TREM-2 agonists for the treatment of Marfan syndrome

Abstract

Marfan syndrome is a condition caused by mutations in the FBN1 gene (15q21), which encodes the essential connective tissue protein fibrillin-1, and is responsible for significant morbidity and mortality. Apart from surgery, treatment options are limited. Therefore, it is essential to develop new pharmacological approaches to limit aortic dilatation and / or rupture. We have demonstrated the critical role of TREM-2 in the pathophysiology of ascending aortopathy associated with Marfan disease. Loss of TREM-2 actually exacerbates ascending aortic dilatation and rupture. Stimulating the TREM-2 receptor with peptides or agonist monoclonal antibodies represents a novel therapeutic approach for Marfan syndrome.

Description

[Technical Field]

[0001] Field of the invention: The present invention is in the field of medicine, particularly vascular disease.

[0002] Background of the invention: Marfan syndrome is caused by mutations in the FBN1 gene (15q21), which encodes the essential connective tissue protein fibrillin-1. The borderline form is associated with mutations in the TGFBR2 gene, located on chromosome 3, which encodes the TGF-beta receptor. Its prevalence is estimated at 1 / 5000, or 12,000 patients in France. Transmission is autosomal dominant. Therefore, the disease affects both men and women indiscriminately, and affected individuals have a 50% risk of transmitting the mutation. Symptoms can appear at any age and can vary greatly from person to person, even within the same family. Skeletal signs are often warning signs and may include arachnodactyly (excessively long limbs), tall stature, arachnodactyly, and joint hypermobility. Ophthalmologic damage includes axial myopia, which can lead to retinal detachment and ectopia (displacement or dislocation, a characteristic sign) of the lens. Skin signs (striae), risk of pneumothorax, and dural ectasia are also possible. More importantly, cardiovascular complications determine the prognosis of patients with Marfan syndrome, who have progressive dilatation of the ascending aorta with a high risk of potentially fatal aortic dissection. Mitral (prolapse) or bicuspid aortic valve abnormalities have also been described. Pregnancy increases the risk of complications and should therefore be carefully monitored (Keanemg, Pyeritz, Circulation, 2008). Although significant progress has been made in the management of Marfan patients (Pyeritz et al, Heart 2009), morbidity and mortality remain too high.

[0003] The only treatment currently recommended by experts is the beta-blocker propranolol, which limits the risk of aortic dilatation and dissection (Shores et al., New Engl J Med 1994; Ladouceur et al., Am J Cardiol 2007). This treatment is recommended upon confirmation of a diagnosis of Marfan syndrome or related syndromes in cases of aortic dilatation, or in patients aged 4 years or older who do not have aortic dilatation but who have a mutation. Medical treatment should continue even after cardiac surgery and during pregnancy. Treatment should not be discontinued after delivery (Omnes et al., Int J Gynaecol Obstet. 2013). In women with Marfan syndrome, pregnancy is associated with an excess risk of aortic dissection. Pregnancy is contraindicated if the aortic diameter exceeds 45 mm. Angiotensin 1 receptor blockers have been investigated, but their beneficial effects remain controversial. In animal models of Marfan disease, losartan, an AT1R antagonist, improves arterial wall architecture and reduces aortic dilatation (Hibashi et al., Science, 2006). However, the benefit of this molecule in humans is less clear. A randomized, double-blind, multicenter trial reported no benefit (Milleron et al., Eur Heart J), whereas another study (N=192) using similar methodology reported a benefit of losartan on the progression of aortic dilatation (Mullen et al., Lancet 2019). Surgical (±endovascular) treatment of the ascending aorta, either alone or with aortic valve manipulation, is considered if the ascending aorta is larger than 50 mm in diameter or if the increase in dilatation is rapid (more than 3 mm in 1 year when verified by two techniques) (European Society of Cardiology 2014 recommendations apply (Erbel et al, Eur Heart J 2014)). Surgical procedures are associated with significant perioperative complications, such as distal anastomotic leak or dissection.

[0004] In summary, Marfan syndrome is a condition that causes high morbidity and mortality. Treatment options other than surgery are limited. Therefore, it is essential to develop new pharmacological approaches to limit the dilatation and / or rupture of the ascending aorta.

[0005] The pathophysiological mechanisms underlying aortic dilatation and dissection in Marfan syndrome are not clearly understood. Marfan syndrome is the result of a quantitative or qualitative genetic deficiency in fibrillin-1. Fibrillin-1 is a glycoprotein abundant in the extracellular matrix and ensures tissue elasticity, a key mechanism for regulating biomechanical stresses associated with aortic ejection at the aortic root level (Dingemans et al., Anat Rec. 2000). Fibrillin binds to extracellular matrix proteins such as elastin and is involved in the organization of microfibrils. In the aortas of patients with Marfan syndrome, the structure of the aortic wall is disorganized due to disruption of elastic blades and a paucity of smooth muscle cells. Overexpression of genes encoding contractile proteins also alters the phenotype of myocytes (Crosas-Molist et al., ATVB 2015). The rate and activity of TGF-b is increased, and some recent studies suggest that this is a compensatory and unexplained mechanism in aortic disease (Mallat et al, Circ Res).

[0006] Only a few pathophysiological studies have evaluated the involvement of immune inflammatory responses in Marfan syndrome. However, several factors support a pathogenic role of immunity in disease progression and complications. First, M-CSF levels in the blood of Marfan patients are higher in patients with high aortic dilation rates. There is a significant increase in the infiltration of T cells and CD68+ macrophages in the aortic media and adventitia of Marfan patients compared with the aortas of control subjects (Radonic et al. PlosOne 2012) (D'amico, Int J Mol Sci. 2020; He, J ThoracCardiovasc Surg 2006). In a mouse model mimicking Marfan syndrome, there is also infiltration of inflammatory cells into the aortic wall with local overexpression of genes encoding chemokines (CCL-2, CCL-5), chemokine receptors (CX3CR1), and cytokines (IL-1b). However, the mechanisms regulating the recruitment and activation of these macrophages are unknown, as is their involvement in vascular disease.

[0007] TREMs, identified in 2000 as novel activating receptors of the immunoglobulin superfamily expressed on human myeloid cells, include inhibitory and activating isoforms encoded by a gene cluster linked to the major histocompatibility complex (MHC) (Bouchon, J Immunol 2000; Colonna, Nat Rev Immunol 2003). Currently, research has investigated several members of the TREM family of proteins, including TREM1 (also known as CD354), TREM-2, TREM3, TREM4, plasmacytoid dendritic cell (pDC)-TREM, TREM-like transcript (TLT-1), and TLT-2. Among them, TREM-2 is an immunoinhibitory receptor that has recently attracted the attention of oncologists. Research has shown that TREM-2 is expressed on several myeloid cell types, including DCs, monocytes, osteoclasts, Kupffer cells, alveolar macrophages, and microglia (Qi, Front Immunol 2021). To date, research has demonstrated that TREM-2 has several biological functions, including but not limited to regulating cell maturation, cell proliferation, cell survival, phagocytosis, and inflammation (Deczkowska Cell 2020). After TREM-2 ligand binds to TREM-2, TREM-2 interacts with the adaptor proteins DAP12 and DAP10. The main kinase recruited by the ITAM region of DAP12 is spleen tyrosine kinase (SYK), which activates downstream signaling molecules such as PI3K, Akt, mTOR, and MAPK, ultimately leading to cell activation, cell survival, and increased intracellular calcium levels (Mocsai, Nat Rev Immunol 2010). In addition to the above signaling pathways, TREM-2 also negatively regulates the toll-like receptor (TLR) signaling pathway, which plays an important role in the innate immune system by recognizing pathogen-associated molecular patterns (Kawasaki, Front Immunol 2014). Long et al. found that TREM-2 could attenuate neuroinflammation by downregulating the TLR signaling pathway (Long Neurochem Res 2019).Recently, Binder's group reported that Trem2 deletion induces increased production of chemokines and inflammatory cytokines in a model of NASH, exacerbating liver disease (Hendrikx, J Hepatol 2022). Recently, agonistic activity of anti-TREM-2 antibodies has been described (Price, BR, Sudduth, TL, Weekman, EM, Johnson, S., Hawthorne, D., Woolums, A., & Wilcock, DM (2020). Therapeutic Trem2 activation ameliorates amyloid-beta deposition and improves cognition in the 5XFAD model of amyloid deposition. Journal of Neuroinflammation, 17(1), 238; Schlepckow, K., Monroe, KM, Kleinberger, G., Cantuti-Castelvetri, L., Parhizkar, S., Xia, D., … Haass, C. (2020). Enhancing protective microglial activities with a dual function TREM-2 antibody to the stalk region. EMBO Molecular Medicine, 12(4), e11227; Wang, S., Mustafa, M., Yuede, CM, Salazar, SV, Kong, P., Long, H., … Colonna, M. (2020). Anti-human TREM-2 induces microglia proliferation and reduces pathology in an Alzheimer's disease model. Journal of Experimental Medicine, 217(9), e20200785.) The role of TREM-2 in Marfan syndrome and the interest of agonistic TREM-2 antibodies for the treatment of this disease have never been explored.

[0008] Summary of the Invention: The invention is defined by the claims. In particular, the invention relates to the use of TREM-2 agonists for the treatment of Marfan syndrome.

[0009] Detailed description of the invention: A first object of the present invention relates to a method for treating Marfan syndrome in a patient in need thereof, comprising administering a therapeutically effective amount of a TREM-2 agonist.

[0010] As used herein, the term "Marfan syndrome" has its common meaning in the art and refers to a systemic disease of connective tissue characterized by a variable combination of cardiovascular, musculoskeletal, ophthalmic, and pulmonary manifestations. Symptoms may appear at any age and vary widely between individuals, even within the same family. Cardiovascular involvement is characterized by 1) progressive dilatation of the aorta with an increased risk of aortic dissection, which impacts prognosis (aortic dilatation may result in leaky aortic valve); and 2) mitral regurgitation, which may be complicated by arrhythmias, endocarditis, or heart failure. Skeletal involvement is often the first sign of the disease and may include arachnoid limbs (excessively long limbs), large size, arachnodactyly, joint hypermobility, scoliotic deformity, protruding acetabulum, chest deformity (pectus carinatum or pectus excavatum), long head in the anteroposterior axis, micrognathia, or zygomatic hypoplasia. Ocular involvement results in axial myopia, which can lead to retinal detachment and ectopia of the lens (dislocation or dislocation is a characteristic sign). Ocular complications, particularly ectopia of the lens, can lead to blindness. Skin signs (striae), risk of pneumothorax, and dural ectasia can also occur. In the majority of cases, Marfan syndrome is caused by mutations in the FBN1 gene (15q21), which encodes fibrillin-1, a protein essential for connective tissue. A frontier form has been identified secondary to mutations in the TGFBR2 gene, located on chromosome 3, which encodes the TGF-beta receptor.

[0011] As used herein, the term "treatment" or "treating" refers to both preventative or prophylactic treatment and curative or disease-modifying treatment, including treatment of patients who are at risk of or suspected of being affected, as well as patients who are ill or have been diagnosed with a disease or medical condition, including the suppression of clinical recurrence. Treatment may be administered to a subject with a medical disorder or a subject who may ultimately acquire a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of disease treatment, e.g., a dosing pattern used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient early in the treatment regimen. The induction regimen may employ (in part or in whole) a "loading regimen," which may involve administering a drug at a dose greater than the dose the physician would employ during the maintenance regimen, administering the drug at a frequency greater than the frequency the physician would administer the drug during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used to maintain a patient while treating a disease, for example, to keep the patient in remission for an extended period of time (e.g., months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of a specific predetermined criterion (e.g., disease onset, etc.)).

[0012] In particular, the TREM-2 agonists of the present invention are particularly suitable for preventing rupture of the ascending aorta.

[0013] As used herein, "TREM-2" has its common meaning in the art and refers to triggering receptor 2 expressed on myeloid cells. TREM-2 is variously referred to as TREM-2, TREM-2a, TREM-2b, TREM-2c, triggering receptor-2a expressed on myeloid cells, and triggering receptor-2 expressed on monocytes. TREM-2 is a 230 amino acid membrane protein. TREM-2 is an immunoglobulin-like receptor expressed primarily on myeloid lineage cells, including, but not limited to, macrophages, dendritic cells, monocytes, dermal Langerhans cells, Kupffer cells, osteoclasts, and microglia. An exemplary amino acid sequence is represented by SEQ ID NO:1. The extracellular domain of TREM-2 spans from amino acid residue 19 to amino acid residue 174 of SEQ ID NO:1.

[0014] [ka]

[0015] As used herein, the term "TREM-2 agonist" refers to any compound, chemical, antibody, or peptide, natural or synthetic, that directly or indirectly increases one or more TREM-2 activities. In certain embodiments, a TREM-2 agonist directly binds to TREM-2 and increases one or more TREM-2 activities. The one or more TREM-2 activities are: (a) binding of TREM-2 to DAP12; (b) phosphorylation of DAP12; (c) activation of Syk kinase; (d) modulation of one or more pro-inflammatory mediators selected from the group consisting of IFN-β, IL-1α, IL-1β, TNF-α, IL-6, IL-8, CRP, CD86, MCP-1 / CCL2, CCL3, CCL4, CCL5, CCR2, CXCL-10, Gata3, members of the IL-20 family, IL-33, LIF, IFN-gamma, OSM, CNTF, CSF-1, OPN, CD11c, GM-CSF, IL-11, IL-12, IL-17, IL-18, and IL-23, wherein optionally the modulation is in the presence of a cytotoxic effect on macrophages, M1 macrophages, activated M1 macrophages, or IL-23. (e) recruitment of Syk to the DAP12 / TREM-2 complex; (f) increasing the activity of one or more TREM-2-dependent genes, optionally wherein the one or more TREM-2-dependent genes comprise nuclear factor of activated T cells (NFAT) transcription factor; (g) increasing the viability of dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, dermal Langerhans cells, Kupffer cells, microglia, M1 microglia, activated M1 microglia, and M2 microglia, or any combination thereof; and (h) increasing the viability of CD83, CD86 Modulated expression of one or more stimulatory molecules selected from the group consisting of MHC class II, CD40, and any combination thereof, wherein optionally CD40 is expressed on dendritic cells, monocytes, macrophages, or any combination thereof, and optionally the dendritic cells include bone marrow-derived dendritic cells.In some embodiments, an increase in one or more TREM-2 activities may be measured by any suitable in vitro cell-based assay or suitable in vivo model described herein or known in the art, for example, by using a luciferase-based reporter assay to measure TREM-2-dependent gene expression, by using Western blot analysis to measure increases in TREM-2-induced phosphorylation of downstream signaling partners such as Syk, or by using flow cytometry such as fluorescence-activated cell sorting (FACS) to measure changes in cell surface levels of markers of TREM-2 activation. Any in vitro cell-based assay or suitable in vivo model described herein or known in the art may be used to measure the interaction (e.g., binding) between TREM-2 and one or more TREM-2 ligands. One of skill in the art can readily determine whether a TREM-2 agonist enhances, increases, or activates one or more TREM-2 activities.

[0016] In some embodiments, the TREM-2 agonist is an agonist TREM-2 antibody.

[0017] As used herein, the term "antibody" has its common meaning in the art and refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule, so long as the antibody exhibits the desired biological activity. Antibodies may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain three (α, δ, γ) to five (μ, ε) domains: a variable domain (VH) and three to four constant domains (CH1, CH2, CH3, and CH4, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine antigen binding recognition and specificity. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-combining site and an antigenic determinant. The antibody-combining site is primarily composed of residues from the hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) may participate in the antibody combining site or influence the overall domain structure and hence the combining site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. The framework region (FR) refers to the amino acid sequence intervening between the CDRs. Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al., as set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues within the SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to shortening of, or insertion into, structural components of the basic variable domain structure, whether framework regions or complementarity-determining regions (CDRs). The exact Kabat numbering of residues can be determined for a given antibody by alignment of homologous residues in the sequence of that antibody with a "standard" sequence numbered as Kabat. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0018] As used herein, the terms "agonist TREM-2 antibody" and "activating TREM-2 antibody" refer to an antibody that induces (e.g., increases) one or more activities or functions of TREM-2 after the antibody binds to TREM-2. For example, an agonist TREM-2 antibody may have not only the precise epitope specificity compatible with receptor activation, but also the ability to induce or maintain receptor clustering on the cell surface. Moreover, agonist anti-TREM-2 antibodies of the present disclosure may exhibit the ability to bind to TREM-2 without blocking the simultaneous binding of one or more TREM-2 ligands. Anti-TREM-2 antibodies of the present disclosure may further exhibit additive and / or synergistic functional interactions with one or more TREM-2 ligands. In some embodiments, the increase in one or more TREM-2 activities induced by binding of one or more TREM-2 ligands to the TREM-2 protein is measured in primary cells, including but not limited to, dendritic cells, bone marrow-derived dendritic cells, monocytes, microglia, macrophages, neutrophils, NK cells, osteoclasts, dermal Langerhans cells, and Kupffer cells, or in cell lines, e.g., using an in vitro cell assay. In some embodiments, the anti-TREM-2 antibodies of the present disclosure have a human antibody isotype, such as IgG2, which, due to their unique structure, have the intrinsic ability to cluster receptors or retain receptors in a clustered arrangement, thereby activating receptors such as TREM-2 without binding to Fc receptors (e.g., White et al., (2015) Cancer Cell 27, 138-148).

[0019] In some embodiments, the agonist TREM-2 antibody binds to human TREM-2 at an epitope within the extracellular domain of human TREM-2. In some embodiments, the agonist TREM-2 antibody binds to human TREM-2 at an epitope within amino acids 19-174 of SEQ ID NO: 1. In some embodiments, the agonist TREM-2 antibody binds to human TREM-2 at an epitope within amino acids 23-128 of SEQ ID NO: 1, or an epitope within amino acids 131-148 of SEQ ID NO: 1.

[0020] In some embodiments, the agonist TREM-2 antibodies of the invention do not specifically bind to human TREM1.

[0021] Agonistic TREM-2 antibodies known in the art typically include those described in US10508148B2, US10676525B2, US11084875B2, US11124567B2, US11186636B2, WO2017062672, WO2018195506, WO2019055841, WO2019079529, WO2020055975, and WO2020121195.Other examples of agonist TREM-2 antibodies include Fassler, M., Rappaport, MS, Cuno, CB et al. Engagement of TREM-2 by a novel monoclonal induce antibodies activation of microglia and improves cognitive function in Alzheimer's disease models. J Neuroinflammation 18, 19 (2021); Okuzono Y, Sakuma H, Miyakawa S, Ifuku M, Lee J, Das D, Banerjee A, Zhao Y, Yamamoto K, Ando T, Sato S. Reduced TREM-2 activation in microglia of patients with Alzheimer's disease. FEBS Open Bio. 2021 Nov;11(11):3063-3080; and Ibach M, Mathews M, Linnartz-Gerlach B, Theil S, Kumar S, FeederleR, Bruestle O, Neumann H, Walter J. A reporter cell system for The triggering receptor expressed on myeloid cells 2 reveals differential effects of disease-associated variants on receptor signaling and activation by antibodies against the stalk region. Glia. 2021 May;69(5):1126-1139.

[0022] In some embodiments, the agonist TREM-2 antibodies of the invention comprise a light chain variable region having complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region having complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 2); CDRL2 comprises the amino acid sequence of GASTRAT (SEQ ID NO: 3); CDRL3 comprises the amino acid sequence of LQDNNFPPT (SEQ ID NO: 4); CDRH1 comprises the amino acid sequence of SWIG (SEQ ID NO: 5); CDRH2 comprises the amino acid sequence of IIYPGDADARYSPSFQG (SEQ ID NO: 6); and CDRH3 comprises the amino acid sequence of RRQGIFGDALDF (SEQ ID NO: 7).

[0023] In some embodiments, an agonist TREM-2 antibody of the invention comprises a light chain having the amino acid sequence of SEQ ID NO:8 and a heavy chain having the amino acid sequence of SEQ ID NO:9.

[0024] [ka]

[0025] In some embodiments, the agonist TREM-2 antibodies of the invention comprise a light chain variable region having complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region having complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 10); CDRL2 comprises the amino acid sequence of WASTRES (SEQ ID NO: 11); CDRL3 comprises the amino acid sequence of QQYYNYPFT (SEQ ID NO: 12); CDRH1 comprises the amino acid sequence of DYNIH (SEQ ID NO: 13); CDRH2 comprises the amino acid sequence of YIYPKNGGTGYTQKFK (SEQ ID NO: 14); and CDRH3 comprises the amino acid sequence of RTARASWFAF (SEQ ID NO: 15).

[0026] In some embodiments, the agonist TREM-2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the agonist TREM-2 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the agonist TREM-2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 16 and a VL comprising the amino acid sequence of SEQ ID NO: 17.

[0027] [ka]

[0028] In some embodiments, the TREM-2 agonist antibody of the invention comprises a VH and a VL, wherein the VH comprises the same amino acid sequence as the VH of the antibody produced by the CGX-c hybridoma deposited with the ATCC® on November 14, 2018 under accession number PTA-125491.

[0029] As used herein, the term "therapeutically effective amount" refers to an amount of a TREM-2 agonist sufficient to treat Marfan syndrome in a subject. However, it will be understood that the total daily dosage of the drug will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the particular composition employed; the subject's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the particular compound employed; the duration of treatment; drugs used in combination with or concomitantly with a particular drug; and similar factors well known in the medical arts. For example, it is well within the skill of one of ordinary skill in the art to initiate doses of the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the drug can vary over a wide range, from 0.01 to 1,000 mg / day per adult. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the drug, with dosages adjusted symptomatically to suit the subject being treated. Medicaments typically contain from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg of body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0030] Typically, the TREM-2 agonists of the present invention are combined with pharmaceutically acceptable excipients and, optionally, sustained-release matrices such as biodegradable polymers to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to mammals, particularly humans, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation adjuvant. Typically, pharmaceutical compositions contain a pharmaceutically acceptable vehicle for injectable formulations. These can be in specific isotonic sterile saline solutions (such as monosodium phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts) or in dried, particularly lyophilized, compositions that allow the constitution of an injectable solution upon addition of sterile water or saline, as the case may be. Pharmaceutical preparations suitable for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the required amount of active ingredient in an appropriate solvent, along with some of the other ingredients mentioned above, as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients and the required other ingredients from those mentioned above into a sterile vehicle containing a basic dispersion medium. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from its previously sterilized, filtered solution.

[0031] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0032] [Figure 1] A. qPCR quantification of Trem-2 mRNA in the ascending aorta of 10-week-old (right, N=7-8 / group) control (Fibrillin WT) or Marfan (Fibrillin mgR) mice. ***, P<0.001. B. qPCR quantification of Trem2 mRNA in the spleen of 8-week-old (right, N=8-12 / group) control (Fibrillin Wt / Wt) or MFS (Fibrillin mgR / mGr) mice. [Figure 2] qPCR quantification of Trem-2 mRNA in the ascending and abdominal aorta of 10-week-old Marfan mice (fibrillin-mgR) (right, N=7–8 / group). *, P<0.05. [Figure 3] Representative images of ascending aortic sections from fibrillin WT / WT mice, fibrillin mgR / mgR Trem-2+ / + mice, and fibrillin mgR / mgR Trem-2- / - mice, and quantification of ascending aortic diameter at 10 weeks of age. *, P<0.05; **, P<0.01. [Figure 4] Survival curves for fibrillin-mgR / mgR / Trem-2+ / + mice (N=20) and fibrillin-mgR / mgR / Trem-2- / - mice (N=10–14 / group). [Figure 5]A. Quantification of collagen content in the ascending aorta of fibrillin-mgR / mgRTrem2+ / + and fibrillin-mgR / mgRTrem2- / - mice (N=7 / group). Bar scale: 50 μm. B. Quantification of ascending aortic sections from 10-week-old fibrillin-mgR / mgRTrem2+ / + and fibrillin-mgR / mgRTrem2- / - mice. Cartography of MMP-2, MMP-3, MMP-9, and MMP-13 activity in the aorta of 10-week-old fibrillin-mgR / mgRTrem2+ / + and fibrillin-mgR / mgRTrem2- / - mice. MMP activity was measured using MMPsense 680 (NEV 10126, PerkinElmer), and images were acquired using a fluorescence molecular imaging system (FMT 2500™, VisEnMedical) (N=5). Bar scale: 50 μm. **, P<0.01; ***, P<0.001. [Figure 6] Five-week-old fibrillin-mgR / mgR mice were orally treated with either PBS or Ki20227, a CSF1-receptor inhibitor, for 15 weeks in a pulse protocol (1 week of KI20227 treatment alternating with 1 week of PBS). A. Quantification of CD68+ macrophage content in the ascending aorta. Bar scale: 50 μm. B. Monitoring of ascending aortic diameter using ultrasonography. C. Monitoring of survival rate. [Figure 7] A. Quantification of CD68+ macrophages in ascending aortic sections from fibrillin-mgR / mgRTrem2+ / + and fibrillin-mgR / mgRTrem2- / - mice (N=6 / group, nonparametric test). Il6 and Il1b mRNA levels in the ascending aorta from 8-week-old fibrillin-mgR / mgRTrem2+ / + and fibrillin-mgR / mgRTrem2- / - mice (N=6 / group). *, P<0.05.

[0033] Working Example: TREM-2 is expressed in the aortic wall of Marfan mice. First, we analyzed Trem2 gene expression in the whole aorta of fibrillin-1-deficient mice, designated fibrillin-mgR / mgR mice, by qPCR. This is a haploinsufficient mouse model that mimics Marfan syndrome, and we compared it with littermate control mice (fibrillin WT). We showed that Trem-2 mRNA levels were fivefold higher in the aorta of fibrillin-mGr / mGr mice compared with 10-week-old control mice (Figure 1A). Upregulation of Trem2 transcripts was observed in the aorta but not in other organs, such as the spleen, supporting its specificity to the vascular phenotype (Figure 1B). Single-cell RNA sequencing (scRNA-seq) analysis demonstrated higher total and Trem-2+ macrophage content in the aorta of Marfan mice compared with control mice, with TREM2 expression in the aortic wall largely restricted to the macrophage population (data not shown). We also used scRNA-seq to find that TREM-2 was specifically expressed by macrophages in human aortic tissue from patients with MFS (data not shown).

[0034] TREM-2 expression is higher in the ascending aorta Next, we compared Trem-2 mRNA levels in two different aortic regions (the ascending aorta and abdominal aorta, where aneurysms occur) in fibrillin-mgR / mgR mice by qPCR. Interestingly, Trem2 transcript levels were twofold higher in the ascending aorta (Fig. 2).

[0035] 2.3. TREM-2 deficiency exacerbates ascending aortic dilation in a mouse model of Marfan syndrome. To investigate the role of TREM-2 in the aortic pathology of Marfan syndrome, we transformed fibrillin mgR / mgR mice (Pereira et al. PNAS 1999) into Trem-2-deficient mice (Trem-2 - / - ) (Seno, PNAS 2009) and fibrillinmgR / mgR Mouse Trem-2 + / + and fibrillin mgR / mgR Mouse Trem-2 - / - We demonstrated that Trem2 deficiency significantly exacerbated MFS aortic disease by increasing ascending aortic dilation by threefold (n = 7-8 / group, Figure 3).

[0036] TREM-2 deficiency exacerbates lethal aortic rupture in a mouse model of Marfan syndrome. To study the role of TREM-2 in the complications of aortic pathology in Marfan syndrome, animals (male and female) were followed and survival was recorded. As shown in Figure 4, we found that Trem-2 deficiency exacerbated aortic aneurysm rupture and higher premature mortality in both males and females (n = 10-14 / group, Figure 4), which was accompanied by exacerbation of non-cardiovascular abnormalities associated with MFS, including skeletal defects and rectal prolapse (observation and CT scan).

[0037] Adverse remodeling of the extracellular matrix Several studies have reported alterations in the extracellular matrix in the MFS aortic wall, leading to dilatation and rupture. Here, we showed that Trem2 deficiency exacerbated adverse aortic wall remodeling, accompanied by a decrease in local collagen content (Fig. 5A), which was associated with significantly increased local MMP-2, MMP-3, MMP-9, and MMP-13 activity (Fig. 5B).

[0038] Local immune response To assess the contribution of aortic resident macrophages to disease severity, fibrillin mgR / mgRMice were treated with Ki20227, an inhibitor of macrophage colony-stimulating factor 1 (CSF1) receptor tyrosine kinase. Pulse Ki20227 treatment depleted tissue macrophages (Figure 6A) and limited both aortic dilation and rupture, supporting the pathogenic role of local macrophages in adverse vascular remodeling in MFS mice (Figures 6B-C). We found that Trem2 gene deletion was associated with a massive increase in macrophage content in both the media and adventitia (Figure 7A), which was associated with a bias of the local inflammatory response toward a pro-inflammatory phenotype (Figure 7B).

[0039] conclusion For the first time, we have demonstrated a critical role for TREM-2 in the pathophysiology of ascending aortopathy associated with Marfan disease. Loss of TREM-2 exacerbates ascending aortic dilation and rupture. Stimulating the TREM-2 receptor with peptides or agonist monoclonal antibodies represents a novel therapeutic approach for Marfan syndrome.

[0040] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated herein by reference into the present disclosure.

Claims

1. A method of treating Marfan syndrome in a patient in need thereof comprising administering a therapeutically effective amount of a TREM-2 agonist.

2. 10. The method of claim 1, wherein the TREM-2 agonist is suitable for preventing ascending aortic rupture.

3. 3. The method of claim 1 or 2, wherein the TREM-2 agonist is an agonist TREM-2 antibody.

4. The method of claim 3, wherein the agonist TREM-2 antibody binds to human TREM-2 at an epitope within amino acids 19-174 of SEQ ID NO:

1.

5. The method of claim 3, wherein the agonist TREM-2 antibody binds to human TREM-2 at an epitope within amino acids 23-128 of SEQ ID NO:1, or to an epitope within amino acids 131-148 of SEQ ID NO:

1.

6. The method of claim 3, wherein the agonist TREM-2 antibody comprises a light chain variable region having complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region having complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 2); CDRL2 comprises the amino acid sequence of GASTRAT (SEQ ID NO: 3); CDRL3 comprises the amino acid sequence of LQDNNFPPT (SEQ ID NO: 4); CDRH1 comprises the amino acid sequence of SWIG (SEQ ID NO: 5); CDRH2 comprises the amino acid sequence of IIYPGDADARYSPSFQG (SEQ ID NO: 6); and CDRH3 comprises the amino acid sequence of RRQGIFGDALDF (SEQ ID NO: 7).

7. The method of claim 6, wherein the TREM-2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:8 and a heavy chain having the amino acid sequence of SEQ ID NO:

9.

8. The method of claim 3, wherein the agonist TREM-2 antibody comprises a light chain variable region having complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region having complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 10); CDRL2 comprises the amino acid sequence of WASTRES (SEQ ID NO: 11); CDRL3 comprises the amino acid sequence of QQYYNYPFT (SEQ ID NO: 12); CDRH1 comprises the amino acid sequence of DYNIH (SEQ ID NO: 13); CDRH2 comprises the amino acid sequence of YIYPKNGGTGYTQKFK (SEQ ID NO: 14); and CDRH3 comprises the amino acid sequence of RTARASWFAF (SEQ ID NO: 15).

9. The method of claim 8 , wherein the agonist TREM-2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:16 and / or a VL comprising the amino acid sequence of SEQ ID NO:

17.

10. 4. The method of claim 3, wherein the TREM-2 agonist antibody comprises a VH and a VL, and the VH comprises the same amino acid sequence as the VH of the antibody produced by the CGX-c hybridoma deposited with the ATCC (registered trademark) on November 14, 2018 under accession number PTA-125491.

Images