CN116554343A

CN116554343A - Long-acting recombinant human growth hormone and application thereof

Info

Publication number: CN116554343A
Application number: CN202210113780.5A
Authority: CN
Inventors: 韩照中; 潘红芽; 彭孟凡
Original assignee: Linnuo Shanghai Pharmaceutical Technology Co ltd
Current assignee: Linnuo Shanghai Pharmaceutical Technology Co ltd
Priority date: 2022-01-30
Filing date: 2022-01-30
Publication date: 2023-08-08

Abstract

The present application relates to a fusion protein comprising: a transferrin binding protein comprising a polypeptide, antibody or antigen-binding fragment thereof capable of binding transferrin, and a growth hormone or functionally active fragment thereof. The application also provides a preparation method and application of the fusion protein.

Description

Long-acting recombinant human growth hormone and application thereof

Technical Field

The present application relates to the field of biological medicine, in particular to a fusion protein comprising transferrin binding protein and growth hormone, and uses thereof.

Background

At present, growth hormone in the domestic market is divided into a short-acting type and a long-acting type, and the improvement of patient compliance is brought while better treatment effect is realized through iteration from short-acting to long-acting. The 2014 gold-race pharmaceutical industry promotes the independently developed global first-branch polyethylene glycol long-acting growth hormone, so that the growth hormone injection frequency of the infant suffering from the dwarf disease is changed from once daily to once weekly, and from 365-day injection administration to 52-time injection administration. To date, no second long-acting growth hormone has been marketed in batches. However, because the price of the long-acting dosage form is relatively high, the effect of 'later on' is not achieved, the short-acting water injection is still a main current scheme in China, and according to the statistics of related data, the first to third quarters of 2020, in the sales of growth hormone in a domestic sample hospital, the common water injection accounts for 73%, the powder injection accounts for 26% and the long-acting water injection accounts for 1%. Meanwhile, according to the report of Frost & Sullivan, the total sales of recombinant growth hormone in China is 59 hundred million yuan, and the sales of long-acting growth hormone is about 6 hundred million. Therefore, the existing long-acting dosage forms cannot be used by most of children suffering from growth hormone deficiency, and nearly 300 tens of thousands of children in China still suffer from the growth hormone deficiency.

Thus, there is a need to develop more efficient and less costly depot formulations to improve patient compliance and quality of life and reduce healthcare costs.

Disclosure of Invention

The present application provides a fusion protein comprising: a transferrin binding protein comprising a polypeptide, antibody or antigen-binding fragment thereof capable of binding transferrin, and a growth hormone or functionally active fragment thereof. Fusion proteins described herein achieve an extension of half-life and maximum bioavailability of the protein based on the process of binding and circulating inside and outside the cell of transferrin and transferrin receptor. The fusion protein is produced by using prokaryotic escherichia coli, the purity of the fusion protein can reach more than 95% through SEC detection, the protein production is stable, and the molecular weight is as small as 37KD. The protein is more stable with lower coupling cost of PEG compared to vinca Jin Saizeng. Compared with the hybrid FC fusion rhGH protein of the environmental organism TJ101, the molecular weight is 103KD, and the production cost of the escherichia coli is lower when the recombinant rhGH protein is produced by using mammalian cells. Furthermore, the in vitro activity (EC 50 nM) of the rhGH-VHH fusion protein produced in E.coli was reduced by about 1-fold in comparison with native rhGH (EC 50 nM) on a cell model. The fusion protein rhGH-VHH has a better maintenance of biological activity and a low molecular weight and low immunogenicity compared to VRS-317 of Versartis (XTEN 1-rhGH-XTEN 2) having a molecular weight of 119kD, an EC50 of 0.6nM for rhGH, and a 6.8nM for VRS-317EC50, which is reduced by about a factor of 10. In addition, from the analysis of pharmacokinetic studies, 150nmol/kg of the rhGH-VHH fusion protein is administrated to have a half-life of 11.5 hours in mice, while 50nmol/kg half-life of 50nmol/kg of a once-weekly preparation of Novanordisk currently on the market is 5.3 hours in rats, and Pfizer MOD-4023 of BLA application filed 1 month 2021 has a half-life of 3.7 hours in rats, the rhGH-VHH fusion protein of the present application also exhibits superior characteristics in terms of pharmacokinetics for achieving a long-acting dosage form.

In one aspect, the present application provides a fusion protein comprising: a transferrin binding protein comprising a polypeptide, antibody or antigen-binding fragment thereof capable of binding transferrin, and a growth hormone or functionally active fragment thereof.

In certain embodiments, the fusion protein has one or more of the following properties:

(1) Is capable of extending the in vivo half-life of said growth hormone;

(2) Can cross the blood brain barrier;

(3) Can be delivered orally; and

(4) The growth hormone can be delivered to cells expressing transferrin receptor.

In certain embodiments, the transferrin is human transferrin.

In certain embodiments, the antibody is selected from one or more of the following groups: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In certain embodiments, the antigen binding fragment comprises a Fab, fab ', fv fragment, F (ab') ₂ ，F(ab) ₂ scFv, VHH, di-scFv and/or dAb.

In certain embodiments, the transferrin binding protein in the fusion protein is a VHH.

In certain embodiments, the transferrin binding protein in the fusion protein comprises a CDR3, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO. 3.

In certain embodiments, the transferrin in the fusion protein binds to protein CDR2, and the CDR2 comprises the amino acid sequence shown in SEQ ID NO. 2.

In certain embodiments, the transferrin binding protein in the fusion protein comprises CDR1, and the CDR1 comprises the amino acid sequence shown in SEQ ID No. 1.

In certain embodiments, the transferrin binding protein in the fusion protein comprises a VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO. 8.

In certain embodiments, the growth hormone or functionally active fragment thereof in the fusion protein is human growth hormone or functionally active fragment thereof.

In certain embodiments, the growth hormone or functionally active fragment thereof in the fusion protein comprises the amino acid sequence shown in SEQ ID NO. 10.

In certain embodiments, in the fusion protein, the transferrin binding protein and the growth hormone or functionally active fragment thereof are linked directly or indirectly.

In certain embodiments, in the fusion protein, the N-terminus of the transferrin binding protein and the C-terminus of the growth hormone or functionally active fragment thereof are directly or indirectly linked.

In certain embodiments, in the fusion protein, the C-terminus of the transferrin binding protein and the N-terminus of the growth hormone or functionally active fragment thereof are directly or indirectly linked.

In certain embodiments, in the fusion protein, the transferrin binding protein and the growth hormone or functionally active fragment thereof are linked by a linker.

In certain embodiments, in the fusion protein, the linker comprises the amino acid sequence shown in SEQ ID NO. 9.

In certain embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO. 11.

In certain embodiments, the fusion protein is capable of binding to transferrin, and is capable of binding to and/or activating a growth hormone receptor.

In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the fusion proteins described herein.

In another aspect, the present application provides a vector comprising a nucleic acid molecule as described herein.

In another aspect, the present application provides a cell comprising said nucleic acid molecule, and/or said vector.

In another aspect, the present application provides a pharmaceutical composition comprising said fusion protein, said nucleic acid molecule, said vector or said cell, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present application provides the use of the fusion protein, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of a disease and/or disorder.

In certain embodiments, the disease and/or condition comprises a disease and/or condition caused by abnormal growth hormone.

In certain embodiments, the disease and/or condition comprises growth hormone deficiency.

In another aspect, the present application provides a method of preventing and/or treating a disease and/or disorder comprising administering to a subject in need thereof the fusion protein, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition.

In another aspect, the present application also provides a method for increasing the half-life of growth hormone in vivo comprising administering said fusion protein.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The drawings are briefly described as follows:

FIGS. 1A-1C show the binding capacity (A) pH7.4 of a single domain antibody VHH binding transferrin to hTf; (B) pH6.0. VHH binding well to hTf significantly prolonged the half-life of recombinant ovalbumin OVA in mouse blood;

FIGS. 2A-2B show SDS-PAGE (A) and SEC-HPLC (B) analysis of rhGH. The theoretical molecular weight of rhGH is 22kD.

FIGS. 3A-3B show SDS-PAGE (A) and SEC-HPLC (B) analysis of rhGH-VHH. The theoretical molecular weight of rhGH-VHH is 37kD.

FIGS. 4A-4B show the binding capacity assays of rhGH-VHH to human growth hormone receptor (hGH) and human transferrin (hTf).

FIGS. 5A-5B show assays of the binding activity of rhGH-VHH to the cell membrane surface transferrin (hTf)/transferrin receptor (hTfR 1) complex or human growth hormone receptor (hGH).

FIG. 6 shows the establishment and identification of 293F-GAS-GHR stably transformed cell lines.

FIG. 7 shows the biological activity assay of rhGH-VHH.

FIG. 8 shows the development and validation of a pharmacokinetic ELISA method.

FIG. 9 shows the metabolic half-life comparison of rhGH and rhGH-VHH in mouse blood.

FIGS. 10A-10B show pharmacokinetic studies of rhGH-VHH in mice.

FIGS. 11A-11D show the growth phenotype of GHRH deficient mice.

Figures 12A-12D show the therapeutic effect of rhGH in GHRH gene-deficient mice.

FIGS. 13A-13D show the therapeutic effect of rhGH-VHH in mice deficient in the GHRH gene.

Detailed Description

Further advantages and effects of the invention of the present application will be readily apparent to those skilled in the art from the disclosure of the present application by describing embodiments of the invention with specific examples.

Definition of terms

In this application, the term "fusion protein" generally refers to a protein that is fused from two or more proteins or polypeptides. Fusion proteins can be prepared artificially by recombinant DNA techniques. For example, genes or nucleic acid molecules encoding the two or more proteins or polypeptides may be linked to each other to form a fusion gene or fused nucleic acid molecule, which may encode the fusion protein. Translation of the fusion gene may result in a single polypeptide, which may have the properties of at least one, and even each, of the two or more proteins or polypeptides prior to fusion.

In the present application, the term "transferrin" generally refers to a glycoprotein that is capable of binding and transporting multivalent ions. For example, transferrin can be a single chain glycoprotein. For example, transferrin can have at least one ion binding site. For example, the ion binding sites may have different affinities for iron ions. For example, the multivalent iron ion may be an iron ion, a chromium ion, a manganese ion, a cadmium ion, or a nickel ion thereof. For example, each molecule of transferrin can bind two ferric atoms. For example, the transferrin may be iron-containing Holo-transferrin, or iron-free apo-transferrin. For example, the transferrin may be mouse transferrin. For example, the amino acid sequence of mouse transferrin can be specified in GenBank. EDL21066.1, AAL34533.1, or AAL34533.1. For example, the transferrin may be human transferrin. For example, the amino acid sequence of human transferrin may be specified in GenBank. AAH59367.1, AAH59367.1, or AAB22049.1. In the present application, the term "transferrin" may comprise functionally active fragments, homologues, analogues and/or variants thereof.

In the present application, the term "transferrin binding protein" generally refers to a protein comprising a transferrin binding moiety, and optionally allows the antigen binding moiety to adopt a scaffold or backbone moiety that facilitates the conformation of the antigen binding protein to bind an antigen. For example, transferrin binding proteins described herein can include, but are not limited to, antibodies, antigen binding fragments (Fab, fab ', F (ab) 2, fv fragments, F (ab') 2, VHH, scFv, diav, and/or dAb), immunoconjugates, multispecific antibodies, antibody fragments, antibody derivatives, antibody analogs, or fusion proteins so long as they exhibit the desired antigen binding activity. For example, antigen binding proteins are capable of specifically binding to transferrin. For example, the transferrin binding protein may not interfere with the interaction between transferrin and transferrin receptor 1. For example, the transferrin binding protein may not affect Tf/TfR1 binding. For example, transferrin binding proteins can maintain normal physiological functions of iron transport.

In this application, the term "antibody" generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. For example, immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. For example, light chains can be classified as kappa or lambda, which can define immunoglobulin types, respectively: igκ and igλ. Heavy chains can be classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes, respectively: igG, igM, igA, igD and IgE. For example, an antibody may have structural units comprising tetramers, each tetramer may be composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy chain" (about 50-70 kD), each member having an N-terminal end that may define a variable region of about 100 to 110 amino acids or more, which is primarily responsible for antigen recognition. For example, the terms "light chain variable region (VL)" and "heavy chain variable region (VH)" generally refer to the variable region regions of the light and heavy chains, respectively. Antibodies may exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases or de novo expression.

In the present invention, the term "antigen-binding fragment" generally refers to one or more portions of a full-length antibody that retain substantially the ability to bind to the same antigen (e.g., CD 38) to which the antibody binds, capable of competing with the full-length antibody for specific binding to the antigen. In some cases, antigen binding fragments include Fab, fab ', F (ab') 2, (Fab) 2, fd, fv, dAb and Complementarity Determining Region (CDR) fragments, VHH, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabodies) and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide.

In the present application, the term "VHH" generally refers to an antibody comprising the variable antigen binding domain of a heavy chain antibody (see Vanland choot P. Et al 2011,Antiviral Research 92, 389-407). VHH may also be referred to as Nanobody (Nb) and/or single domain antibodies.

In this application, the term "functionally active fragment" generally refers to a fragment that has a partial region of a full-length protein or nucleic acid, but retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment may retain or partially retain the ability of a full-length protein to bind to another molecule. For example, a functionally active fragment of growth hormone may retain or partially retain the biologically active function of full length growth hormone that causes cell proliferation.

In the present application, the term "growth hormone" may comprise wild-type growth hormone, as well as modified growth hormone. For example, the growth hormone may comprise a homologue, analogue, derivative and/or functional variant thereof. For example, the growth hormone may comprise full length growth hormone and the growth hormone may comprise a functionally active fragment of growth hormone.

In this application, the term "Fab" generally refers to an antibody fragment consisting of VL, VH, CL and CH1 domains.

In the present application, the term "Fab'" generally refers to an antibody fragment having several additional residues at the carboxy terminus of the CH1 domain compared to the Fab fragment. For example, fab' may include one or more cysteines from the antibody hinge region.

In the present application, the term "F (ab) 2" generally refers to antigen binding fragments resulting from cysteine-linked pairs of Fab fragments.

In this application, the term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain (Ward et al Nature341:544-546 (1989)).

In this application, the term "complementarity determining region CDR" generally refers to the 3 hypervariable regions (HVRs) of the light chain variable region (VL) and the heavy chain variable region (VH), which are also known as complementarity determining regions due to their spatial structure which form a precise complementarity with an epitope.

In this application, the term "Fv fragment" generally refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

In this application, the term "scFv" generally refers to a molecule consisting of an antibody heavy chain variable region and a light chain variable region linked by a short peptide linker (linker), also known as a single chain antibody.

As used herein, the term "sequence homology" generally refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When a program (e.g., an Emboss Needle or BestFit) is used to determine sequence identity, similarity or homology between two different amino acid sequences, a default setting may be used, or an appropriate scoring matrix (such as blosum45 or blosum 80) may be selected to optimize the identity, similarity or homology score. In some embodiments, homologous polynucleotides are those sequences that hybridize under stringent conditions and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity compared to those sequences. When aligned with sequences of substantial length, homologous polypeptides have at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity, or at least 99% sequence identity.

Typically, in a polypeptide chain, an amino group is linked to another carboxyl group in the polypeptide chain so that it becomes a chain, but at both ends of the protein, amino acid residues not forming a peptide bond remain, respectively, the end of the polypeptide chain carrying a free amino group and the end of the polypeptide chain carrying a carboxyl group. In the present application, the term "N-terminal" generally refers to the end of a polypeptide chain in which the amino acid residue carries a free amino group. In the present application, the term "C-terminal" generally refers to the end of a polypeptide chain in which the amino acid residue carries a free carboxyl group.

In this application, the term "nucleic acid molecule" generally refers to any length of isolated form of nucleotide, deoxyribonucleotide or ribonucleotide or analog thereof, either isolated from the natural environment or synthesized.

In the present invention, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. For example, the carrier comprises: a plasmid; a bacteriophage; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-papilloma-vacuolated viruses (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these.

In the present application, the term "pharmaceutical composition" generally refers to a formulation in a form that allows the biological activity of the active ingredient to be effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. For example, these formulations may be sterile.

In this application, the term "pharmaceutically acceptable" generally refers to adjuvants suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment. For example, pharmaceutically acceptable adjuvants may refer to those adjuvants for animals (more particularly for humans) approved by a regulatory agency (e.g., the united states food and drug administration, the chinese food and drug administration, or the european pharmaceutical administration) or listed in a generally accepted pharmacopeia (e.g., the united states pharmacopeia, chinese pharmacopeia, or european pharmacopeia).

Detailed Description

In one aspect, the present application provides a fusion protein comprising: transferrin binding proteins, growth hormone or functionally active fragments thereof. For example, the transferrin binding protein may comprise a polypeptide, antibody or antigen-binding fragment thereof capable of binding transferrin.

In the present application, the fusion protein can have one or more properties. For example, the fusion protein is capable of extending the in vivo half-life of the growth hormone. For example, the fusion protein is capable of achieving drug delivery across the blood brain barrier. For example, the fusion protein can be delivered orally. For example, the fusion protein is capable of delivering the growth hormone to a cell expressing a transferrin receptor.

In the present application, the transferrin may be human transferrin.

In the present application, the antibody capable of binding to transferrin may comprise one or more selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In the present application, the antigen binding fragment capable of binding transferrin may include Fab, fab ', fv fragment, F (ab') ₂ ，F(ab) ₂ scFv, VHH, di-scFv and/or dAb.

In the present application, the transferrin binding protein may comprise a VHH capable of binding transferrin.

For example, the transferrin binding protein may comprise at least one CDR in a VHH, and the VHH may comprise the amino acid sequence shown in SEQ ID NO. 8.

In this application, the CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be contacted with an antigen or epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, abM, a combination of Kabat/Chothia et al. These coding systems are known in the art and can be found, for example, in http:// www.bioinf.org.uk/abs/index. The CDR regions can be determined by one skilled in the art using different coding systems depending on the sequence and structure of the antibody. Using different coding systems, CDR regions may differ. In this application, the CDRs encompass CDR sequences partitioned according to any CDR partitioning scheme; variants thereof are also contemplated, including amino acid substitutions, deletions and/or additions to the amino acid sequence of the CDRs. Such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; homologues thereof are also contemplated, which may be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequences of the CDRs. In certain embodiments, the CDRs of the transferrin binding proteins described herein can be defined by the Kabat coding system.

For example, the transferrin binding protein may comprise a CDR3, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO. 3.

For example, the transferrin binding protein may comprise CDR2, and the CDR2 may comprise the amino acid sequence shown in SEQ ID NO. 2.

For example, the transferrin binding protein may comprise CDR1, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO. 1.

For example, the transferrin binding protein may comprise CDR1, CDR2 and CDR3, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.

For example, the transferrin binding protein may comprise the framework region FR1 of a VHH, and the FR1 may comprise the amino acid sequence shown in SEQ ID NO. 4. For example, the transferrin binding protein may comprise the framework region FR2 of a VHH, and the FR2 may comprise the amino acid sequence shown in SEQ ID NO. 5. For example, the transferrin binding protein may comprise the framework region FR3 of a VHH, and the FR3 may comprise the amino acid sequence shown in SEQ ID NO. 6. For example, the transferrin binding protein may comprise the framework region FR4 of a VHH, and the FR4 may comprise the amino acid sequence shown in SEQ ID NO. 7.

In the present application, the transferrin binding protein may comprise a VHH capable of binding transferrin, and the VHH comprises the amino acid sequence shown in SEQ ID NO. 8.

In the present application, the growth hormone or functionally active fragment thereof in the fusion protein may be human growth hormone or functionally active fragment thereof. In this application, the growth hormone or functionally active fragment thereof may be engineered to retain the function and/or activity of growth hormone.

In this application, the growth hormone or functionally active fragment thereof in the fusion protein may comprise the amino acid sequence shown in SEQ ID NO. 10.

In the present application, the transferrin binding protein may be linked directly or indirectly to the growth hormone or functionally active fragment thereof in the fusion protein.

In the present application, the N-terminus of the transferrin binding protein may be directly or indirectly linked to the C-terminus of the growth hormone or functionally active fragment thereof in the fusion protein.

In the present application, the C-terminus of the transferrin binding protein may be directly or indirectly linked to the N-terminus of the growth hormone or functionally active fragment thereof in the fusion protein.

In the present application, in the fusion protein, the transferrin binding protein may be linked to the growth hormone or a functionally active fragment thereof via a linker. For example, the connector may comprise a flexible joint. For example, the linker may comprise a peptide linker.

In the present application, in the fusion protein, the linker may comprise the amino acid sequence shown in SEQ ID NO. 9.

In this application, the fusion protein may comprise the amino acid sequence shown in SEQ ID NO. 11.

In the present application, the fusion protein is capable of binding to transferrin and is capable of binding to and/or activating growth hormone receptors.

Nucleic acid molecule, vector, cell, pharmaceutical composition and preparation method

In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the fusion proteins.

The nucleic acid molecules described herein may be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, or (iv) synthesized, e.g. by chemical synthesis.

Recombinant DNA and molecular Cloning techniques include those described by Sambrook, j., fritsch, e.f., and manitis, t.molecular Cloning: A Laboratory Manual; cold Spring Harbor Laboratory Press: cold Spring Harbor, (1989) (Maniatis) and those described by T.J.Silhavy, M.L.Bennan and L.W. Enquist, experiments with Gene Fusions, cold Spring Harbor Laboratory, cold Spring Harbor, N.Y. (1984) and by Ausubel, F.M. et al, current Protocols in Molecular Biology, pub.by Greene Publishing assoc. And Wiley-Interscience (1987). Briefly, the nucleic acids may be prepared from genomic DNA fragments, cDNA and RNA, all of which may be extracted directly from the cell or recombinantly produced by various amplification methods, including but not limited to PCR and RT-PCR.

Direct chemical synthesis of nucleic acids typically involves sequential addition of 3' -blocked and 5' -blocked nucleotide monomers to the terminal 5' -hydroxyl group of the growing nucleotide polymer chain, wherein each addition is accomplished by nucleophilic attack of the terminal 5' -hydroxyl group of the growing chain at the 3' -position of the added monomer, which is typically a phosphorus derivative such as a phosphotriester, phosphoramidite, or the like. See, e.g., matteuci et al, tet. Lett.521:719 (1980); U.S. patent No. 4,500,707 to Caruthers et al; and U.S. Pat. Nos. 5,436,327 and 5,700,637 to Southern et al; in another aspect, the present application provides vectors comprising the isolated polynucleotides of the present application. The vector may be any linear nucleic acid, plasmid, phage, cosmid, RNA vector, viral vector, or the like. Non-limiting examples of viral vectors can include retroviruses, adenoviruses, and adeno-associated viruses.

In another aspect, the present application provides one or more vectors comprising the nucleic acid molecule. For example, the vector may comprise one or more nucleic acid molecules described herein. Each vector may comprise one or more of the nucleic acid molecules. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequences may vary depending on the species or cell type function, but typically comprises 5' non-transcribed and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, a 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional attachment nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering.

In another aspect, the present application provides a cell comprising said fusion protein, said nucleic acid molecule, or said vector. The cell may be a host cell. For example, the cells may include a number of cell types such as prokaryotic cells like E.coli or Bacillus subtilis, fungal cells like yeast cells or Aspergillus, insect cells like S2 Drosophila cells or Sf9, or animal cells like fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK293 cells or human cells.

For example, the vector may be stably or transiently introduced into a host cell by a variety of established techniques. For example, one method involves a calcium chloride treatment in which the carrier is introduced by calcium precipitation. Other salts, such as calcium phosphate, may also be used in a similar manner. In addition, electroporation (i.e., the application of an electrical current to increase the permeability of cells to nucleic acids) may be used. Other examples of transformation methods include microinjection, DEAE dextran-mediated transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes and dendrimers can also be used to transfect host cells.

In another aspect, the present application provides a method of preparing the fusion protein, which may include culturing the cell under conditions that enable expression of the fusion protein. For example, such methods are known to those of ordinary skill in the art by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like.

In another aspect, the present application provides a composition comprising said fusion protein, or said nucleic acid molecule, and optionally a pharmaceutically acceptable carrier.

For example, the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes, and/or nonionic surfactants, and the like.

In this application, the composition may be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients in accordance with conventional techniques in the art, such as in accordance with Remington: the Science and Practice of Pharmacy, nineteenth edition, gennaro editions, mack Publishing co., easton, PA, 1995.

In the present application, the pharmaceutical composition may be formulated for oral administration in the form of tablets, capsules, pills, powders, sustained release formulations, solutions, suspensions. The pharmaceutical composition may be in unit dosage form suitable for single administration of precise doses. The pharmaceutical composition may further comprise conventional pharmaceutical carriers or excipients. In addition, the pharmaceutical composition may include other drugs or agents, carriers, adjuvants, and the like.

The pharmaceutical compositions described herein may comprise a therapeutically effective amount of the fusion protein. The therapeutically effective amount is that amount which is required to be able to prevent and/or treat (at least partially treat) a disorder or condition (e.g., growth hormone deficiency) and/or any complications thereof in a subject suffering from or at risk of developing the disorder or condition. The specific amount/concentration of the dose may vary depending on the method of administration and the patient's needs, and may be determined based on, for example, patient volume, viscosity, and/or weight, among others. It will be appreciated that these particular dosages may be readily adjusted by one skilled in the art (e.g., a physician or pharmacist) based on the particular patient, formulation, and/or condition of the disease.

Use of the same

In another aspect, the present application provides the fusion protein, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition for use in preventing and/or treating a disease and/or disorder.

In the present application, the diseases and/or disorders may include diseases and/or disorders caused by abnormal growth hormone.

In this application, the disease and/or condition may include growth hormone deficiency.

The application also provides the following technical scheme:

1. a fusion protein comprising: a transferrin binding protein comprising a polypeptide, antibody or antigen-binding fragment thereof capable of binding transferrin, and a growth hormone or functionally active fragment thereof.

2. The fusion protein of embodiment 1 having one or more of the following properties:

(1) Is capable of extending the in vivo half-life of said growth hormone;

(2) Can cross the blood brain barrier;

(3) Can be delivered orally; and

3. The fusion protein according to any one of embodiments 1-2, wherein the transferrin is human transferrin.

4. The fusion protein according to any one of embodiments 1-3, wherein the antibody is selected from one or more of the following group: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

5. The fusion protein according to any one of embodiments 1-4, wherein the antigen-binding fragment comprises a Fab, fab ', fv fragment, F (ab') 2,F (ab) 2, scFv, vhh, di-scFv, and/or dAb.

6. The fusion protein of any one of embodiments 1-5, wherein the transferrin binding protein is a VHH.

7. The fusion protein according to any one of embodiments 1-6, wherein the transferrin binding protein comprises CDR3, and the CDR3 comprises the amino acid sequence shown in SEQ ID No. 3.

8. The fusion protein according to any one of embodiments 1-7, wherein the transferrin binding protein comprises CDR2, and the CDR2 comprises the amino acid sequence shown in SEQ ID No. 2.

9. The fusion protein according to any one of embodiments 1-8, wherein the transferrin binding protein comprises CDR1, and the CDR1 comprises the amino acid sequence shown in SEQ ID No. 1.

10. The fusion protein according to any one of embodiments 1-9, wherein the transferrin binding protein comprises a VHH and the VHH comprises the amino acid sequence shown in SEQ ID NO. 8.

11. The fusion protein according to any one of embodiments 1-10, wherein the growth hormone is human growth hormone.

12. The fusion protein according to any one of embodiments 1-11, wherein the growth hormone or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 10.

13. The fusion protein according to any one of embodiments 1-12, wherein the transferrin binding protein and the growth hormone or functionally active fragment thereof are directly or indirectly linked.

14. The fusion protein according to any one of embodiments 1-13, wherein the N-terminus of the transferrin binding protein and the C-terminus of the growth hormone or functionally active fragment thereof are directly or indirectly linked.

15. The fusion protein according to any one of embodiments 1-13, wherein the C-terminus of the transferrin binding protein and the N-terminus of the growth hormone or functionally active fragment thereof are directly or indirectly linked.

16. The fusion protein according to any one of embodiments 1-15, wherein the transferrin binding protein and the growth hormone or functionally active fragment thereof are linked by a linker.

17. The fusion protein according to embodiment 16, wherein the linker comprises the amino acid sequence shown in SEQ ID NO. 9.

18. The fusion protein according to any one of embodiments 1-17, comprising the amino acid sequence shown in SEQ ID NO. 11.

19. The fusion protein according to any one of embodiments 1-18, which is capable of binding to transferrin and of binding to and/or activating a growth hormone receptor.

20. One or more isolated nucleic acid molecules encoding the fusion protein of any one of embodiments 1-19.

21. A vector comprising the nucleic acid molecule of embodiment 20.

22. A cell comprising the nucleic acid molecule of embodiment 20, and/or the vector of embodiment 21.

23. A pharmaceutical composition comprising the fusion protein of any one of embodiments 1-19, the nucleic acid molecule of embodiment 20, the vector of embodiment 21 or the cell of embodiment 22, and optionally a pharmaceutically acceptable carrier.

24. A method of making the fusion protein of any one of embodiments 1-19, comprising culturing the cell of embodiment 22 under conditions in which the fusion protein is expressed.

25. Use of the fusion protein of any one of embodiments 1-19, the nucleic acid molecule of embodiment 20, the vector of embodiment 21, the cell of embodiment 22, and/or the pharmaceutical composition of embodiment 23 in the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.

26. The use according to embodiment 25, wherein the disease and/or condition comprises a disease and/or condition caused by abnormal growth hormone.

27. The use according to any one of embodiments 25-26, wherein the disease and/or disorder comprises growth hormone deficiency.

28. A method of preventing and/or treating a disease and/or disorder comprising administering the fusion protein of any one of embodiments 1-19, the nucleic acid molecule of embodiment 20, the vector of embodiment 21, the cell of embodiment 22, and/or the pharmaceutical composition of embodiment 23 to a subject in need thereof.

29. The method of embodiment 28, wherein the disease and/or condition comprises a disease and/or condition caused by abnormal growth hormone.

30. The method according to any one of embodiments 28-29, wherein the disease and/or disorder comprises growth hormone deficiency.

31. A method of extending the half-life of growth hormone in vivo comprising administering the fusion protein of any one of embodiments 1-19.

Without intending to be limited by any theory, the following examples are meant to illustrate the various aspects of the present invention and are not intended to limit the scope of the present invention.

Examples

Example 1 discovery of transferrin-specific single domain antibodies VHH

1.1 construction of phage display libraries

Two asian camels of 2-3 years old were immunized with purified human transferrin from human plasma to specific serum titers of 10000 or more (ELISA method). 200 ml of peripheral blood is taken for separating and purifying mononuclear cells (PBMC), mRNA is extracted, reverse transcription is carried out to cDNA, and a VHH-specific primer is used for amplifying a nucleotide sequence encoding VHH, and the VHH is cloned into a phage display vector to establish a VHH phage display library. From nearly 100 randomly selected clones DNThe A sequence measurement results were deduced, and the effective capacity of the phage display library constructed was about 1 hundred million (10) ⁸ ) Individual VHH sequences.

1.2 panning of VHH library

1-10 micrograms of biotinylated human transferrin was incubated with 1012CFU (colony forming units) of phage in phosphate buffer (PBST-1% BSA) containing 1% bovine serum albumin, 0.05% Tween 20 for 1 hour, and pre-washed, blocked, streptavidine-coupled magnetic beads (Invitrogen, dynabeads M-280 streptavidine) with PBST-1% BSA were added. After 1 hour incubation at room temperature, the beads were washed 10 times with PBST. Phages bound to magnetic beads were dissociated with 10. Mu.g/ml of pancreatin for infection expansion in E.coli, for the next round of panning, or were picked up for monoclonal selection of VHH sequences specifically binding to human transferrin but not affecting transferrin binding to human transferrin receptor by ELISA.

1.3 recombinant ovalbumin (VHH-OVA)

In the specific VHH sequence, SLN9056 (SEQ ID NO: 8) shows better stability compared with SLN0066 (SEQ ID NO: 12) which does not bind hTF, has stronger binding capacity with hTf under the conditions of pH 7.4 (figure 1A) and pH 6.0 (figure 1B), and can remarkably prolong the half-life of recombinant Ovalbumin (OVA) fused with the SLN9056 in the blood of mice (figure 1C).

SLN9056 was used to prepare rhGH-VHH fusion proteins in the following steps, the amino acid sequence of which is shown in SEQ ID NO. 11.

Example 2 production of fusion proteins

2.1 protein expression

Both mammalian cell systems and E.coli systems are used for the production of recombinant proteins rhGH and rhGH-VHH. In mammalian cell systems, HEK293F cells were used for transient expression transfection of recombinant expression plasmids and cell culture supernatants were used for purification of recombinant proteins. In the E.coli expression system, an expression vector plasmid (pET 28 a) comprising DNA encoding the nucleotide sequences of rhGH and rhGH-VHH was transformed into E.coli BL21 (DE 3) strain. Coli harboring the expression plasmid was cultured at 37℃and 1mM isopropyl β -D-1 thiopyran galactoside (IPTG) (GENERAL-Reagent, 367-93-1) was used to induce expression of the target protein. Bacterial inclusion bodies were collected and dissolved in 2M urea, 25ml 100mM Tris buffer (pH 12.5) for purification of recombinant proteins.

2.2 protein purification

Ion exchange column chromatography and molecular sieve chromatography are used for purification of recombinant proteins, respectively. In the ion exchange column chromatography, the protein solution was first adjusted to a 25mM Tris buffer system (solution A) containing 5mM EDTA, 0.4M urea, pH 8.5, and then loaded onto a DEAE-Sepharose anion exchange column (GE Healthcare) pre-equilibrated with the same solution A at a flow rate of 5 ml/min. The column was washed with 5 column volumes of solution A, then with 3 column volumes of solution B (0.1M NaCl was added to solution A), the recombinant protein was eluted at a flow rate of 5ml/min using a 0.1-0.5M NaCl gradient of solution A, urea was removed by dialysis in 5mM EDTA, pH 8.5 25mM Tris buffer (solution C), the precipitate was removed by centrifugation at 5000rpm, and the supernatant was concentrated by ultrafiltration (Millipore, 10 kDa) and then used for gel filtration column chromatography. In the gel filtration purification process, the protein solution was loaded onto a Sephadex G75 column (GE Healthcare) equilibrated with solution C at a flow rate of 1ml/min, and the recombinant protein was eluted with solution C.

2.3 purity identification of recombinant proteins

Purified recombinant proteins were purified by SDS-PAGE and SEC-HPLC (Waters, CAT#E2695), respectively, and the results were shown in FIG. 2 (recombinant human growth hormone, rhGH) and FIG. 3 (recombinant human growth hormone-VHH fusion protein, rhGH-VHH).

EXAMPLE 3 biochemical characterization of recombinant human growth hormone

Recombinant proteins were tested for their ability to bind to transferrin, transferrin/transferrin receptor complex, human growth hormone receptor, and their ability to activate human growth hormone receptor, respectively, using the ELISA, FACS, luciferase reporter gene method.

3.1 ELISA detection of the binding Capacity of recombinant proteins to growth hormone receptors

96-well plates were coated with growth hormone receptor (HEK 293 cells recombinantly expressed GHR-Fc, 0.5. Mu.g/well) and incubated overnight at 4℃and after 3 washes with PBST (phosphate buffer, 0.1% Tween-20, pH 7.4) were blocked with 200. Mu.l 1% BSA/PBST for 1 hour at room temperature. The cells were washed 3 times with PBST, serial dilutions of rhGH or rhGH-VHH protein were added and incubated for 1 hour at room temperature. After washing the plates 3 times with PBST, PBST (100 μl/well) containing biotinylated goat anti-human growth hormone antibody (R & D, cat#af 1201) was added and incubated for 1 hour at room temperature. After washing 3 times with PBST, 100. Mu.l of PBST containing streptavidin-HRP (Sigma, CAT#S5512-1 MG) was added to each well and incubated for 1 hour at room temperature. Then 100. Mu.l of TMB substrate solution was added after washing as before and incubated for 15 minutes at room temperature. After adding 100. Mu.l/Kong Zhongzhi solution, the absorbance at 450nm was read with an ELISA reader. The results are shown in FIG. 4A, and FIG. 4A shows the binding capacity analysis of recombinant protein to human growth hormone receptor (hGH), comparing the in vitro binding activity of different concentrations of rhGH and rhGH-VHH to purified hGH-Fc recombinant protein, and the results show that rhGH-VHH has similar binding capacity to human growth hormone receptor to rhGH.

3.2 ELISA detection of the binding Capacity of recombinant proteins to transferrin

96-well plates were coated with streptavidin (0.1. Mu.g/well) and incubated overnight at 4℃and after 3 washes with PBST, blocked with 200. Mu.l of PBST containing 1% Bovine Serum Albumin (BSA) for 1 hour at room temperature. Washed 3 times with PBST, and incubated for 1 hour at room temperature after the addition of biotinylated human transferrin (1. Mu.g/ml, 100. Mu.l/well). Plates were washed 3 times with PBST, serial dilutions of rhGH or rhGH-VHH protein were added and incubated for 1 hour at room temperature. PBST 3 times after washing PBST containing murine anti-human growth hormone mab (Abcam, cat#ab 9821) was added to each well and incubated for 1 hour at room temperature before washing. PBST wash 3 times before adding PBST containing HRP conjugated goat anti-mouse Fc monoclonal antibody (Abcam, cat#ab 9871) to each well, after incubation for 1 hour at room temperature, before washing. After addition of 100. Mu.l/well TMB substrate solution, incubation was carried out for 15 minutes at room temperature. After addition of 100. Mu.l/well of stop solution, the absorbance at 450nm was read with an ELISA reader. The results are shown in FIG. 4B, and FIG. 4B shows an analysis of the binding capacity of recombinant proteins to human transferrin (hTf). Comparing the in vitro binding activity of different concentrations of rhGH and rhGH-VHH to purified native hTf protein, the results show that rhGH-VHH is able to bind to human transferrin and rhGH is unable to bind to human transferrin.

3.3 FACS detection of binding of recombinant proteins to cell membrane surface transferrin, transferrin receptor complexes

293F cell lines stably expressing human transferrin receptor 1 (hTfR 1, uniProt P02786) were used to detect binding of recombinant proteins to cell membrane surface transferrin/transferrin receptor complexes. At 0.5x10 per well ⁶ 293F/hTfR1 cells were plated in 96-well U-bottom serum plates, centrifuged at 1000rpm for 5 min at 4℃and washed with 200ul of 1 XPBS (pH 7.4) followed by resuspension and blocking of the cells with 200ul of PBS (blocking solution) containing 2% fetal bovine serum (FBS, gibco, cat No. 10099-141C). After incubation at 4 ℃ for 30min, washed as above, resuspended in blocking solution containing serial dilutions of recombinant protein samples, with or without native transferrin (Sigma, cat No. t 3309). After incubation at 4℃for 30min, murine anti-VHH antibody (Genscript) and AF 647-labeled goat anti-murine antibody (Abcam, CAT#ab 150115) were added sequentially. After incubation at 4 ℃ for 30min, the cells were analysed for binding to recombinant proteins by flow cytometry after centrifugation, washing and re-suspension in a blocking solution. The results are shown in FIG. 5A, and FIG. 5A shows the binding activity of the recombinant protein to the cell membrane surface transferrin (hTf)/transferrin receptor (hTfR 1) complex. The binding curves of different concentrations of rhGH and rhGH-VHH to HEK293F cells stably expressing hTfR1 (293F/hTfR 1 stable cell line) were compared and the results showed that rhGH-VHH could bind to human transferrin receptor on the surface of cell membrane through the mediation of human transferrin.

3.4 FACS detection of binding of recombinant proteins to human growth hormone receptors on cell membrane surfaces

293F cell lines stably expressing human growth hormone receptor (hGH R, uniProt P10912) were used to detect binding of recombinant proteins to human growth hormone receptor on cell membrane surfaces. At 0.5x10 per well ⁶ 293F/hGH R cells were plated in 96-well U-bottom serum plates, centrifuged at 1000rpm for 5 min at 4℃and washed with 200ul of 1 XPBS (pH 7.4) followed by resuspension and blocking of the cells with 200ul of PBS (blocking solution) containing 2% fetal bovine serum (FBS, gibco, cat No. 10099-141C). Incubation at 4℃for 30min followed by washing as above, resuspension in blocking solution containing serial dilutions of recombinant protein samples, with or without native transferrin (Sigma, cat NO. T3309). After incubation at 4℃for 30min, murine anti-human growth hormone antibodies (Abcam, CAT#Ab9821) and fluorescently labeled goat anti-murine antibodies (Abcam, CAT#ab 150115) were added sequentially. After incubation at 4 ℃ for 30min, the cells were analysed for binding to recombinant proteins by flow cytometry after centrifugation, washing and re-suspension in a blocking solution. The results are shown in FIG. 5B, and FIG. 5B shows the binding activity of the recombinant protein to the human growth hormone receptor (hGH R) on the surface of the cell membrane. The binding curves of different concentrations of rhGH and rhGH-VHH to HEK293F cells stably expressing hGH (293F/hGH R stable cell line) were compared and the results show that rhGH-VHH and rhGH possess the binding capacity to human growth hormone receptor expressed on the surface of cell membrane as well.

EXAMPLE 4 analysis of biological Activity of recombinant proteins

The biological activity test of the recombinant protein was performed in a cell line stably expressing a luciferase (luciferase) reporter gene.

4.1 Establishment of a stable transgenic cell line of 293T/GAS-luminescence/hGH R

First, pSLN-0134 (pNL2.2-SG-MinP)/GAS vector was constructed: a stable HEK293T cell line of luciferase (luciferase) gene (pnl 2.2-SG-minip, promega, N104A) with an interferon activated site (GAS) regulatory element inserted into the genome. Co-expressing a human growth hormone receptor (hGH R) and a plasmid containing puromycin resistance gene in a 293T/GAS-luciferase stable cell line, obtaining a stable cell bank of 293T/GAS/hGH R by continuous 20-day puromycin pressure screening, obtaining a monoclonal cell line clone #15 by a limiting dilution method, and as a result, as shown in FIG. 6, FIG. 6 shows an expression analysis of the human growth hormone receptor (hGH R) in the stable cell bank, specifically, hGH positive cells are detected by using goat anti-hGH antibodies (Abcam, CAT#AF 11210) and donkey anti-goat IgG-PE (Abcam, CAT#AF 109), and scanning analysis by using a flow cytometer; and the stability of the monoclonal stable transgenic cell line and the reactivity of the monoclonal stable transgenic cell line to human growth hormone treatment are respectively compared with the reactivity of cells of different generations (original 0, 12, 16 and 20 generations) to recombinant human growth hormone.

4.2 biological function detection of recombinant proteins

DMEM medium (Gibco, cat No. C119955500 BT) containing 10% FBS and 2ug/ml puromycin was used for the culture of 293T/GAS/hGH R cell lines. Cells with a density of about 75% were digested with 0.25% trypsin for 5 min at 37 degrees, diluted with medium, blown into single cell suspension and washed 3 times with DMEM medium containing 1% fbs. The washed cells were resuspended in DMEM medium containing 1% FBS and placed in 96-well cell culture plates at a cell density of 20000 cells/50. Mu.l, and serial dilutions of the samples (rhGH or rhGH-VHH) in medium were added. After incubation at 37℃for 4 hours with 5% CO2, the expression of luciferases was quantitatively determined by addition of Nanoglo reagent (Promega, N1110). As shown in FIG. 7, the rhGH and rhGH-VHH have similar biological activity, or fusion of VHH has no effect on the biological activity of GH. Recombinant proteins derived from mammalian cell expression systems and E.coli expression systems have similar biological activities, both at the protein level (ELISA) and at the cellular level (FACS and luciferase reporter gene tests).

EXAMPLE 5 Pharmacokinetic (PK) studies of recombinant proteins in mice

5.1 Establishment of PK methods

Mouse anti-human growth hormone monoclonal antibody (Abcam, CAT#Ab9821) was used to coat 96-well plates and after 3 washes with PBST (Tween-20, 0.1%) was blocked with 200. Mu.l of PBST containing 1% BSA for 1 hour at room temperature. After 3 washes of PBST, PBST containing 1% mouse serum (as sample matrix) and serial dilution standards (rhGH or rhGH-VHH) were added. After 1 hour incubation at room temperature, the mixture was washed 3 times with PBST, and a biotin-labeled goat anti-human growth hormone polyclonal antibody (R & D, CAT#AF 1201) was added thereto, and incubated at room temperature for 1 hour. Streptavidin-labeled horseradish peroxidase (HRP) (Sigma, cat#s5512-1 MG) was added after 3 washes with PBST and incubated for 1 hour at room temperature. Then washed as before, incubated with 100. Mu.l TMB substrate solution for 15 minutes, and after addition of stop solution, absorbance at 450nm was read. Fig. 8 shows the validation results of the PK method, with the results of validation with three standard concentrations (high, medium, low) indicating that the accuracy (CV% < 20%) and the accuracy (RE% +/-25%) of the method both meet the sample detection requirements.

5.2 PK study with Single dose administration

PK studies were performed in 6-8 week old male C57BL/6 mice. The animals were grouped according to body weight and 10mg/kg of human transferrin was subcutaneously injected daily on the day prior to dosing and during the study. The single subcutaneous doses of rhGH and rhGH-VHH were 50, 150, 500nmol/kg, respectively. Blood samples were collected at 2, 4, 8, 24, 32 and 48 hours post-dose (rhGH group), or 2, 6, 24, 30, 48, 54, 72, 78 and 96 hours post-dose (rhGH-VHH group), serum was isolated, quantitative analysis of the drug in serum was performed using the PK method described above, and PK parameters were calculated using PKsovler software. The results shown in FIG. 9 demonstrate a significant improvement in the blood half-life (11.5 hours) of rhGH-VHH over rhGH (blood half-life less than 1 hour) at a dose of 150 nmol/kg. In the different dose groups, it was found that binding of transferrin by VHH significantly prolonged the blood half-life of rhGH (fig. 10A).

5.3 PK study with multiple dosing

PK studies were performed in 6-8 week old male C57BL/6 mice. The animals were grouped according to body weight and 10mg/kg of human transferrin was subcutaneously injected daily on the day prior to dosing and during the study. The subcutaneous doses of rhGH-VHH were all 50, 150, 500nmol/kg, once every 48 hours (two days) and three consecutive doses. Blood samples were collected at 2, 6, 24, 48, 54 (6 hours after the second administration), 72 (24 hours after the second administration), 96 (48 hours after the second administration), 102 (6 hours after the third administration), 120 (24 hours after the third administration), and 7 th day (72 hours after the third administration) and 9 th day (120 hours after the third administration), respectively, for quantitative analysis of drugs in blood by the above-mentioned PK method, and PK parameters were calculated by PKsovler software. The result is shown in FIG. 10B.

EXAMPLE 6 Pharmacokinetic (PK) studies of recombinant proteins in mice

6.1 GHRH deficient mouse model

A GHRH gene (GenBank: M31658.1) deficient mouse model is purchased from Nanjing's Jixiaokang, and the genotype of the mouse is verified by PCR and sequencing. Body Weight (BW), body length (nose-anus distance, N-A), femur and tibiA length were measured in 1-5 week old mice using calibrated electronic balance and electronic digital calipers. The results showed no significant differences between heterozygote mice and wild-type mice, but the homozygous GHRH-deficient mice were significantly reduced in body length, body weight, femur and tibia length (fig. 11). Homozygous mice were used for pharmacodynamic studies of recombinant proteins.

6.2 Pharmacodynamic studies of rhGH

Mice deficient in the GHRH gene of homozygous for male and female (5 animals each) were subcutaneously administered 150nmol/kg or 500nmol/kg daily, respectively, and the animals were continuously treated for 4 weeks, and Body Weight (BW) and body length (nose-anus distance, N-A) were measured 2 times A week. Mice were euthanized at week 5 (day 44) post-treatment and serum was collected for determination of IGF-1 levels. The results in fig. 12 demonstrate that daily administration significantly improves the growth status of GHRH gene deficient mice.

6.3 Pharmacodynamic studies of rhGH-VHH

Animals were measured for Body Weight (BW) and body length (nose-anus distance, N-A) by subcutaneously (twice weekly), 500nmol/kg (twice weekly), or 1500nmol/kg (once weekly) for 4 weeks continuously in one week-old male and female (5 each) homozygous GHRH gene-deficient mice, respectively. Mice were euthanized at week 5 (day 44) after treatment and serum was collected for the determination of IGF-1 levels. The results in FIG. 13 demonstrate that in GHRH gene deficient mice, a similar therapeutic effect of 150nmol/kg per day can be achieved in the rhGH-VHH treated group administered twice a week or 1500nmol/kg per week.

The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Numerous variations of the presently exemplified embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and equivalents thereof.

Sequence listing

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Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu

65 70 75 80

Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val

85 90 95

Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp

100 105 110

Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu

115 120 125

Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser

130 135 140

Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr

145 150 155 160

Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe

165 170 175

Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly

180 185 190

Gly Gly Gly Ser Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Glu Val

195 200 205

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

210 215 220

Arg Leu Ser Cys Ala Ala Ser Gly His Ala Tyr Gly Gly Asn Tyr Met

225 230 235 240

Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Val

245 250 255

Leu Tyr Thr Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly

260 265 270

Arg Phe Thr Ile Ser Glu Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

275 280 285

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Leu

290 295 300

Ala Leu Gly Ser Ala Arg Trp Tyr Thr Ser Ser Leu Asp Ala Arg Ala

305 310 315 320

Tyr Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

325 330

<210> 12

<211> 129

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> SLN0066

<400> 12

His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ile Gly His Gly Phe Asn

20 25 30

Asn Asn Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Gly Val Ala Ala Val Tyr Thr Gly Gly Gly Thr Pro Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Leu Ser Gln Asp Asn Ala Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Gly Leu Asp Pro Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Ala Asp Ile Trp Arg Thr Tyr Arg Cys Gly Ala Gly Asp

100 105 110

Thr Thr Val Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

Claims

2. The fusion protein of claim 1, having one or more of the following properties:

(1) Is capable of extending the in vivo half-life of said growth hormone;

(2) Can cross the blood brain barrier;

(3) Can be delivered orally; and

3. The fusion protein of any one of claims 1-2, wherein the antigen binding fragment comprises Fab, fab ', fv fragment, F (ab') ₂ ，F(ab) ₂ scFv, VHH, di-scFv and/or dAb.

4. A fusion protein according to any one of claims 1-3, wherein the growth hormone is human growth hormone.

5. The fusion protein according to any one of claims 1 to 4, comprising the amino acid sequence shown in SEQ ID NO. 11.

6. One or more isolated nucleic acid molecules encoding the fusion protein of any one of claims 1-5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A cell comprising the nucleic acid molecule of claim 6, and/or the vector of claim 7.

9. A pharmaceutical composition comprising the fusion protein of any one of claims 1-5, the nucleic acid molecule of claim 6, the vector of claim 7 or the cell of claim 8, and optionally a pharmaceutically acceptable carrier.

10. Use of the fusion protein of any one of claims 1-5, the nucleic acid molecule of claim 6, the vector of claim 7, the cell of claim 8, and/or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.