New compounds for treating obesity

Abstract

The present invention relates to constructs comprising compounds targeting areas involved in weight regulation in the brain and allosteric ligands for receptors located in the blood-brain barrier (BBB). The present invention also relates to compositions and uses of such constructs, such as for use in preventing or treating overweight and obesity. Preferred compounds for regulating body weight include GLP‑1 receptor agonists (GLP‑1RA), while preferred receptors located in the BBB include transferrin receptors (TfR). Exemplary fusions and conjugates of GLP‑1RA and anti-TfR‑Fab show increased binding to brain regions expressing GLP‑1 receptors, particularly in brain regions protected by the BBB, compared to fusions or conjugates with inactive control Fabs. In vivo mouse studies have confirmed that active constructs lead to enhanced reductions in food intake and weight loss compared to inactive constructs.

Description

Technical Field

[0001] The present invention relates to novel compounds useful in the treatment of obesity and related conditions.

[0002] Incorporation by Reference of Sequence Listing

[0003] The sequence listing entitled "SEQUENCE LISTING" is 9676 bytes, was created on May 23, 2018, and is incorporated herein by reference. Background Art

[0004] Obesity is the result of a chronic positive energy balance in which energy intake exceeds expenditure. The central nervous system (CNS) plays an important role in maintaining body weight within a narrow range by regulating energy intake. To regulate energy intake, both neuronal and humoral signals from peripheral organs involved in food intake convey information about hunger and / or satiety to the brain. Gut-derived hormones, such as dietary secreted glucagon-like peptide 1 (GLP-1), have been identified as playing a role in feeding regulation by conveying meal-related information about nutritional status to the brain (Journal of Endocrinology (2014), vol. 221(1), p. T1-T16).

[0005] The presence of GLP-1 receptors in the CNS and findings in animal and human studies suggest that the satiety and weight effects induced by GLP-1 receptor agonists (GLP-1RAs) are at least partially mediated by their actions on the brain; however, brain penetration of GLP-1 and GLP-1RAs is severely limited by the essentially impenetrable blood-brain barrier (BBB) ​​(Journal of Clinical Investigation (2014), vol. 124(10), p. 4473-4488).

[0006] One approach to facilitate the delivery of protein therapeutics across the BBB is to exploit receptor-mediated transcytosis, an endogenous endocytic process in which ligands are transported across the endothelial cell barrier. One of the many strategies to overcome this obstacle is to exploit the transcytotic trafficking pathway of endogenous receptors such as transferrin receptor (TfR) expressed at the brain capillary endothelium (Science Translational Medicine (2011), vol. 3 (84), p. 1-8).

[0007] WO 2004 / 019872 A2 relates to pharmaceutical compositions comprising a transferrin (Tf) protein exhibiting reduced glycosylation fused to at least one therapeutic protein or peptide, and methods of use thereof. Prophetic Example 1 is entitled "GLP-1 -Transferrin Fusion Protein".

[0008] Byung-Joon Kim et al. disclosed in the article "Transferrin Fusion Technology: A Novel Approach to Prolonging Biological Half-Life of Insulinotropic Peptides" in Journal of Pharmacology and Experimental Therapeutics, Vol. 334 No. 3 (2010), p. 682-691 that transferrin fusion proteins were prepared using analogs of GLP-1 and exendin-4. These fusions are called GLP-1-Tf and Ex-4-Tf, respectively, and are made with non-glycosylated forms of human transferrin. According to the abstract, GLP-1-Tf activates the GLP-1 receptor, is resistant to inactivation by peptidases, and has a half-life of about 2 days. The abstract also clearly shows that these fusion proteins do not cross the blood-brain barrier, while, for example, GLP-1-Tf retains the acute glucose-dependent insulin secretion properties of native GLP-1 in diabetic animals and has a profound effect on the proliferation of pancreatic beta cells.

[0009] US 2010 / 0077498 A1 relates to compositions and methods for blood-brain barrier delivery in mice. Summary of the invention

[0010] The present invention relates to novel compounds and their use in the treatment of obesity and related conditions.

[0011] In one aspect, the invention relates to a construct comprising a compound targeting an area of ​​the brain involved in body weight regulation and an allosteric ligand for a receptor located in the blood-brain barrier (BBB), and pharmaceutically acceptable salts, amides or esters thereof.

[0012] In other aspects, the invention relates to compositions comprising such constructs and at least one pharmaceutically acceptable excipient, said constructs being generally useful as medicaments, and in particular for the prevention or treatment of obesity and related conditions.

[0013] In one aspect, the constructs of the invention exhibit improved binding to brain regions involved in body weight regulation. The binding is particularly improved in regions protected by the BBB.

[0014] In one aspect, the compound targeting an area of ​​the brain involved in body weight regulation is a GLP-1 receptor agonist (GLP-1RA).

[0015] Thus, in one aspect, the invention is used to increase the accessibility of a systemically administered GLP-1RA to appetite regulatory regions in the brain.

[0016] Additionally, or alternatively, the present invention is used to target additional GLP-1 receptor (GLP-1R) populations in the brain, such as those protected by the BBB.

[0017] In one aspect, these effects on appetite and body weight are achieved while maintaining glucose homeostasis at levels comparable to systemic treatment with classical GLP-1 therapies (eg, with GLP-1 derivatives such as liraglutide and semaglutide).

[0018] Additionally, or alternatively, the constructs of the invention enhance the binding of systemically administered GLP-1RA to brain regions expressing GLP-1R. This can be demonstrated, for example, by the studies described in Example 16 (see Fig.16A ).

[0019] Additionally, or alternatively, the constructs of the invention enhance the binding of systemically administered GLP-1RA to brain regions expressing GLP-1R that are protected by the BBB. This can be demonstrated, for example, by the studies described in Example 16 (see Figure 16C).

[0020] Additionally, or alternatively, in one aspect, the construct has the effect of reducing food intake. For example, this can be demonstrated by the studies described in part I of Example 20.

[0021] Additionally, or alternatively, in one aspect, the construct has the effect of causing weight loss. For example, this can be demonstrated by the studies described in part III of Example 20.

[0022] In one aspect, the receptor located in the BBB is the transferrin receptor (TfR).

[0023] TfR is highly expressed in the BBB endothelium and is a carrier protein of transferrin. Transferrin is an iron-binding plasma glycoprotein that controls the level of free iron (Fe) in biological fluids. Transferrin is required for the import of iron into cells and is regulated by intracellular iron concentration. It imports iron by internalizing transferrin-iron complexes via receptor-mediated endocytosis. Therefore, complete transcytosis via this receptor pathway seems unlikely, which is one of the challenges to be overcome using TfR as a delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] A better understanding of the features of the present invention will be obtained by referring to the following detailed description of the invention and the accompanying drawings, in which:

[0025] Figure 1A The structure of the anti-TfR-Fab prepared as described in Example 1 is shown,

[0026] Figure 1B The method used in Example 19 is shown. Figure 1A The structure of the fluorescently labeled form of the Fab,

[0027] Figure 2 The structure of a control Fab that does not bind to TfR, prepared as described in Example 2, is shown.

[0028] Figure 3 The structure of the anti-TfR-Fab prepared as described in Example 3 is shown,

[0029] Figure 4 The structure of a control Fab that does not bind to TfR, prepared as described in Example 4, is shown.

[0030] Figure 5 The structure of the fusion protein of the control Fab and the GLP-1 analog prepared as described in Example 5 is shown.

[0031] Figure 6 shows the structure of the GLP-1 anti-TfR-Fab fusion protein prepared as described in Example 6,

[0032] Figure 7 The structure of the GLP-1 control Fab fusion protein prepared as described in Example 7 is shown.

[0033] Figure 8 shows the structure of the fluorescently labeled GLP-1 anti-TfR-Fab fusion protein prepared as described in Example 8,

[0034] Fig. 9 shows the structure of the fluorescently labeled GLP-1 control Fab fusion protein prepared as described in Example 9,

[0035] Fig.10 shows the structure of the GLP-1 analog prepared as described in Example 10,

[0036] Fig.11 shows the structure of the GLP-1 anti-TfR-Fab conjugate prepared as described in Example 11,

[0037] Fig.12 Shown is the structure of the GLP-1 control Fab conjugate prepared as described in Example 12,

[0038] Fig.13 shows the structure of the derivative of the GLP-1 analogue prepared as described in Example 13,

[0039] Fig.14 Shown is the structure of the GLP-1 control Fab conjugate prepared as described in Example 14,

[0040] Fig.15 shows the structure of the GLP-1 anti-TfR-Fab conjugate prepared as described in Example 15,

[0041] Fig.16A The in vivo imaging results of fluorescently labeled GLP-1Fab fusion protein after acute administration to mice are shown, more specifically the total intensity of the fluorescent signal after quantification in all brain regions expressing GLP-1R for an "active" construct that binds to TfR and an "inactive" control construct that does not bind to TfR,

[0042] FIG16B shows the in vivo imaging results of fluorescently labeled GLP-1 Fab fusion proteins after acute administration to mice, more specifically the total intensity of the fluorescent signal after quantification in a periventricular apparatus expressing GLP-1R but lacking the BBB for an "active" construct that binds to TfR and an "inactive" control construct that does not bind to TfR,

[0043] FIG16C shows the in vivo imaging results of fluorescently labeled GLP-1 Fab fusion proteins after acute administration to mice, more specifically the total intensity of the fluorescent signal after quantification in an exemplary brain structure protected by the BBB for an "active" construct that binds to TfR and an "inactive" control construct that does not bind to TfR,

[0044] Figure 16D shows the in vivo imaging results of fluorescently labeled GLP-1Fab fusion protein after acute administration to mice, more specifically, the total intensity of the fluorescent signal after quantification in the brain area that does not express GLP-1R for the "active" construct that binds to TfR and the "inactive" control construct that does not bind to TfR,

[0045] Figure 17A shows the results of an acute in vivo study in lean mice, more specifically the cumulative food intake (FI) in g for up to 24 hours after administration of an "active" GLP-1 anti-TfR-Fab conjugate,

[0046] Figure 17B shows the results of an acute in vivo study in lean mice, more specifically the plasma exposure levels in pM measured up to 24 h after administration of the "active" GLP-1 anti-TfR-Fab conjugate,

[0047] Figure 18A shows the results of an acute in vivo study in lean mice, more specifically the cumulative food intake (FI) in g during 24 hours after administration of an "active" GLP-1 anti-TfR-Fab conjugate or an "inactive" GLP-1 control Fab conjugate,

[0048] Figure 18B shows the results of an acute in vivo study in lean mice, more specifically the plasma exposure levels in pM measured up to 24 h after administration of an "active" GLP-1 anti-TfR-Fab conjugate or an "inactive" GLP-1 control Fab conjugate,

[0049] Figure 19A shows the results of a subchronic in vivo study in diet-induced obese (DIO) mice, more specifically the body weight loss in % measured over a 19-day treatment period with once-daily administration of an "active" GLP-1 anti-TfR-Fab conjugate or an "inactive" control Fab conjugate (for more details, see Part III of Example 20), and

[0050] Figure 19B shows the results of a subchronic in vivo study in diet-induced obese (DIO) mice, more specifically plasma exposure in pM 2 hours after dosing on the last day of a 19-day treatment period with once-daily administration of an "active" GLP-1 anti-TfR-Fab conjugate or an "inactive" control Fab conjugate. DETAILED DESCRIPTION

[0051] In the following, Greek letters may be represented by their symbols or corresponding written names, for example: α = alpha; β = beta; ε = epsilon; γ = gamma; δ = delta; ω = omega, etc. In addition, the Greek letter μ can also be represented by "u", for example μl = ul or μM = uM.

[0052] In the following, chemical terms are conventional in the art, for example the name -C(O)- refers to a divalent carbonyl group:

[0053] Chemical formula 7:

[0054]

[0055] And, for example, (COOH) used in the name -CH(COOH)- refers to a monovalent carboxylic acid group of Chemical Formula 8:

[0056]

[0057] It is attached to the carbon atom shown on the left (CH).

[0058] When used herein, the word "a" generally means "one or more." For example, a construct of the invention, which is defined as comprising "a" compound that targets an area of ​​the brain involved in weight regulation and "an" allosteric ligand for a receptor located in the blood-brain barrier (BBB), may incorporate one or more of such compounds and ligands.

[0059] Unless otherwise specified in the specification, terms presented in the singular form generally also include the plural case.

[0060] The present invention also relates to constructs, compounds, ligands, GLP-1RA, GLP-1 analogs, methods of preparation, compositions and uses as disclosed herein, wherein open terms such as "comprising" and "including" can be replaced with closed terms such as "consisting of..."

[0061] The present invention relates to novel compounds and their use in the treatment of obesity and related conditions. The compounds of the present invention show improved binding to brain regions involved in weight regulation. The binding is particularly improved in areas protected by the blood-brain barrier (BBB).

[0062] In one aspect, the invention relates to a construct comprising a compound that targets an area of ​​the brain involved in body weight regulation and an allosteric ligand for a receptor located in the BBB, and pharmaceutically acceptable salts, amides or esters thereof.

[0063] Constructs

[0064] The term "construct" - comprising a compound targeting an area of ​​the brain involved in body weight regulation and an allosteric ligand directed against a receptor located in the BBB - refers to a chemical entity (molecule) in which the compound and the ligand are covalently linked, optionally via a linker.

[0065] In one embodiment, the construct consists of a compound targeting an area of ​​the brain involved in body weight regulation, an allosteric ligand for a receptor located in the BBB, and an optional linker.

[0066] In one embodiment, the compound and the ligand are directly attached to each other (no linker is present).

[0067] In another embodiment, the compound and the ligand are linked to each other via a linker.

[0068] Fusion Protein

[0069] In one embodiment, the construct is a fusion protein.

[0070] A fusion protein is a compound that can be produced by a process that includes the step of recombinantly expressing the entire fusion protein in a host cell.

[0071] In one embodiment, the amino acid sequence of the fusion protein consists only of the encoded amino acids.

[0072] In one embodiment, the protein obtained by the recombinant expression step is the same as the desired fusion protein, in which case the fusion protein is or can be produced by a "completely recombinant" process. In this case, the fusion protein does not require further chemical modification, and the expressed fusion protein can be isolated and purified to form the final desired product.

[0073] A non-limiting example of a fusion protein is the fusion protein of Example 6 ( Figure 6 ), wherein the GLP-1 analog is linked to the V of the anti-TfR-Fab via a peptide linker (4x(GlyGlyGlySer)). L The N-terminal fusion of the domain. Each amino acid sequence of each of the three parts consists entirely of the encoded amino acids.

[0074] Conjugate

[0075] In one embodiment, the construct is a conjugate.

[0076] In one embodiment, the "conjugate" is not a fusion protein.

[0077] In one embodiment, the covalent bond between the compound targeted to an area of ​​the brain involved in body weight regulation, the allosteric ligand, and the optional linker can be formed by any chemical process, except a completely recombinant process.

[0078] In one embodiment, the structural formula of the conjugate includes at least one non-coded amino acid. A non-limiting example of a non-coded amino acid is the amino acid Aib (see, e.g., the conjugate of Example 11 ( Fig.11 )).

[0079] Additionally, or alternatively, the structural formula of the conjugate includes at least one element that is not an amino acid. A non-limiting example of such an element is a chemical linker, such as Formula 1: -CH2-C(O)- (see, e.g., the conjugate of Example 11 ( Fig.11 ), wherein the linker of Formula 1 links the epsilon amino group of Lys 485 to the thiol group of Cys 377. Another non-limiting example of such an element is the chemical linker of Formula 2: -CH2-C(O)-Ado-gGlu-Ado (see, e.g., the conjugate of Example 15, wherein the linker of Formula 2 links the epsilon amino group of Lys 438 to the thiol group of Cys 377.

[0080] In some embodiments, the conjugate is or can be produced by a partially recombinant process (e.g., the protein portion of the desired conjugate is or can be produced in a recombinant expression step, while the remainder of the desired conjugate is or can be added in one or more separate process steps, after the recombinant expression step, in one or more chemical reaction steps outside of the host cell. Such a production process may be referred to as a "semi-recombinant" process.

[0081] Brain regions involved in weight regulation

[0082] Bloemendaal et al., “Effects of glucagon-like peptide 1 on appetite and body weight: focus on the CNS”, Thematic Review, Journal of Endocrinology (2014), 221, T1-T16 (see, e.g., Figure 1 on page T8), provide non-limiting examples of brain regions involved in body weight regulation.

[0083] In some embodiments, regions of the brain involved in weight regulation include, but are not limited to, the lateral septal nucleus (LS), the dentate gyrus (DG), the medial habenular nucleus (MH), and / or the parabrachial nucleus (PB) (see FIG. 16C and Example 16).

[0084] In some embodiments, regions of the brain involved in weight regulation include, but are not limited to, the nucleus accumbens (ACB), the dorsomedial hypothalamic nucleus (DMH), the lateral hypothalamic area (LHA), and / or the lateral habenular nucleus (LH).

[0085] Weight Regulation

[0086] In one embodiment, regulation of body weight refers to weight loss.

[0087] In one embodiment, weight loss may be due to one or more of: decreased food intake, decreased calorie intake, decreased appetite, increased energy expenditure, and / or changes in food preferences.

[0088] Thus, in some embodiments, the compound portion of the construct of the invention targets areas of the brain involved in: a) decreased food intake, b) decreased calorie intake, c) decreased appetite, d) increased energy expenditure, and / or e) altered food preferences.

[0089] In some embodiments, the present invention relates to methods of weight management. In some embodiments, the present invention relates to methods of reducing appetite. In some embodiments, the present invention relates to methods of reducing food intake.

[0090] Generally, all subjects suffering from obesity are also considered to be overweight. In some embodiments, the present invention relates to methods for treating or preventing obesity. In some embodiments, the present invention relates to the use of the constructs of the present invention in treating or preventing obesity. In some embodiments, the subject suffering from obesity is a human, such as an adult or a pediatric patient (including infants, children and adolescents). Body mass index (BMI) is a measure of body fat based on height and weight. The calculation formula is BMI=weight in kilograms / height in meters 2 A human subject suffering from obesity may have a BMI of ≥30; the subject may also be referred to as obese. In some embodiments, a human subject suffering from obesity may have a BMI of ≥35 or a BMI in the range of ≥30 to <40. In some embodiments, the obesity is severe obesity or morbid obesity, wherein the human subject may have a BMI of ≥40.

[0091] In some embodiments, the present invention relates to a method for treating or preventing overweight, optionally in the presence of at least one weight-related comorbidity. In some embodiments, the present invention relates to the use of the construct of the present invention, optionally in the presence of at least one weight-related comorbidity, for treating or preventing overweight. In some embodiments, the subject suffering from overweight is a human, such as an adult or a pediatric patient (including infants, children, and adolescents). In some embodiments, a human subject suffering from overweight may have a BMI of ≥25, such as a BMI of ≥27. In some embodiments, a human subject suffering from overweight has a BMI in the range of 25 to <30 or a BMI in the range of 27 to <30. In some embodiments, the weight-related comorbidity is selected from hypertension, diabetes (such as type 2 diabetes), dyslipidemia, high cholesterol, and obstructive sleep apnea.

[0092] In some embodiments, the present invention relates to methods of reducing weight. In some embodiments, the present invention relates to the use of the constructs of the present invention in reducing weight. A person experiencing weight loss according to the present invention may have a BMI of ≥25, such as a BMI of ≥27 or a BMI of ≥30. In some embodiments, a person experiencing weight loss according to the present invention may have a BMI of ≥35 or a BMI of ≥40. The term "weight loss" may include the treatment or prevention of obesity and / or overweight.

[0093] Compound

[0094] Compounds that target areas of the brain involved in body weight regulation include, but are not limited to, gut-derived hormones, which have been identified to play a role in the regulation of eating by conveying meal-related information about nutritional status to the brain.

[0095] In one embodiment, the compound that targets an area of ​​the brain involved in weight regulation is a protein. In one embodiment, the compound is a polypeptide. In one embodiment, the compound is a peptide.

[0096] In one embodiment, the compound targeting an area of ​​the brain involved in weight regulation is a hormone. In one embodiment, the compound is a gut-derived hormone. In one embodiment, the hormone is selected from eicosanoids, steroids, and amino acid / protein derivatives. In one embodiment, the hormone is an amino acid / protein derivative.

[0097] In one embodiment, the compound targeting an area of ​​the brain involved in body weight regulation is a GLP-1 receptor agonist (GLP-1RA).

[0098] In one embodiment, the compound targeted to an area of ​​the brain involved in weight regulation is an insulinotropic agent.The term refers to the ability to initiate a signal transduction pathway resulting in an insulinotropic effect or physiological effect known in the art.

[0099] In one embodiment, the compound targeting an area of ​​the brain involved in weight regulation targets a pathway in the brain involved in weight regulation. In one embodiment, the compound targets a receptor in the brain involved in weight regulation.

[0100] Receptors located in the BBB

[0101] Receptors located in the blood-brain barrier (BBB) ​​are endogenous receptors expressed at the brain capillary endothelium.

[0102] In one embodiment, the receptor located in the BBB is the transferrin receptor (TfR).

[0103] Allosteric / orthosteric

[0104] An "orthosteric ligand" of a receptor is the endogenous or primary ligand that mediates the activation of that receptor. The site where an orthosteric ligand binds to a receptor is called the "orthosteric site" of that receptor.

[0105] An "allosteric ligand" for a receptor is a ligand that binds to a different receptor site (ie, the "allosteric site" of the receptor) than the endogenous (orthosteric) ligand.

[0106] As an example, transferrin (Tf) is an orthosteric ligand that regulates activation of the transferrin receptor (TfR), and Tf binds to the orthosteric site of TfR. An allosteric ligand of TfR binds to a different receptor site (i.e., the allosteric site of TfR) than the orthosteric ligand (Tf).

[0107] GLP-1 receptor agonists

[0108] A receptor agonist can be defined as a compound that binds to a receptor and elicits a response typical of a natural ligand. A full agonist can be defined as an agonist that elicits a response of the same magnitude as a natural ligand (see, e.g., "Principles of Biochemistry", AL Lehninger, DL Nelson, MM Cox, 2nd edition, Worth Publishers, 1993, p. 763).

[0109] Thus, a "GLP-1 receptor agonist" (GLP-1RA) can be defined as a compound that is capable of binding to and activating a GLP-1 receptor (GLP-1R). And a "full" GLP-1RA can be defined as a GLP-1 receptor agonist that is capable of eliciting a GLP-1 receptor response of a similar magnitude to native GLP-1. In one embodiment, the GLP-1 receptor is a human GLP-1 receptor.

[0110] The ability to activate the human GLP-1 receptor may suitably be determined in culture medium containing membranes expressing the human GLP-1 receptor, and / or in an assay using whole cells expressing the human GLP-1 receptor.

[0111] For example, purified plasma membranes from a stably transfected cell line expressing the human GLP-1 receptor can be stimulated with the compound in question and the efficacy of cAMP production measured, e.g., based on competition between endogenously formed cAMP and exogenously added biotin-labeled cAMP, which can be captured using a specific antibody.

[0112] Additionally, or alternatively, the response of the human GLP-1 receptor can be measured in a reporter gene assay, for example in a stably transfected BHK cell line that expresses the human GLP-1 receptor and contains a cAMP response element (CRE) DNA coupled to a promoter and a firefly luciferase (CREluciferase) gene. When cAMP is produced due to activation of the GLP-1 receptor, this in turn results in the expression of luciferase. Luciferase can be assayed by adding luciferin, which is converted by the enzyme to oxyluciferin and produces bioluminescence, which is measured and used as a measure of in vitro potency. A non-limiting example of such an assay is described in Example 17.

[0113] The term half maximal effective concentration (EC 50 ) is usually the concentration that induces a response that is halfway between baseline and maximum, referring to a dose-response curve. EC 50 It is used as a measure of compound potency and is the concentration at which 50% of its maximal effect is observed.

[0114] The in vitro potency of the constructs and GLP-1RA compounds of the invention can be determined as described above and the EC of the compound or construct in question determined. 50 .EC 50 The lower the value, the better the efficacy.

[0115] In some embodiments, the GLP-1RA of the constructs of the invention is a GLP-1 analog. In some embodiments, the GLP-1RA of the constructs of the invention is a GLP-1 derivative.

[0116] Non-limiting examples of GLP-1 analogs are those of Examples 6, 10 and 13 ( Figure 6 , Fig.10 , Fig.13 ), i.e., amino acids 1-31 of SEQ ID NO: 11, amino acids 1-28 of SEQ ID NO: 13, and amino acids 1-31 of SEQ ID NO: 15, respectively.

[0117] Non-limiting examples of GLP-1 derivatives are disclosed in Examples 10 and 13 (SEQ ID NO: 13, SEQ ID NO: 15). These are GLP-1 analogs with a covalently attached linker.

[0118] Connector

[0119] The constructs of the invention may comprise a linker connecting the compound targeted to the area of ​​the brain involved in body weight regulation with the allosteric ligand directed to a receptor located in the BBB. In other words, the linker is optional.

[0120] In one embodiment, the linker is a peptide linker. The peptide linker consists of amino acids. In one embodiment, the peptide linker consists of amino acids. In one embodiment, the linker consists of encoded amino acids.

[0121] Non-limiting examples of peptide linkers are: GQAPGQAPGQAPGQAPGQAPK (SEQ ID NO: 13) and GGGSGGGSGGGSGGGS (residues 32-47 of SEQ ID NO: 11), see Examples 11 and 6 (see, respectively). Fig.11 and Figure 6 ).

[0122] In another embodiment, the linker is a non-peptide linker or a chemical linker.Although a non-peptide linker may comprise amino acid residues, it is not entirely composed of amino acids.

[0123] Non-limiting examples of non-peptide linkers are: Formula 1: -CH2-C(O)-, see Example 11 ( Fig.11 ), and Chemical Formula 2: -CH2-C(O)-Ado-gGlu-Ado-, wherein Ado is a divalent group of 8-amino-3,6-dioxaoctanoic acid, Chemical Formula 3: -NH-(CH2)2-O-(CH2)2-O-CH2-C(O)-, and gGlu is a divalent group of L-γ-glutamic acid, Chemical Formula 4: -NH-CH(COOH)-(CH2)2-C(O)-, see Example 15 ( Fig.15 ).

[0124] Amino acids, peptides, proteins

[0125] Amino acids can be defined as compounds comprising an amino group and a carboxylic acid group and optionally one or more additional groups commonly referred to as side chains. For example, the amine group can be a primary amino group or a secondary amino group. Proline is a non-limiting example of an amino acid comprising a secondary amino group.

[0126] A subgroup of amino acids are the α-amino acids, in which the nitrogen atom of the primary or secondary amino group is bonded to an α-carbon atom.

[0127] An amino acid residue is a group of amino acids incorporated into a peptide or protein.

[0128] Peptides are distinguished from polypeptides and proteins according to size. In this article, unless otherwise stated, peptides are composed of up to and including 50 amino acid residues, while polypeptides and proteins are composed of more than 50 amino acid residues. Proteins can be composed of one or more polypeptide chains that can be arranged in a biological function manner. In a specific embodiment, a peptide is composed of (i) at least 2 amino acid residues, (ii) at least 5 amino acid residues, (iii) at least 10 amino acid residues, (iv) at least 20 amino acid residues, (v) at least 30 amino acid residues, or (vi) at least 40 amino acid residues. In a further specific embodiment, a polypeptide is composed of (i) no more than 2000 amino acid residues, (ii) no more than 1500 amino acid residues, (iii) no more than 1000 amino acid residues, or (iv) no more than 600 amino acids. In further specific embodiments, the protein consists of (i) no more than 2000 amino acid residues, (ii) no more than 1500 amino acid residues, (iii) no more than 1000 amino acid residues, (iv) no more than 600 amino acids, or (v) no more than 500 amino acid residues.

[0129] Amino acids can be classified as coded amino acids or non-coded amino acids based on their source. The term "coded" amino acids (sometimes also referred to as "natural" amino acids) can be defined with reference to IUPAC (Table 1 in Section 3AA-1): (http: / / www.chem.qmul.ac.uk / iupac / AminoAcid / AA1n2.html#AA1), which gives the structures, common names, systematic names, single-letter and three-letter symbols for the 20 amino acids. These are all α-amino acids.

[0130] A non-limiting example of a non-coded amino acid is Aib, also known as α-aminoisobutyric acid, having the following structure:

[0131] Chemical formula 6:

[0132]

[0133] GLP-1 peptides and analogs

[0134] As used herein, the term "GLP-1 peptide" refers to human glucagon-like peptide-1 (GLP-1 (7-37)) or an analog thereof, the sequence of which is contained in the sequence listing as SEQ ID NO: 16. The peptide having the sequence of SEQ ID NO: 16 may also be referred to as "native" GLP-1.

[0135] As used herein, the term "GLP-1 analog" or "analog of GLP-1" refers to a peptide or compound that is a variant of GLP-1 (7-37) (SEQ ID NO: 16).

[0136] In the sequence listing, the first amino acid residue (histidine) of SEQ ID NO: 16 is designated as number 1. However, hereinafter, in accordance with established practice in the art, the histidine residue is referred to as number 7, and subsequent amino acid residues are numbered accordingly, ending at glycine number 37. Thus, generally, any reference herein to the amino acid residue number or position number of the GLP-1 (7-37) sequence refers to the sequence starting with His at position 7 and ending with Gly at position 37.

[0137] The GLP-1 analogs of the constructs and derivatives of the present invention can be described by reference to i) the numbering of the amino acid residues in native GLP-1 (7-37) corresponding to the altered amino acid residue (i.e. the corresponding position in native GLP-1), and ii) the actual alteration.

[0138] In other words, a GLP-1 analog is a GLP-1 (7-37) peptide in which a number of amino acid residues have been altered compared to native GLP-1 (7-37) (SEQ ID NO: 16). These alterations may independently represent one or more amino acid substitutions, additions and / or deletions.

[0139] It is obvious from the above examples that amino acid residues can be represented by their full name, their one-letter code and / or their three-letter code. These three methods are completely equivalent.

[0140] The expression "position equivalent to..." or "position corresponding to..." can be used to characterize the position of variation in the variant GLP-1 (7-37) sequence with reference to the native GLP-1 (7-37) (SEQ ID NO: 16). Equivalent or corresponding positions, as well as variation numbers, can be easily inferred, for example, by simple writing and visual inspection; and / or standard protein or peptide alignment programs can be used, such as "align" based on the Needleman-Wunsch algorithm. The algorithm is described in Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48: 443-453, and the align program is described by Myers and W. Miller in "Optimal Alignments in Linear Space" CABIOS (computer applications in the biosciences) (1988) 4: 11-17. For the alignment, the default scoring matrix BLOSUM62 and the default identity matrix can be used, and the penalty for the first residue in a gap can be set to -12, or preferably -10, and the penalty for other residues in a gap can be set to -2, or preferably -0.5.

[0141] The term "peptide" as used, for example, in the context of the GLP-1 analogues of the derivatives of the invention refers to a compound comprising a series of amino acids interconnected by amide (or peptide) bonds.

[0142] The GLP-1 peptide of the present invention comprises at least five constituent amino acids linked by peptide bonds. In a specific embodiment, the GLP-1 peptide comprises at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 25, or most preferably at least 28 amino acids.

[0143] In further specific embodiments, the peptide a) comprises i) 29, ii) 30, iii) 31 or iv) 32 amino acids, or b) consists of i) 29, ii) 30, iii) 31 or iv) 32 amino acids.

[0144] In a further specific embodiment, the peptide consists of amino acids linked to each other by peptide bonds.

[0145] In the following, all amino acids of the GLP-1 peptide for which the optical isomers thereof are not specified should be understood to mean the L-isomer (unless otherwise specified).

[0146] Pharmaceutically acceptable salts, amides, esters

[0147] The constructs of the present invention may be in the form of pharmaceutically acceptable salts, amides or esters, as may the constituent compounds and ligand moieties themselves.

[0148] For example, salts are formed by chemical reactions between bases and acids, such as: 2NH3+H2SO4→(NH4)2SO4.

[0149] The salt may be a basic salt, an acidic salt, or it may be neither (ie, a neutral salt). In water, basic salts produce hydroxide ions and acidic salts produce hydronium ions.

[0150] Salts may be formed by adding cations or anions between anionic or cationic groups, respectively. These groups may be located in the compound portion, the ligand portion and / or the optional linker portion of the construct of the invention.

[0151] Non-limiting examples of anionic groups of the constructs of the invention include free carboxyl groups, such as the free carboxyl group at the C-terminal amino acid of the construct, and free carboxyl groups at internal acidic amino acid residues such as Asp and Glu.

[0152] Non-limiting examples of cationic groups include the free amino group at the N-terminus (if present), and any free amino group of internal basic amino acid residues such as His, Arg and Lys.

[0153] Esters of the constructs of the invention can be formed, for example, by reaction of a free carboxylic acid group with an alcohol or phenol, which reaction results in replacement of at least one hydroxyl group by an alkoxy or aryloxy group.

[0154] Ester formation may involve the free carboxyl group at the C-terminus, and / or any free carboxyl group within the construct.

[0155] Amides of the constructs of the invention can be formed, for example, by reaction of a free carboxylic acid group with an amine or substituted amine, or by reaction of a free or substituted amino group with a carboxylic acid.

[0156] Amide formation can involve the free carboxyl group at the C-terminus of the construct, any free carboxyl group within the construct, the free amino group at the N-terminus of the construct, and / or any free or substituted amino group within the construct.

[0157] In a specific embodiment, the construct is in the form of a pharmaceutically acceptable salt. In another specific embodiment, the construct is in the form of a pharmaceutically acceptable amide, preferably with an amide group at the C-terminus. In a further specific embodiment, the construct is in the form of a pharmaceutically acceptable ester.

[0158] Production process

[0159] The production of proteins and peptides such as control Fab, anti-TfR-Fab and GLP-1 analogs disclosed herein is well known in the art.

[0160] For example, GLP-1 analogs can be produced by classical peptide synthesis, such as solid phase peptide synthesis using t-Boc or Fmoc chemistry or other established techniques, see, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza et al. “Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH, 2000, and “Fmoc Solid Phase Peptide Synthesis” edited by WC Chan and PD White, Oxford University Press, 2000.

[0161] In addition, or alternatively, it can be produced by recombinant methods, i.e., by culturing a host cell containing a DNA sequence encoding the analog and capable of expressing the peptide in a suitable nutrient medium under conditions that allow the peptide to be expressed. Non-limiting examples of host cells suitable for expressing these peptides are: Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines.

[0162] For example, those constructs of the invention comprising non-encoded amino acids can be generated as described in the experimental section. Alternatively, see, for example, Hodgson et al.: "The synthesis of peptides and proteins containing non-natural amino acids", Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430; and WO 2009 / 083549A1 entitled "Semi-recombinant preparation of GLP-1 analogues".

[0163] Specific examples of methods for making various constructs are included in the experimental section.

[0164] Pharmaceutical composition

[0165] In one embodiment, the composition of the invention is a pharmaceutical composition.

[0166] Pharmaceutical compositions comprising a construct of the invention or a pharmaceutically acceptable salt, amide or ester thereof and a pharmaceutically acceptable excipient may be prepared as known in the art.

[0167] The term "excipient" broadly refers to any component other than the active therapeutic ingredient. An excipient can be an inert substance, an inactive substance, and / or a non-pharmaceutically active substance.

[0168] Excipients may serve various purposes, for example as carriers, vehicles, diluents, tableting aids and / or to improve administration and / or absorption of the active substance.

[0169] The formulation of pharmaceutically active ingredients with various excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th Edition (1995) and any subsequent editions).

[0170] Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity modifiers, chelating agents and stabilizers.

[0171] Examples of formulations include liquid formulations, i.e. aqueous formulations comprising water. Liquid formulations may be solutions or suspensions. Aqueous formulations typically comprise at least 50% w / w water, or at least 60%, 70%, 80% or even at least 90% w / w water.

[0172] Alternatively, the pharmaceutical composition may be a solid preparation, for example, a freeze-dried or spray-dried composition, which may be used as is, or to which a solvent and / or diluent may be added by the physician or the patient prior to use.

[0173] The pharmaceutical composition may comprise a buffer.

[0174] The pharmaceutical compositions may contain a preservative.

[0175] The pharmaceutical composition may comprise a chelating agent.

[0176] The pharmaceutical composition may comprise a stabilizer.

[0177] The pharmaceutical composition may comprise one or more surfactants.

[0178] The pharmaceutical composition may comprise one or more protease inhibitors.

[0179] The constructs of the present invention can be administered in the form of a pharmaceutical composition. It can be administered to several sites of a patient in need thereof.

[0180] The route of administration can be, for example, parenteral.

[0181] The composition can be administered in several dosage forms, for example, solutions; suspensions; emulsions; microemulsions; multiple emulsions; injectable solutions; infusible solutions.

[0182] Systemic or parenteral administration can be carried out by subcutaneous, intramuscular, intraperitoneal or intravenous injection with the aid of a syringe, optionally a pen-like syringe, or with the aid of an infusion pump.

[0183] Treatment with the constructs according to the invention can also be combined with one or more further pharmacologically active substances.

[0184] Treatment with the constructs according to the invention may also be combined with surgery affecting glucose levels and / or lipid homeostasis, such as gastric banding or gastric bypass.

[0185] Specific implementation plan

[0186] The present invention is further described by the following non-limiting embodiments.

[0187] 1. A construct comprising a compound that targets an area of ​​the brain involved in body weight regulation and an allosteric ligand for a receptor located in the blood-brain barrier (BBB); or a pharmaceutically acceptable salt, amide or ester thereof.

[0188] 2. The construct of embodiment 1, wherein the compound targets a pathway in the brain involved in body weight regulation.

[0189] 3. The construct of any one of embodiments 1-2, wherein the compound targets a receptor in the brain involved in body weight regulation.

[0190] 4. The construct of any one of embodiments 1-3, wherein the compound is a peptide, a polypeptide or a protein.

[0191] 5. The construct of any one of embodiments 1-4, wherein the compound is a hormone.

[0192] 6. The construct of embodiment 5, wherein the hormone is selected from the group consisting of eicosanoids, steroids, and amino acid / protein derivatives.

[0193] 7. The construct of embodiment 5, wherein the hormone is an amino acid / protein derivative.

[0194] 8. The construct of any one of embodiments 1-7, wherein the compound is a gut-derived hormone.

[0195] 9. The construct of any one of embodiments 1-8, wherein the compound is an insulinotropic agent.

[0196] 10. The construct of any one of embodiments 1-9, wherein the compound is a GLP-1 receptor agonist (GLP-1RA).

[0197] 11. The construct of any one of embodiments 1-10, wherein the receptor located in the BBB is the transferrin receptor (TfR).

[0198] 12. The construct of embodiment 11, wherein the allosteric ligand binding to TfR is a peptide, polypeptide or protein.

[0199] 13. The construct of any one of embodiments 11-12, wherein the allosteric ligand binding to TfR is an anti-transferrin receptor Fab (anti-TfR-Fab).

[0200] 14. The construct of any one of embodiments 1-13, which is a fusion protein or a conjugate.

[0201] 15. The construct of any one of embodiments 1-14, further comprising a linker.

[0202] 16. The construct of embodiment 15, wherein the linker is a peptide linker or a non-peptide linker.

[0203] 17. The construct of any one of embodiments 1-16, which is a GLP-1RA.

[0204] 18. The construct of any one of embodiments 1-17, which is a complete GLP-1RA.

[0205] 19. The construct of any one of embodiments 1-18, which has an EC of equal to or lower than 1000 pM. 50 The corresponding in vitro potency was determined using the test of Example 17.

[0206] 20. The construct according to any one of embodiments 1-19, which is capable of binding to GLP-1R.

[0207] 21. The construct of any one of embodiments 1-20, which has an IC of equal to or lower than 100 nM. 50 The corresponding GLP-1R binding affinity was determined using the assay of Example 18.

[0208] 22. The construct of any one of embodiments 1-21, which is capable of binding to transferrin receptor (TfR).

[0209] 23. The construct of any one of embodiments 1-22, which is capable of inhibiting the uptake of a TfR targeting antibody.

[0210] 24. The construct of embodiment 23, which inhibits the uptake of the TfR targeting antibody corresponding to an IC of equal to or less than 100 nM 50 , which was determined using the experiment of Example 19.

[0211] 25. The construct of embodiment 24, wherein the IC 50 The values ​​were lower than the IC of the control construct 50 The control construct incorporates the same GLP-1RA but a ligand that does not bind to TfR, where the two IC 50 The values ​​are determined using the test of Example 19.

[0212] 26. The construct of any one of embodiments 11-25, which exhibits increased binding to a brain region expressing GLP-1R compared to a control construct, the control construct differing from the construct only in the incorporation of a ligand that does not bind to TfR.

[0213] 27. The construct of embodiment 26, wherein the binding to the GLP-1R expressing brain region is determined using in vivo imaging of a fluorescently labeled construct.

[0214] 28. The construct of embodiment 27, which produces a higher fluorescent signal than a control construct in at least a majority of brain regions expressing GLP-1R, wherein the brain regions expressing GLP-1R are Fig.16A shown.

[0215] 29. The construct of embodiment 28, wherein the fluorescence signal is higher in at least 50%, 60%, 70%, 80% or preferably at least 90% of the brain regions expressing GLP-1R.

[0216] 30. The construct of any one of embodiments 26-29, wherein the binding to a brain region expressing GLP-1R is determined essentially as described in Example 16.

[0217] 31. The construct of any one of embodiments 11-30, which exhibits increased binding to brain regions expressing GLP-1R protected by the BBB compared to a control construct, wherein the control construct differs from the construct only in the incorporation of a ligand that does not bind to TfR.

[0218] 32. The construct of embodiment 31, which produces a higher fluorescent signal in at least most BBB-protected GLP-1R-expressing brain regions compared to a control construct.

[0219] 33. The construct of embodiment 32, wherein the BBB-protected GLP-1R-expressing brain regions are LS, DG, MH and PB as shown in Figure 16C.

[0220] 34. The construct of embodiment 32, wherein the BBB-protected GLP-1R-expressing brain regions are ACB, DMH, LHA and LH.

[0221] 35. The construct of any one of embodiments 32-34, wherein the fluorescent signal is higher in at least 50%, 60%, 70%, 80% or at least 90% of the brain regions expressing GLP-1R.

[0222] 36. The construct of any one of embodiments 32-35, wherein the fluorescent signal is at least two times, three times, four times, five times, six times, seven times, eight times, nine times or at least ten times the fluorescent signal of the control construct.

[0223] 37. The construct of any one of embodiments 32-36, wherein the binding to the GLP-1R expressing brain region protected by the BBB is determined essentially as described in Example 16.

[0224] 38. The construct of any one of embodiments 1-37, which reduces food intake.

[0225] 39. The construct of embodiment 38, which reduces the food intake dose in a food-dependent manner.

[0226] 40. The construct of any one of embodiments 38-39, which results in lower food intake compared to a control, the control differing from the construct only by the incorporation of a ligand that does not bind to TfR.

[0227] 41. The construct of any one of embodiments 38-40, wherein the food intake is the cumulative food intake in g over a 24 hour period.

[0228] 42. The construct of any one of embodiments 38-41, wherein food intake is determined in an acute study in lean mice, essentially as described in part I or part II of Example 20.

[0229] 43. The construct of any one of embodiments 1-42, which results in weight loss.

[0230] 44. The construct of embodiment 43, which results in greater weight loss compared to a control, the control differing from the construct only by the incorporation of a ligand that does not bind to TfR.

[0231] 45. The construct of any one of embodiments 43-44, wherein weight loss is determined in a subchronic study in DIO mice, essentially as described in part III of Example 20.

[0232] 46. ​​A composition comprising the construct of any one of embodiments 1-45 and at least one pharmaceutically acceptable excipient.

[0233] 47. The construct of any one of embodiments 1-45 or the composition of embodiment 46 for use as a medicament.

[0234] 48. The construct of any one of embodiments 1-45 or the composition of embodiment 46, for use in preventing or treating obesity.

[0235] 49. A method of preventing or treating obesity comprising administering the construct of any one of embodiments 1-45 or the composition of embodiment 46.

[0236] Example

[0237] The experimental section begins with a list of abbreviations, followed by a section covering general methods for testing and characterizing the constructs of the invention. This is followed by multiple examples involving the preparation of specific construct parts and constructs, and finally multiple examples involving the activities and properties of these analogs and derivatives (a section entitled Pharmacological Methods).

[0238] These examples serve to illustrate the invention.

[0239] List of abbreviations

[0240] Ac Acetyl

[0241] ACB Nucleus accumbens

[0242] ad lib randomly

[0243] Ado 8-amino-3,6-dioxaoctanoic acid

[0244] AOB accessory olfactory bulb

[0245] AP Final Area

[0246] APN anterior tectal nucleus

[0247] ARH Arcuate nucleus of the hypothalamus

[0248] AU Arbitrary Unit

[0249] AUC Area under the curve

[0250] BBB blood-brain barrier

[0251] BBB-R blood-brain barrier receptor

[0252] BGG bovine gamma globulin

[0253] BHK Baby Hamster Kidney

[0254] BMA basomedial amygdala

[0255] BID twice a day

[0256] Boc tert-Butyloxycarbonyl

[0257] BrAc Bromoacetamide

[0258] BST Bed nucleus of the stria terminalis

[0259] cAMP

[0260] CeA Central amygdala

[0261] C H Constant region heavy chain

[0262] CHO Chinese Hamster Ovary

[0263] C L Constant region light chain

[0264] CLND Chemiluminescent Nitrogen Detection

[0265] CNS Central Nervous System

[0266] COA Cortical amygdaloid area

[0267] CRE cAMP response element

[0268] CU cuneate nucleus

[0269] CV Coefficient of variation

[0270] Dalton

[0271] DAPI 4',6-diamidino-2-phenylindole

[0272] DBE dibenzyl ether

[0273] DCM Dichloromethane

[0274] DF filtration

[0275] DG dentate gyrus

[0276] dH2O Demineralized Water

[0277] DIC Diisopropylcarbodiimide

[0278] DIO diet-induced obesity

[0279] DMEM Dulbecco's Modified Eagle's Medium

[0280] DMF Dimethylformamide

[0281] DMH Dorsomedial nucleus of the hypothalamus

[0282] DMSO Dimethyl sulfoxide

[0283] DMX Dorsal motor nucleus of the vagus nerve

[0284] DPP-4 dipeptidyl peptidase 4

[0285] DR Dorsal raphe nucleus

[0286] ECU outer wedge core

[0287] EDTA Ethylenediaminetetraacetic acid

[0288] Fab fragment antigen binding (Fab)

[0289] FI Food intake

[0290] Fmoc 9-fluorenylmethoxycarbonyl

[0291] G418 Geneticin

[0292] GENd Geniculate group, dorsal thalamus

[0293] gGlu for incorporated glutamic acid / when linked to another molecule via an amide bond involving the carboxylic acid group at the gamma position of the Glu side chain

[0294] GLP-1 Glucagon-like peptide-1

[0295] GLP-1R Glucagon-like peptide-1 receptor

[0296] GLP-1RA Glucagon-like peptide-1 receptor agonist

[0297] H Hour

[0298] HBSS Hanks Balanced Salt Solution

[0299] HEK Human Embryonic Kidney

[0300] HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

[0301] HFIP 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol

[0302] HPLC High Performance Liquid Chromatography

[0303] HSA Human serum albumin

[0304] IgG4 Immunoglobulin G subclass 4

[0305] IO Lower olive pit

[0306] iv intravenous

[0307] L Liter

[0308] LCMS Liquid chromatography mass spectrometry

[0309] LH Lateral habenular nucleus

[0310] LHA lateral hypothalamic area

[0311] lib random

[0312] LOCI Luminescent Oxygen Channeling Immunoassay

[0313] LoQ Lower limit of quantitation

[0314] LPO Lateral preoptic area

[0315] LRNm Lateral reticular nucleus, magnocellular

[0316] LS lateral septal nucleus

[0317] LSFM Light Sheet Fluorescence Microscopy

[0318] ME median eminence

[0319] MH medial habenular nucleus

[0320] MM medial nucleus of the mammillary body

[0321] MPT medial pretectal area

[0322] MQ Deionized Water

[0323] dRN midbrain reticular nucleus

[0324] Mtt 4-Methyltrityl

[0325] MV Medial vestibular nucleus

[0326] NaP Sodium Phosphate

[0327] NBF Neutral Buffered Formalin

[0328] NI undetermined nucleus (nucleus incertus)

[0329] NLL Lateral lemniscal nucleus

[0330] NTS Nucleus of the solitary tract

[0331] OEG is equivalent to Ado

[0332] O / N Overnight

[0333] OtBu tert-butyl ester

[0334] OV organelle of terminalis

[0335] Oxyma Ethyl cyano-hydroxyimino-acetate

[0336] PB parabrachial nucleus

[0337] Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

[0338] PBS Phosphate buffered saline

[0339] Pen / Strep Penicillin Streptomycin

[0340] PFA Paraformaldehyde

[0341] QC Quality Control

[0342] QD once a day

[0343] QW Once a week

[0344] Rt retention time;

[0345] RT Room temperature

[0346] sc subcutaneous

[0347] SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0348] SEM Standard error of the mean

[0349] SEC-HPLC Size Exclusion High Performance Liquid Chromatography

[0350] SFO Subforniceal Organ

[0351] SPPS Solid Phase Peptide Synthesis

[0352] tBu tert-butyl

[0353] TCEP Tris(2-carboxyethyl)phosphine

[0354] TEA Triethanolamine

[0355] Tf transferrin

[0356] TFA Trifluoroacetic acid

[0357] TfR transferrin receptor

[0358] TFT Tangential Flow Filtration

[0359] THF Tetrahydrofuran

[0360] TIC Total Ion Count

[0361] TIPS Triisopropylsilane

[0362] TNP 2,4,6-Trinitrophenol

[0363] Tris tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethyl-propane-1,3-diol

[0364] Trt Triphenylmethyl (trityl)

[0365] Tween 20 Polyethylene glycol sorbitan monolaurate

[0366] UF Ultrafiltration

[0367] ULoQ Upper limit of quantification

[0368] V L Light chain variable domain

[0369] WGA wheat germ agglutinin

[0370] General methods for detection and characterization

[0371] 1. LC-MS method

[0372] Method: LCMS_34

[0373]

[0374]

[0375]

[0376] Method: LCMS_36

[0377] This protocol summarizes the MS method described in the protocol title and serves to link the method description to the analytical results. The method is used to measure the purity of the analytes by chromatographic separation of the samples and to identify and quantify the components based on their mass spectra and total ion counts (TIC).

[0378] LC-system: Waters Acquity UPLC H-class. Step gradient: 5% to 25% B 1 min 25-65% B 6 min

[0379]

[0380]

[0381] Embodiment 1:

[0382] Transient expression of anti-TfR-Fab

[0383] This embodiment describes Figure 1A Transient expression of anti-Tfr-Fab (SEQ ID NO: 3) as shown.

[0384] Light chain (SEQ ID NO: 1):

[0385] DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGVPS

[0386] RFSGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKLELKRTVAAPSVFIFPP

[0387] SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

[0388] LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0389] Heavy chain (SEQ ID NO: 2):

[0390] EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSSKMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWG QGVSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGCLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK

[0391] The anti-TfR-Fab was designed with variable domains as disclosed in US 2010 / 0077498 A1 and human C L and IgG4C H 1 domain chimeric Fab. H The 1 domain carries an A158C mutation to generate a cysteine ​​handle for chemical conjugation.

[0392] The nucleic acid sequence encoding anti-TfR-Fab was cloned into an expression vector derived from pTT5. In order to express Fab, HEK293 6E cells were cultured to a cell density of 1x10E6 cells / ml and transfected with expression vector plasmid DNA using lipofectin (Invitrogen, catalog number 12347-500). 5 days after transfection, the expression supernatant was harvested by centrifugation and filtration of cell culture.

[0393] The cell culture supernatant containing secreted anti-TfR-Fab was loaded onto a protein G Sepharose 4FF affinity column (GE Healthcare) balanced in phosphate buffered saline (PBS). The bound Fab was eluted with 0.1M glycine-HCl (pH 2.8). The fractions were collected and immediately neutralized with 1 / 20 volume of 2M Tris-HCl (pH 9.0). The combined fractions were then diluted into 25mM NaOAc pH 5.0 and loaded onto a SP HP column (GE Healthcare). The bound Fab was eluted with a 100-400mM NaCl linear gradient in 25mM NaOAc pH 5.0, and the buffer was exchanged with PBS on a G25 desalting column (GE Healthcare). The purified protein was sterilized by filtering through a 0.2mm filter unit (Sartorius). The purity of anti-TfR-Fab was analyzed by SDS-PAGE and size exclusion HPLC. The identity of Fab was confirmed by mass spectrometry.

[0394] The purity was higher than 95% by SDS-PAGE and higher than 99.1% by SEC-HPLC (A280 nm).The observed mass was 47198.3 Da, which was consistent with the theoretical mass (47198.5 Da).

[0395] Embodiment 2:

[0396] Transient expression of control Fab

[0397] This embodiment describes Figure 2 Transient expression of a control Fab that does not bind to TfR as shown in FIG. Figure 2 The control Fab has the amino acid sequence of SEQ ID NO:5.

[0398] Light chain (SEQ ID NO:4):

[0399] DIVMTQTPLSLSVTPGQPASISCRSSQSLLHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGQGTKVEIKRTVAAPSV

[0400] FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0401] SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0402] The control Fab was designed to have the variable domain of the light chain of the anti-TNP antibody (SEQ ID NO: 4) and the same heavy chain as the anti-TfR antibody of Example 1 (SEQ ID NO: 2).

[0403] A similar procedure as described in Example 1 was used to clone and express a control Fab.

[0404] The purity was higher than 95% by SDS-PAGE and higher than 99.2% by SEC-HPLC (A280 nm).The observed mass was 47787.4 Da, which was consistent with the theoretical mass (47787.0 Da).

[0405] Embodiment 3:

[0406] Generation of stable cell lines expressing anti-TfR-Fab

[0407] This example describes the expression Figure 3 The anti-TfR-Fab shown in Figure 1A Generation of a stable cell line expressing the same protein as shown in , SEQ ID NO: 3).

[0408] Stable cell lines expressing the anti-TfR-Fab of Example 1 were generated using the CHO K1SV GS KO cell line (Lonza) with GS selection. The Fab light chain (SEQ ID NO: 1) and heavy chain (SEQ ID NO: 2) with an N-terminal CD33 signal peptide (SEQ ID NO: 17) were subcloned into the pEE17.4 vector (Lonza). The DNA was linearized and transfected into the CHO K1SV GS KO cell line by electroporation. The transfected cell micropools were propagated for two weeks in CD CHO medium (Thermo Fisher Scientific) containing MSX (Sigma-Aldrich) at a concentration of 25 μM. The cells were then transferred to a 96-deep well plate without MSX, and the 24 micropools with the highest expression were selected by Fortebio analysis using a protein G sensor (Fortebio).

[0409] In order to achieve higher expression stability, single cell clones were isolated with selected high expression micropools by limiting dilution. Cells were grown for 2-3 weeks, 96 monoclones were picked out, and screened by Fortebio. The first 24 clones were further propagated in 24-well plates and ranked by Fortebio and SDS PAGE analysis using protein G sensors. The six highest expressed cell clones were each used for further stability testing. Cells before and after 30 days of proliferation were cultured in fed-batch cultures to evaluate expression levels by HPLC (fed-batch cultures were 12-day cultures supplemented with two nutrients on the 3rd and 7th days). Two monoclones were stable, and the expression level after 30 days of passage was higher than 70%.

[0410] The stable cell line expressing Fab is revived and propagated. Then the cell culture is inoculated into the CD-CHO culture medium with an initial volume of 10.5L in a 13L Sartorius bioreactor, and the cell density is 0.3x10E6 individual cells / ml. The fed batch cell culture is maintained at 36.5°C, the dissolved oxygen level is 40%, and the pH is 7 (± 0.3). Nutrient feed is carried out according to cell density and glucose level. After 13 days, the Millipore depth filter MDOHC054H1 is used to harvest the cell culture.

[0411] Anti-TfR Fab was purified, sterilized and analyzed as described in Example 1.

[0412] The purity was higher than 95% by SDS-PAGE and higher than 96.7% by SEC-HPLC (A280 nm).The observed mass was 47199.3 Da, which was consistent with the theoretical mass (47198.5 Da).

[0413] Embodiment 4:

[0414] Generation of stable cell lines expressing control Fab

[0415] This example describes the expression Figure 4 The control Fab shown in Figure 2 Generation of a stable cell line expressing the same protein as shown in , SEQ ID NO: 5).

[0416] A stable cell line expressing the control Fab of Example 2 was generated as described in Example 3, except that the Fab light chain of SEQ ID NO: 4 was used instead of the Fab light chain of SEQ ID NO: 1. The measures to achieve higher expression stability described in Example 3 were also followed. Three monoclones were stable, with expression levels above 70% after 30 days of passaging.

[0417] As described in Example 3, a stable cell line expressing a control Fab was further cultivated in a 13 L Sartorius bioreactor.

[0418] The cell culture supernatant containing secreted control Fab was concentrated and the buffer was exchanged to 25 mM NaOAc pH 5.0 using ultrafiltration / diafiltration (UF / DF) in a tangential flow filtration (TFF) system (Millipore) and then loaded onto a SPHP column (GE Healthcare). The bound Fab was eluted with a linear gradient of 0-350 mM NaCl in 25 mM NaOAc pH 5.0 and the buffer was exchanged to PBS using UF / DF in a TFF system (Millipore).

[0419] Purified control Fab was sterilized by filtration through a 0.2 mm filter unit (Sartorius). Purity was analyzed by SDS-PAGE and size exclusion HPLC. The identity of the Fab was confirmed by mass spectrometry.

[0420] The purity was higher than 95% by SDS-PAGE and higher than 98.7% by SEC-HPLC (A280 nm).The observed mass was 47787.5 Da, which was consistent with the theoretical mass (47787.0 Da).

[0421] Embodiment 5:

[0422] Transient expression of GLP-1 anti-TNP-Fab fusion protein

[0423] This example describes the expression of control Fab and GLP-1 analogs. Figure 5 Expression of the indicated fusion protein (SEQ ID NO: 8). The control Fab was an anti-TNP Fab, wherein TNP refers to 2,4,6-trinitrophenol.

[0424] Light chain (SEQ ID NO:6):

[0425] HGEGTFTSDVSSYLEEQAAREFIAWLVKGRPGGGSGGGSGGGSGGGSDIVMTQTPLSLSVTPGQPASISCRSSQSLLHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCSQSTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0426] Heavy chain (SEQ ID NO:7):

[0427] QVQLQESGPGLVKPSETLSLTCTVSGGSISSGYWNWIRQPPGKGLEWIGTISYSGDTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARYGSYVFDYWGQGTTVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKHHHHHHHHHH

[0428] The double-chain Fab fusion protein consists of a light chain (SEQ ID NO: 6) and a heavy chain (SEQ ID NO: 7). In the light chain, the active GLP-1 analog is linked to the V of the anti-TNP-Fab of Example 2 via a flexible 4x (GlyGlyGlySer) linker. L The N-terminal fusion of the domain (SEQ ID NO: 4). H The C-terminus of the 1 domain was extended to include a 10x histidine purification tag. Thus, the light chain can be referred to as "GLP-1-(GGGS)4-aTNP light chain" and the heavy chain can be referred to as "aTNP-(His)10 Heavy chain".

[0429] As described in Example 1, the nucleic acid sequence encoding the anti-TNP-Fab fusion was cloned into a pTT-derived expression vector.

[0430] To express fusion proteins, Expi293F cells (ThermoFisher Scientific, catalog number A14527) were used in the Expi293 Expression TM Growth in suspension culture in culture medium (ThermoFisher Scientific, catalog number A1435101) to a cell density of 3x10E6 cells / ml. TM 293 reagent (ExpiFectamine TM Transient transfection was performed with a mixture of plasmid DNA encoding the two protein chains and 293 transfection kit (ThermoFisher Scientific, catalog number A14525). The day after transfection, ExpiFectamine TM 293 Transfection Kit Transfection Enhancers 1 and 2. 4 days after transfection, the expression supernatant was harvested by centrifugation and filtration of the cell culture.

[0431] The cell culture supernatant containing secreted GLP-1 anti-TNP-Fab fusion protein was loaded onto 2x 5ml HisTrap Excel columns (GE Healthcare) balanced in 50mM NaP (sodium phosphate), 300mM NaCl, 10mM imidazole (pH 7.5). The bound fusion protein was eluted with 50mM NaP, 300mM NaCl, 500mM imidazole (pH 7.5). The fractions were combined and loaded onto a Superdex 200 16-600 column (GE Healthcare) balanced in PBS. The purified protein was sterilized by filtering through a 0.2 μm filter unit. The purity of the fusion protein was analyzed by non-reducing SDS-PAGE, and a single band indicating purity>90% was produced, and size exclusion HPLC showed 12% of high molecular weight substances. The identity of the fusion protein was confirmed by mass spectrometry. The measured intact average mass of 52977 Da and the peptide mass spectrum of the tryptic digested peptides (data not shown) indicated that V H The N-terminal glutamic acid on the 1 domain (SEQ ID NO:7) is actually pyroglutamic acid in the purified protein.

[0432] Embodiment 6:

[0433] Transient expression of GLP-1 anti-TfR-Fab fusion protein

[0434] This example describes the anti-TfR-Fab and GLP-1 analogs Figure 6 Expression of the fusion protein (SEQ ID NO: 11).

[0435] Light chain (SEQ ID NO:9):

[0436] HGEGTFTSDVSSYLEEQAAREFIAWLVKGRPGGGSGGGSGGGSGGGSDIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGVPSRFSGSRSGTQFSLKISRVQVEDI GIYYCLQAYNTPWTTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0437] Heavy chain (SEQ ID NO: 10):

[0438] EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSSKMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQGVSVT VSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGHHHHHHHHHHH

[0439] The double-chain Fab fusion protein consists of a light chain (SEQ ID NO: 9) and a heavy chain (SEQ ID NO: 10). In the light chain, the active GLP-1 analog is linked to the V of the anti-TfR-Fab via a flexible 4x (GlyGlyGlySer) linker. L In the heavy chain, C H 1 The C-terminus of the Fab domain was extended to include a 10x histidine purification tag. Therefore, the light chain can be referred to as "GLP-1-(GGGS)4-aTfR light chain" and the heavy chain can be referred to as "aTfR-(His) 10 Heavy chain".

[0440] As described in Example 1, the nucleic acid sequence encoding the anti-TfR-Fab fusion was cloned into a pTT-derived expression vector.

[0441] As described in Example 5, the fusion protein was expressed in expi293F cells and the supernatant was harvested.

[0442] As described in Example 5, cell culture supernatant containing secreted GLP-1 anti-TfR-Fab fusion protein was loaded onto 2x 5ml HisTrap Excel columns (GE Healthcare) and further purified and sterilized, with an increased stringency wash using 50mM NaP (sodium phosphate), 300mM NaCl, 30mM imidazole (pH 7.5) prior to elution from the HisTrapExcel column.

[0443] The purity of the fusion protein was analyzed by SDS-PAGE and size exclusion HPLC, showing a purity greater than 95%. The identity of the fusion protein was confirmed by mass spectrometry. The average intact mass was measured by mass spectrometry to be 53243.7 Da, thus confirming the identity of the purified protein.

[0444] Embodiment 7:

[0445] Transient expression of GLP-1 control Fab fusion protein

[0446] This example describes another control Fab and GLP-1 analog Figure 7 Expression of the indicated fusion protein (SEQ ID NO: 12).

[0447] The plasmid DNA encoding the light chain of the protein (SEQ ID NO:6) was the same as used in Example 5, while the plasmid DNA encoding the heavy chain of the protein (SEQ ID NO:10) was the same as used in Example 6. This resulted in a hybrid construct that had no TfR binding, although still had an active GLP-1 element similar to the GLP-1 anti-TfR Fab of Example 6 (SEQ ID NO:11).

[0448] As described in Example 1, the nucleic acid sequence encoding the control Fab fusion was cloned into a pTT-derived expression vector.

[0449] As described in Example 5, the fusion protein was expressed in expi293F cells and the supernatant was harvested.

[0450] As described in Example 5, cell culture supernatant containing secreted fusion protein was loaded onto 2 x 5 ml HisTrap Excel columns (GE Healthcare) and further purified and sterilized.

[0451] The purity of the Fab fusion protein was analyzed by SDS-PAGE and size exclusion HPLC, showing a purity greater than 95%. The identity of the fusion protein was confirmed by mass spectrometry. The average intact mass was measured by mass spectrometry to be 53662.9 Da, thus confirming the identity of the purified protein.

[0452] Embodiment 8:

[0453] Preparation of fluorescently labeled GLP-1 anti-TfR-Fab fusion protein

[0454] This example describes the fluorescent labeling of the GLP-1 anti-TfR-Fab fusion protein (SEQ ID NO: 11) of Example 6. The fluorescently labeled compound is Figure 8 (note that the labels are randomly attached, not just at the N-terminus as shown).

[0455] To 2 ml of the GLP-1 anti-TfR-Fab fusion protein of Example 6 (containing approximately 92 nmol), 120 nmol VivoTag750-NHS was added. The latter is an amine-reactive (NHS ester) near-infrared fluorescent dye that is used to label biological molecules for in vivo imaging applications and is commercially available from PerkinElmer.

[0456] Typically, this fluorescent label is attached to the Fab quite quantitatively, but in this case, only very limited labeling can be obtained. 600nmol VivoTag750-NHS was added, and the coupling was carried out overnight. The next day, the solution was loaded onto a PD-10 column (GE Healthcare) running in PBS, and the labeled material was collected in 1.6ml PBS. Most of the blue was later eluted as a hydrolyzed label. The concentration was determined to be 32nmol / ml by CLND (chemiluminescent nitrogen detection), and the solution was aliquoted into 5 Eppendorf tubes, each 320 μL, and frozen.

[0457] LCMS showed a mixture of 0, 1, 2, 3 labels per molecule (average about 1).

[0458] Embodiment 9:

[0459] Preparation of fluorescently labeled GLP-1 control Fab fusion protein

[0460] This example describes the fluorescent labeling of the GLP-1 control Fab fusion protein (SEQ ID NO: 12) of Example 7. The fluorescently labeled compound is Fig. 9 (note that the labels are randomly attached, not just at the N-terminus as shown).

[0461] To 2 ml of the GLP-1 control Fab fusion protein (SEQ ID NO: 12) of Example 7 (containing about 138 nmol) was added 160 nmol of VivoTag750-NHS (PerkinElmer).

[0462] As described in Example 8, an additional amount of fluorescent dye must be added, in this case 900 nmol. Coupling and collection of labeled material was performed as described in Example 8 (in this case, the labeled material was collected in 2 ml PBS). Most of the blue color was later eluted as hydrolyzed tags. The concentration was determined to be 49 nmol / ml by CLND as described in Example 8, and the solution was aliquoted into 5 Eppendorf tubes, 400 μL each, and frozen.

[0463] LCMS showed a mixture of 0, 1, 2, 3 labels per molecule (average about 1).

[0464] Embodiment 10:

[0465] Synthesis of GLP-1 peptides for conjugation

[0466] This embodiment describes Fig.10 Synthesis of the indicated GLP-1 peptides.

[0467] There is no bromoacetamide (BrAc) derivatization on the epsilon amino group of the C-terminal Lys residue at position 58. Fig.10 The peptide has SEQ ID NO: 13 (position 58 corresponds to position 52 of SEQ ID NO: 13).

[0468] The peptide of SEQ ID NO:13 can be defined as an analog of GLP-1(7-37) (8Aib, 22E, 26R, 34R) having a C-terminal peptide linker (i.e., residues 38-58 added to GLP-1(7-37), wherein residues 38-58 correspond to residues 32-52 of SEQ ID NO:13). The peptide linker is: GQAPGQAPGQAPGQAPGQAPK, and has SEQ ID NO:14.

[0469] The Fmoc-protected amino acid derivatives used were the recommended standard: for example, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-H supplied by Anaspec, Bachem, IrisBiotech or NovabioChem. Is(Trt)-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH, Fmoc-Aib-OH. In the absence of any other instructions, the natural L-form of amino acids was used. The N-terminal amino acid was protected with N-terminal Boc at the alpha amino group (e.g., Boc-His(Trt)-OH). SPPS was performed on a SymphonyX Solid Phase PeptideSynthesizer from Protein Technologies (Tucson, AZ 85714U.SA) using Fmoc-based chemistry. Preloaded Fmoc-Lys(Mtt)-wang LL resin (Novabiochem, loading 0.33mmol / g) was used for peptide synthesis. Fmoc deprotection was achieved with 20% piperidine in DMF. Double peptide couplings were performed for 1 h in the first 20 cycles, and single couplings including capping (using acetic anhydride) were performed for 2 h in the remaining cycles. A mixture of amino acid / Oxyma Pure / DIC solutions (0.3M / 0.3M / 0.3M in DMF, 4-fold molar excess) was used for all couplings.

[0470] The resin was transferred to a glass reactor equipped with a filter. HFIP / DCM / TIPS 75:22:3 solution (70 ml) was added, stirred for 20 min, filtered, repeated once with 60 ml, and washed with DCM (3 x 65 ml) and DMF (3 x 65 ml).

[0471] A mixture of bromoacetic acid (3.0 g, 12 eq., 22 mmol) and DIC (1.39 g, 6 eq., 11 mmol) in DMF / DCM 1:1 (50 ml) was allowed to stand for 10 min and poured onto the resin. Allowed to stand overnight and stirred. Filtered and washed with DMF (4 x 60 ml) and DCM (4 x 60 ml).

[0472] The peptide was cleaved from the resin by stirring in a mixture of (95 TFA:2.5 TIPS:2.5 water, 80 ml) for 3 h at room temperature. Filter and wash the resin with a small amount of TFA.

[0473] The solution was divided into 12 plastic vials, dry ice-cooled ether (25 ml) was added to each vial and filled with approximately 20 ml of additional ether. The precipitate was centrifuged.

[0474] The supernatant was removed and the product was purified on preparative HPLC.

[0475] LCMS_34: Rt=2.37min, M / 4=1378.3

[0476] Embodiment 11:

[0477] Synthesis of GLP-1 anti-TfR-Fab conjugate

[0478] This example describes the synthesis of a conjugate of a GLP-1 peptide and an anti-TfR-Fab. The structure of the conjugate is Fig.11 Shown in.

[0479] element Refers to His-Aib-Glu tripeptide ( Fig.11 residues 434-436).

[0480] Fig.11 The N-terminal portion (residues 1-433) is identical to the anti-TfR-Fab of SEQ ID NO: 3 (see Examples 1 and 3).

[0481] Fig.11 The C-terminal portion (residues 434-485) is identical to SEQ ID NO: 13 (see Example 10).

[0482] like Fig.11 As shown, the epsilon amino group of Lys 485 and the thiol group of Cys 377 are connected to each other through the chemical formula 1 -CH2-C(O)-.

[0483] To the solution of the anti-TfR-Fab of Example 3 (41 ml, containing 252.4 mg, 5348 nmol of anti-TfR-Fab) dissolved in PBS buffer (pH 7.4), 522 ul of 200 mM EDTA buffer was added.

[0484] TCEP (5.05 μmol / ml, 1.05 eq, 1112 μL, 5615 nmol) was added to the protein solution.

[0485] Preparation of TCEP solution: TCEP, 0.5 M, pH 7, MW: 286 g / mol.

[0486] Dilute 25 μL of 0.5M TCEP solution with 2475 μL of buffer (20 mM TEA, 2 mM EDTA, pH 8.5 buffer) and let stand for about 2-3 hours or until fully reduced.

[0487] 3.6 equivalents of peptide from Example 10 were dissolved in DMSO (1.5 ml) and added to the Fab solution, and the flask was rinsed with a small amount of TRIS buffer pH 9. The pH was adjusted to pH 8.3 with TRIS buffer pH 9 (starting pH = 6.5) and left overnight.

[0488] The reaction mixture was concentrated by centrifugation in a Vivaspin 20, 10K filter to give 13 ml and was added to a 10K filter cartridge from GE Healthcare. Purification was performed on a purifier.

[0489] Column: Hiload 26 / 600, superdex 200pg SEC

[0490] Buffer: PBS-buffer, pH 7.4

[0491] The following properties of the obtained product were determined:

[0492] LCMS_36: Rt=3.52min; m / 30=1751.4; m / 32=1642.0

[0493] Embodiment 12:

[0494] Synthesis of GLP-1 control Fab conjugate

[0495] This example describes the synthesis of a conjugate of a GLP-1 peptide and a control Fab. The structure of the conjugate is Fig.12 Shown in.

[0496] element Refers to His-Aib-Glu tripeptide ( Fig.12residues 439-441).

[0497] Using the same protocol as described in Example 11, the bromoacetyl peptide ( Fig.10 ) The conjugate was prepared by Cys alkylation of the control Fab of Example 4.

[0498] Fig.12 The N-terminal portion (residues 1-438) of is identical to the control Fab of SEQ ID NO: 5 (see Examples 2 and 4).

[0499] Fig.12 The C-terminal portion (residues 439-490) is identical to SEQ ID NO: 13 (see Example 10).

[0500] like Fig.12 As shown, the epsilon amino group of Lys 490 and the thiol group of Cys 382 are connected to each other through the chemical formula 1: -CH2-C(O)-.

[0501] The following properties of the obtained product were determined:

[0502] LCMS_36: Rt=3.01min; m / 29=1832.1; m / 31=1713.9

[0503] Embodiment 13:

[0504] Synthesis of GLP-1 peptides for conjugation

[0505] This embodiment describes Fig.13 Synthesis of GLP-1 peptide derivatives.

[0506] Fig.13 A GLP-1 analog (8G, 22E, 26R, 34R, 36K) incorporating GLP-1(7-37) (SEQ ID NO: 15).

[0507] Fig.13A non-peptide linker is also incorporated, which can be defined as Formula 5: -Ado-gGlu-Ado-, wherein Ado refers to 8-amino-3,6-dioxaoctanoic acid, and gGlu refers to the amino acid Glu linked to another molecule via an amide bond involving a carboxylic acid group at the γ position of the Glu side chain. The bivalent group of Ado has Formula 3: -NH-(CH2)2-O-(CH2)2-O-CH2-C(O)-. The bivalent group of gGlu has Formula 4: -NH-CH(COOH)-(CH2)2-C(O)-. One Ado of the linker is linked to the epsilon amino group of the Lys residue at position 36 of the GLP-1 analog. The other Ado of the linker is derivatized with bromoacetamide for subsequent conjugation with an anti-TfR Fab or a control Fab, as described in Examples 15 and 14, respectively.

[0508] The Fmoc-protected amino acid derivatives used were the recommended standard: for example, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-H supplied by Anaspec, Bachem, IrisBiotech or NovabioChem. is(Trt)-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH, Fmoc-Aib-OH. Unless otherwise specified, the natural L-form of amino acids was used. The N-terminal amino acid was protected at the α-amino group with N-terminal Boc (e.g., Boc-Tyr(tBu)-OH for peptides with Tyr at the N-terminus). Use the chemical method based on Fmoc, carry out SPPS on the SymphonyX Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714U.SA).The resin for preparing C-terminal peptide is Fmoc-Gly-wang LL resin (Novabiochem, for example, loading is 0.29mmol / g).Realize Fmoc deprotection with 20% piperidine in DMF.Use amino acid / Oxyma Pure / DIC solution (0.3M / 0.3M / 0.3M in DMF, 4 times of molar excess) to carry out peptide coupling 1h.Before washing with DCM (5x 10mL), remove Mtt group by washing resin with HFIP / TIS / DCM (75:2.5:22.5, v / v / v) (3x 10mL x 30min).As mentioned above, continue synthesis with Fmoc-Ado-OH and Fmoc-gGlu (OtBu)-OH. The resin was transferred to a glass reactor equipped with a filter.

[0509] A mixture of bromoacetic acid (0.840 g, 12 eq., 6 mmol) and DIC (0.400 ml, 5.2 eq., 2.6 mmol) in DMF (16 ml) was allowed to stand for 10 min and poured onto the resin. Allowed to stand overnight. Filtered and washed with DMF (4 x 6 ml) and DCM (4 x 6 ml).

[0510] By stirring at room temperature in a mixture of (95TFA:2.5TIPS:2.5 water, 80ml) for 3h, the peptide was cut off from the resin. Filter and wash the resin with a small amount of TFA. The solution was divided into 12 plastic bottles, and dry ice-cooled ether (25ml) was added to each bottle, and filled with about 20ml of other ether. Centrifugal precipitation. Remove the supernatant, and purify the product on preparative HPLC.

[0511] LCMS_34: Rt=2.45min, M / 3=1328.2

[0512] Embodiment 14:

[0513] Synthesis of GLP-1 control Fab conjugate

[0514] This example describes the synthesis of a conjugate of a GLP-1 peptide and a control Fab. The structure of the conjugate is Fig.14 Shown in.

[0515] The same protocol as described in Example 11 was used, except that the peptide derivative of Example 13 was used in an amount of 3.6 equivalents, by using the bromoacetyl peptide derivative of Example 13 ( Fig.13 ) The conjugate was prepared by Cys alkylation of the control Fab of Example 4.

[0516] Fig.14 The N-terminal portion (residues 1-438) of is identical to the control Fab of SEQ ID NO: 5 (see Examples 2 and 4).

[0517] Fig.14 The C-terminal portion (residues 439-469) of Fig.13 Same: Chemical formula 5——-Ado-gGlu-Ado-The distal Ado of the linker, that is Fig.13 The bromoacetamide derivatized in is linked to the sulfhydryl group of Cys 382 via a chemical linker of Formula 1 (Formula 1: -CH2-C(O)-). Thus, the entire linker is Formula 2: -CH2-C(O)-Ado-gGlu-Ado-.

[0518] The following properties of the obtained product were determined:

[0519] LCMS_36: Rt=3.14min; m / 30=1720.0; m / 32=1612.6

[0520] Example 15: Synthesis of GLP-1 anti-TfR-Fab conjugate

[0521] This example describes the synthesis of a conjugate of a GLP-1 peptide and an anti-TfR-Fab. The structure of the conjugate is Fig.15 Shown in.

[0522] Using the same protocol as described above for Example 14, the bromoacetyl peptide derivative of Example 13 ( Fig.13 ) The anti-TfR-Fab of Example 3 was subjected to Cys alkylation to prepare the conjugate.

[0523] Fig.15 The N-terminal portion (residues 1-433) is identical to the anti-TfR-Fab of SEQ ID NO: 3 (see Examples 1 and 3).

[0524] Fig.15 The C-terminal portion (residues 434-464) of Fig.13 Same: Chemical formula 5——-Ado-gGlu-Ado-The distal Ado of the linker, that is Fig.13 The bromoacetamide derivatized in is linked here to the sulfhydryl group of Cys 377 via a chemical linker (Chemical Formula 1: -CH2-C(O)-). Therefore, the entire linker is Chemical Formula 2: -CH2-C(O)-Ado-gGlu-Ado-.

[0525] The following properties of the obtained product were determined:

[0526] LCMS_36: Rt=3.55min; m / 30=1700.4; m / 32=1594.2

[0527] Pharmacological methods

[0528] Embodiment 16:

[0529] In vivo imaging of fluorescently labeled GLP-1TfR fusion protein

[0530] This example examines the access of an "active" and "inactive" GLP-1-Fab fusion protein ("active" means it binds to TfR, "inactive" means it does not bind to TfR) to various areas of the brain that express the GLP-1 receptor. The two fusion proteins contain an identical GLP-1 receptor agonist compound, but differ in the Fab portion. This was to determine if there were substantial differences between the active and inactive fusion proteins in their access to areas of the brain that are protected by the blood-brain barrier (BBB). The study was an acute study in mice.

[0531] Compound Administration:

[0532] Mice (C57BL / 6J, male, Taconic, n=4 / group) received a single intravenous dose of the fluorescently labeled GLP-1 anti-TfR-Fab fusion protein of Example 8 ("GLP-1-active TfR"), 120nmol / kg, or the fluorescently labeled GLP-1 control Fab fusion protein of Example 9 ("GLP-1-inactive TfR"), 120nmol / kg. Six hours after compound administration ("6H"), mice were anesthetized with isoflurane and euthanized by transcardial perfusion of 10ml heparinized (10U / ml) saline and 10ml 10% neutral buffered formalin (NBF) sequentially. The brain was removed, immersed in 10% NBF, and stored overnight at 4°C until further processing.

[0533] Cleaning of tissues:

[0534] The brain was dehydrated and saturated in dibenzyl ether (DBE) to minimize light scattering during scanning by removing water and matching the refractive index. The brain tissue was dehydrated at room temperature with graded tetrahydrofuran (THF) 50 / 80 / 96 / 99% / 2x 100% diluted in demineralized H2O (w / v), each step for 6-12 hours. The brain was then cleared with 3x DBE at room temperature, each step lasting 6-12 hours.

[0535] Scanning using Light Sheet Fluorescence Microscopy (LSFM):

[0536] Brain samples were scanned at 10.32 μm isotropic resolution using an UltraMicroscope II LSFM system (Lavision Biotec, Bielefeld, Germany). For autofluorescence imaging, a 620 / 60 nm excitation filter and a 680 / 30 nm emission filter were used, while for specific signal imaging, a 710 / 75 nm excitation filter and a 780 / 40 nm emission filter were used for data acquisition.

[0537] Image analysis:

[0538] Images were single plane images (2D) of the z-plane. Spectral decomposition was performed to minimize the contribution of tissue autofluorescence in the images of the presented specific signal. Decomposition was performed on the input tiff files in Imaris (version 7.6.5, Zurich, Switzerland). The estimated autofluorescence contribution in the specific channel was calculated and removed based on the voxel intensity ratio between the selected voxels in the non-specific recording and the corresponding voxels in the specific channel. The ratios were calculated for 40 groups of voxels selected based on the histogram of the non-specific recording. The decomposition algorithm was written in Matlab (version 2012b, MathWorks, Natick, Massachusetts, USA) and used as an XT plugin in Imaris.

[0539] Using the Elastix software library version 4 (Klein, Stefan et al., "Elastix: a toolbox for intensity-based medical image registration." IEEE transactions on medical imaging 29.1 (2010): 196-205), images were registered to a common reference atlas space for visualization and quantification. S (T(x)) is spatially related to I T (x) aligned coordinate transformation T(x), transforming the source image I S (x) Register to the target atlas image I T (x). We initialized using an affine coordinate transformation, followed by a non-rigid b-spline coordinate transformation that iteratively optimizes for mutual information between the source and the target. The registration parameters used to map the LSFM data were similar to those used by Renier et al. After image registration, a manual quality check was performed for correct registration. If parts of the registered brain were not aligned, the measurements associated with these individual regions were deleted. Therefore, the number of samples in each group varied between 3 and 4 for some brain regions. Quantification was performed by summing the unmixed intensity values ​​of all voxels within the individual brain regions, indicating the total fluorescence signal from the compounds in these regions. The total signal is reported in arbitrary units ("AU"). The total signal is the sum of the unmixed voxel intensity values ​​in the individual brain regions. Therefore, the total signal indicates the total fluorescence signal from the compounds in these regions. (Renier, Nicolas et al., "Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes." Cell (2016)).

[0540] result:

[0541] The results of image analysis are Fig.16A , 16B, 16C and 16D.

[0542] exist Fig.16A In the Figures, the total intensity of fluorescently labeled compounds ("GLP-1-active TfR" and "GLP-1-inactive TfR") was quantified in brain regions expressing the GLP-1 receptor (GLP-1R). The total signal is reported in arbitrary units ("AU") and represents the average. Figure 16B shows the intensity of fluorescently labeled compounds in an example of periventricular apparatus that expresses GLP-1R but lacks a BBB. Figure 16C shows the intensity of fluorescently labeled compounds in an example of brain structures expressing GLP-1R that are protected by the BBB. Figure 16D shows the intensity of fluorescently labeled compounds in an example of brain regions that do not express GLP-1R. The abbreviations for each brain region are defined in the list of abbreviations.

[0543] Fig.16A It is shown that in the area where GLP-1R is expressed, the fluorescence signal from GLP-1-active TfR is higher compared to GLP-1-inactive TfR.

[0544] In periventricular organs that express GLP-1R but lack the BBB, the difference between active and inactive TfR compounds was smaller (Figure 16B). TfR is expressed in both capillaries and brain parenchyma. Therefore, the slightly higher contribution of active TfR compounds in these areas indicates that in addition to being bound by GLP-1R, there is also a contribution from TfR binding of the compound.

[0545] The signal in brain regions that do not express GLP-1R was also quantified (Figure 16D). Here, the signals for both compounds were nearly identical, with a slightly higher contribution from the active TfR compound again indicating a contribution from TfR binding of these compounds.

[0546] Examples of brain structures protected by the BBB are given in Figure 16C. Compared with GLP-1-inactive TfR, the signal from GLP-1-active TfR is highly increased, indicating that the GLP-1 component is carried to areas that are not easily accessible to GLP-1 itself. Although binding to TfR may also produce this signal, the difference between the two compounds in areas expressing GLP-1R is much greater than that in areas without GLP-1R (Figure 16D). This suggests that most of the signal comes from binding to GLP-1R.

[0547] in conclusion:

[0548] The clear and significant differences in signal intensity between active and inactive TfR compounds seen in brain structures protected by the BBB (Figure 16C) suggest that conjugation of GLP-1R agonists to anti-TfR Fabs can be used as a means to target GLP-1R agonists to brain regions that are not easily accessible to GLP-1R agonists. Some of these regions, such as the ACB, DMH, LHA, and LH, have been shown to be critical in body weight regulation and reward-related food intake.

[0549] Example 17: GLP-1 in vitro efficacy

[0550] The purpose of this example is to test the GLP-1 activity of the active and inactive GLP-1 fusions and conjugates disclosed herein (wherein the terms active / inactive refer to the ability to bind / not bind to the transferrin receptor (TfR), respectively). GLP-1 activity is determined as the in vitro potency of GLP-1 and is a measure of human GLP-1 receptor activation in a whole cell assay.

[0551] principle:

[0552] In vitro efficacy is determined by measuring the response of the human GLP-1 receptor in a reporter gene assay. The assay is performed in a stably transfected BHK cell line expressing the human GLP-1 receptor and containing a cAMP response element (CRE) DNA coupled to a promoter and a firefly luciferase (CRE luciferase) gene. When the human GLP-1 receptor is activated, it leads to the production of cAMP, which in turn leads to the expression of luciferase protein. When the assay incubation is complete, a luciferase substrate (luciferin) is added, and the enzyme converts luciferin into oxyluciferin to produce bioluminescence. The luminescence is measured as the readout of the assay.

[0553] To test the binding of compounds to albumin, the assay can be performed in the absence of serum albumin as well as in the presence of relatively high concentrations of serum albumin (1.0% final assay concentration). 50 An increase in the values ​​indicates affinity for serum albumin and represents a method for predicting a prolonged pharmacokinetic profile of a test substance in an animal model.

[0554] Cell culture and preparation:

[0555] The cells used in this test (clone FCW467-12A / KZ10-1) are BHK cells with BHKTS13 as the parental cell line. The cells are derived from a clone expressing the human GLP-1 receptor (FCW467-12A) and were established by further transfection with CRE luciferase to obtain this clone. The cells were cultured in cell culture medium at 5% CO2. The cells were aliquoted and stored in liquid nitrogen. Aliquots were taken before each assay and washed twice in PBS before being suspended in the assay-specific buffer at the desired concentration. For a 96-well plate, the suspension was prepared to give 5x10 3 The final concentration is cells / well.

[0556] Material:

[0557] The following chemicals were used in this assay: Pluronic F-68 (10% v / v) (Gibco 2404), human serum albumin (HSA) (Sigma A9511), ovalbumin (Sigma A5503), DMEM without phenol red (Gibco 11880-028), 1 M Hepes (Gibco 15630), Glutamax 100 (Gibco 35050) and steadylite plus (PerkinElmer 6016757).

[0558] Buffer:

[0559] The cell culture medium consisted of DMEM medium containing 10% w / v FBS (fetal bovine serum; Invitrogen 16140-071), 1 mg / ml G418 (Invitrogen 15140-122), 240 nM MTX (methotrexate; Sigma M9929) and 1% w / v pen / strep (penicillin / streptomycin; Invitrogen 15140-122).

[0560] The assay medium consisted of DMEM without phenol red, 10 mM Hepes, and 1x Glutamax. The 1% assay buffer consisted of 2% ovalbumin, 0.2% Pluronic F-68, and 2% HSA in assay medium. The 0% assay buffer consisted of 2% w / v ovalbumin and 0.2% v / v Pluronic F-68 in assay medium.

[0561] program:

[0562] 1. Thaw the cell stock solution in a 37°C water bath.

[0563] 2. Wash cells three times in PBS.

[0564] 3. Count the cells and adjust to 5x10 in assay medium. 3 cells / 50μl (1x10 5 Cells / ml). 50 μl aliquots of cells were transferred to each well of the assay plate.

[0565] 4. Dilute the stock solution of test compound to a concentration of 0.2 μM in 0% assay buffer for the 0% HSA CRE luciferase assay and in 1% assay buffer for the 1% HSA CRE luciferase assay. Dilute the compound 10-fold to obtain the following concentrations: 2x10 -7 M, 2x10 -8 M, 2x10 -9 M, 2x10 -10 M, 2x10 -11 M, 2x10 -12 M, 2x10 -13 M and 2x10 -14 M.

[0566] 5. Transfer 50 μl aliquots of compound or blank from the dilution plate to the assay plate. Test compounds at the following final concentrations: 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M and 1x10 -14 M.

[0567] 6. Incubate the assay plate at 37°C in a 5% CO2 incubator for 3 h.

[0568] 7. Remove the assay plate from the incubator and allow it to stand at room temperature for 15 min.

[0569] 8. Add 100 μl aliquots of steadylite plus reagent to each well of the assay plate (the reagent is light sensitive).

[0570] 9. Cover each assay plate with aluminum foil to protect from light and shake at room temperature for 30 min.

[0571] 10. Read each assay plate in a microtiter plate reader.

[0572] Calculation and results:

[0573] Data from the plate reader were transferred to GraphPad Prism software. The software performed nonlinear regression (log(agonist) vs. response). EC was calculated using the software and reported in pM. 50 The values ​​are shown in Table 1 below. A minimum of two replicates were measured for each sample on separate assay plates. The reported values ​​are the average of the replicates. For some instances, more than one experiment was run, and in this case, the reported data are the average of the average replicates.

[0574] Table 1: In vivo efficacy

[0575]

[0576]

[0577] All compounds had potency data demonstrating that they were GLP-1 receptor agonists. All compounds had good GLP-1 in vitro potency corresponding to EC values ​​below 1000 pM at 0% HSA. 50 .

[0578] Example 18: GLP-1 receptor binding

[0579] The purpose of this example is to test the in vitro GLP-1 receptor binding activity of the active and inactive GLP-1 fusions and conjugates disclosed herein (wherein the terms active / inactive refer to the ability to bind / not bind to the transferrin receptor (TfR), respectively). Receptor binding is a measure of the affinity of a compound for the human GLP-1 receptor.

[0580] principle:

[0581] Binding to the human GLP-1 receptor is measured in a competitive binding assay. In this type of assay, a labeled ligand (in this case 125 Each test compound is added to isolated membranes containing human GLP-1 receptors at a range of concentrations and displacement of labeled ligand is monitored. Receptor binding is reported as the concentration at which half of the labeled ligand is displaced from the receptor - IC 50 To test the binding of the derivatives to albumin, the assay can be performed in the presence of very low concentrations of serum albumin (up to 0.001% (w / v) final assay concentration) as well as in the presence of relatively high concentrations of serum albumin (2.0% (w / v) final assay concentration). 50 An increase in the value indicates affinity for serum albumin.

[0582] Material:

[0583] The following chemicals were used in this assay: human serum albumin (HSA) (Sigma A1653), DMEM without phenol red (Gibco 11880-028), Pen / strep (Invitrogen 15140-122), G418 (Invitrogen 10131-027), 1 M HEPES (Gibco 15630), EDTA (Invitrogen 15575-038), PBS (Invitrogen 14190-094), fetal bovine serum (Invitrogen 16140-071), EGTA, MgCl2 (Merck 1.05832.1000), Tween 20 (Amresco 0850C335), SPA particles (wheat germ agglutinin (WGA) SPA beads, Perkin Elmer RPNQ0001), [ 125 I]-GLP-1]-(7-36)NH2 (produced in-house), OptiPlate TM -96 (Packard 6005290).

[0584] Buffer 1 consisted of 20 mM Na-HEPES plus 10 mM EDTA and the pH was adjusted to 7.4. Buffer 2 consisted of 20 mM Na-HEPES plus 0.1 mM EDTA and the pH was adjusted to 7.4. Assay buffer consisted of 50 mM HEPES supplemented with 5 mM EGTA, 5 mM MgCl2, 0.005% w / v Tween 20 and the pH was adjusted to 7.4. The 8% albumin stock solution consisted of HSA dissolved at 8% (w / v) in assay buffer. The 0.02% albumin stock solution consisted of HSA dissolved at 0.02% (w / v) in assay buffer.

[0585] Cell culture and membrane preparation:

[0586] The cells used in this experiment (clone FCW467-12A) are BHK cells with BHKTS13 as the parental cell line. The cells express the human GLP-1 receptor. The cells were grown in DMEM, 10% w / v fetal bovine serum and 1% w / v pen / strep (penicillin / streptomycin) under 5% CO2. For membrane preparation, the cells were grown to about 80% confluence. The cells were washed twice in phosphate buffered saline and harvested. The cells were pelleted using a brief centrifugation and the cell pellet was stored on ice. The cell pellet was washed with ULTRA-THURRAX TMDispersion instrument Homogenize in an appropriate amount of buffer 1 (e.g., 10 ml) for 20-30 seconds. Centrifuge the homogenate for 15 minutes. Resuspend the precipitate in 10 ml buffer 2 (homogenize) and centrifuge. Repeat this step once more. Resuspend the resulting precipitate in buffer 2 and determine the protein concentration. Divide the membrane into equal portions and store at -80°C.

[0587] program:

[0588] 1. For receptor binding assay in the presence of low HSA (0.001%), add 50 μl of assay buffer to each well of the assay plate. Continue with step 3.

[0589] 2. For receptor binding assays in the presence of high HSA (2.0%), add 50 μl of 8% albumin stock solution to each well of the assay plate. Continue with step 3.

[0590] 3. Serially dilute the test compound to obtain the following concentrations: 8x10 -7 M, 8x10 -8 M, 8x10 -9 M, 8x10 -10 M, 8x10 -11 M, 8x10 -12 M and 8x10 -13 M. Add 25 μl to appropriate wells of the assay plate.

[0591] 4. Thaw cell membrane aliquots and dilute to their working concentrations. Add 50 μl to each well of the assay plate.

[0592] 5. Suspend WGA SPA beads at 20 mg / ml in assay buffer. Dilute this suspension to 10 mg / ml in assay buffer just before adding to the assay plate. Add 50 μl to each well of the assay plate.

[0593] 6. Add 25 μl of [ 125 Incubation was started with a 480 pM solution of [I]-GLP-1]-(7-36)NH2. A 25 μl aliquot was reserved for measuring total counts / well.

[0594] 7. Incubate the assay plate at 30°C for 2h.

[0595] 8. Centrifuge the assay plate for 10 min.

[0596] 9. Read the assay plate in a Packard TopCount NXT instrument.

[0597] calculate:

[0598] Data from the TopCount instrument were transferred to GraphPad Prism software. The software averaged the values ​​of the replicates and performed nonlinear regression. IC 50 Values ​​are reported in nM.

[0599] result:

[0600] The following results are obtained:

[0601] Table 2: GLP-1 receptor binding

[0602]

[0603]

[0604] IC of all derivatives 50 (Low albumin) are all below 60nM.

[0605] Example 19: Transferrin Receptor Uptake Inhibition

[0606] The purpose of this example is to test the in vitro transferrin receptor binding activity (wherein the terms active / inactive refer to the ability to bind / not bind to the transferrin receptor (TfR), respectively) of the exemplary active and inactive GLP-1 fusions and conjugates disclosed herein. Transferrin receptor binding is a measure of the affinity of the compound for the mouse transferrin receptor.

[0607] principle:

[0608] The ability of the conjugates of Examples 11-12 and 14-15 to inhibit the uptake of transferrin receptor-targeted antibodies was measured in a competitive uptake assay. The two compounds in each group of Fab conjugates (Examples 11 and 12; and Examples 15 and 14) contained the same GLP-1R agonist portion but differed in the Fab portion, being "active" and "inactive" in binding to TfR, respectively.

[0609] Each compound was added to a commercially available mouse fibroblast cell line (MEF-1) at a range of concentrations together with a set concentration of fluorescently labeled Fab fragments (described below). The inhibition of cellular uptake of the labeled Fab fragments by each compound was reported as the concentration at which half-maximal signal was obtained (IC 50 value).

[0610] Material:

[0611] Preparation of fluorescently labeled Fab fragments:

[0612] The fluorescently labeled Fab fragment was prepared by Cy5 labeling of the anti-TfR-Fab of Example 1. Cy5-labeled anti-TfR-Fab was Figure 1B , where Cy5 dye is shown attached to Cys at position 377.

[0613] 226uL 200mM EDTA buffer was added to the anti-TfR-Fab solution (6.15mg / ml, 17.5ml) of Example 1. 25uL TCEP solution (0.5M, pH 7.0) was diluted with 2475ul buffer (20mM TEA, 2mM EDTA, pH 8.5). 517uL (1.25 equivalents, 2.9umol) of the diluted TCEP solution was added to the anti-TfR-Fab solution. The solution was stored at -80°C.

[0614] A 1 mL aliquot (6.15 mg, 131 nmol) of the above generated solution was bubbled with argon and 1 mg Cy5 (Amersham Cy5 maleimide mono-reactive dye) dissolved in 100 uL anhydrous DMF was added. The mixture was kept at 5°C under argon overnight.

[0615] The product was purified on a Zeba Spinn desalting column, 7K MWCO, 5 ml using the following protocol: Column was spun at 1000G for 2 min. 2.4 ml was washed out. Washed 3 times with 2.4 ml PBS buffer, each time spun at 1000G for 2 min. After 3 times, the product (1 ml + 0.7 ml wash) was placed on top of the column and spun at 1000G for 2 min. The product (1.7 ml) was collected. Calculated accurate mass (m+z) / z (dimensionless): 47859.88. Measured accurate mass (m+z) / z (dimensionless): 47859.83.

[0616] The following additional chemicals were used in this experiment:

[0617] DMEM (Gibco 31966-026), Pen / strep (Invitrogen 15140-122), G418 (Invitrogen 10131-027), 1M HEPES (Gibco 15630), EDTA (Invitrogen 15575-038), fetal bovine serum (Invitrogen 16140-071), PFA 4% (Chem Cruz Sc-281692), human serum albumin (Sigma A9511), collagen-coated imaging plates (Becton Dickinson 734-0319), DAPI Hoechst 33342 (Sigma B2261), wheat germ agglutinin-Alexa 488 (Invitrogen W32464), HBSS (Gibco 14025).

[0618] program:

[0619] 1. MEF-1 cells were grown in DMEM, 10% w / v fetal bovine serum and 1% w / v penicillin / streptomycin at 5% CO2 before seeding into cell plates. The day before each assay, cells were seeded at 5000 cells / well.

[0620] 2. On the day of the assay, cells were washed with HBSS containing 0.1% (w / v) HSA.

[0621] 3. Serially dilute the test compound to obtain the following concentrations: 2x10 -6 M, 2x10 -7 M, 2x10 -8 M, 2x10 -9 M, 2x10 -10 M, 2x10 -11 M, 2x10 -12 M and 2x10 -13 M. Add 100 μl of each to the appropriate wells of the assay plate.

[0622] 4. Add fluorescently labeled Fab fragment to each well to a final concentration of 5 nM.

[0623] 5. Incubate the plate for 15 min at 37° C. After incubation, remove the buffer and wash the cells with HBSS containing 0.1% (w / v) HSA.

[0624] 6. Add 50 μl of cold 4% PFA in a fume hood. Incubate the plate at room temperature for 10 min, then remove the PFA.

[0625] 7. Add 50 μl of labeling solution to the plate and incubate for 10 min. The labeling solution contains 1:200 (v / v; from 10 mg / ml stock) of DAPI and 1:200 (v / v; from 5 mg / ml stock) of WGA-Alexa488. Incubate the assay plate at room temperature for 10 min.

[0626] 8. Wash the assay plate 3 times with 100 μl HBSS and add 100 μl HBSS to each well after the washing steps.

[0627] 9. Read the assay plate in a high content imaging instrument (IN Cell 2200 instrument, GE).

[0628] calculate:

[0629] Data from the IN Cell instrument were analyzed using the software provided with the instrument. Cell nuclei were identified using DAPI-stained nuclei and cell boundaries were constructed based on WGA-Alexa488 staining. The amount of internalized fluorescence was calculated by defining particles that were visible in the Cy5 channel of the instrument and corresponded to internalized Cy5-labeled Fab fragments. The number and intensity of particles in each cell were calculated. The readout of the instrument used for further calculations was the total area of ​​particles divided by the number of cells in each acquisition area. A typical experiment contained 32 fields of view per well using a 20x microscope objective. The data were transferred to GraphPad Prism software and plotted as a concentration response curve with log M (M is the molar concentration of the test compound) on the x-axis and the total particle area per cell on the y-axis. The software performs nonlinear regression. IC was calculated using the software 50 Values ​​are reported in nM.

[0630] result:

[0631] The following results are obtained:

[0632] Table 3: Transferrin receptor uptake inhibition

[0633]

[0634] IC of all derivatives with active Fab (TfR-Fab) 50 values ​​were all below 20 nM, whereas the IC values ​​of all derivatives with inactive Fab (control Fab) 50 All greater than 1000nM.

[0635] Example 20: In vivo studies in mice

[0636] The purpose of this example was to evaluate whether the GLP-1 anti-TfR-Fab conjugate was biologically active in vivo, focusing on features associated with obesity.

[0637] Part I of this Example reports studies on the ability of an "active" GLP-1 anti-TfR-Fab conjugate of the invention to reduce food intake in lean mice following acute intravenous administration.

[0638] Part II reports a comparison of this "active" conjugate with an "inactive" GLP-1 control Fab conjugate to determine whether the TfR arm contributes to the results obtained in Part I.

[0639] Part III reports studies in subchronically treated diet-induced obese mice to investigate whether the reduction in food intake observed in Parts I and II translates into weight loss.

[0640] I. In vivo study of the GLP-1 anti-TfR-Fab conjugate of Example 11

[0641] Study Design:

[0642] Food intake was studied in male lean C57Bl6J mice (Taconic, Denmark) acutely intravenously administered (0-100nmol / kg) with the GLP-1 anti-TfR-Fab conjugate of Example 11 over a 24-hour period using the biodaq automated monitoring system (Research Diets, New Brunswick, NJ, USA). Briefly, mice were kept under a 12-hour light-dark cycle, with food (Altromin, catalog number 1314, Lippe, Germany) and tap water available ad libitum. Prior to dosing, mice (n=4-6 per group) were assigned to one of five treatment groups evenly matched in weight and placed in the biodaq system 8 days before dosing to adapt to the testing and handling procedures. On the day of the experiment, the vehicle or the compound of Example 11 was administered intravenously to mice at time 0 according to the following table:

[0643] Table 4: Groups

[0644]

[0645] *PBS + 0.05% Polysorbate 80

[0646] Plasma Exposure:

[0647] After 24 h, blood samples were collected via the sublingual plexus into EDTA tubes containing a protease inhibitor cocktail (3.097 g K3EDTA (MW 406.53), Fluka 03665, dissolved in 50 ml Trasylol, 10.000 KIU (kallikrein inactivator units), 0.5 ml 20 mM Val-Pyr (16.48 mg Val-pyr / 4 ml H2O, pH adjusted to 7.4 with 1 M HCl) to stabilize the peptide during plasma analysis. Afterwards, the animals were euthanized by exsanguination under anesthesia followed by cervical dislocation. Blood samples were kept on ice and centrifuged within 30 minutes at 4°C (4 min x 4000 g). 50 μl of plasma were then aliquoted into appropriately labeled tubes, placed on dry ice, and stored at -80°C until analysis.

[0648] The plasma concentration of the compound of Example 11 (for Parts II and III, the compound of Example 12) was measured by LOCI (luminescent oxygen channel immunoassay). Briefly, the LOCI reagent includes two latex bead reagents (donor and acceptor beads) and a biotinylated monoclonal antibody that recognizes the N-terminal epitope of GLP-1. The donor bead reagent containing a photosensitive dye is coated with streptavidin. The second bead reagent - the acceptor bead - is conjugated with another monoclonal antibody specific for the C-terminus of GLP-1, which constitutes a sandwich structure. During the assay, these three reactants are combined with the compound of Example 11 (or, in Parts II and III, the compound of Example 11 or the compound of Example 12) in plasma to form a bead-aggregate immune complex. Excitation of the complex releases singlet oxygen molecules from the donor beads, which are guided into the acceptor beads and trigger a chemiluminescent reaction that is measured in the Envision plate reader. The amount of light generated (reported in counts per second (cps)) is proportional to the concentration of the analyte.

[0649] All calibrators, QC samples and unknown samples in EDTA plasma were analyzed in quadruplicate.

[0650] The analysis procedure is as follows:

[0651] Prepare working solutions of acceptor beads / biotinylated antibody and donor beads in LOCI buffer.

[0652] 1 μL of plasma sample / calibrator / QC was applied per well.

[0653] 15 μL of a working solution containing a biotinylated monoclonal antibody that recognizes the N-terminal epitope of GLP-1 and acceptor beads coated with a monoclonal antibody specific for the C-terminus of GLP-1 were added to each well.

[0654] The plate was sealed and covered with a black lid.

[0655] The assay was incubated at 18-22°C for 1 hour.

[0656] Add 30 μL of streptavidin-coated donor beads diluted in LOCI buffer under green light.

[0657] The plate was covered with a black lid and incubated at 18-22°C for 30 minutes.

[0658] Finally, the plate was read in Envision.

[0659] Analysis was performed using an Envision reader controlled by Envision software. The instrument response (counts per second) was exported to the LOCI Calculator where plasma concentrations were calculated.

[0660] Calibrators: A series of dilutions of Example 11 compound calibrators (for Parts II and III, Example 12 compound calibrators) were prepared in a mouse plasma pool covering the range of 400.000 to 42 pmol / L. Mouse plasma pool was included as a 0 pM calibrator (blank). Calibrators were stored in Micronic tubes at -18°C.

[0661] performance:

[0662] The lower limit of quantitation (LoQ) was determined to be 40 pmol / L. The upper limit of quantitation (ULoQ) was determined to be 25.000 pM. The imprecision (CV) was assessed by measuring three samples in 12 consecutive analytical runs and performing 21 determinations on each of the three samples in the same analytical run; the overall CV was <10%.

[0663] Material:

[0664] Reagents:

[0665] LOCI buffer: 25 mM Hepes (Sigma H-3375), 50 mM NaCl (Merck 8.22184), 10 mM K-EDTA (Fluka 03664), 2 mg / ml dextran (Pharmacosmos 551005009007), 0.5% ovalbumin OA (Sigma-A 5573), 0.05% BGG (Sigma G-7516), 0.1% Tween 20 (Merck 8.22184), 0.01% Proclin 300 (Sigma-Aldrich 48912-U), 0.01% gentamicin (Biol. Indust. 03-035-1), 0.2 mg / ml HBR1 (Scan. Lab. 3KC533), pH 7.4

[0666] Mouse plasma for dilutions and calibrators:

[0667] Female mouse plasma (C57BL / 6) from Bioreclamation, K2 EDTA, Cat.MSEPLEDTA2-C57-F

[0668] Assay Plate:

[0669] PerkinElmer, PPN 6005359.

[0670] Data Analysis:

[0671] Graph The software was used for all analyses; data are presented as mean values. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test comparing all groups.

[0672] result:

[0673] The results are shown in Figure 17, where Figure 17A shows the cumulative food intake (FI) in grams, and Figure 17B shows the plasma exposure in pM measured 24 hours after dosing. Data are shown as mean values.

[0674] As can be clearly seen from Figure 17A, the compound of Example 11 reduces food intake in a dose-dependent manner. And Figure 17B shows that the higher the dose, the higher the plasma exposure. This confirms that the compound has biological activity in vivo.

[0675] II. Head-to-head in vivo studies of the "active" and "inactive" compounds of Examples 11 and 12

[0676] To determine whether the TfR arm in the GLP-1 anti-TfR-Fab conjugate of Example 11 contributes to the food intake reducing ability determined above, a head-to-head comparison was performed with the GLP-1 control Fab conjugate of Example 12, in which the same GLP-1R agonist was conjugated to an inactive Fab to serve as a control compound.

[0677] Briefly, lean C57B16J mice were maintained under a 12 h light-dark cycle (lights off at 11 am) with free access to food and water as described in Section I above. Also similar to Section I above, mice were adapted to the biodaq food intake monitoring system for 7 days before being assigned to one of three treatment groups evenly matched for body weight. On the day of the experiment, mice were administered vehicle or 100 nmol / kg of the compound of Example 11 or the compound of Example 12 according to the following table:

[0678] Table 5: Groups

[0679]

[0680] *PBS + 0.05% Polysorbate 80

[0681] Food intake was monitored hourly for the next 24 h, after which mice were sacrificed by exsanguination under anesthesia followed by cervical dislocation.

[0682] A set of satellite mice was used to generate exposure curves for each compound. Blood samples were collected via the sublingual plexus into EDTA tubes containing a cocktail of protease inhibitors, and plasma was collected and analyzed for exposure as described in Section I above. For each compound, blood was drawn by sparse sampling at 1 h, 4 h, 8 h, 10 h, 16 h, 18 h, and 24 h after intravenous administration, with n=3 mice per time point. Plasma samples were analyzed for exposure as described in Section I above.

[0683] Data Analysis:

[0684] Graph The software was used for all analyses; data are presented as mean values. Statistical analysis was performed using two-way ANOVA followed by Bonferroni's multiple comparison test comparing all groups.

[0685] result:

[0686] The results are shown in FIG. 18 , where FIG. 18A shows the cumulative food intake (FI) in g, and FIG. 18B shows the plasma exposure levels (plasma concentrations in pM) measured over 24 h.

[0687] As can be seen from Figure 18A, both GLP-1 compounds reduced food intake relative to vehicle-treated mice over the 24-hour observation period. However, relative to mice treated with Example 12 compounds, the reduction in food intake was significantly greater in mice treated with Example 11 compounds. Figure 18B shows that in mice treated with the control compound of Example 12, plasma exposure levels were similar or higher, suggesting that the differences in food intake reduction between the analogs were not related to differences in PK. On the contrary, these data show that the active TfR Fab in the Example 11 compound contributes to a greater reduction in food intake.

[0688] III. Subtypes of the "active" and "inactive" compounds of Examples 11 and 12 in diet-induced obese mice Chronic in vivo studies, determination of weight loss

[0689] To determine whether the reduction in acute food intake in a preclinical model of obesity translates into weight loss, we next compared the compounds used in Part I and Part II above in DIO mice treated subchronically for 19 days. Briefly, male C57Bl6J mice (Charles River, France) (n = 79) were maintained on a 60% high-fat diet (D12492, Research Diets, New Brunswick, NJ, USA) from approximately 4 weeks of age. Obese mice were transported to the testing facility at approximately 20 weeks of age and housed in a temperature and humidity controlled environment under a 12-12h light-dark cycle (lights off at 11:00 p.m.), n = 10 per cage. After 4 days, mice in the high-fat diet-fed group were switched from a 60% high-fat diet to a 45% high-fat diet (HFD D12451, Research Diets, New Brunswick, NJ, USA) to prevent crushing and spilling and to more accurately measure food intake. All mice were housed individually at this time and allowed to adapt to the new environment for another two weeks. Food and water were available ad libitum at all times.

[0690] Body composition was measured on day 5 before dosing (Echo MRI 700, Houston, TX, USA), and mice were assigned to one of three treatment groups evenly matched for fat and lean tissue on day 3 before dosing.

[0691] Starting from the day before dosing, food intake and body weight were monitored daily. Starting from day 1, and according to Table 6 below, compounds were administered daily starting at 12:30 p.m. each day. Mice were initially dosed intravenously on days 1-4 and then subcutaneously on days 5-19. Blood samples were collected 2 h after dosing on day 19 for plasma exposure and analyzed as described in Section I above.

[0692] Table 6: Groups

[0693]

[0694] *PBS + 0.05% Polysorbate 80

[0695] result:

[0696] The results are shown in FIG. 19 , wherein FIG. 19A shows the average body weight loss (%), and FIG. 19B shows the average plasma concentration (pM) measured 2 h after administration on day 19.

[0697] As is evident from Figure 19A, both compounds reduced body weight compared to vehicle treated mice. Figure 19B shows similar differences in plasma exposure levels between groups, however, DIO mice treated with the "active" compound of Example 11 had greater weight loss than DIO mice treated with the "inactive" compound of Example 12, indicating that the "active" compound with TfR Fab provides additional benefit on weight loss compared to the control compound.

[0698] Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and changes that fall within the true scope of the invention.

Claims

1. A construct consisting of a compound targeting an area of ​​the brain involved in weight regulation and an allosteric ligand for a transferrin receptor (TfR) located in the blood-brain barrier (BBB), and optionally the compound and the allosteric ligand are linked to each other via a linker; or a pharmaceutically acceptable salt or amide thereof; wherein the compound is a GLP-1 receptor agonist (GLP-1RA), and the GLP-1 receptor agonist (i) capable of binding to and activating the GLP-1 receptor; (ii) is a polypeptide, and (iii) comprising GLP-1(7-37) (SEQ ID NO: 16) or a variant thereof, wherein the variant has one or more amino acid substitutions, additions and / or deletions compared to GLP-1(7-37) (SEQ ID NO: 16); or comprising amino acids 1-31 of SEQ ID NO: 11, amino acids 1-31 of SEQ ID NO: 13, or amino acids 1-31 of SEQ ID NO: 15; and The allosteric ligand is an anti-transferrin receptor Fab (anti-TfR-Fab).

2. The construct according to claim 1, characterized in that The structure of the GLP-1 receptor agonist is shown below: or .

3. The construct according to claim 1, wherein the anti-TfR-Fab comprises a light chain as shown in SEQ ID NO: 1 and a heavy chain as shown in SEQ ID NO:

2.

4. The construct according to any one of claims 1 to 3, which is a fusion protein or a conjugate.

5. The construct of claim 1, wherein the linker is a peptide linker or a non-peptide linker.

6. The construct according to any one of claims 1 to 3, characterized in that The construct is capable of reducing food intake.

7. A construct according to any one of claims 1 to 3, characterized in that The construct is capable of inducing weight loss.

8. A construct according to any one of claims 1 to 3, characterized in that The construct is capable of exhibiting increased binding to a BBB-protected GLP-1R-expressing brain region compared to a control construct, the control construct differing from the construct only in the incorporation of a ligand that does not bind to TfR, wherein the BBB-protected GLP-1R-expressing brain region is selected from one or more of the following regions: (i) the lateral septal nucleus, the dentate gyrus, the medial habenular nucleus, and the parabrachial nucleus; and / or (ii) the nucleus accumbens, the dorsomedial hypothalamic nucleus, the lateral hypothalamic region, and the lateral habenular nucleus.

9. A composition comprising the construct of any one of claims 1 to 8 and at least one pharmaceutically acceptable excipient.

10. Use of the construct according to any one of claims 1 to 8 or the composition according to claim 9 for the preparation of a medicament for preventing or treating obesity.

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