Ultra-long acting glp1 or glp1 / gip analog drug for type 2 diabetes and obesity

Abstract

The present disclosure provides a GLP1 analogue and a GLP1 / GIP receptor co-agonist analogue, a fusion protein comprising the GLP1 analogue or the GLP1 / GIP receptor co-agonist analogue, and methods of use thereof. In various embodiments, the fusion protein comprises the GLP1 analogue or the GLP1 / GIP receptor co-agonist analogue, which is fused to a protective antibody, or further fused to a stabilizing domain. In one embodiment, the fusion protein can be used to treat or ameliorate symptoms or indications of diseases such as obesity and diabetes.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 558,021 and U.S. Patent Application No. 19 / 060,067, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to human glucagon-like peptide-1 (GLP1) or GLP1 / glucose-dependent insulinotropic peptide (GIP) analogues, and treatment methods using said drugs. Background Technology

[0003] Obesity has become a major health problem in the United States. By modern standards, more than 40% of Americans are considered overweight or obese due to poor diet, lack of exercise, or other unhealthy behaviors. Obesity is a significant potential risk factor for developing other diseases such as heart disease, stroke, and diabetes. Generally, moderate weight loss can reduce the risk of developing certain obesity-related diseases, such as heart disease and diabetes.

[0004] Diabetes is a disorder in which the body cannot produce enough insulin or cannot respond properly to insulin, resulting in abnormally high blood sugar levels. Long-term high blood sugar levels often lead to undesirable long-term health consequences, including heart disease, stroke, poor circulation, and, in severe cases, lower limb amputation. Treatment for diabetes typically involves a combination of regular exercise and dietary control, along with certain medications such as insulin and / or metformin, to control and / or lower blood sugar levels.

[0005] Mature glucagon-like peptide-1 (GLP1(7-37)), with the sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:1), is a 31-amino acid peptide hormone released by intestinal "L" cells after nutrient intake. It enhances glucose-induced insulin secretion from pancreatic β-cells, increases insulin expression, inhibits β-cell apoptosis, promotes β-cell regeneration, reduces glucagon secretion, delays gastric emptying, promotes satiety, and increases peripheral glucose disposal. These multiple effects have generated considerable interest in identifying long-acting GLP1 receptor (GLP1R) agonists for the treatment of type 2 diabetes.

[0006] One approach used to treat diabetes and for glycemic control involves the use of GLP-1R agonists that target the incretin pathway. GLP1 exerts its primary effect by stimulating the release of glucose-dependent insulin from the islets of Langerhans. GLP1 has also been shown to slow gastric emptying, inhibit inappropriate postprandial glucagon release, and reduce food intake. However, GLP1 is rapidly inactivated and / or degraded by enzymes such as dipeptidyl peptidase 4 (DPP4), resulting in a very short half-life of approximately 1.5 minutes. This is undesirable because it severely limits its effectiveness in regulating glycemic levels. Longer-acting GLP1 derivatives and GLP1 receptor agonists, including GLP1-containing fusion proteins, offer improved incretin action and have therefore been investigated for diabetes control. Such GLP1 analogs, fusion proteins, and GLP1 receptor agonists are well known in the art, with exemplary examples disclosed in, for example, U.S. Patent No. 9,409,966 and published applications including US20160194371, US20170114115, US20170112904, US20160361390, US20150259416, WO2017074715, WO2016127887, EP3034514, EP2470198, and EP2373681.

[0007] GLP1 peptide variants and GLP1 receptor agonists resistant to degradation by DPP4 and other factors have also been explored. Previous work has shown that various amino acid substitutions at position 8 of GLP1 (7-37) make such peptides more resistant to DPP4, thus conferring a longer half-life. Several other GLP1 receptor agonists are also disclosed in US 2019 / 0091296A1 by Wei et al., which is incorporated herein by reference in its entirety. While these variants provide some protection against DPP4 cleavage, they only extend the half-life of GLP1 to several hours in mice, while GLP1 remains subject to cleavage by DPP4 and other proteases. Therefore, there is a need to develop new molecules that are more resistant to DPP4 and other proteases.

[0008] Glucose-dependent insulinotropic peptides (GIPs) are 42-amino acid gastrointestinal regulatory peptides that play a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic β-cells in the presence of glucose and protecting these cells. GIPs and GLP1 are collectively known as incretins; incretin receptor signaling plays a physiologically relevant role crucial for glucose homeostasis. In type 2 diabetes, the hypoglycemic effect of GIPs alone is very limited. Both native GIPs and GLP1 are rapidly inactivated by the ubiquitous protease DPP4, thus limiting their use to short-term metabolic control. Certain GIP analogues have been described in WO 2013 / 164483, WO 2014 / 192284, and WO 2011 / 119657 as exhibiting simultaneous GIP and GLP1 activity. GLP1 / GIP receptor co-agonists are also disclosed in US9474780B2. These GLP1 / GIP receptor co-agonists share the same short half-life problem as GLP1 and are also subject to protease degradation.

[0009] Therefore, there is a need to develop new molecules that are more resistant to DPP4 and other proteases and have better glycemic and weight loss effects. Summary of the Invention

[0010] According to a first aspect, this disclosure provides a glucagon-like peptide-1 (GLP1) analog SP01, the amino acid sequence of which is SEQ ID NO:2. The amino acid sequence changes compared to mature glucagon-like peptide-1 (GLP1(7-37)) include A8G and R36G. This disclosure also provides a GLP1 / GIP receptor co-agonist analog SP02 or SP03, the amino acid sequences of which are SEQ ID NO:4 and SEQ ID NO:5, respectively. The amino acid changes compared to Tirzepatide (SEQ ID NO:3) involve replacing the non-natural amino acid α-aminoisobutyric acid at positions 2 and 30 with glycine and tyrosine, respectively, and glycine and glutamine. SP01, SP02, or SP03 can enhance resistance to proteolytic cleavage and confer a longer half-life. Fusion proteins containing SP01, SP02, and SP03 can be synthesized by CHO cells and are easily purified.

[0011] According to a second aspect, this disclosure provides a fusion protein comprising: a) a GLP1 analog SP01, a GLP1 / GIP receptor co-agonist analog SP02, or a GLP1 / GIP receptor co-agonist analog SP03; and b) a protective antibody fused to the N-terminus of the GLP1 analog or the GLP1 / GIP receptor co-agonist analog via a linker. The protective antibody specifically binds to the GLP1 analog or the GLP1 / GIP receptor co-agonist analog and protects it from proteolytic cleavage. The fusion protein may enhance resistance to proteolytic cleavage and confer a longer half-life. In such embodiments, the fusion protein is inactive and remains inactive until proteolytic cleavage removes the protective antibody. In some embodiments, the protective antibody is a nanobody. In some embodiments, the linker is a commonly used short amino acid sequence, preferably 3XG4S (GGGGSGGGGSGGGGS), followed by a proteolytic cleavage sequence. Preferred proteolytic cleavage sequences are factor Xa and its variants (i.e., RGER, RKR, RGR, RSR, RR, etc.). In a particular embodiment, the fusion protein comprises: a) a GLP1 analog SP01; and b) a nanobody fused to the N-terminus of the GLP1 analog. The sequence of the nanobody has at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO:21, 58, 59, and 61 to 66. The sequence of the nanobody is preferably any one of SEQ ID NO:21, 58, 59, and 61 to 66, more preferably SEQ ID NO:21. The sequence of the nanobody is preferably a humanized modified sequence of any one of SEQ ID NO:21, 58, 59, and 61 to 66, more preferably a humanized modified sequence of SEQ ID NO:21. In one particular embodiment, the fusion protein comprises: a) a GLP1 / GIP receptor co-agonist analog SP02; and b) a nanobody fused to the N-terminus of the GLP1 / GIP analog. The sequence of the nanobody has at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO:34 and 67 to 70. The sequence of the nanobody is preferably any one of SEQ ID NO:34 and 67 to 70, more preferably SEQ ID NO:34. The sequence of the nanobody is preferably a humanized modified sequence of any one of SEQ ID NO:34 and 67 to 70, more preferably a humanized modified sequence of SEQ ID NO:34.

[0012] In some embodiments, the fusion protein described in the second aspect further comprises a stabilizing domain fused via a linker to the C-terminus of a GLP1 analog or a GLP1 / GIP receptor co-agonist analog. The stabilizing domain may be an Fc portion; or an antibody or antigen-binding fragment thereof that specifically binds to the GLP1 receptor or GIP receptor; or a GIP receptor blocking antibody or antigen-binding fragment thereof; or a bispecific antibody or antigen-binding fragment thereof of both the GLP1 receptor and GIP receptor; or a nanobody that specifically binds to albumin; or an albumin-binding domain (ABD). When the stabilizing domain is an antibody (e.g., anti-GIP receptor), it not only increases the stability of SPO2 but also enhances the activity of SPO2 against the GIP receptor due to the tethering effect. In a particular embodiment, the GLP1 analog or GLP1 / GIP receptor co-agonist analog is further fused at its C-terminus to the following via a linker: c) the N-terminus of the Fc portion of an immunoglobulin, preferably derived from IgG4 or IgG2 or variants thereof; d) the N-terminus of the light or heavy chain of an antibody that specifically binds to the GLP1 receptor or an antigen-binding fragment thereof; e) the N-terminus of the light or heavy chain of an antibody that specifically binds to the GIP receptor or specifically blocks GIPR activity or an antigen-binding fragment thereof; or f) the N-terminus of the light or heavy chain of a bispecific antibody that specifically binds to both the GLP1 and GIP receptors or an antigen-binding fragment thereof; g) the N-terminus of a nanobody that specifically binds to albumin; h) the N-terminus of an albumin-binding domain (ABD) that specifically binds to albumin. The linker is preferably 3xG4S (GGGGSGGGGSGGGGS).

[0013] According to a third aspect, this disclosure provides a nucleic acid molecule encoding the fusion protein described in the second aspect. The invention also provides recombinant expression vectors capable of expressing the fusion protein described in the second aspect. For example, this disclosure includes recombinant expression vectors comprising any of the aforementioned nucleic acid molecules. The scope of this disclosure also includes host cells to which such vectors have been introduced, and methods for producing proteins or fragments thereof by culturing host cells under conditions allowing the production of proteins or fragments thereof and recovering the proteins and fragments thus produced.

[0014] According to a fourth aspect, this disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of at least one agent selected from the fusion protein described in the second aspect, and a drug carrier or excipient. The pharmaceutical composition may further comprise a second therapeutic agent. This disclosure provides a treatment method for preventing, improving, or treating GLP1-related diseases or conditions (e.g., diabetes, obesity, type 2 diabetes) in a subject, wherein the treatment method comprises administering a therapeutically effective amount of at least one agent selected from the fusion protein described in the second or third aspect, or the pharmaceutical composition described above. In some embodiments, this disclosure provides a method for lowering blood glucose levels or weight in a subject, wherein the method comprises administering a therapeutically effective amount of at least one agent selected from the fusion protein described in the second or third aspect, or the pharmaceutical composition described above. The above methods may also include combination with a second therapeutic agent or therapy. Attached Figure Description

[0015] The above and other objects and advantages of this disclosure will become apparent when considered in conjunction with the following detailed description, wherein like reference numerals always refer to like parts, and wherein: Figure 1 The pharmacokinetic profiles of P93 and P99 in C57BL mice after subcutaneous (SC) administration of 100 or 300 nmol / kg are presented. All data are expressed as mean ± SEM.

[0016] Figure 2 The pharmacokinetic profiles of P95 in C57BL mice after subcutaneous (SC) administration of 100 or 300 nmol / kg are presented. All data are expressed as mean ± SEM.

[0017] Figure 3 The IPGTT results in C57BL mice are shown. On day 0, the drug was administered subcutaneously at 100 or 300 nmol / kg. On days 1 (A, E), 7 (B, F), 14 (C, G), 21 (D, H), and 28 (I), intraperitoneal glucose tolerance tests (IPGTTs) were performed after a 6-hour fast, with blood glucose concentrations measured at 0, 15, 30, 60, 90, and 120 minutes. All data are expressed as mean ± SEM.

[0018] Figure 4The IPGTT results in C57BL mice are shown. On day 0, drugs P46, P86, and P98 were administered subcutaneously at 30, 100, or 300 nmol / kg. On days 1 (A, D), 5 (B), 7 (E), 10 (C), 14 (F), and 21 (G), after a 6-hour fast, the intraperitoneal glucose tolerance test (IPGTT) was performed, with blood glucose concentrations measured at 0, 15, 30, 60, 90, and 120 minutes. All data are expressed as mean ± SEM.

[0019] Figure 5 The IPGTT results in C57BL mice are shown. On day 0, drugs P19, P28, and P100 were administered subcutaneously at 15 nmol / kg P19, 10 or 30 nmol / kg P28, and 60 nmol / kg P100. On days 1 (A and C) and 7 (B and D), intraperitoneal glucose tolerance tests (IPGTTs) were performed after a 6-hour fast, with blood glucose concentrations measured at 0, 15, 30, 60, 90, and 120 minutes. All data are expressed as mean ± SEM.

[0020] Figure 6 Weight loss was demonstrated with long-term administration of the following products to 18-week-old DIO mice: duraglutide (marketed under the brand name Trulicity®), 10 or 30 nmol / kg, twice weekly; telpolide and P46, 30 nmol / kg, twice weekly; P99, P93, P98, P86, and P28, 100 nmol / kg, once weekly; and P100, 60 nmol / kg, once weekly. The initial weight of the DIO mice was approximately 45 g. At 10 nmol / kg, P93 showed comparable weight loss to duraglutide (TRULICITY® brand) (12% vs. 11%), while at 30 nmol / kg, P99 showed better weight loss than duraglutide (TRULICITY® brand) (20.7% vs. 16.6%).

[0021] Figure 7 The results showed blood glucose levels in mice subjected to long-term administration of the following DIO ( Figure 7 A) and cumulative food intake ( Figure 7 B): Duraglutide (TRULICITY® brand), 10 and 30 nmol / kg, twice weekly; telpotide and P46, 30 nmol / kg, twice weekly; P99, P93, P98, and P86, 100 nmol / kg, once weekly. P93, P99, P86, and P98 all showed significant glycemic control. P99 showed a significant reduction in food intake, similar to telpotide. Detailed Implementation

[0022] Before describing the method of the present invention, it should be understood that the invention is not limited to the specific methods or experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive, as the scope of this disclosure will be limited only by the appended claims.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in practice or testing of this disclosure, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

[0024] As used herein, a "stabilizing domain" is any macromolecule that enhances the in vivo activity and / or stability of a peptide upon fusion with it. For example, a stabilizing domain could be a domain containing immunoglobulin C. H 3. Peptides with stabilizing domains. In some embodiments, the stabilizing domain prolongs the serum half-life of the peptide. In some embodiments, the stabilizing domain increases the in vivo potency of the peptide. Non-limiting examples of stabilizing domains are the Fc portion of immunoglobulins, such as the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype within each isotype group. In some embodiments, the stabilizing domain is an Fc fragment, or an amino acid sequence of 1 to about 200 amino acids containing at least one cysteine ​​residue. As another example, the stabilizing domain can be an immunoglobulin or an antigen-binding fragment thereof. In some embodiments, the stabilizing domain is an immunoglobulin comprising a heavy chain variable region and a light chain variable region, wherein the immunoglobulin binds to a specific antigen. In some embodiments, the stabilizing domain comprises an antigen-binding domain and (e.g., an Fc domain of an IgG1 or IgG4 antibody), or may comprise only an antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment), and may be modified to affect its functionality. In one particular embodiment, the stabilizing domain is an immunoglobulin comprising a heavy chain variable region and a light chain variable region, wherein the immunoglobulin binds to a GLP1 receptor and / or a GIP receptor. In other embodiments, the stabilizing domain is a cysteine ​​residue or a short peptide containing cysteine. Other stabilizing domains include peptides or polypeptides that comprise or consist of a leucine zipper, a helical ring motif, or a coiled helical motif.

[0025] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule comprising four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), as well as its multimers (e.g., IgM) or its antigen-binding fragments. Each heavy chain contains a heavy chain constant region (including the C domain). H 1. C H 2 and C H 3) and lg variable region, wherein the lg variable region can be a heavy chain variable region (“HCVR” or “V”). H ) or light chain variable region (“LCVR” or “V”) L Each light chain includes a light chain variable region (“LCVR” or “V”). L ") and light chain constant region (C L V H and V L The region can be further subdivided into highly variable regions called complementary determinant regions (CDRs) and more conservative regions scattered throughout, called framing regions (FRs). Each V H and V L It consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the invention, the FRs of the antibody (or its antigen-binding fragment) may be identical to human germline sequences, or may be naturally or artificially modified. The common amino acid sequence can be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antigen-binding protein" also includes antibodies.

[0026] It is possible to replace one or more CDR residues, or to omit one or more CDRs. Some antibodies have been described in the scientific literature that omit one or two CDRs upon binding. Padlan et al. (1995 FASEBJ. 9:133-139) analyzed the contact region between the antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered many antibodies in which one or two CDRs do not contact the antigen with any amino acid (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0027] Methods and techniques for identifying CDRs within VR amino acid sequences are well known in the art and can be used to identify CDRs within the specified VR amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop region, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within the antigen-binding domain of antigen-binding proteins or antibodies.

[0028] CDR residues that do not come into contact with the antigen can be identified based on previous studies (e.g., residues H60-H65 in CDRH2 are generally not required), from the Kabat CDR region located outside the Chothia CDR, through molecular modeling, and / or empirically. If the CDR or its residues are omitted, they are usually replaced with amino acids that occupy the corresponding positions in another human antibody sequence or in a common sequence of such sequences. The position of the substitution within the CDR and the amino acid to be substituted can also be selected empirically. Empirical substitutions can be either conserved or non-conserved.

[0029] As used herein, the term "antigen-binding fragment" includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antigen-binding protein that retain the ability to specifically bind to a GLP1 receptor or GIP receptor. Antigen-binding fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing a CDR, or isolated CDRs. In some embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments can be derived from whole protein molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and (optionally) constant domains of antigen-binding proteins. This DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthetic. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0030] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the smallest recognition unit consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules (such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent and bivalent nanobodies), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains) are also included in the term "antigen-binding fragments" as used herein.

[0031] The antigen-binding fragments disclosed herein will typically include at least one immunoglobulin (Ig) variable domain. The variable domain can have any size or amino acid composition and will typically contain at least one CDR adjacent to or in frame one or more frames. Within which V H Domain and V L In the antigen-binding fragment associated with the structural domain, V H Domain and V LDomains can be positioned relative to each other in any suitable arrangement. For example, variable regions can be dimer and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of an antigen-binding protein may contain monomer V. H or V L Structural domain.

[0032] In some embodiments, the antigen-binding fragment may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be found within the antigen-binding fragment of the antibody disclosed herein include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v)V H C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3;(vii)V H -C L (viii)V L -C H 1; (ix)V L -C H 2; (x)V L -C H 3;(xi)V L C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3; (xiii)V L -C H 2-C H 3; and (xiv)V L -C LIn any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or may be connected via complete or partial hinge or linker regions. The hinge region may consist of at least two amino acids (e.g., 5, 10, 15, 20, 40, 60, or more), thereby creating flexible or semi-flexible bonds between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of this disclosure may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, which are non-covalently associated with each other and / or with one or more monomers V H or V L Non-covalent association of structural domains (e.g., through one or more disulfide bonds).

[0033] For a complete protein molecule, the antigen-binding fragment can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment will typically include at least two distinct variable domains, each capable of specifically binding to a single antigen or different epitopes on the same antigen. Any form of multispecific antigen-binding protein, including the exemplary bispecific antigen-binding protein forms disclosed herein, can be adapted using conventional techniques available in the art to suit the context of the antigen-binding fragments disclosed herein.

[0034] As used in this article, the term "protective antibody" includes the Fab region of an antibody, nanobodies, and bispecific T-cell connective "BiTE" antibodies.

[0035] As used in this article, "nanobody" is a novel type of antibody characterized by its small relative molecular weight, high stability, strong solubility, excellent antigen-binding properties, and low immunogenicity. In camels, there exists a naturally occurring heavy chain antibody lacking a light chain. Cloning its variable region yields a single-domain antibody composed solely of the heavy chain variable region, called a VHH (variable region of a heavy chain antibody), also known as a nanobody (single-domain antibody), which is the smallest functional antigen-binding fragment. Compared to conventional antibodies, nanobodies have smaller molecular weights, simpler structures, easier gene modification, smaller size, better antigen specificity, stronger tissue penetration, and higher stability. Humanized nanobodies exhibit over 90%, 95%, or 99% homology.

[0036] To confer better resistance to proteolytic cleavage, the first step is to modify the sequence of the GLP1 or GLP1 / GIP1 receptor co-agonist to provide enhanced resistance. This disclosure has demonstrated herein that novel GLP1 analogs or GLP1 / GIP1 receptor co-agonist analogs do indeed exhibit high resistance to protease degradation. Optionally, this resistance is further enhanced by fusing a protective antibody (such as a nanobody) at its N-terminus via a linker, which can specifically bind to the receptor. The second step compensates for any weakened or reduced GLP1 activity by fusing the GLP1 analog or GLP1 / GIP1 receptor co-agonist analog with a corresponding stable domain, thus binding the weakened GLP1 or GLP1 / GIP1 receptor co-agonist to the receptor and thereby increasing its potency. Furthermore, this disclosure has found that these fusion proteins have a prolonged serum half-life and result in a longer-lasting decrease in blood glucose levels.

[0037] The GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or their fusion proteins disclosed herein may be used to treat and / or prevent diseases or disorders or conditions associated with hyperglycemia (such as diabetes), and / or to relieve at least one symptom associated with such diseases, disorders or conditions. In one embodiment, the GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or their fusion proteins disclosed herein may be administered at a therapeutic dose to a patient with diabetes (e.g., type 2 diabetes).

[0038] In some embodiments, the GLP1 analogs or GLP1 / GIP1 receptor co-agonist analogs or their fusion proteins disclosed herein may be used to treat patients suffering from diseases or disorders selected from the group consisting of: diabetes, obesity, insulin resistance, hypertension, dyslipidemia, type 2 diabetes, type 1 diabetes, prediabetes, cardiovascular disease, atherosclerosis, congestive heart failure, coronary artery disease, arteriosclerosis, peripheral artery disease, stroke, respiratory dysfunction, kidney disease, fatty liver disease, non-alcoholic steatohepatitis (NASH), and metabolic syndrome.

[0039] In some embodiments, the GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or fusion proteins thereof disclosed herein may be used to treat overweight or obese subjects and / or to prevent or treat one or more obesity-related disorders, such as heart disease, stroke and diabetes.

[0040] In some embodiments, the GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or their fusion proteins disclosed herein may be used to treat subjects with diabetes and / or to prevent one or more complications of diabetes, such as heart disease, stroke, kidney disease, retinopathy, blindness and nerve damage.

[0041] This document also envisions the prophylactic use of one or more of the GLP1 analogs or GLP1 / GIP1 receptor co-agonist analogs or their fusion proteins selected from the present disclosure in subjects at risk of developing diabetes (e.g., type 2 diabetes). Subjects at risk include, but are not limited to, elderly subjects, pregnant women, and subjects with one or more risk factors, including a family history of obesity, high blood cholesterol, smoking, excessive alcohol consumption, and / or lack of exercise.

[0042] In another embodiment, the GLP1 analogue or GLP1 / GIP1 receptor co-agonist analogue or its fusion protein of this disclosure is used to prepare a pharmaceutical composition or medicament for treating patients with diseases or disorders such as diabetes and obesity. In another embodiment of the invention, the GLP1 analogue or GLP1 / GIP1 receptor co-agonist analogue or its fusion protein of this disclosure is used as an adjunct therapy to any other agent or therapy known to those skilled in the art for the treatment or improvement of diseases or disorders associated with hyperglycemia, such as diabetes (e.g., type 2 diabetes).

[0043] Combination therapies may include the GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or their fusion proteins disclosed herein, and any additional therapeutic agents that may be advantageously combined with the GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or their fusion proteins. The GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or their fusion proteins disclosed herein may be synergistically combined with one or more drugs or therapies for treating any disease or disorder (e.g., diabetes) associated with hyperglycemia. In some embodiments, the GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or their fusion proteins disclosed herein may be combined with a second therapeutic agent to lower the subject's blood glucose level or improve one or more symptoms of diabetes.

[0044] The GLP1 analogues or GLP1 / GIP1 receptor co-agonist analogues or their fusion proteins disclosed herein can be used in combination with: insulin (or insulin analogues); insulin sensitizers, such as biguanides (e.g., metformin) and thiazolidinediones (e.g., rosiglitazone); insulin secretagogues, such as sulfonylureas (e.g., chlorpropamide) and meglitinides (e.g., nateglinide); α-glucosidase inhibitors (e.g., acarbose); dipeptidyl peptidase 4 (DPP4) inhibitors (e.g., sitagliptin); pramlinide; bromoergot. Cyclic peptides; sodium-glucose cotransporter 2 (SGLT-2) inhibitors (e.g., canagliflozin); antihypertensive drugs (e.g., angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, diuretics, calcium channel blockers, alpha-adrenergic receptor blockers, endothelin receptor blockers, organic nitrates, and protein kinase C inhibitors); statins; aspirin; different GLP1 receptor agonists; different GLP1 / GIP receptor co-agonists; dietary supplements; or any other therapy for treating or managing diabetes (e.g., exercise). In some embodiments, the GLP1 analogue or GLP1 / GIP1 receptor co-agonist analogue or its fusion protein of this disclosure may be administered in combination with a second therapeutic agent or therapy selected from the group consisting of: insulin, insulin analogues, metformin, rosiglitazone, pioglitazone, chlorpropamide, glibenclamide, glimepiride, glipizide, tolazamide, tolbutamide, nateglinide, repaglinide, acarbose, miglitol, exenatide, liraglutide, albiglutide, dulaglutide, sitagliptin, saxagliptin, linagliptin, alogliptin, pramlinide, bromoergot cyclopeptide immediate release, canagliflozin, dapagliflozin, empagliflozin, dietary modifications, and exercise.

[0045] As used herein, the term “in combination with” means that one or more additional therapeutically active components may be administered before, simultaneously with or after administration of the GLP1 analogue or GLP1 / GIP1 receptor co-agonist analogue or its fusion protein disclosed herein.

[0046] This disclosure includes pharmaceutical compositions in which the GLP1 analog or GLP1 / GIP1 receptor co-agonist analog or fusion protein of the present disclosure is co-administered with one or more additional therapeutically active components as described elsewhere herein.

[0047] For example, in embodiment 1, this disclosure provides a glucagon-like peptide-1 (GLP1) analog SP01 (the amino acid sequence of which is SEQ ID NO:2), a GLP1 / GIP receptor co-agonist analog SP02 or SP03 (the amino acid sequences of SP02 and SP03 are SEQ ID NO:4 and SEQ ID NO:5, respectively).

[0048] In embodiment 2, this disclosure provides a fusion protein comprising: a) a GLP1 analog SP01, a GLP1 / GIP receptor co-agonist analog SP02, or a GLP1 / GIP receptor co-agonist analog SP03; and b) a protective antibody fused to the N-terminus of the GLP1 analog or the GLP1 / GIP receptor co-agonist analog via a linker. The protective antibody specifically binds to the GLP1 analog or the GLP1 / GIP receptor co-agonist analog and protects it from proteolytic cleavage. The fusion protein may enhance resistance to proteolytic cleavage and confer a longer half-life.

[0049] In embodiment 3, this disclosure provides the fusion protein of embodiment 2, wherein the protective antibody is a nanobody.

[0050] In embodiment 4, this disclosure provides the fusion protein of embodiment 3, wherein the linker is 3XG4S (GGGGSGGGGSGGGGS), followed by a proteolytic cleavage sequence, preferably a factor Xa and its variants (i.e., RGER, RKR, RGR, RSR, RR, etc.).

[0051] In embodiment 5, this disclosure provides a fusion protein of any one of embodiments 3-4, wherein the fusion protein comprises: a) a GLP1 analog SP01; and b) a nanobody fused to the N-terminus of a GLP1 analog. The sequence of the nanobody has at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO:21, 58, 59, and 61 to 66. The sequence of the nanobody is preferably any one of SEQ ID NO:21, 58, 59, and 61 to 66, more preferably SEQ ID NO:21.

[0052] In embodiment 6, this disclosure provides a fusion protein of any one of embodiments 3-4, wherein the fusion protein comprises: a) a GLP1 / GIP receptor co-agonist analog SP02; and b) a nanobody fused to the N-terminus of the GLP1 / GIP analog, wherein the sequence of the nanobody has at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO:34 and 67 to 70. The sequence of the nanobody is preferably any one of SEQ ID NO:34 and 67 to 70, more preferably SEQ ID NO:34.

[0053] In embodiment 7, this disclosure provides a fusion protein of any one of embodiments 2-6, wherein a GLP1 analog or a GLP1 / GIP receptor co-agonist analog is further fused to a stable domain at the C-terminus via a linker.

[0054] In embodiment 8, this disclosure provides the fusion protein of embodiment 7, wherein the GLP1 analog or GLP1 / GIP receptor co-agonist analog is further fused at its C-terminus to: c) the N-terminus of the Fc portion of an immunoglobulin, preferably derived from IgG4 or IgG2 or variants thereof; or d) the N-terminus of the light or heavy chain of an antibody that specifically binds to the GLP1 receptor or an antigen-binding fragment thereof; or e) the N-terminus of the light or heavy chain of an antibody that specifically binds to the GIP receptor or specifically blocks GIPR activity or an antigen-binding fragment thereof; or f) the N-terminus of the light or heavy chain of a bispecific antibody that specifically binds to both the GLP1 receptor and the GIP receptor or an antigen-binding fragment thereof.

[0055] In embodiment 9, this disclosure provides a fusion protein of embodiment 13 or 14, wherein the stable domain is fused to the GLP1 analog or GLP1 / GIP receptor co-agonist analog via a linker (preferably 3xG4S(GGGGSGGGGSGGGGS)).

[0056] In embodiment 16, this disclosure provides a fusion protein of embodiment 7 or 8, wherein the linker is preferably 3xG4S (GGGGSGGGGSGGGGS).

[0057] In Implementation Scheme 10, this disclosure provides a nucleic acid molecule encoding the fusion protein of any one of Implementation Schemes 2-9.

[0058] In Implementation Scheme 11, this disclosure provides a recombinant expression vector capable of expressing the fusion protein of any one of Implementation Schemes 2-9.

[0059] In embodiment 12, this disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of at least one agent selected from any one of embodiments 2-9 of a fusion protein, and a pharmaceutical carrier or excipient, the pharmaceutical composition possibly further comprising a second therapeutic agent.

[0060] In embodiment 13, this disclosure provides a treatment method for preventing, improving or treating GLP1-related diseases or conditions (e.g., diabetes, obesity, type 2 diabetes) in a subject, wherein the treatment method comprises administering a therapeutically effective amount of at least one agent selected from any one of embodiments 2-9 of the fusion protein or the pharmaceutical composition of claim 12.

[0061] In embodiment 14, this disclosure provides a method for lowering blood glucose levels or reducing weight in a subject, the method comprising administering a therapeutically effective amount of at least one agent selected from any one of embodiments 2-9 of the fusion protein or the pharmaceutical composition according to claim 12.

[0062] In embodiment 15, this disclosure provides a method of embodiment 13 or 14, the method further comprising a combination with a second therapeutic agent or therapy.

[0063] Example The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the methods and compositions characterized in this invention, and are not intended to limit the scope of what the inventors consider to be their invention. While every effort has been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), some experimental errors and deviations should still be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, room temperature is about 25°C, and pressure is atmospheric pressure or close to atmospheric pressure.

[0064] Reagents and batch numbers used 1. SP01: Exemplary GLP1 analog (SEQ ID NO:2) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG.

[0065] 2. SP02: GLP1 / GIP receptor co-agonist analog (SEQ ID NO:4) YGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPS.

[0066] 3.SP03: GLP1 / GIP receptor co-agonist analog (SEQ ID NO:5) YGEGTFTSDYSIQLDKIAQKAFVQWLIAGGPSSGAPPPS.

[0067] 4. Eli Lilly's Trulicity: Manufactured by Vetter Pharma-Fertigung, Lilly France.

[0068] 5. P19: Eli Lilly's telpolide, synthesized by Zhejiang Paipai Biotechnology Co., Ltd., China, catalog number 603733.

[0069] 6. P26 (SEQ ID NO:40-43): SP02 bispecific antibody against GLP1R and GIPR.

[0070] 7.P28 (SEQ ID NO:38-39): SP02-amG2.

[0071] 8.P46 (SEQ ID NO:29-27): SP01-amG3.

[0072] 9. (P62-P73)-hIgG1Fc (SEQ ID NO:6-15): 10 SP01 nanobodies fused with hIgG1Fc.

[0073] 10. (P49-59)-hIgG1Fc (SEQ ID NO:16-20): Five SP02 nanobodies fused with hIgG1Fc.

[0074] 11.P86 (SEQ ID NO:26-27):P67-RGER-SP01-amG3.

[0075] 12. P93 (SEQ ID NO: 22): P67-RGER-SP01-hlgG4Fc.

[0076] 13.P95 (SEQ ID NO:35-36):P56-RGER-SP02-amG2.

[0077] 14.P98 (SEQ ID NO:28-27):P67-RKR-SP01-amG3.

[0078] 15. P99 (SEQ ID NO: 23): P67-RKR-SP01-hlgG4Fc.

[0079] 16.P100 (SEQ ID NO:37-36):P56-RKR-SP02-amG2.

[0080] Example 1: Design of GLP-1 analogs and GLP-1 / GIP receptor co-agonist analogs Mature GLP-1 is a 31-amino acid peptide hormone, which contains amino acids 7 to 37 of the full-length GLP-1 and has the amino acid sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:1).

[0081] GLP-1 analogs are generated by modifying mature GLP-1 through amino acid mutations. An exemplary GLP-1 analog SP01 has the amino acid sequence: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO:2). Compared to mature GLP-1, the amino acid sequence changes are A8G, G22E, and R36G.

[0082] GLP1 / GIP screening: Eli Lily’s telpokines (SEQ ID NO:3) is a dual GLP1 / GIP receptor agonist with the amino acid sequence: Y[Aib]EGTFTSDYSI[Aib]LDKIAQK[diacid-γ-Glu-(AEEA)2]AFVQWLIAGGPSSGAPPPS (SEQ ID NO:3).

[0083] By replacing two non-coding amino acid residues (α-aminoisobutyric acid) at positions 2 and 13 with glycine and tyrosine or glutamine, respectively, two new GLP1 / GIP receptor co-agonists were obtained.

[0084] Exemplary GLP1 / GIP receptor co-agonist analogs SP02 and SP03 have the following amino acid sequences: SP02 (SEQ ID NO:4):YGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPS, SP03 (SEQ ID NO: 5): YGEGTFTSDYSIQLDKIAQKAFVQWLIAGGPSSGAPPPS.

[0085] Example 2: HTRF cAMP bioassay targeting GLP1 / GIP receptor co-agonist analogs The ability of GLP1 / GIP receptor co-agonist analogs to stimulate cAMP production in HEK293 / hGLP1R / CRE-LUC and HEK293 / hGIPR / CRE-LUC cell lines was tested.

[0086] For homogeneous time-resolved fluorescence (HTRF) cAMP bioassay (Cisbio, catalog number 62AM4PEJ), cells were cultured at 37°C and 5% CO2 in 90% DMEM, 10% FBS, and 100 μg / mL hygromycin B, and then seeded at 1000 cells / well into 384-well assay plates (Greiner, catalog number 784075) containing assay buffer (0.5 mM IBMX and 0.1% BSA) for DPBS.

[0087] To determine the dose-response of the test peptides, human GLP1, human GIP, dulaglutide (TRULICITY® brand), P19, SP02, and SP03 were added to cells at concentrations ranging from 0.02 pM to 100 nM. After incubation at 37°C for 30 min, 5 μL of cAMP Eu-Cave Complex Working Solution (Promega) and 5 μL of anti-cAMP-d2 Working Solution were added to each well of the assay plate. The plate was covered and incubated at room temperature for 1 h, and fluorescence was read at 665 nm and 620 nm using an EnVision plate reader (PerkinElmer) with a time-resolved fluorescence laser (TRF LASER). The results were analyzed using nonlinear regression (three-parameter) with Prism 6 software (GraphPad) to obtain EC50. 50 value.

[0088] As shown in Table 1, SP02 and SP03 show EC2 activation for GLP1R. 50 The values ​​were 0.024 nM and 0.225 nM, respectively, for ECGs activated by GIPR. 50 The values ​​are 3.69 nM and 22 nM, respectively. SP02's EC 50 It was superior to SP03 and was selected for subsequent research.

[0089] Table 1 EC5 of GLP1 / GIP receptor co-agonist analogs 50

[0090] Example 3: Assay of agonist-stabilizing domain fusion protein and luciferase To prolong the half-life of the agonist and compensate for any potential decrease in activity, SP01 and SP02 were fused with a stabilizing domain.

[0091] AmG3 fused with GLP1 analog SP01 or fused as follows: P46 (SEQ ID NO:29-27): SP01 is fused with the N-terminus of the amG3 light chain via the connector GGGGSGGSGGSGGGGS.

[0092] The GLP1 / GIP receptor co-agonist analog SP02 is fused with either a bispecific antibody against amG2 or GLP-1R (ahG1) and GIPR (amG2), as shown below: P28 (SEQ ID NO:38-39): SP02 is fused to the N end of the amG2 light chain via the following connector: GGGGSGGGGSGGGGSGGS; P26 (SEQ ID NO:40-43): SP02 is fused to the N-terminus of a bispecific antibody with the ahG1 and amG2 light chains via the linker GGGGSGGGGSGGGGS.

[0093] The ability of the aforementioned fusion protein to stimulate cAMP production in human GLP-1R cell lines (HEK293 / GLP1R / CRE-LUC), human GIPR cell lines (HEK293 / hGIPR / CRE-LUC), and mouse GIPR cell lines (HEK293 / mGIPR / CRE-LUC) was tested. The results of the HTRF cAMP bioassay are shown in Table 2.

[0094] Table 2 EC of agonist-stabilizing domain fusion proteins 50

[0095] As shown in Table 2, since amG2 is an mGIPR-specific binding antibody, P26 and P28 show ECG activation against hGIP1R. 50 The values ​​were 48 nM and 69 nM, respectively, while due to the tethering effect, EC2 activated by mGIP1R... 50 The values ​​increased to 0.35 nM and 0.42 nM, respectively. P46 contains an mGIPR-specific blocking antibody, which showed an IC50 value against mGIPR. 50 The value is 11.2 nM.

[0096] Example 4: Screening nanobodies by immunizing alpacas with SP01 or SP02 GLP1(7-37) has a short half-life (1-2 minutes) because it is rapidly inactivated by dipeptidyl peptidase 4 (DPP4) and other proteases. GLP-1 and GIP receptor co-agonists have been shown to effectively reduce body weight and food intake; and these effects are significantly greater than those of GLP-1 receptor agonists. Telboride is currently the only available GLP1 / GIP dual agonist drug, requiring weekly administration, and has a half-life of less than one day in mice. To confer better resistance to protease degradation, protective antibodies against SP01 and SP02 were first screened.

[0097] Methods for preparing nanobodies that specifically bind to antigens or epitopes (heavy chain single-domain antibodies, VHH) are described in the references, see, for example, R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J. Biol. Chem., 287 (2012): 13713-13721; Deffar et al., African Journal of Biotechnology, 8 (12), p. 2645, June 17, 2009; and WO 94 / 04678.

[0098] Antigens SP01 and SP02, conjugated to the C-terminus of bovine serum albumin (BSA) or hemocyanin (KLH) and produced by QYAOBIO (China), were synthesized for immunization of alpacas. A phage display library was constructed using phage display technology. Phages expressing antigen-specific nanobodies were enriched after three rounds of biopanning and following the detailed experimental procedures previously reported (US2022 / 0396616, 3U Biopharmaceuticals (Shanghai) Co., Ltd.).

[0099] Ten nanobodies that bind well to SP01 (see Table 3) and five nanobodies that bind well to SP02 (see Table 4) were selected. All nanobodies were fused with hIg1Fc, and EC was measured by ELISA. 50 .

[0100] Table 3 EC of SP01 nanobody 50

[0101] Table 4 EC of SP02 nanobodies 50

[0102] The amino acid sequence of P67 is as described in SEQ ID NO:21. The amino acid sequences of P62-P65, P66, and P68-73 are as described in SEQ ID NO:58-66.

[0103] The amino acid sequence of P56 is as described in SEQ ID NO:34. The amino acid sequences of P49, P55, P58 and P59 are as described in SEQ ID NO:67-70.

[0104] Example 5: Pharmacokinetic Study of Fusion Protein in C57BL Mice Pharmacokinetic profiles were investigated in C57BL mice (male, approximately 25 g, Beijing Vital River Laboratory Animal Technology Co., Ltd.) following subcutaneous (SC) administration of the fusion protein at 30 nmol / kg. Blood was drawn from test subjects (6 mice per group) at different time points at 6 hours and days 1, 2, 4, 7, 10, and 14 post-administration. Plasma was collected from each sample and analyzed by: i) a nanobody-specific detection antibody (GenScript MonoRab™ rabbit anti-cameloid VHH mixture, catalog number A02016), which detects only the intact fusion protein; or ii) an N-terminal specific ELISA, which detects only the released active GLP-1 (Novus Biologicals, catalog number NBP1-05180H).

[0105] For the initial screening, all nine SP01 nanobodies were fused to the N-terminus of SP01 via a 3x(G4S) linker without the Xa factor sequence, and then further fused to the N-terminus of the hIgG4 Fc fragment via the 3x(G4S) linker. Pharmacokinetic results are shown in Table 5.

[0106] Table 5. Pharmacokinetics of the nanobody-SP01 fusion protein in mice.

[0107] In the initial screening, all five SP02 nanobodies were fused to the N-terminus of SP02 via a 3x(G4S) linker without the Xa factor sequence, and then further fused to the N-terminus of the amG2 light chain via the 3x(G4S) linker. Pharmacokinetic results are shown in Table 6.

[0108] Table 6. Pharmacokinetics of the nanobody-SP02 fusion protein in mice

[0109] Based on the preliminary screening results of the nanobodies, nanobodies P56 and P67 were selected for more detailed research.

[0110] Example 6: Exemplary fusion protein containing SP01 To confer better resistance to protease degradation, the first part of the technology involves using a linker containing a factor Xa digestion site to introduce a protective antibody against a GLP1 analog at its N-terminus. This protective antibody specifically binds to the GLP1 analog and protects it from proteolytic cleavage. Because the N-terminus of GLP1 is hidden, the fusion is completely resistant to DPP4 cleavage. The active GLP-1 analog can be released by digestion with factor Xa in the blood. To further extend the half-life of the GLP1 fusion protein, the second part of the technology involves fusing the protected GLP1 analog with an immunoglobulin Fc fragment or a GIPR blocking antibody (GIPR antagonist) to form a bispecific agonist of GLP1R and GIPR.

[0111] For example, SP01-binding nanobody P67 (SEQ ID NO:21) was fused with the GLP1 analog SP01 (SEQ ID NO:2) to protect it from proteolytic cleavage. To further extend its half-life, the aforementioned nanobody-GLP1 fusion protein was fused with the immunoglobulin Fc fragment of IgG4, as shown below: P93 (SEQ ID NO:22): Nanobody P67 (SEQ ID NO:21) is fused to the N-terminus of GLP1 analog SP01 via a linker (GGGGSGGGGSGGSGGSRGER) with the factor Xa protein hydrolysis cleavage sequence RGER. Then, the above P67-GLP1 fusion protein is further fused to the N-terminus of the Fc fragment of human IgG4 via a linker (GGGGSGGGGSGGGS).

[0112] P99 (SEQ ID NO:23): Nanobody P67 (SEQ ID NO:21) is fused to the N-terminus of GLP1 analog SP01 via a linker (GGGGSGGGGGGSGGSGSRKR) with the factor Xa protein hydrolysis cleavage sequence RKR. Then, the above P67-GLP1 fusion protein is further fused to the N-terminus of the Fc fragment of human IgG4 via a linker (GGGGSGGGGSGGGGS).

[0113] Bispecific antibodies against GLP1 agonists and GIP antagonists have shown good hypoglycemic and weight loss effects (Amgen patent). To achieve dual specificity as both a GLP1 agonist and a GIP antagonist, the aforementioned P67-GLP1 fusion protein was further linked to the N-terminus of the light chain of a GIP receptor blocking antibody, including the heavy chain variable region shown in SEQ ID NO:24 and the light chain variable region shown in SEQ ID NO:25 (hereinafter referred to as "amG3", MAbs. 2020 Jan-Dec; 12(1):1710047). P86 (SEQ ID NO:26-27): Nanobody P67 (SEQ ID NO:21) is fused to the N-terminus of GLP1 analog SP01 via a linker (GGGGSGGGGSGGSGGSRGER), and then the fusion protein is further fused to the N-terminus of the amG3 light chain via a linker (GGGGSGGGGSGGSGGSGGS).

[0114] P98 (SEQ ID NO:28-27): Nanobody P67 (SEQ ID NO:21) is fused to the N-terminus of GLP1 analog SP01 via a linker (GGGGSGGGGGGGGSGGSRKR), and then the fusion protein is further fused to the N-terminus of the amG3 light chain via a linker (GGGGSGGGGSGGGSGGSGGS).

[0115] Example 7: Exemplary fusion protein containing SP02 To confer better resistance to protease degradation, the SP02 dual agonist (SEQ ID NO:4) is fused to its bound nanobody P56 via a linker containing a factor Xa digestion site to protect it from proteolytic cleavage. To further extend its half-life and compensate for potential reduced activity, the aforementioned nanobody-SP02 fusion protein is further fused with an antibody containing the heavy chain variable region shown in SEQ ID NO:30 and the light chain variable region shown in SEQ ID NO:31 (hereinafter referred to as "amG2"; Xiaoshan Min et al. Molecular mechanism of an antagonistic antibody against glucose-dependent insulinotropic polypeptide receptor. Mabs, Nov-December 2020; 12(1):1710047); or further fused with a bispecific antibody containing the heavy chain variable region shown in SEQ ID NO:32 and the light chain variable region shown in SEQ ID NO:32 (hereinafter referred to as "ahG1"; U.S. Patent Application No. 20060275288 [Abbott Laboratories]).

[0116] P95 (SEQ ID NO:35-36): Nanobody P56 (SEQ ID NO:34) is fused to the N-terminus of SP02 via a linker (GGGGSGGSGGGSGGSRGER), and then the fusion protein is further fused to the N-terminus of the amG2 light chain via a linker (GGGGSGGGSGGSGGS).

[0117] P100 (SEQ ID NO:37-36): Nanobody P56 (SEQ ID NO:34) is fused to the N-terminus of SP02 via a linker (GGGGSGGGGSGGGSRKR), and then further fused to the N-terminus of the amG2 light chain via a linker (GGGGSGGGGSGGSGGS).

[0118] Example 8: Pharmacokinetic Study of the Exemplary Fusion Proteins in Examples 6-7 Pharmacokinetic studies of the fusion proteins P93 and P99 were performed in C57BL mice using methods similar to those described in Example 5, at doses of 100 and 300 nmol / kg. The intact fusion proteins and released active GLP1 in collected plasma samples were detected by ELISA, and the results are shown below. Figure 1 In the middle. At doses of 100 or 300 nmol / kg, the half-lives of the fusion protein P93 with the slower factor Xa digestive linker (RGER) were 143 h and 175 h, respectively. Figure 1 A, 1B); while at doses of 100 or 300 nmol / kg, the half-life of the fusion protein P99 with the rapid factor Xa digestion linker (RKR) was 36 hours or 35 hours, respectively. Figure 1 C, 1D). Figure 1 E and 1F showed that P93 could stably release the active GLP1 fusion protein at a constant level of approximately 2 ng / ml; while P99 could rapidly release the active GLP1 fusion protein ( Figure 1 (G, 1H). Therefore, the half-life of P99 is shorter than that of P93.

[0119] Pharmacokinetic studies of the fusion protein P95 were performed in C57BL mice using a method similar to that described in Example 5, at doses of 100 and 300 nmol / kg. The concentration of intact fusion protein P95 in collected plasma samples was determined by ELISA, and the results are shown below. Figure 2 In the middle. At doses of 100 or 300 nmol / kg, the half-life of the fusion protein P95 was 95.3 hours and 115 hours, respectively. Figure 2 A, 2B).

[0120] Notably, all protected fusion proteins exhibited superior pharmacokinetic properties in C57BL mice compared to dulaglutide (TRULICITY® brand) and the unprotected P46.

[0121] Example 9: Effect of fusion protein on glucose tolerance in C57BL mice The effect of the fusion protein on glucose tolerance was determined in C57BL mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). Each group (n=6 per group) received a single subcutaneous injection of PBS-mediated control, dulaglutide (TRULICITY® brand) (Vetter Pharma-Fertigung KG, Lilly France), P46, P28, P93, P99, P86, P98, P95, or P100 at doses of 100 or 300 nmol / kg. Intraperitoneal glucose tolerance tests (IPGTT) were performed on days 1, 7, 14, 21, and 28, after a 6-hour fast, with tail vein blood glucose measurements taken at 0, 15, 30, 60, and 120 minutes. The mean ± SEM of blood glucose levels at each time point is shown in [Figure / Image / Text]. Figure 3 .

[0122] In C57BL mice, three administrations of the protected fusion proteins P93 and P99 at doses of 100 or 300 nmol / kg resulted in a significant decrease in glucose levels that lasted for up to 28 days. Figure 3 I). P93 and P99 showed very stable blood glucose-lowering effects on days 1, 7, and 14. Figure 3 E, 3F, 3G), while dulaglutide (TRULICITY® brand) showed a very strong reduction in glucose on day 1 ( Figure 3 A), but it weakened rapidly on days 7 and 14 ( Figure 3 B, 3C), and completely lost their effect on the 21st day ( Figure 3 (D), which is consistent with pharmacokinetic results that P93 and P99 can release active GLP1 in a controlled manner and maintain steady-state plasma concentrations, while dulaglutide (TRULICITY® brand) is rapidly degraded.

[0123] A single administration of the protected fusion proteins P86 and P98 at a dose of 100 nmol / kg showed a longer duration of hypoglycemic effect than the unprotected fusion protein P46. Figure 4 P86 and P98 are in sections 1 and 7 (). Figure 4 D, 4E) and 14 days ( Figure 4 F) improved glucose tolerance, while P98 still had some effect on day 21. P46 only showed improvement on days 1 and 5. Figure 4 On days A and 4B, there was a blood sugar lowering effect, while on day 10 ( Figure 4 C) Completely loses its effect. P86 and P98 have lower blood sugar lowering ability than P93 and P99 because they contain GIP blocking antibodies that block the activity of GIP receptors.

[0124] Only a slight decrease in plasma glucose was observed with the fusion protein P95 (data not shown), possibly because active SP02 is more difficult to release due to differences in the protective nanobody and the different N-terminus of SP02 adjacent to the digestion site of factor Xa. A 60 nmol / kg dose of P100 showed improvement on day 1 ( Figure 5 C) and day 7 ( Figure 5 D) has a glucose-lowering effect, while the unprotected fusion proteins P28 and telpoeptide P19 showed only a decrease in glucose on day 1. Figure 5 A) has a glucose-lowering effect, while on the 7th day ( Figure 5 B) Loss of all effect. In our experiments, the concentration of released active SPO2 could not be measured due to the lack of a specific detection antibody for N-terminal SPO2.

[0125] Example 10: Weight Loss Experiment in DIO Mice To further characterize and understand the efficacy of the fusion protein, body weight and metabolic parameters were measured in DIO mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) throughout the 28-day study. Each group (n=6 mice per group) received dulaglutide (TRULICITY® brand), telposide (P19), and P46 twice weekly at doses of 10 or 30 nmol / kg; and P93, P86, P95, P98, and P99 once weekly at doses of 100 nmol / kg.

[0126] Treatment with LY3298176 (telpotetide, a GLP1 / GIP dual receptor agonist) resulted in more significant dose-dependent weight loss compared to dulaglutide (TRULICITY® brand). Figure 6 The weight loss achieved with LY3298176 was statistically significant compared to dulaglutide (TRULICITY® brand).

[0127] After 28 days, mice treated with P93 or P99 at a dose of 100 nmol / kg once weekly showed a weight loss of 12.2% and 20.7%, respectively. Figure 6 Mice treated with Trulicity at 10 or 30 nmol / kg twice weekly experienced a weight loss of 10.9% or 16.6%, respectively. Figure 6 Mice treated with 30 nmol / kg Tirzepatide showed a 26% reduction in body weight. Mice treated with 100 nmol / kg P86 or P98 showed a 7.8% and 12.2% reduction in body weight, respectively.

[0128] The effects of all fusion proteins on glycemic reduction and food intake were determined in the DIO mice described above. Figure 7All mice underwent blood sampling for 6 hours after fasting to measure blood glucose levels twice weekly. The mean SEM of blood glucose levels at each time point for each group was calculated and is shown in the figure. Figure 7 In group A, all tested samples showed a significant reduction in glucose, with effects comparable to 30 nmol / kg Trulicity or Telborpeptide, and superior to 10 nmol / kg Trulicity. Food intake was measured every two days. Figure 7 B), and the cumulative food intake reduction was greatest with a dose of P99 of 100 nmol / kg per week.

[0129] As can be seen, the implementation scheme of this application verifies that the fusion protein of this application has an extended half-life and has the technical effects of weight loss and blood sugar reduction.

[0130] The scope of this disclosure is not limited to the specific embodiments described herein. In fact, various modifications to the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

[0131] sequence list 1. SEQ ID NO:1: GLP1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 2. SEQ ID NO:2: SP01 GLP1 analogue HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG 3. SEQ ID NO:3: Telborpeptide Y[Aib]EGTFTSDYSI[Aib]LDKIAQK[diacid-γ-Glu-(AEEA)2] AFVQWLIAGGPSSGAPPPS 4. SEQ ID NO:4: SP02 GLP1 / GIP dual agonist YGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPS 5. SEQ ID NO:5: SP03 GLP-1 / GIP dual agonist YGEGTFTSDYSIQLDKIAQKAFVQWLIAGGPSSGAPPPS 6. SEQ ID NO:6: P62-hIgG1Fc QVQLVETGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAVISRSGDVTTYAHSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAGLAANTANYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 7.SEQ ID NO:7: P64-hIgG1Fc EVQLQESGGGLVQAGGSLRLSCAASGSISSLNGIWYRQAPGKQREVVAGITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNGLKPEDTAIYYCNTVRNWDIRTALSRSGGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 8.SEQ ID NO:8: P65-hIgG1Fc QVQLVESGGGLVQPGGSLRLSCAASGSIFAINVMGWYRQASGNQRELVAGITTGGARDYADSVKGRFTISRDNAKNTVYLSMNSLKPEDTAVYYCNLHAPKYIDSRYDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9.SEQ ID NO:9: P66-hIgG1Fc EVQVQESGGGLVQAGGSLRLSCAASGRSFRSDRMGWFRQAPGKEREFVAVISRGGGVTTYADSVKGRFTVSRDDAKNTVDLQMNSLKPEDTAVYYCAAGITTKAANYWGMGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10.SEQ ID NO:10: P67-hIgG1Fc QVQLVESGGGLVQPGGSLRLSCAASGSVFSRNFMGWYRQAPGKQRELVAYITVDGGTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNRAGTTPYAWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11.SEQ ID NO:11: P68-hIgG1Fc QVRLVESGGGLVQAGGSLRLSCTASGRTFSSYSVGWFRQAPGKERAFVAVISKSGGITTYADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCAASGSSLRLADSGSWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 12.SEQ ID NO:12: P70-hIgG1Fc EVQLVESGGGLVQAGGSLRLSCVASGRTFGNYRMAWFRQAPGKEREFVAVISRNGGITTYADSAKGRFTLSRDNAKNAVYLQMNSLKPEDTAVYYCTGGRNLGTWDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 13.SEQ ID NO:13: P71-hIgG1Fc QVQLVESGGGLVQAGGSLRLSCVASGRTFSSGRMAWFRQAPGKEREFVAVISQNGGVTTYAASVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYCAAGVTTNTANYWGQGTRVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14.SEQ ID NO:14: P72-hIgG1Fc EVQLQESGGGLVQAGGSLRLSCVVSGRTFGSLRMAWFRQAPGKEREFVSIISKSGGITTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADASIRPRTNPSDYDGWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15.SEQ ID NO:15: P73-hIgG1Fc QVQLVESGGGLVQPGGSLRLSCVASGNIFSSSIMGWYRQAPGKQREAVAFITNGGSTDYADSVKGRFTISRDNAKNTYLQMNSLKFEDTAVYYCNLRNQRNFQEGSLGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 16.SEQ ID NO:16: P49-hIgG1Fc EVQLVESGGGLVQAGGSLRLSCAASGIDFRADVMAWYRQAPGKQREVVAAITAYGDTDYVDSVKGRFTISRDNAKNTVYLLMNSLKPEDTAVYYCNVPVRRYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 17.SEQ ID NO:17: P55-hIgG1Fc EVQVVESGGGLVQPGGSLRLSCAASGSYFSINVMAWYRQAPGKQREVVAAISRGGFADYAQFAKGRFTISGDNAKNTVSLQMNSLKPEDTAVYYCNARRAWDTAINYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 18.SEQ ID NO:18: P56-hIgG1Fc QVQLVESGGGLVQPGGSLRLSCAASGSIHSINILGWYRQAPGDHRELVAGITTGGSTDYADSVKDRFTISRDNAKNTVYLQMNDLKPADTAVYYCNLRQSSTRLTWRPYDSWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19.SEQ ID NO:19: P58-hIgG1Fc QVQLVESGGGLVQPGGSLRLSCAASGFTFSSTSMTWVRQRPGRGLEWVSSIFSDGRSTYYADSLKGRVTISRDNAENTLYLQMNSLQSEDTAVYYCAKGLPSLRVGVMGLLRGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20.SEQ ID NO:20: P59-hIgG1Fc EVRLVESGGGLVQAGGSLRLSCAASGISFRADVMAWYRQAPGKQREVVAAITSYGDADYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVPARRYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 21. SEQ ID NO: 21: Nanobody of P67 SP01 QVQLVESGGGLVQPGGSLRLSCAASGSVFSRNFMGWYRQAPGKQRELVAYITVDGGTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNRAGTTPYAWGQGTLVTVSS 22. SEQ ID NO: 22: P93:P67-RGER-SP01- hIgG4Fc QVQLVESGGGLVQPGGSLRLSCAASGSVFSRNFMGWYRQAPGKQRELVAYITVDGGTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNRAGTTPYAWGQGTLVTVSSGGGGSGGGGSGGGGSRGERHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 23.SEQ ID NO: 23: P99:P67-RKR-SP01-hIgG4Fc QVQLVESGGGLVQPGGSLRLSCAASGSVFSRNFMGWYRQAPGKQRELVAYITVDGGTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNRAGTTPYAWGQGTLVTVSSGGGGSGGGGSGGGGSRKRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 24.SEQ ID NO: 24: amG3 Heavy chain variable region QVQLQQPGAELVKPGASVKLSCRASGYTFTSNWMHWVKQRPRQGLEWIGEINPSNGRSNYNEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARFYYGTSWFAYWGQGTLVAVSA 25.SEQ ID NO: 25: amG3 Light chain variable region DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAADLGVYFCSQSTHVPPFTFGGGTKLEIK 26.SEQ ID NO: 26: P86:P67-RGER-SP01-amG3 light chain QVQLVESGGGLVQPGGSLRLSCAASGSVFSRNFMGWYRQAPGKQRELVAYITVDGGTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNRAGTTPYAWGQGTLVTVSSGGGGSGGGGSGGGGSRGERHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAADLGVYFCSQSTHVPPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 27. SEQ ID NO: 27: P86 heavy chain, P46 heavy chain, amG3 heavy chain pcDNA3.4-SIP008-amG3-HC(IgG4SP-AA-no K) QVQLQQPGAELVKPGASVKLSCRASGYTFTSNWMHWVKQRPRQGLEWIGEINPSNGRSNYNEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARFYYGTSWFAYWGQGTLVAVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 28. SEQ ID NO: 28: P98:P67-RKR-SP01-amG3 light chain QVQLVESGGGLVQPGGSLRLSCAASGSVFSRNFMGWYRQAPGKQRELVAYITVDGGTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNRAGTTPYAWGQGTLVTVSSGGGGSGGGGSGGGGSRKRHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAADLGVYFCSQSTHVPPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 29. SEQ ID NO: 29: P46:SP01-amG3 light pcDNA3.4-SIP008-SP01-amG3-LC(κ) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAADLGVYFCSQSTHVPPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 30. SEQ ID NO: 30: amG2 heavy chain variable region DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARGKYPTWIAYWGQGTLVTVSA 31.SEQ ID NO: 31: amG2 light chain variable region DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELK 32.SEQ ID NO: 32: ahG1 heavy chain variable region QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSS 33.SEQ ID NO: 33: ahG1 light chain variable region QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKR 34.SEQ ID NO: 34: P56: Antibody against SP02 QVQLVESGGGLVQPGGSLRLSCAASGSIHSINILGWYRQAPGDHRELVAGITTGGSTDYADSVKDRFTISRDNAKNTVYLQMNDLKPADTAVYYCNLRQSSTRLTWRPYDSWGQGTQVTVSS 35.SEQ ID NO: 35: P95: P56-RGER-SP02-amG2 light chain QVQLVESGGGLVQPGGSLRLSCAASGSIHSINILGWYRQAPGDHRELVAGITTGGSTDYADSVKDRFTISRDNAKNTVYLQMNDLKPADTAVYYCNLRQSSTRLTWRPYDSWGQGTQVTVSSGGGGSGGGGSGGGGSRGERYGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 36. SEQ ID NO: 36: P95 heavy chain, P100 heavy chain, pcDNA3.4-SIP009-amG2-HC(IgG4SP-AA-No K) DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARGKYPTWIAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 37. SEQ ID NO: 37: P100: P56-RKR-SP02-amG2 Light Chain QVQLVESGGGLVQPGGSLRLSCAASGSIHSINILGWYRQAPGDHRELVAGITTGGSTDYADSVKDRFTISRDNAKNTVYLQMNDLKPADTAVYYCNLRQSSTRLTWRPYDSWGQGTQVTVSSGGGGSGGGGSGGGGSRKRYGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 38. SEQ ID NO: 38: P28 Light Chain: SP02-amG2 Light Chain YGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 39. SEQ ID NO: 39: P28 Heavy Chain pcDNA3.4-SIP003-amG2-HC(IgG4) DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARGKYPTWIAYWGQG TLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 40.SEQ ID NO:40:P26:L1:pcDNA3.4-SIP004- SP02-(G4S)3- amG2-LC(κ) YGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISS VQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 41.SEQ ID NO:41:P26:L2:pcDNA3.4-SIP004-SP02-(G4S)3-ahG1(Abt)-LC(κ) YGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 42.SEQ ID NO: 42: P26: H1: pcDNA3.4-SIP004-amG2-HC(IgG1)-Knob DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARGKYPTWIAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 43.SEQ ID NO: 43: P26: H2: pcDNA3.4-SIP004-ahG1(Abt)-HC(IgG1)-Hole QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 44.SEQ ID NO:44: P62-3x(G4S)-SP01-3x(G4S)- hIgG4Fc QVQLVETGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAVISRSGDVTTYAHSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAGLAANTANYWGQGTQVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 45.SEQ ID NO:45: P64-3x(G4S)-SP01-3x(G4S)- hIgG4Fc EVQLQESGGGLVQAGGSLRLSCAASGSISSLNGIWYRQAPGKQREVVAGITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNGLKPEDTAIYYCNTVRNWDIRTALSRSGGQGTQVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 46.SEQ ID NO:46: P66-3x(G4S)-SP01-3x(G4S)- hIgG4Fc EVQVQESGGGLVQAGGSLRLSCAASGRSFRSDRMGWFRQAPGKEREFVAVISRGGGVTTYADSVKGRFTVSRDDAKNTVDLQMNSLKPEDTAVYYCAAGITTKAANYWGMGTQVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 47.SEQ ID NO:47: P67-3x(G4S)-SP01-3x(G4S)- hIgG4Fc QVQLVESGGGLVQPGGSLRLSCAASGSVFSRNFMGWYRQAPGKQRELVAYITVDGGTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNRAGTTPYAWGQGTLVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 48.SEQ ID NO:48: P68-3x(G4S)-SP01-3x(G4S)- hIgG4Fc QVRLVESGGGLVQAGGSLRLSCTASGRTFSSYSVGWFRQAPGKERAFVAVISKSGGITTYADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCAASGSSLRLADSGSWGQGTQVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 49.SEQ ID NO:49: P70-3x(G4S)-SP01-3x(G4S)- hIgG4Fc EVQLVESGGGLVQAGGSLRLSCVASGRTFGNYRMAWFRQAPGKEREFVAVISRNGGITTYADSAKGRFTLSRDNAKNAVYLQMNSLKPEDTAVYYCTGGRNLGTWDYWGQGTQVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 50.SEQ ID NO:50: P71-3x(G4S)-SP01-3x(G4S)- hIgG4Fc QVQLVESGGGLVQAGGSLRLSCVASGRTFSSGRMAWFRQAPGKEREFVAVISQNGGVTTYAASVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAGVTTNTANYWGQGTRVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 51.SEQ ID NO:51: P72-3x(G4S)-SP01-3x(G4S)- hIgG4Fc EVQLQESGGGLVQAGGSLRLSCVVSGRTFGSLRMAWFRQAPGKEREFVSIISKSGGITTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADASIRPRTNPSDYDGWGQGTQVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 52.SEQ ID NO: 52: P73-3x(G4S)-SP01-3x(G4S)-hIgG4Fc QVQLVESGGGLVQPGGSLRLSCVASGNIFSSSIMGWYRQAPGKQREAVAFITNGGSTDYADSVKGRFTISRDNAKNTTYLQMNSLKFEDTAVYYCNLRNQRNFQEGSLGQGTQVTVSSGGGGSGGGGSGGGGSHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 53.SEQ ID NO: 53: P49-3x(G4S)-SP02-3x(G4S)-amG2 light chain EVQLVESGGGLVQAGGSLRLSCAASGIDFRADVMAWYRQAPGKQREVVAAITAYGDTDYVDSVKGRFTISRDNAKNTVYLLMNSLKPEDTAVYYCNVPVRRYWGQGTQVTVSSGGGGSGGGGSGGGGSYGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 54. SEQ ID NO: 54: P55-3x(G4S)-SP02-3x(G4S)-amG2 light chain EVQVVESGGGLVQPGGSLRLSCAASGSYFSINVMAWYRQAPGKQREVVAAISRGGFADYAQFAKGRFTISGDNAKNTVSLQMNSLKPEDTAVYYCNARRAWDTAINYWGQGTQVTVSSGGGGSGGGGSGGGGSYGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 55. SEQ ID NO: 55: P56-3x(G4S)-SP02-3x(G4S)-amG2 light chain QVQLVESGGGLVQPGGSLRLSCAASGSIHSINILGWYRQAPGDHRELVAGITTGGSTDYADSVKDRFTISRDNAKNTVYLQMNDLKPADTAVYYCNLRQSSTRLTWRPYDSWGQGTQVTVSSGGGGSGGGGSGGGGSYGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 56. SEQ ID NO: 56: P58-3x(G4S)-SP02-3x(G4S)-amG2 light chain QVQLVESGGGLVQPGGSLRLSCAASGFTFSSTSMTWVRQRPGRGLEWVSSIFSDGRSTYYADSLKGRVTISRDNAENTLYLQMNSLQSEDTAVYYCAKGLPSLRVGVMGLLRGQGTLVTVSSGGGGSGGGGSGGGGSYGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 57. SEQ ID NO: 57: P59-3x(G4S)-SP02-3x(G4S)-amG2 light chain EVRLVESGGGLVQAGGSLRLSCAASGISFRADVMAWYRQAPGKQREVVAAITSYGDADYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVPARRYWGQGTQVTVSSGGGGSGGGGSGGGGSYGEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 58. SEQ ID NO: 58: P62: Antibody against SP01 QVQLVETGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAVISRSGDVTTYAHSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAGLAANTANYWGQGTQVTVSS 59. SEQ ID NO: 59: P64: Antibody against SP01 EVQLQESGGGLVQAGGSLRLSCAASGSISSLNGIWYRQAPGKQREVVAGITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNGLKPEDTAIYYCNTVRNWDIRTALSRSGGQGTQVTVSS 60. SEQ ID NO: 60: P65: Antibody against SP01 QVQLVESGGGLVQPGGSLRLSCAASGSIFAINVMGWYRQASGNQRELVAGITTGGARDYADSVKGRFTISRDNAKNTVYLSMNSLKPEDTAVYYCNLHAPKYIDSRYDYWGQGTQVTVSS 61. SEQ ID NO: 61: P66: Antibody against SP01 EVQVQESGGGLVQAGGSLRLSCAASGRSFRSDRMGWFRQAPGKEREFVAVISRGGGVTTYADSVKGRFTVSRDDAKNTVDLQMNSLKPEDTAVYYCAAGITTKAANYWGMGTQVTVSS 62. SEQ ID NO: 62: P68: antibody against SP01 QVRLVESGGGLVQAGGSLRLSCTASGRTFSSYSVGWFRQAPGKERAFVAVISKSGGITTYADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCAASGSSLRLADSGSWGQGTQVTVSS 63. SEQ ID NO: 63: P70: antibody against SP01 EVQLVESGGGLVQAGGSLRLSCVASGRTFGNYRMAWFRQAPGKEREFVAVISRNGGITTYADSAKGRFTLSRDNAKNAVYLQMNSLKPEDTAVYYCTGGRNLGTWDYWGQGTQVTVSS 64. SEQ ID NO: 64: P71: antibody against SP01 QVQLVESGGGLVQAGGSLRLSCVASGRTFSSGRMAWFRQAPGKEREFVAVISQNGGVTTYAASVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAGVTTNTANYWGQGTRVTVSS 65. SEQ ID NO: 65: P72: antibody against SP01 EVQLQESGGGLVQAGGSLRLSCVVSGRTFGSLRMAWFRQAPGKEREFVSIISKSGGITTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADASIRPRTNPSDYDGWGQGTQVTVSS 66. SEQ ID NO: 66: P73: antibody against SP01 QVQLVESGGGLVQPGGSLRLSCVASGNIFSSSIMGWYRQAPGKQREAVAFITNGGSTDYADSVKGRFTISRDNAKNTTYLQMNSLKFEDTAVYYCNLRNQRNFQEGSLGQGTQVTVSS 67. SEQ ID NO: 67: P49: Antibody against SP02 EVQLVESGGGLVQAGGSLRLSCAASGIDFRADVMAWYRQAPGKQREVVAAITAYGDTDYVDSVKGRFTISRDNAKNTVYLLMNSLKPEDTAVYYCNVPVRRYWGQGTQVTVSS 68. SEQ ID NO: 68: P55: Antibody against SP02 EVQVVESGGGLVQPGGSLRLSCAASGSYFSINVMAWYRQAPGKQREVVAAISRGGFADYAQFAKGRFTISGDNAKNTVSLQMNSLKPEDTAVYYCNARRAWDTAINYWGQGTQVTVSS 69. SEQ ID NO: 69: P58: Antibody against SP02 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSTSMTWVRQRPGRGLEWVSSIFSDGRSTYYADSLKGRVTISRDNAENTLYLQMNSLQSEDTAVYYCAKGLPSLRVGVMGLLRGQGTLVTVSS 70. SEQ ID NO: 70: P59: Antibody against SP02 EVRLVESGGGLVQAGGSLRLSCAASGISFRADVMAWYRQAPGKQREVVAAITSYGDADYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVPARRYWGQGTQVTVSS.

Claims

1. A glucagon-like peptide-1 (GLP1) analog SP01 having the amino acid sequence SEQ ID NO:2; or a GLP1 / GIP receptor co-agonist analog SP02 or SP03 having the amino acid sequences SEQ ID NO:4 or SEQ ID NO:5, respectively.

2. A fusion protein comprising: a) a GLP1 analog SP01 or a GLP1 / GIP receptor co-agonist analog SP02 or SP03 as described in claim 1; and b) a protective antibody fused to the N-terminus of the GLP1 analog or the GLP1 / GIP receptor co-agonist analog via a linker.

3. The fusion protein of claim 2, wherein the protective antibody is a nanobody.

4. The fusion protein of claim 3, wherein the linker is 3XG4S (GGGGSGGGGSGGGGS), followed by a protein hydrolysis cleavage sequence.

5. The fusion protein of claim 4, wherein the protein hydrolysis cleavage sequence is factor Xa and its variants, the variants being selected from the group consisting of RGER, RKR, RGR, RSR and RR.

6. The fusion protein of claim 3, wherein the fusion protein comprises: a) the GLP1 analog SP01; and b) the nanobody fused to the N-terminus of the GLP1 analog, and the sequence of the nanobody has at least 90% identity with any one of SEQ ID NO: 21, 58, 59 and 61 to 66.

7. The fusion protein of claim 6, wherein the sequence of the nanobody is any one of SEQ ID NO: 21, 58, 59 and 61 to 66.

8. The fusion protein of claim 3, wherein the fusion protein comprises: a) the GLP1 / GIP receptor co-agonist analog SP02; and b) the nanobody fused to the N-terminus of the GLP1 / GIP analog, the nanobody having a sequence identity of at least 90% with any one of SEQ ID NO:34 and 67 to 70.

9. The fusion protein of claim 8, wherein the sequence of the nanobody is any one of SEQ ID NO:34 and 67 to 70.

10. The fusion protein of claim 2, wherein the GLP1 analog or the GLP1 / GIP receptor co-agonist analog is further fused to the stable domain at the C-terminus via a linker.

11. The fusion protein of claim 10, wherein the GLP1 analog or the GLP1 / GIP receptor co-agonist analog is further fused at its C-terminus to: c) the N-terminus of the Fc portion of an immunoglobulin; or d) the N-terminus of the light or heavy chain of an antibody that specifically binds to the GLP1 receptor or an antigen-binding fragment thereof; or e) the N-terminus of the light or heavy chain of an antibody that specifically binds to the GIP receptor or specifically blocks GIPR activity or an antigen-binding fragment thereof; or f) the N-terminus of the light or heavy chain of a bispecific antibody that specifically binds to both the GLP1 receptor and the GIP receptor or an antigen-binding fragment thereof; or g) the N-terminus of a nanobody that specifically binds to albumin; or h) the N-terminus of an albumin-binding domain (ABD) that specifically binds to albumin.

12. The fusion protein of claim 11, wherein the immunoglobulin is derived from IgG4 or IgG2 or variants thereof.

13. The fusion protein of claim 10, wherein the linker is 3xG4S (GGGGSGGGGSGGGGS).

14. A nucleic acid molecule encoding the fusion protein as described in claim 2.

15. A recombinant expression vector capable of expressing the fusion protein as described in claim 2.

16. A pharmaceutical composition comprising a therapeutically effective amount of at least one agent selected from the fusion protein of claim 2, and a pharmaceutical carrier or excipient.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition further comprises a second therapeutic agent.

18. A method of treatment for preventing, improving or treating GLP1-related diseases or conditions in a subject, wherein the method of treatment comprises administering a therapeutically effective amount of at least one agent selected from the fusion protein of claim 2.

19. The treatment method of claim 18, wherein the GLP1-related disease or condition is diabetes, obesity, or type 2 diabetes.

20. A method of treatment for preventing, improving or treating GLP1-related diseases or conditions in a subject, wherein the method of treatment comprises administering a therapeutically effective amount of at least one agent selected from the pharmaceutical compositions of claim 16.

21. The treatment method of claim 20, wherein the GLP1-related disease or condition is diabetes, obesity, or type 2 diabetes.

22. A method for lowering blood glucose levels or reducing weight in a subject, wherein the method comprises administering a therapeutically effective amount of at least one agent selected from the fusion protein of claim 2.

23. The method of claim 22, wherein the method further comprises a combination with a second therapeutic agent or therapy.

24. A method for lowering blood glucose levels or reducing weight in a subject, wherein the method comprises administering a therapeutically effective amount of at least one agent selected from the pharmaceutical compositions of claim 16.

25. The method of claim 24, wherein the method further comprises a combination with a second therapeutic agent or therapy.

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