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 for producing 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 a review, see Hazenberg, Rheum. Dis. Clin. North Am. 39 (2013), 323–345. These rigid, unbranched fibrils, approximately 10 nm in diameter, are characterized by a molecular beta-pleated sheet structure, usually composed of peptides arranged in an antiparallel configuration. This structure of the fibrils accounts for their insolubility, resistance to proteolysis, and binding affinity for the amyloid-specific Congo Red and Thioflavin S dyes. Three mechanisms appear to operate, 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 forms of the protein (transthyretin); and proteolytic remodeling of the precursor protein (β-amyloid precursor protein). Interaction with the extracellular matrix also appears 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, leading 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 region of the body, such as Alzheimer's disease with amyloid-β protein plaques in the brain or type 2 diabetes with amyloid amylin deposits in the islets of Langerhans in the pancreas, and systemic forms in which fibril deposits occur in various organs and tissues throughout the body. One of the most common systemic amyloidoses is amyloid transthyretin (ATTR) amyloidosis, in which ATTR fibrils accumulate in multiple organs and tissues, leading to multiple organ dysfunction with rapid disease progression and fatal outcomes.

[0004] ATTR amyloidosis presents in two major clinical manifestations: 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 an 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, the immune system is hypothesized to have the ability to eliminate amyloid fibrils; however, for reasons that are not yet fully understood, amyloid fibrils escape immune surveillance and accumulate to toxic levels in ATTR patients. No specific treatment is currently 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 it is difficult to apply to elderly and vulnerable patient populations. Liver transplantation was considered an option for mutant ATTR, but both peripheral neuropathy and cardiac amyloidosis can progress after liver transplantation. The transthyretin stabilizer tafamidis (Vyndaqel®) was approved in Europe in November 2011 for patients with early-stage ATTR-PN. However, in a post-approval trial, neurological disability scores worsened by 55% one year after tafamidis treatment, 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 resulting in symptomatic remission in patients.

[0005] In the development of new drug therapies, animal models are essential for evaluating the efficacy and safety of drugs. Not only in ATTR, but also in most, if not all, amyloidosis drug treatments have so far been studied using transgenic animal models, particularly transgenic mouse models, which rely largely on mutations in the respective precursor proteins observed in human patients. For example, several transgenic mouse models have been presented to the scientific community in an attempt to generate models of ATTR amyloidosis; however, 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] Characterization analysis of transgenic mouse models has shown 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 can adequately 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 edition (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, and 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 present 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 into 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, a non-human mammal in general.

[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 an amyloid-related disease, the method 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 upon administration of the drug or its variant compared to a control indicates suitability for the anti-amyloid drug. In one embodiment, the method comprises collecting tissue biopsies at first and second time points after administration and analyzing and quantifying amyloid fibrils, including immunohistochemistry. Preferably, the amount of amyloid fibrils is expressed as a percentage of the graft tissue area, and the amyloid-stained area of ​​the graft tissue area in the group treated with the drug or its variant is significantly lower than that in the control group. In further 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 present 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 of human memory B cell origin, 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 a further embodiment 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 pharmaceutically acceptable carrier, the process 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 to be a suitable anti-amyloid drug with a pharmaceutically 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 an amyloid-related disease, 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, contractual 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 an amyloid-related disease. 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 an amyloid-related disease.

[0021] In another aspect, the present invention relates to a method for generating a patient-derived amyloid xenograft (PDAX) non-human animal model of the present invention, comprising isolating ATTR fibrils from a tissue biopsy obtained from a patient suffering from ATTR amyloidosis or an ATTR-related disease, and implanting 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 explanation 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 cardiac 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 of ATTR fibril extracts per lane. B: Western blot of antibody NI-301.37F1 (10 nM) binding to ATTR fibrils extracted from postmortem cardiac tissue (samples 4, 67, 87, and 70) obtained from four different patients with ATTR amyloidosis, using a 30-second exposure time. Binding to sample 70 was very faint, consistent with the very low amount of ATTR detected 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]Figure 1 shows the characterization of subcutaneous ATTR fibril implants in mice. A: 24 hours after implantation, fibril implants were recognizable by their characteristic shape and morphology, which differed from adjacent skin tissue. Fibril implants were Congo red-positive and stained strongly by IHC with the human TTR antibody Dako A0002, indicating the presence of ATTR fibrils. Fibril implants were infiltrated with cells that were negative for the 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 removing 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: Forty-eight hours after subcutaneous fibril implantation and intravenous antibody injection, fluorescently labeled NI-301.37F1 (NI-301.37F1-VT680) accumulated on ATTR fibril implants in a dose-dependent manner as detected by Thioflavin S fluorescence. This was not observed with the isotype antibody (Isotype-VT680). Representative images from 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 graft in vivo. A dose-dependent increase in NI-301.37F1-VT680 concentration in plasma (upper left panel) and NI-301.37F1-VT680 fluorescence intensity on the fibril graft (upper right panel) over the dose range of 0.05-15 mg / kg is shown. A linear correlation between NI-301.37F1-VT680 concentration and fluorescence intensity over the dose range of 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 shows that treatment with mouse chimeric NI-301.37F1 accelerates ATTR fibril clearance in vivo. IHC quantification of remaining human TTR in ATTR fibril implants 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. [Figure 5] Figure 1 shows that treatment with mouse chimera NI-301.37F1 dose-dependently accelerates ATTR fibril clearance in vivo. ATTR fibril quantification using TTR IHC. N=5 per group, one-way ANOVA combined with Dunnett's multiple comparison test: ns: not significant (p>0.05), **: p=0.001, ***: p<0.001. [Figure 6A] Figure 1 shows the characterization of subcutaneous ATTR fibril implants in mice upon treatment with mouse chimera NI-301.37F1. A: IBA1 staining on subcutaneous ATTR fibril implants 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] Figure 1 shows the 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 implants 6 and 96 hours after implantation and treatment with 5 mg / kg ch.NI-301.37F1 or isotype intravenously. Representative images from three different mice per treatment group and time point. Staining performed on five mice per group and two non-consecutive sections per animal. DETAILED DESCRIPTION OF THE INVENTION

[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 yet highly effective model for this purpose, which is advantageously not necessarily based on transgenic animals. The PDAX model is characterized by implants of amyloid transthyretin (ATTR) fibrils isolated from the tissues or organs of patients suffering from ATTR amyloidosis or ATTR-related diseases. Surprisingly, the amyloid fibril implants are maintained in the animals for several days and are amenable to drug treatment. In particular, as exemplified by mice implanted with ATTR fibrils in Examples 1 and 2, the amyloid fibril implants are tolerated in experimental animals and, after histological analysis, display characteristic amyloid features in tissue biopsies. Furthermore, as shown in Examples 3 and 4, the PDAX model has been validated with an anti-ATTR-specific antibody, which has been shown to actively promote ATTR fibril clearance in vivo in another mouse model. The PDAX model is therefore well suited to testing compounds or drugs for their suitability as anti-amyloid agents.

[0024] Previously, in a poster presentation at the XVIth International Symposium on Amyloidosis (March 26–29, 2018) in 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 can 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 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 great deal of variation in its phenotype and ATTR fibril composition; for example, there are different types of amyloid fibrils; type A consists of a C-terminal ATTR fragment and full-length TTR, while other types, type B, consist 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] Furthermore, it is known that in various types of systemic amyloidosis caused by amyloid fibril protein, the deposition of amyloid fibril protein in tissue and organ is co-localized and / or co-aggregated with one or more different amyloid fibril proteins.In fact, as known in the art and published in, for example, Higashi et al., Brain Research 1184 (2007) 284-294, τ- and α-synuclein and TAR-DNA binding protein 43 (TDP43) have been found to coexist in the brain pathology of Alzheimer's disease and Lewy body dementia; also see 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 (Aλ amyloidosis), Ig kappa light chain amyloidosis (Aκ amyloidosis), and Aβ2M amyloidosis; see Sakata et al., J. Histochem. Cytochem. 53 (2005), 237-242. Therefore, artificial in vitro generated TTR-V30M aggregates may not reflect the actual structure of ATTR fibrils in vivo, i.e., in patients with ATTR amyloidosis.

[0029] Additionally, the suitability of ADCP for in vivo therapeutic applications of anti-TTR antibodies is 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 have only been characterized in vitro to date; 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 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) that express 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 for orthotopic implantation of amylin (islet amyloid polypeptide (IAPP)) aggregates and amylin imaging probes, particularly 99m A mouse model of pancreatic islet amyloidosis has been described, designed to study the development of Tc-labeled pyridylbenzofuran derivatives. 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."

[0032] However, while the implantation method can, in principle, be used to generate a PDAX animal model according to the present invention, Yoshimura et al.'s islet amyloid model mouse may not be suitable for the intended purpose, even though it represents a suitable animal model for the purposes of the present invention. This is because, as with the in vivo model of ATTR by Higaki et al. (2018), it is not possible to predict that amylin aggregates prepared in vitro will (completely) reflect the in vivo structure of amylin. For example, an association between phosphorylated TDP43 (pTDP43) and IAPP was observed, which was particularly strong and significant in subjects with DM and high levels of IAPP in the pancreas; see Leino et al., Journal of Alzheimer's Disease 59 (2017), 43-56.

[0033] Therefore, one reason why most previous approaches to providing 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, does not take into account the actual environment and three-dimensional structure of amyloid fibrils in the affected tissue or organ, 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, kidney 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, 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 blotting. As described in Example 1 and shown in Figure 1, amyloid transthyretin (ATTR) fibrils can be effectively isolated from postmortem frozen cardiac tissue.

[0035] According to the present invention, amyloid fibrils are implanted subcutaneously or subcapsularly, and implanted in the kidney, peritoneum, muscle, brain, ventricle, nerve, eye, tongue, or heart. As shown in the examples, amyloid fibrils are conveniently injected subcutaneously into the thighs of mice, where they form deposits that are amenable to drug treatment and further analysis in skin biopsies. 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 the characteristic features of ATTR amyloid in human biopsies, i.e., they are Congo red positive and strongly stained by IHC with the human TTR antibody Dako A0002 (Fig. 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 raise and have a short generation time, i.e., large numbers of 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 strains or other animals always involves cost and considerable time. Therefore, it is highly advantageous to be able to use wild-type animals for the PDAX model according to the present invention. Therefore, in one embodiment of the present invention, the animals are non-transgenic at least for amyloid fibril proteins. Of course, where appropriate, transgenic animals can also be used, for example, to better reflect the situation in actual patients. For example, it is conceivable that amyloid fibrils can be implanted into immunodeficient mice to create a model of a patient suffering from a disease associated with immunodeficiency in addition to amyloidosis. This would allow the analysis of anti-amyloid drugs in a combined patient system for additional diseases, such as immunodeficiency or other diseases for which transgenic animal models are 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 an amyloid-related disease. The method comprises administering a drug or a variant thereof to a model of the present invention and determining amyloid fibrils in the model, wherein accelerated clearance or reduction of amyloid fibrils upon administration of the drug compared to a control indicates suitability for the anti-amyloid drug, and preferably, the accelerated clearance or reduction is observed in a dose-dependent manner. A variant of the drug, rather than the intended 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, which may tend to induce an immune response in animals. For example, as illustrated in the Examples, although the drug ultimately produced is a parent fully human antibody, a chimeric version of an otherwise fully human antibody may be used for testing.

[0039] Those skilled in the art are familiar with various methods for 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 controls, amyloid fibrils are then quantified using appropriate devices and processes, such as automated microscopy and image analysis. Furthermore, as mentioned in Examples 4 and 5, ATTR fibrils undergo a spontaneous fibril clearance process in the model. Therefore, the acceleration of fibril clearance or reduction by treatment with the drug being evaluated is an indicator of its suitability as an anti-amyloid drug. Therefore, 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 present 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 A1, US2017 / 0058023 A1, and US9,879,080 B2. Of course, it is contemplated within the scope of the present invention that particularly novel antibodies or compounds generally can be applied to the method of the present invention to evaluate 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 for the treatment of amyloidosis have involved screening of lead candidate compounds with recombinant amyloid proteins and their fibrils produced in vitro, which, however, does not take into account the actual environment and three-dimensional structure of amyloid fibrils in the affected 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., toxic amyloid deposits, at desired and necessary locations in the patient to be treated.

[0045] In certain preferred embodiments, the candidate antibody is a humanized, human-like, or human antibody, preferably a human-derived antibody, most preferably a human-derived antibody 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, and human constant domains, preferably of IgG1 or IgG4 subtype, but not necessarily identical to the constant domains 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 familiar with various routes for administering drugs to animal models.Therefore, in one embodiment of the method of the present invention, drugs are administered intravenously, intraperitoneally, subcutaneously, or orally.For example, drugs are intravenously injected into the tail vein of mice receiving implants 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 pharmaceutically 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 pharmaceutically 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 compositions of the present invention can be formulated according to methods well known in the art; for example, see Remington: The Science and Practice of Pharmacy (2000), University of Sciences in Philadelphia, ISBN 0-683-306472; Vaccine Protocols 2nd Edition, edited by Robinson et al., 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 compositions containing such carriers can be formulated according to well-known conventional methods. These pharmaceutical compositions can be administered to subjects at appropriate doses. Administration of suitable compositions can be achieved in a variety of ways. Examples include administering compositions containing a pharmaceutically acceptable carrier orally, intranasally, rectally, topically, intraperitoneally, intravenously, intramuscularly, subcutaneously, subdermally, transdermally, intrathecally, and intracranially. Aerosol formulations, such as nasal spray formulations, contain purified aqueous or other solutions of the active substance along with preservatives and isotonicity agents. Such formulations are preferably adjusted to a pH and isotonicity compatible with the nasal mucosa. Pharmaceutical compositions for oral administration, such as single-domain antibody molecules (e.g., "Nanobodies™"), are also contemplated in the present invention. Such oral formulations can be in 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 suppositories 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 the corresponding latest edition. 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 ATTR-related diseases, whereby amyloidosis is characterized by ATTR deposits in patients, particularly 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, the anti-amyloid drugs are characterized in dose-response studies, target association experiments (drug binding to the target), or characterized for their treatment mode of action, such as the local activation of a specific immune response by the drug at the fibril graft site, or PK / PD relationships, such as dose-response as a function of drug exposure levels. Information about drugs subjected to the above-described methods for determining and / or obtaining anti-amyloid drugs of the present invention is thereby transmitted to clients, contracting parties, or collaboration partners. Furthermore, drugs determined to be suitable anti-amyloid drugs can be selected, and optionally, the anti-amyloid drugs or pharmaceutical compositions containing the anti-amyloid drugs are used to treat amyloidosis or amyloid-related diseases.

[0052] The present invention further relates to the use of the 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 pharmaceuticals for the treatment of amyloidosis or amyloid-related diseases. Methods for using the models of the present invention to characterize drugs, for example, in dose-response studies or target association experiments (drug binding to targets), are well known to those skilled in the art. Furthermore, according to the present invention, the model is used to characterize the therapeutic mode of action of a drug, for example, the local activation of a specific immune response by the drug at the fibril graft site, or the PK / PD relationship, for example, the dose-response as a function of drug exposure level.

[0053] In this context, PDAX model can also be considered to be used in the context of personalized medicine, which means that the amyloid fibrils are isolated from individual patients and used the model of the present invention to analyze their response to anti-amyloid drugs individually.With regard to this use of the model of the present invention, advantageously, before treating patient and before exposing patient to possible adverse effects, it can determine whether treatment is effective in specific patient.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 for producing the patient-derived amyloid xenograft (PDAX) non-human animal model of the present invention, comprising isolating ATTR fibrils from a tissue biopsy obtained from a patient suffering from ATTR amyloidosis or an ATTR-related disease, and implanting the isolated amyloid fibrils in a non-human animal, preferably such that the fibril preparation has a total amyloid fibril protein concentration of about 0.5-5 mg / ml, preferably about 1-4 mg / ml, and most preferably about 2±0.5 mg / ml, as determined preferably 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 produced is depicted in Example 1. As described above, the tissue biopsy from which amyloid fibrils are isolated may be cardiac tissue, kidney 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 containing 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 fibrils are described in detail in Tennent, Methods Enzymol. 309 (1999), 26-47. As shown in Example 1, isolated amyloid fibrils are implanted by subcutaneous injection, although other implantation formats, such as subcapsular implantation, implantation in the kidney, peritoneum, muscle, brain, ventricle, nerve, eye, tongue, or heart, are also contemplated. [Example]

[0055] The following Examples 1-5 help 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 help illustrate embodiments of the present invention. The patient-derived amyloid xenograft (PDAX) mouse model presented in the following examples involves 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 purified water. This has been described to eliminate calcium-dependent binding of SAP protein to amyloid fibrils, allowing for 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 Western blotting with the 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 minutes at 20 V. The membranes were blocked for 1 hour 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 using 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 ATTR fibril extracts at 0.2 μg, 2.0 μg, and 20 μg per lane. The tissue extraction procedure resulted in a fraction highly enriched in 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 (ATTR+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 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 light chain variable domain (VL) of the human monoclonal antibody NI-301.37F1 are disclosed in Figure 1 of WO2015 / 092077 A1, with SEQ ID NOs: 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 such as those described in WO 2015 / 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 tissue containing the fibril implants was collected, fixed, embedded (PPFA), and sectioned for histological analysis with Congo red and IHC with the human TTR antibody Dako A0002, as well as for macrophage markers CD68, F4 / 80, and Iba1. Congo red staining was performed using a modified Putchler method. Briefly, tissue sections were stained with hemalum, destained in water, and 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 and mounted in 100% EtOH with and without 0.01% NaOH. 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 minutes at room temperature, incubation in blocking buffer (PBS + 5% horse / goat serum + 4% BSA) for 1 hour at room temperature, 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 using TTR antibody Dako A0002 at a 1:500 dilution in combination with biotin-conjugated goat anti-rabbit IgG antibody at a 1:400 dilution; CD68 antibody at a 1:400 dilution, F4 / 80 antibody at a 1:200 dilution, and IBA1 antibody at a 1:750 dilution, each combined with HRP-conjugated biotin donkey anti-rabbit IgG at a 1:400 dilution in PBS.

[0061] Amyloid fibril implants were easily recognizable from adjacent skin tissue by their shape and appearance, which were clearly distinct 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 known as integrin αM), indicating that these cells were undoubtedly neutrophils, which, like macrophages, are capable of antibody-mediated phagocytosis. Neutrophils are the most abundant immune cell type in the blood. Subcutaneous fibril implantation resulted in microlesions with local capillary rupture and the release of neutrophils that encapsulated, infiltrated, and completely eliminated the fibril implant over a period of 6–8 days. As shown in Example 4 below, this process is accelerated in the presence of the ATTR antibody NI-301.37F1, demonstrating 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 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 via subcutaneous injection in the thigh under brief gas anesthesia, followed by intravenous administration 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 via tail vein injection. 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 the antibody prepared in carbonate buffer. After a 2-hour incubation in the dark, the labeled antibody was purified by overnight dialysis in PBS buffer at 4°C.

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

[0064] Subcutaneous ATTR fibril implants were clearly recognizable from adjacent skin tissue due to their distinctive shape and appearance and displayed strong thioflavin S fluorescence, indicating the presence of fibrils with a preserved amyloid conformation (Figure 3A). Binding of the fluorescently labeled antibody NI-301.37F1 increased dose-dependently on fibrils but not on surrounding tissue (Figure 3A); fibril binding was selective for NI-301.37F1 and was not observed with isotype antibodies. NI-301.37F1 fluorescence overlapped with thioflavin S fluorescence, indicating NI-301.37F1 binding to ATTR fibrils. These results demonstrate 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 throughout 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 at the highest doses tested (5 mg / kg and 15 mg / kg).

[0066] Plasma levels of 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 room temperature. Plasma samples were diluted 1:500 in duplicate in blocking buffer. Calibration samples were similarly prepared by diluting chimeric NI-301.37F1 antibody in duplicate in blocking buffer over a concentration range from 5 pM to 5 nM. 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 across the dose range of 0.5 to 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 because 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 subcutaneously into wild-type mice, followed by intravenous administration of 5.0 mg / kg of the murine chimeric NI-301.37F1 variant (ch.NI-301.37F1) or the corresponding isotype antibody. After 6 or 96 hours, the 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 distinctive shape and appearance. Six hours after implantation and treatment administration, ATTR fibril implants were completely stained for TTR by IHC, with TTR staining accounting for 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 accounted for only 12% of the implant area in the ch.NI-301.37F1-treated group 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 antibodies.

[0071] To further characterize the activity of NI-301.37F1 in vivo, ATTR fibril-implanted 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 the isotype at 50 mg / kg. One mouse from each group was sacrificed 6 hours after drug administration (t0) for reference; 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 occupied 86% of the graft area at t0, but decreased to 58% after 4 days (96 h) (Figure 5), reflecting spontaneous fibril clearance in this model. Fibril clearance 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 clearance was achieved 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 ATTR fibril clearance in the PDAX model, as shown in Example 4, was further characterized by staining for the myeloid cell markers IBA1, DC11B, and LY6G.

[0075] IBA1, also known as allograft inflammatory factor 1 (AIF1), is a marker for macrophages and activated neutrophils. ATTR graft tissue was stained for IBA1 expression by IHC. Cells present within and around the ATTR graft tissue did not express any IBA1 6 hours after transplantation (Figure 6A). Similarly, 96 hours after transplantation, IBA1 expression was low or absent, detected only 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 not only by monocytes and macrophages but also by granulocytes and natural killer cells. ATTR graft tissue stained for CD11B expression by IHC revealed 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 cells displaying elongated processes. Ninety-six hours after transplantation, we observed a significant 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 primarily present on peripheral neutrophils. ATTR graft sections stained for LY6G expression revealed 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 subcutaneously or subcapsularly implanted into a tissue of the non-human animal, wherein the tissue is selected from the group consisting of skin, kidney, peritoneum, muscle, brain, nerve, eye, tongue, and heart; administering an anti-amyloid drug to said patient-derived amyloid xenograft non-human animal model; and determining that the anti-amyloid drug accelerates the clearance or reduction of amyloid fibrils compared to a control at the post-administration time point.

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, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

5. The method of claim 1, wherein the control is antibody NI-301.37F1.

6. The method of claim 2 , wherein the control is an isotype antibody.

7. 1. A method for predicting the efficacy of treatment with an anti-amyloid drug in a patient with 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 the patient into a tissue of a non-human animal, wherein the tissue of the non-human animal is selected from the group consisting of skin, kidney, peritoneum, muscle, brain, nerve, eye, tongue, and heart; (b) administering an anti-amyloid drug to said patient-derived amyloid xenograft non-human animal model; (c) determining the amount or area of ​​said 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 ​​the amyloid fibrils after administration of the anti-amyloid drug compared to a control 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 to 5 mg / mL.

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

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

11. The method according to any one of claims 7 to 9, wherein the anti-amyloid drug is an antibody.

12. The method described in claim 11, wherein the control is an isotype antibody.

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

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

15. 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 a non-human animal, wherein the tissue of the non-human animal is selected from the group consisting of skin, kidney, peritoneum, muscle, brain, nerve, eye, tongue, and heart; (b) determining the immune response of the non-human animal at a time point after administration of the antibody; (c) evaluating the immune response to the antibody by comparing the immune response of step (b) with a control.

16. The method according to any one of claims 1 to 9 and 15, wherein the non-human animal is a mouse, a rat, or a non-human primate.

17. 16. The method of claim 15, comprising detecting and / or measuring the expression level of one or more myeloid cell markers IBA1, CD11B, and / or LY6G.

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

19. The method described in claim 15, 17 or 18, wherein the control is an isotype antibody.

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