjugated anti-human IgG antibody in combination with a chemiluminescent substrate.

[0057] Figure 1A shows semi-native SDS-PAGE and Western blot analysis with NI-301.37F1 of tetrameric WT-TTR (100 ng, not detected), misfolded aggregated WT-TTR (100 ng), and 0.2 μg, 2.0 μg, and 20 μg of ATTR fibril extracts per lane. The tissue extraction procedure resulted in fractions highly enriched for ATTR fibrils, which were detected by NI-301.37F1 on the semi-native SDS-PAGE gel as high molecular weight aggregates (Figure 1A, left panel) that were absent in the secondary antibody only control (Figure 1A, right panel). Similar experiments were performed on ATTR fibrils prepared from postmortem cardiac tissue obtained from four different donors with ATTR amyloidosis (ATTR+), four donors without amyloidosis (ATTR-), and one donor with amyloidosis unrelated to ATTR (A+TTR-) (Figure 1B). NI-301.37F1 selectively detected patient-derived ATTR fibrils in all ATTR cases, including one with very low amounts of amyloid (sample 70), and did not detect unrelated proteins present in the same tissue fractions prepared from tissue without ATTR amyloidosis.

[0058] Implantation of patient-derived fibrils WT SKH1 female mice were briefly anesthetized with isoflurane and given an implant of 100 μg of ATTR fibril extract by subcutaneous injection in the thigh.

[0059] Anti-TTR antibody The mouse chimeric antibody ch.NI-301.37F1 was generated from the human-derived monoclonal antibody NI-301.37F1 disclosed in WO2015 / 092077 A1. The mouse chimeric variant was designed to contain the human variable domain of NI-301.37F1 in a mouse constant domain backbone. In particular, the amino acid sequences of the heavy chain variable domain (VH) and the light chain variable domain (VL) of the human-derived monoclonal antibody NI-301.37F1 are disclosed in FIG. 1 of WO2015 / 092077 A1, and are SEQ ID NO: 10 and 53, respectively, for the VH chain, and SEQ ID NO: 12 for the VL chain, while the mouse heavy chain constant domain corresponds to Uniprot entry P01863, and the mouse light chain constant domain corresponds to Uniprot entry P01837. Briefly, gene synthesis was used to generate a synthetic heavy chain gene (see sequence mur.37F1 H) containing a sequence encoding the human heavy chain variable domain of NI-301.37F1 followed by a sequence encoding the mouse IgG2a heavy chain constant domain, and a synthetic light chain gene (see sequence mur.37F1 L) containing a sequence encoding the human light chain variable domain of NI-301.37F1 followed by a sequence encoding the mouse kappa light chain constant domain. These two genes were then subcloned into an appropriate expression vector used to transfect CHO cells. The Ch.NI-301.37F1 antibody was purified from the cell culture medium using standard processes as described in WO2015 / 092077 A1, including purification of the antibody by chromatography on a protein A column.

[0060] Example 2: Characterization of the PDAX model Twenty-four hours after implantation of patient-derived ATTR fibrils, mice were sacrificed and skin tissues containing fibril implants were harvested, fixed, embedded (PPFA), and sectioned for histological analysis with Congo Red and IHC with human TTR antibody Dako A0002, as well as macrophage markers CD68, F4 / 80, and Iba1. Congo Red staining was performed using a modification of Putchler. Briefly, tissue sections were stained with hemalum, destained in water, incubated sequentially in solution I (80% EtOH, 30 g / l NaCl, 0.01% NaOH) and solution II (80% EtOH, 30 g / l NaCl, 5 g / l Congo Red, 0.01% NaOH) for 30 min each at room temperature (RT), cleared in 100% EtOH with and without 0.01% NaOH, and mounted. Sections were imaged with a 20x objective in brightfield and polarized light modes. Immunostaining was performed according to standard procedures by quenching endogenous peroxidase activity with 3% H2O2 in methanol for 20 min at RT, incubation in blocking buffer (PBS + 5% serum (horse / goat) + 4% BSA) for 1 h at RT, followed by incubation with primary antibodies overnight at 4°C. Detection was performed using the Vectastain ABC kit (Vector Laboratories) and appropriate secondary antibodies (all from Jackson Immuno-research; 1:400) in combination with diaminobenzidine (Dako). TTR staining was performed with TTR antibody Dako A0002 at 1:500 dilution combined with biotin-conjugated goat anti-rabbit IgG antibody at 1:400 dilution; CD68 antibody at 1:400 dilution, F4 / 80 antibody at 1:200 dilution, and IBA1 antibody at 1:750 dilution were used, each combined with HRP-conjugated biotin donkey anti-rabbit IgG at 1:400 dilution in PBS.

[0061] Amyloid fibril implants were easily recognizable from adjacent skin tissues by their shape and appearance, which is clearly different from the structured and complex morphology of skin. As shown in Figure 2A, amyloid fibril implants were positive for Congo red staining and TTR IHC. Fibril implants were infiltrated with small cells that stained negative for mouse macrophage markers CD68, F4 / 80, and Iba1. Instead, as shown in Figure 2B, the cells infiltrating the amyloid fibril implants stained positive for the monocyte marker CD11b (also named integrin αM), indicating that these cells were undoubtedly neutrophils, which, like macrophages, can perform antibody-mediated phagocytosis. Neutrophils are the most abundant immune cell type in blood. Fibril implants by subcutaneous injection produce, over the course of 6-8 days, microlesions with rupture of local capillaries, as well as the release of neutrophils that encapsulate, infiltrate, and completely eliminate the fibril implant. As shown in Example 4 below, this process is accelerated in the presence of the ATTR antibody NI-301.37F1, indicating that the antibody can activate the immune system in vivo.

[0062] Example 3: In vivo binding of compounds to ATTR fibrils in the PDAX model To characterize the PDAX model, we have analyzed the in vivo binding of ATTR-specific antibodies to implanted patient-derived ATTR fibrils. WT SKH1 female mice were implanted with 100 μg of patient-derived ATTR fibrils by subcutaneous injection in the thigh under brief gas anesthesia, followed by intravenous injection in the tail vein of fluorescently labeled NI-301.37F1 or isotype antibodies at 0.05 mg / kg, 0.5 mg / kg, 5.0 mg / kg, or 15 mg / kg. Antibody labeling with Vivotag-680 was performed according to the manufacturer's instructions (Perkin Elmer). Briefly, Vivotag-680 ester-reactive dye was dissolved in DMSO and mixed with antibodies prepared in carbonate buffer. After 2 h of incubation in the dark, the labeled antibodies were purified by dialysis overnight at 4°C in PBS buffer.

[0063] 48 hours after implantation, mice were sacrificed and skin biopsies were collected, fixed, embedded (PPFA), and cut for histological analysis. Sections were dehydrated, stained with thioflavin S, and mounted with Dapi-containing mounting medium. Slide scanning was performed using an automated fluorescent microscope at 20x magnification using Dapi, FITC, and Cy5 channels.

[0064] Subcutaneous ATTR fibril implants were clearly recognizable from adjacent skin tissue by their specific shape and appearance and displayed strong thioflavin S fluorescence, indicating the presence of fibrils with preserved amyloid conformation (Figure 3A). Binding of the fluorescently labeled antibody NI-301.37F1 was increased in a dose-dependent manner on fibrils but not on surrounding tissues (Figure 3A); fibril binding was selective for NI-301.37F1 and was not observed for isotype antibodies. NI-301.37F1 fluorescence overlapped with thioflavin S fluorescence, indicating binding of NI-301.37F1 on ATTR fibrils. These results show that NI-301.37F1 binds to patient-derived ATTR fibrils implanted in mice in a dose-dependent manner.

[0065] NI-301.37F1-VT680 fluorescence intensity at fibril sites was quantified by software-based image analysis and served as a proxy for antibody density. Fluorescence intensity increased continuously over the entire dose range tested; more specifically, there was a continuous increase in fluorescence intensity, indicating that there was no saturation of NI-301.37F1 binding even between the highest doses tested (5 mg / kg and 15 mg / kg).

[0066] Plasma levels for chimeric NI-301.37F1 were determined using a direct TTR ELISA and a calibration curve with known antibody concentrations. Briefly, 96-well microplates were coated with human wild-type TTR diluted to a concentration of 10 μg / ml in PBS buffer (pH 7.4) for 1 hour at 37°C. Nonspecific binding sites were blocked with blocking buffer (2% BSA, 0.1% tween-20 pH 7.4 in PBS buffer) for 1 hour at RT. Plasma samples were diluted 1:500 in duplicate in blocking buffer. Calibration samples were similarly prepared by diluting chimeric NI-301.37F1 antibody in a concentration range from 5 pM to 5 nM in duplicate in blocking buffer. Diluted plasma samples were quantified in duplicate (total of quadruplicates per sample) as follows: samples were incubated overnight at 4°C, chimeric NI-301.37F1 was detected with an HRP-conjugated anti-mouse IgG2a antibody (Jackson Immunoresearch) at a 1:4000 dilution in blocking buffer, and then HRP activity was measured using standard procedures.

[0067] Analysis of NI-301.37F1-VT680 concentration versus fluorescence intensity on ATTR fibrils revealed a linear correlation over the dose range of 0.5-15 mg / kg, indicating no target saturation at antibody doses up to 15 mg / kg (Figure 3B). Results for the 0.05 mg / kg dose group were excluded from the correlation analysis, as the antibody concentration may have been underestimated due to matrix interference effects at very low concentrations.

[0068] Example 4: ATTR fibril clearance in vivo Patient-derived ATTR fibrils were implanted in WT mice by subcutaneous injection, followed by intravenous administration of mouse chimeric NI-301.37F1 variant (ch.NI-301.37F1) or corresponding isotype antibody at 5.0 mg / kg. After 6 or 96 hours, mice were sacrificed and skin biopsies were collected for histological analysis. ATTR fibrils were detected by immunohistochemistry using the commercially available human TTR antibody Dako A0002. Quantification was performed using automated microscopy and image analysis, and the amount of ATTR fibrils was expressed as a percentage of the implant tissue area.

[0069] Subcutaneous ATTR fibril implants were clearly recognizable from adjacent skin tissue due to their specific shape and appearance. Six hours after implantation and treatment administration, ATTR fibril implants were completely stained for TTR by IHC, with TTR staining occupying an average of 60-70% of the implant tissue area in both treatment groups (Figure 4). Ninety-six hours after fibril implantation, the TTR stained area occupied only 12% of the implant area in the group treated with ch.NI-301.37F1 and 39% in the isotype group (Figure 4). The difference between the two groups was statistically significant at p<0.0001. These results demonstrate that ATTR fibril clearance was accelerated by treatment with ch.NI-301.37F1.

[0070] Similar experiments were performed based on Congo red and Thioflavin S staining, which further confirmed that treatment with ch.NI-301.37F1 accelerated ATTR fibril clearance compared to treatment with isotype antibody.

[0071] To further characterize the activity of NI-301.37F1 in vivo, ATTR fibril-grafted mice received a single intravenous dose of ch.NI-301.37F1 at 0.05 mg / kg, 0.5 mg / kg, 5.0 mg / kg, and 50 mg / kg, or isotype at 50 mg / kg. One mouse from each group was sacrificed 6 hours after drug administration for reference (t0); all other mice were sacrificed 96 hours later and skin biopsies were collected for histological analysis as described above.

[0072] In isotype-treated mice, ATTR fibrils, which occupied 86% of the graft area at t0, decreased to 58% after 4 days (96 h) (Figure 5), reflecting spontaneous fibril elimination in this model. Fibril elimination was accelerated in a dose-dependent manner by treatment with ch.NI-301.37F1. The treatment effect was statistically significant at doses of 0.5 mg / kg and above (Figure 5), but was not significant at the very low dose of 0.05 mg / kg due to the limited effect size and low number of mice per dose group. Complete fibril elimination was obtained at both the 5.0 mg / kg and 50 mg / kg doses.

[0073] These data confirm the activity of NI-301.37F1 in vivo and demonstrate that activation of the immune system for clearance of ATTR fibrils occurs already at low doses.

[0074] Example 5: Characterization of PDAX models with antibody treatment In vivo clearance of ATTR fibrils in the PDAX model, as shown in Example 4, was further characterized by staining for myeloid cell markers IBA1, DC11B, and LY6G.

[0075] IBA1, also known as allograft inflammatory factor 1 (AIF1), is a marker for macrophages as well as activated neutrophils. ATTR graft tissue was stained by IHC for IBA1 expression. Cells present in and around the ATTR graft tissue did not have any IBA1 expression 6 hours after transplantation (Figure 6A). Similarly, IBA1 expression 96 hours after transplantation was low or absent, and was only detected in a few cells within the ATTR graft area.

[0076] Cluster of differentiation molecule 11B (CD11B), also known as integrin alpha M (ITGAM), macrophage-1 antigen (Mac-1), or complement receptor 3 (CR3), is expressed by monocytes and macrophages as well as granulocytes and natural killer cells. ATTR graft tissue stained for CD11B expression by IHC presented CD11B-positive cells within and adjacent to the graft tissue 6 hours after transplantation (Figure 6B). At this time point, CD11B-positive cells were typically small and amoeboid, with some of the cells presenting elongated processes. 96 hours after transplantation, we observed a large increase in CD11B-positive cells infiltrating the entire ATTR graft in both control antibody-treated and ch.NI-301.37F1-treated mice.

[0077] LY6G is a GPI-anchored protein and, together with LY6C, is a component of the myeloid differentiation antigen GR-1. LY6G is present primarily on peripheral neutrophils. ATTR graft sections stained for LY6G expression presented LY6G-positive cells within and around the ATTR graft 6 hours after transplantation, regardless of treatment (Figure 6C). In contrast, LY6G-expressing cells were not detected 96 hours after transplantation.

Claims

1. 1. A method for obtaining an anti-amyloid drug for treating a patient suffering from amyloidosis, comprising: creating a patient-derived amyloid xenograft non-human animal model in which amyloid fibrils containing amyloid transthyretin obtained from a patient suffering from amyloidosis are transplanted into tissue of the non-human animal; administering an anti-amyloid drug to said patient-derived amyloid xenograft non-human animal model; and determining that the anti-amyloid drug eliminates or reduces amyloid fibrils at a time point after administration.

2. The method of claim 1 , wherein the anti-amyloid drug is an antibody.

3. The method of claim 2 , wherein the antibody is a humanized antibody, a human-like antibody, or a human antibody.

4. The method of claim 2 or 3, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

5. The method of any one of claims 1 to 4, wherein the elimination or reduction of amyloid fibrils is compared to a control.

6. The method of claim 5, wherein the control is an isotype antibody or the antibody NI-301.37F1.

7. 1. A method for predicting the efficacy of treatment with an anti-amyloid drug in a patient having amyloidosis or an amyloid-related disease, comprising: (a) generating a patient-derived amyloid xenograft non-human animal model by subcutaneously or subcapsularly transplanting amyloid fibrils containing amyloid transthyretin obtained from a tissue or organ of said patient into tissue of a non-human animal; (b) administering an anti-amyloid drug to said patient-derived amyloid xenograft non-human animal model; (c) determining the amount or area of ​​amyloid fibrils present in tissue sections of said patient-derived amyloid xenograft non-human animal model; The method, wherein a reduction in the amount or area of ​​amyloid fibrils following administration of an anti-amyloid drug indicates efficacy of the treatment.

8. The method of claim 7, wherein the total protein concentration of the amyloid fibrils in step (a) is 0.5-5 mg / mL.

9. 9. The method of claim 7 or 8, wherein the amount or area of ​​amyloid fibrils is determined by Congo red staining, Thioflavin S staining, or immunohistochemistry.

10. The method of any one of claims 1 to 9, wherein the anti-amyloid drug is administered intravenously, intraperitoneally, subcutaneously, or orally to the patient-derived amyloid xenograft non-human animal model.

11. The method of any one of claims 7 to 10, wherein the anti-amyloid drug is an antibody.

12. The method of claim 11 , wherein the antibody is a humanized antibody, a human-like antibody, or a human antibody.

13. The method of claim 11 or 12, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

14. 1. A method for assessing an immune response to an anti-amyloid fibril protein antibody, comprising: (a) administering the antibody to a patient-derived amyloid xenograft non-human animal model prepared by subcutaneously or subcapsularly implanting amyloid fibrils containing amyloid transthyretin obtained from a patient's tissue or organ into the tissue of the non-human animal; (b) determining the immune response of the non-human animal at a time point after administration of the antibody.

15. The method according to any one of claims 1 to 14, wherein the non-human animal is a mouse, a rat, or a non-human primate.

16. The method according to any one of claims 1 to 15, wherein the non-human animal tissue is skin, kidney, peritoneum, muscle, brain, nerve, eye, tongue, or heart.

17. 17. The method of claim 16, comprising detecting and / or measuring the expression level of one or more myeloid cell markers.

18. 18. The method of claim 17, wherein the one or more myeloid cell markers are detected or measured via immunohistochemistry.

19. The method of claim 17 or 18, wherein the one or more myeloid cell markers include IBA1, CD11B, and / or LY6G.

20. An anti-amyloid drug for treating a patient having amyloidosis or an amyloid-related disease, which is determined to exhibit elimination or reduction of amyloid fibrils when administered to a patient-derived amyloid xenograft non-human animal model prepared by transplanting amyloid fibrils containing amyloid transthyretin obtained from a patient suffering from amyloidosis into a non-human animal.

21. The anti-amyloid drug of claim 20 which is an antibody.

22. The anti-amyloid drug of claim 21 , wherein the antibody is a humanized antibody, a human-like antibody, or a human antibody.

23. The anti-amyloid drug of claim 21 or 22, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

24. The anti-amyloid drug according to any one of claims 20 to 23, wherein the non-human animal is a mouse, a rat, or a non-human primate.

25. The anti-amyloid drug according to any one of claims 20 to 24, wherein the tissue of the non-human animal is skin, kidney, peritoneum, muscle, brain, nerve, eye, tongue, or heart.

26. A pharmaceutical composition comprising the anti-amyloid agent according to any one of claims 20 to 25 and a pharma- ceutically acceptable carrier.

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