Transferrin binding antibodies and uses thereof

Abstract

The present application provides a method of transporting a molecule across a cell membrane by using a transferrin binding protein. The transferrin binding protein can specifically bind to transferrin and does not interfere with the interaction between the transferrin and a transferrin receptor.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of December 2, 2021, application number CN2021800983966, and invention name “Transferrin-binding antibodies and their applications”. Background Art

[0002] Transmembrane drug delivery systems can be used for drug delivery across the blood-brain barrier (BBB), oral drug delivery, intracellular drug delivery, etc. Currently, transferrin receptor (TfR) is the most widely studied target protein in non-invasive blood-brain barrier drug delivery technologies such as receptor-mediated trans-blood-brain barrier drug delivery. These TfR-targeted carriers include antibody clones OX26 and 8D3 in the form of Fabs, scFv or dual variable domain immunoglobulins, GYR22 peptides, and engineered Fc regions of human IgG with TfR binding properties. Although most of these approaches are still in the preclinical stage, some studies have shown promising results in clinical trials, even with several limitations.

[0003] The affinity and titer of TfR antibodies can alter the intracellular trafficking of TfR and the delivered therapeutic entity; high-affinity bivalent TfR antibodies can transfer the antibody-TfR complex to lysosomes, resulting in TfR degradation and reduced expression levels. Therefore, TfR antibodies for transmembrane drug delivery need to be optimal and / or monovalent in terms of affinity for TfR to achieve more efficient transcytosis into the brain. The efficiency of TfR targeted delivery technology can vary depending on the biological or biochemical properties of the entity to be delivered, and anti-TfR antibodies can also cause acute clinical symptoms and reduced circulating reticulocyte counts. The ubiquitous expression of TfR throughout the body suggests that TfR targeting strategies will likely lead to increased uptake of TfR targeted drugs into peripheral compartments such as the bone marrow, liver, and spleen.

[0004] With a few exceptions, oral administration is currently only a choice for small drug molecules that show acceptable intestinal absorption. As a class of rapidly developing drugs, biologics are currently mainly administered by injection. Decades of major research efforts have explored the technology that enables oral delivery of biologics, but progress is relatively less impressive. The drug delivery strategy in this field mainly utilizes absorption or penetration enhancers such as SNAC, or focuses on relatively small biologics of mass, such as glucagon-like peptide 1 (GLP-1) analogs. However, safety issues, including problems related to many surfactants used in preparations, have hindered the clinical transformation of these methods. The key challenge in the field of oral delivery of macromolecular biologics involves solving the difficulties of intestinal epithelial barriers that are difficult to overcome, rather than additional barriers that can be solved by relatively mature technologies, such as gastric acid and mucosal enzymes. Safety is a key requirement for the technology that enables the treatment of biologics related oral delivery. Rather than non-selectively destroying and increasing the permeability of the intestinal epithelium (i.e., the effect that classical penetration enhancers will show), it is better to design a delivery system that selectively permeates the intestinal mucosa. Summary of the invention

[0005] The present application provides a method for transporting molecules across cell membranes by using transferrin binding protein. The transferrin binding protein can specifically bind to transferrin and does not interfere with the interaction between transferrin and transferrin receptor.

[0006] In one aspect, the present application provides methods for transporting molecules across a cell membrane by using transferrin binding proteins.

[0007] In some embodiments, the transferrin binding protein comprises a transferrin binding antibody or a fragment thereof.

[0008] In some embodiments, the methods are used to deliver drugs across the cell membranes of polarized and / or unpolarized cells.

[0009] In some embodiments, the polarized and / or unpolarized cells express a transferrin receptor on their cell membrane.

[0010] In some embodiments, drug delivery across the cell membrane of a polarized cell includes delivering the drug intracellularly, and / or recycling the endocytosed drug back into the blood circulation.

[0011] In some embodiments, drug delivery across the cell membrane of an unpolarized cell includes systemically administered drugs targeted to the central nervous system (CNS) across the blood-brain barrier (BBB), orally administered drugs across the intestinal epithelium, and / or drugs penetrating multiple layers of cells in solid tissues.

[0012] In some embodiments, a medicament comprises a therapeutic or diagnostic substance.

[0013] In some embodiments, the drug comprises a small molecule compound, a synthetic peptide, a recombinant protein, an antibody or antibody fragment, an enzyme, a nucleotide sequence fragment, a liposome, a lipid nanoparticle, a drug carrier, a synthetic nucleotide sequence, a modified virus and / or a gene therapy vector.

[0014] In some embodiments, the drug is bound to the transferrin binding protein by chemical coupling or genetic fusion.

[0015] In another aspect, the present application provides a transferrin binding protein that is capable of extending the circulation half-life of an entity to which it binds.

[0016] In some embodiments, the transferrin binding protein enables a drug comprising the transferrin binding protein to cross the blood-brain barrier (BBB).

[0017] In some embodiments, the transferrin binding protein is used for oral delivery of drugs.

[0018] In some embodiments, the transferrin binding protein enables intracellular delivery of entities to which it binds to cells expressing the transferrin receptor.

[0019] In some embodiments, the transferrin binding protein is capable of specifically binding to transferrin and does not interfere with the interaction between transferrin and transferrin receptor 1.

[0020] In some embodiments, the transferrin is human transferrin.

[0021] In some embodiments, the transferrin binding protein has a higher affinity for iron-containing, iron-saturated transferrin than for iron-free, apotransferrin.

[0022] In some embodiments, the transferrin binding protein comprises a monoclonal antibody, a single chain antibody fragment, a single domain antibody fragment, an engineered protein, or a peptide.

[0023] In some embodiments, the antibody or antibody fragment comprises a sequence of animal origin, a humanized sequence, a fully human sequence, a chimeric sequence, or a synthetic sequence.

[0024] In some embodiments, the antibody or antibody fragment is a single domain antibody fragment VHH.

[0025] In some embodiments, the transferrin binding protein comprises a CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 45-69.

[0026] In some embodiments, the transferrin binding protein comprises CDR1, CDR2, and CDR3, and CDR3 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 45-69.

[0027] In some embodiments, the transferrin binding protein comprises a heavy chain variable region VH comprising an amino acid sequence as shown in any one of SEQ ID NOs: 70-104.

[0028] In another aspect, the present application provides a polypeptide comprising a transferrin binding protein and a therapeutic entity.

[0029] In some embodiments, the therapeutic entity comprises engineered cytotoxic Pseudomonas exotoxin A (PE38).

[0030] In some embodiments, the therapeutic entity is glucagon-like peptide-1 (GLP-1) or a variant thereof.

[0031] In some embodiments, a therapeutic entity is linked directly or indirectly to the antigen binding protein.

[0032] In some embodiments, the therapeutic entity is linked to the transferrin binding protein via a linker.

[0033] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding a transferrin binding protein or a polypeptide as shown in any one of SEQ ID NOs: 70-106.

[0034] In another aspect, the present application provides a vector comprising a nucleic acid molecule encoding a polypeptide as shown in any one of SEQ ID NOs: 70-106.

[0035] In another aspect, the present application provides a cell comprising a nucleic acid molecule or a vector expressing a polypeptide as shown in any one of SEQ ID NOs: 70-106.

[0036] In another aspect, the present application provides a method for preparing a transferrin binding protein or polypeptide, which comprises culturing cells under conditions that allow expression of the transferrin binding protein or polypeptide.

[0037] In another aspect, the present application provides a pharmaceutical composition comprising a transferrin binding protein or polypeptide.

[0038] In another aspect, the present application provides a method for extending the circulating half-life of a therapeutic entity, wherein the therapeutic entity is directly or indirectly linked to a transferrin binding protein.

[0039] In another aspect, the present application provides a method for delivering a therapeutic drug or a diagnostic agent to a cell or an organ expressing a transferrin receptor, which comprises using a transferrin binding entity.

[0040] In another aspect, the present application provides a method for delivering a targeted drug across the blood-brain barrier, intestinal epithelium and / or a cell membrane expressing a transferrin receptor, comprising the use of a transferrin binding entity.

[0041] In another aspect, the present application provides a method for oral delivery of a therapeutic or diagnostic entity directly or indirectly linked to the antigen binding protein.

[0042] In another aspect, the present application provides a method for intracellular delivery of a therapeutic entity directly or indirectly linked to the antigen binding protein.

[0043] The present application provides a technical solution that utilizes Tf to replace TfR for targeting as the basis of a drug delivery system. The present application achieves multiple potential advantages in terms of specificity, safety, efficiency, and circulation half-life.

[0044] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive.

[0045] Incorporated by Reference

[0046] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention may be better understood by referring to the following detailed description of illustrative embodiments and the accompanying drawings (also referred to herein as "figures"), which illustrate the principles of the invention, wherein:

[0048] Figure 1 Shown is SDS-PAGE of a representative transferrin binding protein-OVA-his protein expressed in HEK293 cells.

[0049] Figure 2 A- Figure 2 B shows representative selected transferrin binding protein-OVA-His proteins binding to human transferrin at pH 7.4 or pH 6.0.

[0050] Figure 3A- Figure 3 C shows transferrin-mediated binding of selected transferrin binding protein-OVA-His proteins to cells expressing transferrin receptor.

[0051] Figure 4 Epitope identification of representative transferrin binding proteins of the present application is shown.

[0052] Figure 5 A- Figure 5 D shows ELISA data showing binding of representative humanized transferrin binding protein variants to native human transferrin at pH 7.4 or pH 6.0.

[0053] Figure 6 A shows the circulating half-life of OVA fused to transferrin binding proteins cloned in the present application with different epitopes. Figure 6 B shows the circulation half-life of GLP-1-VHH in mice. Figure 6 C shows that the circulation half-life extension achieved by VHH is affinity dependent.

[0054] Figure 7 A- Figure 7 C shows that transferrin binding protein enables transport of conjugated OVA across a monolayer of Caco-2 cells or through the intestinal epithelium via endocytosis.

[0055] Figure 8 A- Figure 8 B, Figure 8 Ca- Figure 8 Cf shows that transferrin binding protein enables the associated OVA to cross the blood-brain barrier in mice and that the dependency of this ability is related to epitope utilization and affinity.

[0056] Fig. 9 A- Fig. 9 B shows the intracellular delivery of transferrin binding protein-PE38 in different cell lines.

[0057] Fig.10 The biodistribution of transferrin binding protein variants after systemic administration is shown. DETAILED DESCRIPTION

[0058] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art may make various changes, modifications and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0059] In this application, the term "cell membrane" generally refers to the interface that separates different media and components inside and outside the cell in the cell structure. The plasma membrane is generally considered to be a basic unit of phospholipid bilayer molecules, i.e., a phospholipid bilayer, on which various types of membrane proteins, as well as sugars and glycolipids bound to the membrane proteins, are embedded. Through the pores and certain properties of transmembrane proteins, selective and controllable material transport can be achieved. For example, the cell membrane can belong to a polarized cell. For example, the cell membrane can belong to an unpolarized cell.

[0060] In the present application, the term "polarized cell" generally refers to a cell that has a stable asymmetry in structure and function, such as a cell that is at a resting potential and is ready to transmit a nerve impulse signal. The term "unpolarized cell" generally refers to a cell that has a change in resting potential and transmits a nerve impulse signal. For example, polarized cells and / or unpolarized cells can belong to the blood-brain barrier of the central nervous system. For example, polarized cells and / or unpolarized cells can belong to the intestinal epithelium. For example, polarized cells and / or unpolarized cells can belong to solid tissue.

[0061] In the present application, the term "transferrin binding protein" generally refers to a protein including an antigen binding portion, and optionally allows the antigen binding portion to adopt a support or skeleton portion in a conformation that promotes the antigen binding protein to bind to the antigen. For example, the antigen binding protein may include but is not limited to an antibody, an antigen binding fragment (Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb), an immunoconjugate, a multispecific antibody (e.g., a bispecific antibody), an antibody fragment, an antibody derivative, an antibody analog or a fusion protein, as long as they show the desired antigen binding activity. For example, the antigen binding protein may be able to specifically bind to transferrin. For example, the antigen binding protein may not interfere with the interaction between transferrin and transferrin receptor 1. For example, the antigen binding protein may not affect the binding of Tf / TfR1. For example, the antigen binding protein can maintain the normal physiological function of iron transport.

[0062] In the present application, the term "transferrin" generally refers to a glycoprotein that can bind multivalent ions and transport them. For example, transferrin can be a single-chain glycoprotein. For example, transferrin can have a molecular weight of about 77,000D. For example, transferrin can have a polysaccharide. For example, transferrin can have two ion binding sites. For example, the ion binding site can have different affinities with iron ions. For example, the multivalent ion can be an iron ion, a chromium ion, a manganese ion, a cadmium ion, or its nickel ion. For example, each transferrin molecule can bind two trivalent iron atoms. For example, transferrin can be an iron-containing iron-saturated transferrin, or an iron-free apotransferrin. For example, transferrin can be a mouse transferrin. For example, the amino acid sequence of mouse transferrin can be as shown in GenBank: EDL21066.1, AAL34533.1 or AAL34533.1. For example, transferrin can be a human transferrin. For example, the amino acid sequence of human transferrin may be as shown in GenBank: AAH59367.1, AAH59367.1 or AAB22049.1.

[0063] In the present application, the term "transferrin receptor" generally refers to a carrier protein of transferrin. For example, transferrin may be a transmembrane glycoprotein. For example, a transferrin receptor may mediate the endocytosis of transferrin bound to two iron ions. For example, a transferrin receptor may maintain the homeostasis of intracellular iron. For example, a transferrin receptor may be transferrin receptor 1 (TfR1) or transferrin receptor 2 (TfR2). For example, TfR1 and TfR2 may show about 45-66% homology in the extracellular domain, but may present different expression patterns in vivo. For example, the affinity of TfR1 for transferrin may be higher than the affinity of TfR2 for transferrin. For example, compared with the affinity of TfR1 for transferrin, the affinity of TfR2 for transferrin is as low as 1 / 25. For example, TfR2 may be expressed primarily in tissues such as the liver and small intestine involved in regulating iron metabolism, while TfR1 is typically present on the surface of most somatic cells. The term "transferrin receptor 1" generally refers to a 97-kDa type 2 membrane protein expressed as a homodimer in a cell membrane. TfR1-mediated transferrin internalization is classically described as a standard iron import pathway. For example, the transferrin receptor can be a mouse transferrin receptor. For example, the amino acid sequence of the mouse transferrin receptor can be as shown in GenBank: AAH54522.1, CAA40624.1 or NP_001344227.1. For example, the transferrin receptor can be a human transferrin receptor. For example, the amino acid sequence of the human transferrin receptor can be as shown in GenBank: AAA61153.1, AAF04564.1 or AAB19499.1.

[0064] In the present application, the term "half-life" generally refers to the time required for the serum concentration of an amino acid sequence, compound or polypeptide to be reduced by 50% due to the degradation of the sequence or compound by natural mechanisms and / or the removal or sequestration of the sequence or compound in vivo. The half-life can be estimated by methods known to those skilled in the art. The in vivo half-life of the amino acid sequence, compound or polypeptide of the present invention can be determined in any known manner, such as by pharmacokinetics. Suitable techniques are obvious to those skilled in the art, for example, as described in paragraph o) of page 57 of WO 08 / 020079. Also as described in paragraph o) of page 57 of WO 08 / 020079, parameters such as t1 / 2-α, t1 / 2-β and area under the curve (AUC) can be used to represent the half-life. In this regard, it should be noted that the term "half-life" in the present application particularly refers to t1 / 2-β or terminal half-life (wherein t1 / 2-α and / or AUC can be ignored). For example, reference is made to the experimental parts and standard manuals such as Kenneth, A et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinete analysis: A Practical Approach (pharmacokinetics) Analysis: Practice Method (1996). Reference is also made to "Pharmacokinetics", M.G.Bibaldi & D.Perron, published by Marcel Dekker, 2nd edition (1982).

[0065] In the present application, the term "fusion protein" generally refers to a chimeric protein comprising the amino acid sequence of two or more different proteins, although the different proteins are not combined in their native state, their respective amino and carboxyl termini are bound by peptide bonds to form a continuous polypeptide. It should be understood that two or more different proteins can be directly bound by a peptide linker or a spacer. For example, a fusion protein may include a preventive or therapeutic drug fused to a heterologous protein, polypeptide or peptide. Wherein, the heterologous protein, polypeptide or peptide may or may not be a different type of therapeutic drug. For example, a fusion protein may include two different proteins, polypeptides or peptides with immunomodulatory activity. For example, compared with the activity of the original polypeptide or protein, the fusion protein can retain or improve the activity. Typically, a fusion protein can be produced by in vitro recombinant techniques known in the art. For example, a fusion protein may include an antigen binding protein. For example, a fusion protein may include a biomolecule.

[0066] In the present application, the term "blood-brain barrier (BBB)" generally refers to the physiological barrier between peripheral circulation and the brain and spinal cord. It is formed by the tight connection in the plasma membrane of brain capillary endothelial cells, and constitutes a tight barrier that restricts molecules, even very small molecules such as urea (60 daltons), to brain transport. For example, brain capillary endothelial cells can have weaker pinocytosis. For example, the blood-brain barrier can include the BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina. For example, the BBB can also include a blood-CSF barrier (choroid plexus), wherein the barrier is composed of ependymal cells rather than capillary endothelial cells.

[0067] In this application, the term "oral delivery of a drug" generally refers to a drug that is absorbed into the blood through the gastrointestinal tract after oral administration and reaches local or systemic tissues through the blood circulation to achieve the purpose of treating or preventing a disease. For example, a drug may include transferrin binding protein. For example, a drug may include a biomolecule.

[0068] In this application, the term "related entity" generally refers to any monomeric or multimeric protein, protein fragment, nucleotide sequence, small molecule compound, delivery vehicle or modified transgenic vector that is specifically connected to an antigen binding protein. Related entities include but are not limited to antibodies and their binding portions, such as immunologically functional fragments. For example, an antigen binding protein can be a transferrin binding protein. In this application, the term "therapeutic entity" generally refers to any monomeric or multimeric protein or protein fragment that has the function of treating or preventing a disease.

[0069] In the present application, the term "heavy chain variable region (VH)" generally refers to the region of the immunoglobulin heavy chain structure, which includes heavy chain complementary determining regions CDR1, CDR2, CDR3 and framework regions FR1, FR2, FR3, FR4. The heavy chain variable region may include a binding domain that interacts with an antigen. For example, the heavy chain variable region of the present application may include a region that specifically binds to transferrin. For example, the heavy chain variable region of the present application may include the VHH of a single domain antibody.

[0070] In this application, the terms "CDR" and "CDRs" generally refer to the complementarity determining regions (CDRs), of which three CDRs constitute the binding properties of the light chain variable region (LCDR1, LCDR2 and LCDR3) and three CDRs constitute the heavy chain variable region (HCDR1, HCDR2 and HCDR3). The CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences containing the backbone or framework regions. The precisely defined CDR boundaries and lengths are subject to different classification and numbering systems.

[0071] In this application, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids. For example, a polypeptide may comprise an antigen binding protein and other molecules. For example, a polypeptide may comprise a fusion antigen. For example, an antigen binding protein and a molecule may be directly or indirectly linked. For example, an antigen binding protein and a molecule may be linked via a linker. For example, a molecule may comprise a therapeutic molecule. For example, a molecule may comprise PE38. For example, a molecule may comprise GLP-1 or an analog.

[0072] In the present application, the term "affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a polypeptide or an antibody) and its binding partner (e.g., a target or an antigen). Unless otherwise indicated, when used herein, "binding affinity" refers to the relationship between members of a binding pair (e.g., in a polypeptide-polynucleotide-complex, or between a polypeptide and its target, or between an antibody and an antibody). The intrinsic binding affinity of a 1:1 interaction between antigens. The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be detected by conventional methods known in the art, such as surface plasmon resonance, and also includes those methods reported in the present application. The higher affinity of a molecule X to a binding partner Y can be seen in lower Kd and / or EC50 values.

[0073] In the present application, the term "isolated nucleic acid molecule" generally refers to a nucleic acid encoding a specific amino acid sequence that selectively binds to transferrin, such as an antibody or an antibody portion (e.g., VH, VL, CDR3), and means a nucleic acid sequence encoding a fusion protein with an antibody or an antibody portion.

[0074] In the present application, the term "vector" generally refers to a composition comprising an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic compounds or amphipathic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmids and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0075] In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a plasmid or vector of a subject, which contains a nucleic acid molecule described in the present application or a nucleic acid molecule described in the present application. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical to the original parental cell (in terms of the morphology or genome of the total DNA complement). Cells may include cells transfected in vitro with the vectors described in the present application.

[0076] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise an antigen binding protein, an immunoconjugate, an isolated nucleic acid molecule, a vector and / or a cell, and an optional pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise one or more (pharmaceutically effective) vehicles and other suitable formulations. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen and lyophilized compositions. DETAILED DESCRIPTION OF THE INVENTION

[0078] transferrin binding protein

[0079] In one aspect, the present application provides a transferrin binding protein that can specifically bind to transferrin and does not interfere with the interaction between transferrin and transferrin receptor 1.

[0080] For example, transferrin-binding proteins may be able to extend the circulation half-life of their associated entities.

[0081] For example, half-life can be estimated by methods known to those skilled in the art. For example, the in vivo half-life of the amino acid sequence, compound or polypeptide of the present invention can be determined in any known manner, such as by pharmacokinetic analysis. Suitable technology is obvious to those skilled in the art, for example, as described in paragraph o) of WO 08 / 020079, page 57. Also as described in paragraph o) of WO 08 / 020079, page 57, parameters such as t1 / 2-α, t1 / 2-β and area under the curve (AUC) can be used to represent half-life. In this regard, it should be noted that the term "half-life" in this application particularly refers to t1 / 2-β or terminal half-life (wherein t1 / 2-α and / or AUC can be ignored). For example, with reference to the following experimental section and standard manuals, such as Kenneth, A et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al.

[0082] For example, related entities can be any monomeric or multimeric protein, protein fragment, nucleotide sequence, small molecule compound, delivery vehicle or modified transgenic vector that is specifically connected to the antigen binding protein. For example, related entities can include but are not limited to antibodies and their binding portions, such as immunologically functional fragments. For example, the antigen binding protein can be transferrin binding protein.

[0083] For example, the transferrin binding protein may enable a fusion protein comprising the transferrin binding protein to cross the blood-brain barrier (BBB).

[0084] For example, BBB can be a physiological barrier between peripheral circulation and the brain and spinal cord. For example, BBB can include the tight connection in the plasma membrane of brain capillary endothelial cells, and constitute a tight barrier for limiting molecules, even very small molecules such as urea (60 daltons), to brain transport. For example, brain capillary endothelial cells can have weaker pinocytosis. For example, the blood-brain barrier can include the blood-spinal barrier in the BBB in the brain, the blood-spinal barrier in the spinal cord and the blood-retinal barrier in the retina. For example, BBB can also include a blood-CSF barrier (choroid plexus), wherein the barrier is composed of ependymal cells rather than capillary endothelial cells.

[0085] For example, the fusion protein can enter the brain tissue by crossing the BBB.

[0086] For example, transferrin binding proteins can be used for oral delivery of drugs.

[0087] For example, the drug may be administered in the form of capsules, granules or tablets for oral delivery.

[0088] For example, a transferrin binding protein may enable intracellular delivery of its associated entity to cells expressing the transferrin receptor.

[0089] For example, the transferrin binding protein may be capable of specifically binding to transferrin and not interfering with the interaction between transferrin and transferrin receptor 1.

[0090] For example, the transferrin can be human transferrin.

[0091] For example, the transferrin may be iron-saturated transferrin containing iron.

[0092] For example, an iron-saturated transferrin containing iron may comprise transferrin and Fe 3+ .

[0093] For example, transferrin can bind Fe 3+ .

[0094] For example, transferrin can bind two Fe 3+ .

[0095] For example, iron-saturated transferrin containing iron can bind to the transferrin receptor.

[0096] For example, iron-saturated transferrin containing iron can bind to transferrin receptor 1.

[0097] For example, iron-saturated transferrin containing iron can be transported to endosomes via the transferrin receptor.

[0098] For example, iron-saturated transferrin containing iron can be transported via endocytosis.

[0099] For example, iron-saturated transferrin containing iron can be transported via endocytosis.

[0100] For example, iron-saturated transferrin containing iron can bind to Fe 3+ Separation.

[0101] For example, the transferrin may be apotransferrin which does not contain iron.

[0102] For example, iron-free apotransferrin can be derived from 3+ Isolated iron-saturated transferrin containing iron.

[0103] For example, iron-free apotransferrin can bind Fe 3+ Combine.

[0104] For example, a transferrin binding protein may have a higher affinity for iron-containing, iron-saturated transferrin than for apotransferrin, which does not contain iron.

[0105] For example, affinity can be detected by a surface plasmon resonance based assay.

[0106] For example, affinity can be measured by enzyme-linked immunosorbent assay (ELISA).

[0107] For example, affinity can be detected by competition assay (RIA).

[0108] For example, affinity can be detected by flow cytometry (FACS).

[0109] For example, affinity can be expressed as a binding equilibrium constant (KD).

[0110] For example, the affinity of the transferrin binding protein for iron-containing, iron-saturated transferrin can be 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold greater than the affinity for iron-free, apotransferrin.

[0111] For example, the transferrin binding protein may comprise a monoclonal antibody.

[0112] For example, the transferrin binding protein may comprise a single chain antibody fragment.

[0113] For example, the transferrin binding protein may comprise a single domain antibody fragment.

[0114] For example, a transferrin binding protein may include an engineered protein.

[0115] For example, a transferrin binding protein may include a peptide.

[0116] For example, an antibody or antibody fragment to transferrin binding protein may comprise a sequence of animal origin.

[0117] For example, an antibody or antibody fragment to transferrin binding protein may comprise a humanized sequence.

[0118] For example, an antibody or antibody fragment to transferrin binding protein may comprise a fully human sequence.

[0119] For example, an antibody or antibody fragment to transferrin binding protein may comprise a chimeric sequence.

[0120] For example, an antibody or antibody fragment to transferrin binding protein may comprise a synthetic sequence.

[0121] For example, the antibody or antibody fragment to transferrin binding protein may be a single domain antibody fragment VHH.

[0122] In the present application, the transferrin binding protein may comprise at least one CDR in the heavy chain variable region VH, wherein VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 70-104.

[0123] For example, VH may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 70-104.

[0124] For example, the transferrin binding protein may comprise HCDR3 of VH having an amino acid sequence as shown in any one of SEQ ID NOs: 45-69.

[0125] For example, the transferrin binding protein may comprise HCDR2 of VH having an amino acid sequence as shown in any one of SEQ ID NOs: 22-44.

[0126] For example, the transferrin binding protein may comprise HCDR1 of VH having an amino acid sequence as shown in any one of SEQ ID NOs: 1-21.

[0127] For example, the transferrin binding protein may comprise HCDR1, HCDR2 and HCDR3, HCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 1-21, HCDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 22-44, and HCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 45-69.

[0128] For example, the transferrin binding protein may comprise an Fc region.

[0129] For example, the Fc region may comprise a human Fc region.

[0130] For example, the Fc region may comprise a human IgG Fc region.

[0131] For example, the Fc region may include a human IgG1 Fc region. For example, the Fc region may include a human IgG4 Fc region.

[0132] For example, transferrin binding protein can include an antibody or its antigen binding fragment. The antibody can be selected from the group consisting of: monoclonal antibody, single chain antibody, chimeric antibody, multispecific antibody, humanized antibody and fully human antibody. The antigen binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, F(ab')2, sdAb, Fv, dAb and ScFv fragment.

[0133] For example, the transferrin binding protein may be a VHH.

[0134] For example, the VHH may comprise a CDR3, and the CDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 45-69.

[0135] For example, the VHH may comprise a CDR2, and the CDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 22-44.

[0136] For example, the VHH may comprise a CDR1, and the CDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 1-21.

[0137] For example, VHH may comprise CDR1, CDR2 and CDR3, CDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 1-21, CDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 22-44, and CDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 45-69.

[0138] For example, the VHH may comprise CDR1, CDR2 and CDR3, and CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 22 and SEQ ID NO: 45, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 22 and SEQ ID NO: 63, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 23 and SEQ ID NO: 46, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 23 and SEQ ID NO: 58, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 40 and SEQ ID NO: 58, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 41 and SEQ ID NO: 58, respectively. NO:41 and SEQ ID NO:58; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:23 and SEQ ID NO:66, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:24 and SEQ ID NO:47, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:25 and SEQ ID NO:48, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:26 and SEQ ID NO:49, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:27 and SEQ ID NO:50, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:27 and SEQ ID NO:50, respectively; NO:27 and SEQ ID NO:59; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:28 and SEQ ID NO:51, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:29 and SEQ ID NO:52, respectively;Or CDR1, CDR2 and CDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:30 and SEQ ID NO:53; or CDR1, CDR2 and CDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:31 and SEQ ID NO:54; or CDR1, CDR2 and CDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:32 and SEQ ID NO:55; or CDR1, CDR2 and CDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:33 and SEQ ID NO:56; or CDR1, CDR2 and CDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:34 and SEQ ID NO:57; or CDR1, CDR2 and CDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:35 and SEQ ID NO:60; or CDR1, CDR2 and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:15, SEQ ID NO:36 and SEQ ID NO:61, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:16, SEQ ID NO:37 and SEQ ID NO:62, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:38 and SEQ ID NO:64, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:18, SEQ ID NO:39 and SEQ ID NO:65, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:42 and SEQ ID NO:67, respectively; or CDR1, CDR2 and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:20, SEQ ID NO:43 and SEQ ID NO:44, respectively. The amino acid sequence shown in SEQ ID NO:68; or CDR1, CDR2 and CDR3 may comprise the amino acid sequences shown in SEQ ID NO:21, SEQ ID NO:44 and SEQ ID NO:69, respectively. ;

[0139] For example, the VHH may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 70-104. For example, SLN9056 comprises an amino acid sequence as shown in SEQ ID NO: 70. For example, SLN9057 comprises an amino acid sequence as shown in SEQ ID NO: 71. For example, SLN0042 comprises an amino acid sequence as shown in SEQ ID NO: 72. For example, SLN0043 comprises an amino acid sequence as shown in SEQ ID NO: 73. For example, SLN0044 comprises an amino acid sequence as shown in SEQ ID NO: 74. For example, SLN0045 comprises an amino acid sequence as shown in SEQ ID NO: 75. For example, SLN0049 comprises an amino acid sequence as shown in SEQ ID NO: 76. For example, SLN0056 comprises an amino acid sequence as shown in SEQ ID NO: 77. For example, SLN0057 comprises an amino acid sequence as shown in SEQ ID NO: 78. For example, SLN0059 comprises an amino acid sequence as shown in SEQ ID NO: 79. For example, SLN0062 comprises the amino acid sequence as shown in SEQ ID NO:80. For example, SLN0064 comprises the amino acid sequence as shown in SEQ ID NO:81. For example, SLN0065 comprises the amino acid sequence as shown in SEQ ID NO:82. For example, SLN0071 comprises the amino acid sequence as shown in SEQ ID NO:83. For example, SLN0072 comprises the amino acid sequence as shown in SEQ ID NO:84. For example, SLN0046 comprises the amino acid sequence as shown in SEQ ID NO:85. For example, SLN0058 comprises the amino acid sequence as shown in SEQ ID NO:86. For example, SLN9008 comprises the amino acid sequence as shown in SEQ ID NO:87. For example, SLN9013 comprises the amino acid sequence as shown in SEQ ID NO:88. For example, SLN9015 comprises the amino acid sequence as shown in SEQ ID NO:89. For example, SLN9025 comprises the amino acid sequence as shown in SEQ ID NO:90. For example, SLN9026 comprises the amino acid sequence as shown in SEQ ID NO: 91. For example, SLN9056 comprises the amino acid sequence as shown in SEQ ID NO: 70.

[0140] The antigen-binding protein of the present application can compete with a reference antibody for binding to Tf, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, HCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 1-21, HCDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 22-44, and HCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 45-69.

[0141] Peptides

[0142] In another aspect, the present application provides a polypeptide comprising a transferrin binding protein.

[0143] For example, a polypeptide may comprise a therapeutic entity.

[0144] For example, the therapeutic entity can be engineered cytotoxic Pseudomonas exotoxin A (PE38). For example, PE38 can comprise the amino acid sequence shown in SEQ ID NO: 109.

[0145] For example, the therapeutic entity may be glucagon-like peptide-1 (GLP-1) or a variant thereof. For example, GLP-1 may comprise the amino acid sequence shown in SEQ ID NO: 108.

[0146] For example, a variant can be a polypeptide that has significant sequence identity with a parent polypeptide and retains the biological activity of the parent polypeptide. For example, the amino acid sequence of a variant can have at least about 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher identity with the amino acid sequence of a parent polypeptide. For example, a variant can also be a protein or polypeptide that replaces, deletes or adds one or more amino acids in the amino acid sequence of a protein and / or polypeptide. For example, a variant can include a protein or polypeptide that has at least one, such as 1-30, 1-20 or 1-10, and such as 1, 2, 3, 4 or 5, amino acid substitutions, deletions or insertions of amino acid changes. For example, substitutions can be conservative.

[0147] For example, the therapeutic entity and the transferrin binding protein can be directly linked.

[0148] For example, the therapeutic entity and the transferrin binding protein can be linked via gene fusion or recombinant DNA technology.

[0149] For example, the therapeutic entity and the transferrin binding protein can be indirectly linked.

[0150] For example, the therapeutic entity and the transferrin binding protein can be linked via a spacer.

[0151] For example, the therapeutic entity and the transferrin binding protein can be linked via a linker.

[0152] For example, the linker can be a peptide having an amino acid sequence.

[0153] For example, the linker may be of artificial synthetic origin.

[0154] Isolated nucleic acid molecules, vectors, cells

[0155] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the isolated transferrin binding protein or polypeptide of the present application.

[0156] The nucleic acid molecules of the present application can be separated. For example, it can be produced or synthesized by the following methods: (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification, (ii) by cloning and recombination, (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesis, such as chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0157] In the present application, nucleic acids encoding antibodies or antigen-binding fragments thereof can be prepared by various methods known in the art, including but not limited to restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides.

[0158] In another aspect, the present application provides a vector, which may contain the nucleic acid molecule of the present application.

[0159] On the other hand, the application provides one or more vectors, which comprise one or more nucleic acid molecules of the application. Each vector may comprise one or more nucleic acid molecules. In addition, the vector may also comprise other genes, such as marker genes that allow the vector to be selected in a suitable host cell and under suitable conditions. In addition, the vector may also comprise expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art, and may comprise, for example, promoters, ribosome binding sites, enhancers and other control elements that regulate gene transcription or mRNA translation. One or more nucleic acid molecules of the application may be operably connected to the expression control elements. The vector may comprise, for example, plasmids, cosmids, viruses, bacteriophages or other vectors that are commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0160] In another aspect, the present application provides a cell, which may contain the nucleic acid molecule of the present application or the vector of the present application.

[0161] On the other hand, the present application provides a host cell, which may contain one or more nucleic acid molecules of the present application and / or one or more vectors of the present application.

[0162] In certain embodiments, every kind or every host cell can comprise one or a kind of nucleic acid molecule or carrier of the present application.In certain embodiments, every kind of host cell or every kind of host cell can comprise multiple (for example, 2 or more) or multiple (for example, 2 or more) nucleic acid molecule or carrier of the present application.For example, the carrier of the present application can be introduced into host cell, for example eukaryotic cell, such as cell, fungal cell or yeast cell from plant etc..The carrier of the present application can be introduced into host cell by methods known in the art (such as electroporation, lipofectine transfection, lipofectamin transfection etc.).

[0163] Pharmaceutical composition

[0164] On the other hand, the present application provides a pharmaceutical composition, which may include the transferrin binding protein of the present application, the nucleic acid molecule of the present application, the vector of the present application and / or the cell of the present application, and optionally a pharmaceutically acceptable adjuvant.

[0165] For example, the pharmaceutical composition of the present application can be used directly for treatment or diagnosis, and thus can be used for the prevention and treatment of diseases. In addition, other therapeutic entities can be used simultaneously.

[0166] The pharmaceutical composition of the present application may include a safe and effective amount (e.g., 0.001-99wt%, 0.01-90wt%, or 0.1-80wt%) of the transferrin-binding protein of the present application and a pharmaceutically acceptable adjuvant (which may include a carrier or excipient). Such carriers may include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the mode of administration. The pharmaceutical composition of the present application may be made into an injectable form, for example, it may be prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as injections or solutions, should be produced under aseptic conditions. The amount of active ingredient applied is a therapeutically effective amount. In addition, the transferrin-binding protein of the present application may also be used together with other therapeutic entities.

[0167] Transferrin binding protein as herein described or pharmaceutical composition can be prepared, applied and administered in a manner that conforms to good medical practice. In this case, factors to be considered include the specific disease being treated, the specific mammal being treated, the clinical symptoms of individual patients, the cause of disease, the drug delivery site, the method of application and other factors known to doctors. The therapeutic entity is not required, but can be selectively prepared and / or applied simultaneously with one or more entities currently used to prevent or treat the disease being paid attention to. The effective amount of such other entities depends on the amount of the therapeutic agent present in the preparation, the type of disease or treatment and other factors mentioned above. Usually, these entities can be used with any dosage appropriately determined according to experience / clinical situation, and by any approach appropriately determined according to experience / clinical use. Compared with monotherapy, the antibody dosage used in conjoint therapy can be reduced. It is easy to monitor the progress of this therapy by conventional techniques.

[0168] method

[0169] In another aspect, the present application provides a method for transporting molecules across a cell membrane by using a transferrin binding protein.

[0170] For example, the methods can be used for drug delivery across cell membranes.

[0171] For example, a cell membrane may belong to a polarized cell.

[0172] For example, a polarized cell can be a functional cell.

[0173] For example, the polarized cell can be a neuron.

[0174] For example, the cell membrane may belong to an unpolarized cell.

[0175] For example, the unpolarized cell can be a neuronal cell.

[0176] For example, unpolarized cells can transmit neural signals.

[0177] For example, polarized and / or unpolarized cells may express transferrin receptors on their cell membranes.

[0178] For example, polarized and / or unpolarized cells may express human transferrin receptor on their cell membrane.

[0179] For example, polarized and / or unpolarized cells may express transferrin receptor 1.

[0180] For example, polarized and / or unpolarized cells may express human transferrin receptor 1.

[0181] For example, unpolarized cells may belong to the blood-brain barrier.

[0182] For example, unpolarized cells may belong to the intestinal epithelium.

[0183] For example, unpolarized cells can belong to multiple layers of cells in a solid tissue.

[0184] In the present application, drug delivery methods across cell membranes may include crossing the blood-brain barrier, crossing the intestinal epithelium, crossing multiple layers of cells in solid tissues, intracellular delivery of drugs, and / or recycling of internalized drugs back into the blood circulation.

[0185] For example, drug delivery across the cell membrane may be pertinent to unpolarized cells.

[0186] For example, drug delivery across a cell membrane may include crossing the blood-brain barrier for systemically administered drugs targeted to the central nervous system.

[0187] For example, drug delivery across a cell membrane may include the passage of an orally administered drug across the intestinal epithelium.

[0188] For example, drug delivery across a cell membrane may involve penetration of the drug through multiple layers of cells in a solid tissue.

[0189] For example, drug delivery across cell membranes can be pertinent to polarizing cells.

[0190] For example, delivery of a drug across a cell membrane may include intracellular delivery of a drug.

[0191] For example, drug delivery across a cell membrane may include recycling of endocytosed drug back into the blood circulation.

[0192] For example, a drug may comprise a small molecule compound.

[0193] For example, a small molecule compound can be a chemically synthesized drug.

[0194] For example, a small molecule compound may have a molecular weight of less than 1000 Daltons.

[0195] For example, small molecule compounds may have therapeutic effects.

[0196] For example, small molecule compounds may have anti-tumor activity.

[0197] For example, the drug may include a synthetic peptide.

[0198] For example, a synthetic peptide may comprise α-amino acids.

[0199] For example, α-amino acids can be linked via a peptide chain.

[0200] For example, synthetic peptides can be synthesized by solid phase peptide synthesis.

[0201] For example, synthetic peptides can be synthesized by liquid phase peptide synthesis.

[0202] For example, a medicament may comprise a recombinant protein.

[0203] For example, a recombinant protein can be a polypeptide of semisynthetic or synthetic origin.

[0204] For example, recombinant proteins can be expressed by joining DNA molecules from different sources using recombinant DNA techniques.

[0205] For example, a recombinant protein may no longer be completely or partially associated with the protein in its native state.

[0206] For example, a recombinant protein may be linked to a peptide that is different from the peptide to which it is naturally linked.

[0207] For example, a recombinant protein may not exist in its native state.

[0208] For example, the drug may comprise an antibody. For example, the drug may comprise an antibody fragment.

[0209] For example, the antigen fragment can be a Fab, Fab', F(ab)2, an Fv fragment, F(ab')2, scFv, di-scFv and / or a dAb.

[0210] For example, a drug may contain an enzyme.

[0211] For example, an enzyme can be a protein that has catalytic activity.

[0212] For example, a drug may comprise a nucleotide sequence.

[0213] For example, the nucleotide sequence may be a DNA sequence.

[0214] For example, the nucleotide sequence may be an RNA sequence.

[0215] For example, the nucleotide sequence can be genomic DNA, cDNA, synthetic DNA, proviral DNA, viral DNA, mRNA, synthetic RNA, or a combination thereof.

[0216] For example, a nucleotide sequence may encode a peptide or a protein.

[0217] For example, synthetic RNA can modulate mRNA stability or translation.

[0218] For example, the synthetic RNA can be a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO).

[0219] For example, the medicament may comprise liposomes.

[0220] For example, a liposome can be a vesicle composed of one or more concentrically arranged lipid bilayers.

[0221] For example, liposomes can comprise an aqueous phase.

[0222] For example, the aqueous phase may contain nucleic acids.

[0223] For example, the drug may comprise nano-sized particles, such as lipid nanoparticles.

[0224] For example, a lipid nanoparticle may comprise a plurality of lipid molecules physically bound to each other by intermolecular forces.

[0225] For example, intermolecular forces can be covalent or non-covalent.

[0226] For example, a lipid nanoparticle may comprise one or more lipids.

[0227] For example, the lipid can be a cationic lipid, a non-cationic lipid, and a PEG-lipid.

[0228] For example, a medicament may include a pharmaceutical carrier.

[0229] For example, the pharmaceutical carrier may be a standard composition suitable for human administration.

[0230] For example, the pharmaceutical carrier may be a typical adjuvant used in animal vaccination.

[0231] For example, a medicament may comprise a modified virus.

[0232] For example, the modified virus can be a genetically engineered virus.

[0233] For example, the modified viruses can inhibit the growth of cancer cells or hyperproliferative cells in vivo or in vitro.

[0234] For example, the modified viruses can induce cancer cell or hyperproliferative cell death in vivo or in vitro.

[0235] For example, the modified virus can be an oncolytic virus.

[0236] For example, a medicament may comprise a gene therapy vector.

[0237] For example, gene therapy vectors are capable of delivering target genes into target cells.

[0238] For example, the target gene can be released in the target cell.

[0239] For example, the target gene can be integrated into the cell nucleus.

[0240] For example, gene therapy vectors can exert the therapeutic function of target genes.

[0241] For example, the gene therapy vector can be a plasmid vector, a phage vector, a viral vector or a non-viral vector thereof.

[0242] For example, the gene therapy vector can be a cloning vector or an expression vector thereof.

[0243] For example, the gene therapy vector can be a temperature-sensitive vector, a fusion expression vector or a non-fusion expression vector.

[0244] For example, the drug may bind to transferrin binding protein.

[0245] For example, the drug can be conjugated to the transferrin binding protein by gene fusion.

[0246] For example, the drug can be bound to the transferrin binding protein by chemical coupling.

[0247] For example, the method can be used to extend the circulation half-life of a therapeutic entity.

[0248] For example, the therapeutic entity can be linked directly or indirectly to the transferrin binding protein.

[0249] For example, the method can be used to deliver therapeutic agents to cells or organs expressing transferrin receptors, containing or using transferrin binding entities.

[0250] For example, the method can be used to deliver targeted drugs across the blood-brain barrier, intestinal epithelium, and / or cell membranes expressing transferrin receptors, including the use of transferrin binding entities.

[0251] For example, the method can be used for oral delivery of a therapeutic or diagnostic entity linked to the antigen binding protein.

[0252] For example, the method can be used for the intracellular delivery of a therapeutic entity that is directly or indirectly linked to the antigen binding protein.

[0253] Example

[0254] The following examples are set forth in order to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors believe to be their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; CPU, colony forming units; rpm, revolutions per minute; RT, room temperature; im, intramuscular injection (Im); ip, intraperitoneal injection (Im); sc, subcutaneous injection (Im); and the like.

[0255] Example 1 Establishment of VHH immune library

[0256] Two healthy and age-appropriate camels were immunized 4 times using human transferrin (human transferrin, Sigma T3309). After the antibody titer was qualified, 200 ml of peripheral blood was extracted from each camel, lymphocytes were separated, and total RNA was extracted. Reverse transcription was used to generate cDNA for the construction of a camel immune VHH antibody library for phage display. The VHH antibody library was quality-checked by sequencing 24 independent phage clone DNAs. The data showed that the sequences of 22 / 24 clones contained standard VHH sequences fused to gp3 proteins for phage display, and each of the 22 clones had a unique VHH sequence, in which the length of CDR3 was 9 or more amino acids. The library was used for subsequent antibody discovery.

[0257] Example 2 Discovery of VHH

[0258] 2.1-Panning

[0259] Take out the phage library from the -80℃ refrigerator, incubate at 37℃ for 10min, and centrifuge at 5000rpm for 5min. 12 ~10 13 CFU phage (input) was transferred to a microtube containing 1 mL PBS solution containing 1% BSA, 10 μg biotinylated human or mouse transferrin. The mixture was incubated at room temperature for 1 hour, and 100 μL of streptavidin-coated magnetic Dyna beads suspension was added. Incubated at room temperature for 30 minutes on a rotating drum, and the microtube was placed on a magnetic stand for 30 seconds to remove the solution mixture. The magnetic beads were washed 10 times with 1 mL PBST (PBS containing 0.05% Tween 20, pH 7.4), and the final wash was performed with PBS.

[0260] Add 1 ml of 10 μg / mL trypsin to PBS, incubate at 37°C for 30 min, elute phage from magnetic beads, and separate on a magnetic stand. Transfer the supernatant (output) to a tube containing 4 mL of TG1 (A600≈0.6) to infect bacteria. Incubate at 37°C for 30 min, and collect infected bacteria by centrifugation at 4,500 rpm for 10 min at 4°C. Resuspend the pellet in 500 μL and spread on a 25×25 cm 2×YT / Amp / Glu plate to amplify phage-infected bacteria. After incubation at 37°C overnight, add 30 mL of 2×YT medium, and collect by centrifugation at 4,500 rpm for 10 min. Add 2 mL of 2×YT medium containing 40% glycerol to resuspend the pellet and store at -80°C, or prepare phage directly as needed (as described below in phage preparation or packaging) for the next round of panning.

[0261] Input and output phage were titrated to determine the phage enrichment efficiency of the panning process. Briefly, serial dilutions (10-fold, usually 10 -1 ~10 -9 ), 10 μL of serially diluted phages were mixed with 90 μL of logarithmic growth phase TG1 in a new tube and incubated at 37°C for 30 min. Thereafter, 10 μL of the mixture was spotted on a pre-warmed 2×YT / Amp plate. Before incubating the bacterial plate at 37°C O / N, the lid was opened to evaporate the solution. The input and output phages were counted and titered, respectively, and the output / input ratio of each round of panning was used to determine the efficiency of the panning process.

[0262] 2.2 Preparation or packaging of bacteriophage

[0263] The phage-infected TG1 bacteria were inoculated into 2×YT / Amp / Glu medium and cultured at 37°C with shaking (250 rpm) until the A600 value reached 0.6 (approximately 1-2 h). Helper phage M13K07 was added at a ratio of phage:bacteria = 1000. The culture was incubated at 37°C without shaking for 30 min, and then cultured with shaking at 180 rpm for 30 min.

[0264] The bacterial culture was centrifuged at 4,500 rpm for 10 min in a sterile centrifuge bottle, and the bacterial pellet was recovered. The bacterial pellet was resuspended in 100 mL 2×YT / Amp / Kan (Amp: 100 μg / mL, Kan: 50 μg / mL), transferred to a 250 mL flask, and incubated at 37°C with shaking (250 rpm) for 4 h.

[0265] The culture was centrifuged at 4,500 rpm for 10 min and the supernatant was collected. Phages were precipitated from the supernatant by adding 1 / 4 volume of PEG solution (20% polyethylene glycol 6000, 2.5 M NaCl) and incubated on ice overnight. After collection by centrifugation at 4,500 rpm for 30 min at 4°C, the phage pellet was resuspended in 5 mL PBS and incubated at 37°C with shaking for 30 min. Insoluble debris was removed by centrifugation at 4,500 rpm for 10 min, and the soluble phage in the supernatant was transferred to a new tube and the PEG precipitation was repeated once. The final phage solution in PBS was used for the next round of panning or mixed with glycerol and stored at -80°C.

[0266] 2.3 Phage-based ELISA screening

[0267] Single colonies from the panning output were picked from the agar plates and inoculated into 2xYT / GA medium (2% glucose, 100 μg / mL ampicillin) in a 96-well deep-well plate and incubated overnight at 37°C with a shaking speed of 320 rpm. 10 μL of the overnight culture was transferred to 400 μL of 2YT / GA medium in a new deep-well plate. The culture was grown at 37°C with shaking (800 rpm) to an A600 of 0.6 (approximately 1 h), and helper phage M13K07 (4.2×10 13 CFU / mL). The culture was incubated at 37°C without shaking for 30 min, and then shaken at 180 rpm for 30 min. The bacterial culture was centrifuged at 4,000 rpm for 30 min and the supernatant was discarded. The bacterial pellet was resuspended in 400 μL 2×YT / AK (Amp: 100 μg / mL, Kan: 50 μg / mL) and cultured at 30°C with shaking (320 rpm) overnight. The bacterial culture was centrifuged at 4,000 rpm for 30 min and the supernatant was collected. The supernatant was used as the phage solution for ELISA screening.

[0268] For ELISA screening, 96-well microtiter plates were coated with 100 μL / well of 1 μg / mL streptavidin (carbonate buffered saline, pH 9.4, or CBS) at 4°C overnight. Blocked with 200 μL / well blocking buffer (PBS pH 7.4 / 0.05% Tween 20 / 1% BSA) at room temperature for 1 hour. Washed 3 times with PBST (PBS / 0.05% Tween 20, pH 7.4), added 100 μL of biotinylated human or mouse transferrin (1 μg / mL in blocking buffer) and incubated at room temperature for 1 hour. Washed 3 times with PBST, added 100 μL of phage solution (as above) and incubated at room temperature for 1 hour. Washed 3 times with PBST, added 100 μL / well mouse anti-M13 IgG-HRP (prepared in blocking buffer) and incubated at room temperature for 1 hour. Washed 3 times with PBST, added 100 μL of TMB solution and incubated at room temperature for 15 minutes in the dark. After the reaction was terminated with 100 μL of stop solution, the microplate was scanned at 450 nm. Positive clones that specifically bound to transferrin were selected for DNA sequencing, and single VHHs were identified based on the deduced amino acid sequence.

[0269] 2.4 Production of recombinant VHH-OVA-his protein

[0270] A single VHH clone was selected for subcloning to construct a recombinant plasmid to produce VHH-OVA-His protein (OVA comprises the amino acid sequence shown in SEQ ID NO: 107). After verifying the plasmid sequence by sequencing, HEK293 cells were transiently transfected with the recombinant plasmid using lipofectamine or PEI as a transfection reagent for small-scale production of recombinant protein. The culture was grown in a shake flask medium at a scale of 30 ml to 100 ml for 5-7 days. The cells were removed by centrifugation and the culture supernatant was used for protein purification by Ni-NTA agarose. The purified protein ( Figure 1 ).

[0271] 2.5 Transferrin binding assay

[0272] In order to detect the binding ability and specificity of the recombinant protein to transferrin, streptavidin (1 μg / ml, in CBS, 100 μl / well) was coated on a 96-well microplate and incubated overnight at 4°C. After washing 3 times with PBST, the microplate was blocked with PBST containing 1% BSA for 1 hr at room temperature. After washing 3 times with PBST, biotinylated human or mouse transferrin (1 ug / ml, in PBS, 100 ul / well) was added and incubated at room temperature for 1 hour. After washing 3 times with PBST, purified VHH-OVA-His protein was added and incubated at room temperature for 1 hour (serial dilution in PBS at pH 7.4 or pH 6.0, starting from 10 ug / ml, 100 ul / well). Rabbit anti-chicken ovalbumin (OVA) and anti-rabbit IgG-HRP secondary antibodies were incubated sequentially, and then the plate was washed 3 times with PBST and incubated with the substrate solution and stop solution as described above for phage ELISA. Representative data are shown in Figure 2. Figure 2 A (pH 7.4, iron-saturated transferrin) or Figure 2 B (pH 6.0, apo-transferrin) is shown to illustrate the binding activity of recombinant VHH-OVA-His to natural human transferrin (Sigma).

[0273] 2.6 VHH binding does not interfere with transferrin / TfR1 interaction on HEK293F-hTFR1 cells

[0274] HEK293F-hTfR1 cells were prepared by transfecting a plasmid expressing hTfR1 with a GFP tag into HEK293F cells. Stably transfected cell pools were used for this assay. Briefly, engineered HEK293F cells expressing hTfR1 were cultured in OPM-293CD05 medium at 37°C, 8% CO2, and saturated humidity with shaking. HEK293F-hTfR1 cells were harvested, counted, and blocked with blocking buffer at 37°C for 30 min. 1 × 106 Cells were incubated with or without transferrin (final concentration 5 μg / mL) and ferric citrate solution (final concentration 0.2 mg / mL) at pH 7.4 or pH 6.0 and mixed with VHH-OVA-His or control protein solution (final concentration 10 μg / mL). After incubation with rabbit anti-OVA antibody and anti-rabbit IgG-AF647 secondary antibody (all prepared in blocking buffer), cells were washed twice by centrifugation at 300 g for 5 min at 4°C. Cells were scanned using CytoFLEX (Beckman). Data were analyzed using CytoExpert 2.4 software to determine 1) differential binding of VHH-OVA-His protein to cells (transferrin binding does not interfere with transferrin / TfR1 interaction on the cell surface); 2) dependence on transferrin (specificity for transferrin); 3) dependence on TfR1 (specificity for TfR1-mediated cell binding); 4) response to pH and Fe 3+ The data (below and Figure 3 ) show that the selected transferrin binding VHHs such as SLN0042, SLN0043, SLN0044, SLN0049, SLN0062, SLN0064, SLN0065, SLN0056, SLN0071 and SLN0072 do not interfere with the transferrin / TfR1 interaction and can only bind to TfR1 on the cell surface by binding to transferrin. The data also validate the results of ELISA, which show that some selected VHHs have different affinities for iron-saturated transferrin and apotransferrin.

[0275] Table 1. Summary of VHH / Tf binding characteristics on the surface of cells overexpressing hTfR1 under different pH conditions.

[0276]

[0277]

[0278] Note: +++≥15%; 15%>++≥10%; 10%>+≥5%; -<5% combination

[0279] 2.7 Epitope binning

[0280] Epitope identification is used to distinguish different “bins” of different proteins, because antibodies in different “bins” bind to different epitopes and display different functional characteristics.

[0281] For epitope identification, 96-well microplates were coated with 5 μg / mL (100 μL / well in CBS buffer) of each VHH-OVA-His recombinant protein and incubated overnight at 4°C. After washing and blocking as described above for ELISA, a mixture of biotinylated human transferrin (final concentration 1 μg / mL) and VHH-OVA-His protein (final concentration 5 μg / mL) was added to cross-check the plate reaction and incubated at room temperature for 1 h. Biotinylated transferrin bound to the coated VHH-OVA-His was detected using streptavidin-HRP and the corresponding method. Figure 4 The data shown indicate that the selected VHHs belong to multiple bins and are particularly different from the reference antibodies (VHH1, VHH2, VHH3).

[0282] 2.8 Transcytosis of VHH-OVA-His protein across Caco-2 cell monolayers

[0283] Caco-2 cells were cultured at 2×10 4 Each well was inoculated in a trans-well 96-well plate and cultured for 12 days to form a cell monolayer in the inner chamber (the culture medium was changed every other day during the first week and every day during the second week). On the 13th day, the monolayer cells were washed with pre-warmed PBS and incubated at 37°C for 30 min in MEM medium containing 0.1% BSA without FBS to remove endogenous Tf. Recombinant VHH-OVA-His protein (10 μg / mL) with or without natural human transferrin (5 μg / mL) was prepared in MEM medium (pH 7.4) containing 0.1% BSA, added to the inner chamber (above the Caco-2 cell monolayer), and incubated at 37°C for 2h or 24h. The culture medium in the outer chamber was collected at the specified time point, and the standard curve formed by referring to the corresponding VHH-OVA-His protein as a control was used for quantitative detection by ELISA method. The integrity of the Caco-2 cell monolayer was tested using the Lucifer yellow (LY) rejection assay according to the manufacturer's instructions. Wells where the Lucifer yellow signal from the outer chamber exceeded 0.1% (basal level for passive transport) compared to the inner chamber signal were considered defective cell monolayers for the assay and were discarded.

[0284] Quantitative ELISA was performed using specific antibodies according to the above procedure. Briefly, a microplate was coated with rabbit anti-OVA polyclonal antibody (5 μg / mL in CBS buffer), a test sample or control (VHH-OVA-His protein) for a standard curve was added, and detection was performed with a biotinylated anti-OVA antibody and a streptavidin-HRP conjugate. A standard curve was prepared by serially diluting recombinant VHH-OVA-His protein from 1000 ng / mL to 0 with a dilution factor of 3. The test was qualified as a CV of less than 20% between test samples and a recovery greater than 90%.

[0285] Figure 7 The data presented in show that VHHs have different abilities to cross Caco-2 cell monolayers, reflecting in part their affinity for human transferrin or transferrin-binding epitopes. The results also suggest a crucial role for transferrin in VHHs enabling transmembrane absorption of biomolecules across intestinal epithelial cells ( Figure 7 B).

[0286] Humanization of selected VHHs

[0287] Select VHH with expected characteristics that enable the fused OVA protein to be transported across the membrane and with fewer development risk factors for humanization. Determine the CDR residues in the selected VHH and annotate them with the Kabat numbering system. N-glycosylation sites, and deamination sites in CDR, N-glycosylation sites or cysteine ​​in or near CDR will be considered as risk factors for future development. VHH humanization is carried out according to standard procedures for CDR transplantation and structural improvement. According to the humanization design, a recombinant DNA construction vector is constructed to produce recombinant VHH-OVA-His, as determined by ELISA and FACS above. Select a humanized sequence with an affinity equal to or better than the original VHH for transferrin, and an acceptable expression and stability level for further development.

[0288] Figure 5 The humanized VHH variants were shown to have a similar profile of transferrin binding as the original VHH.

[0289] Example 3 Using Tf to bind VHH to extend the half-life of the entity it binds

[0290] Fusion proteins were produced by fusion of the specified proteins at the C-terminus or N-terminus of VHH with or without a linker using recombinant DNA technology. Proteins were purified to homogeneity, formulated in appropriate buffers, and quantified by the Bradford method for in vivo testing. All animal procedures were reviewed and approved by the Institutional Review Board (IRB).

[0291] The mice of different genetic strains including C57BL / 6, Balb / c and CR6 are divided into groups according to genetic background, sex and body weight. In the case of co-administration containing or not containing human transferrins,iron complexes (5mg / kg), by IV or SC injection 3mg / kg test article, for example, the albumen fused with transferrin binding protein, and negative control (produced in the same manner, but containing the recombinant protein of VHH not combined with transferrins,iron complexes). For the group of co-administration of transferrins,iron complexes, the human transferrins,iron complexes of 5mg / kg are administered daily, and continue 10 days.

[0292] In order to determine the serum half-life of the test article in mice, 40 μL of whole blood was collected from the subcutaneous vein of the outer corner of the eye at multiple time points. After the collected whole blood was placed at room temperature for 1 hour, the serum was recovered by centrifugation at 10000 rpm for 10 min, and the concentration of VHH fusion protein in the serum was detected by quantitative ELISA. For OVA-VHH-His or GLP-1-VHH-His protein, rabbit anti-OVA polyclonal antibody (Sigma C6534-2mL) or monoclonal anti-GLP-1 antibody was paired with biotinylated rabbit anti-VHH antibody (GeneScript, A02015-200) for specific and quantitative detection, with reference to the qualified scheme with an inter-assay CV of less than 20% and a recovery rate of more than 90%, using purified recombinant protein as the standard curve. PKsovler software calculates PK parameters.

[0293] like Figure 6 The results presented indicate that transferrin-binding VHHs are able to prolong the serum half-life of bound OVA as well as GLP-1 and that this effect is dependent on the affinity of the VHHs for transferrin and its binding epitope.

[0294] Example 4: Oral delivery of binding entities by binding to transferrin VHH

[0295] For the half-life study, mice were anesthetized with 250 mg / kg tribromoethanol, and then the abdominal hair of the mice was shaved and disinfected with iodine and alcohol. The skin of the mid-abdomen was cut into about 0.5-1 cm lengths with surgical scissors to expose the duodenum for injection of transferrin or Fe 3+ The test article or control (purified VHH-OVA-His protein) of the mixture is added. The wound is sutured after disinfection with iodine. Alternatively, the test article or control is administered by intragastric injection by inserting a feeding needle into the mouse stomach. After oral administration, blood samples are collected at multiple time points as described above to recover serum. The concentration of the VHH fusion protein is quantified according to the method described in Example 3.

[0296] Figure 7The data presented in demonstrate that transferrin-binding VHHs enable orally administered VHH-OVA fusion proteins to cross the intestinal epithelium and be absorbed into the bloodstream, thus demonstrating the potential for delivering therapeutic biologics via the oral route of administration.

[0297] Example 5 Binding to transferrin VHH enables binding entities to cross the blood-brain barrier

[0298] With half-life study, mice were given 10 mg / kg purified recombinant protein (e.g., VHH-OVA-His) by IV injection, and 10 mg / kg human transferrin was given or not given by IP injection. At multiple time points after administration, brains were collected after 20 mL PBS perfusion and frozen in liquid nitrogen. Protein was extracted according to the standard procedure of quantitative detection of ELISA. A quantitative ELISA format is coated with rabbit anti-OVA polyclonal antibody (Sigma C6534-2mL), and biotinylated rabbit anti-VHH antibody (GeneScript, A02015-200) is detected. Figure 8 The data shown in the Figures show that the test article (VHHs that demonstrate binding ability to transferrin) significantly increased the levels of the test article in brain tissue (average %ID or percentage of injected dose, for selected VHHs, 24h post-dose was ∼1.03% to ∼3.5%) compared to the negative control (SLN0066, a VHH that does not bind to transferrin, SEQ ID NO: 110). The data also clearly show that VHHs cross the BBB in association with transferrin (%ID in the transferrin co-administered group was approximately 3.2 times higher than in the VHH-OVA-His treated group).

[0299] By using humanized VHH variants that are identical in CDR (and therefore presumably bind to the same epitope on transferrin) but have different affinities, the effect of the affinity of the VHH for transferrin on the ability to mediate crossing the BBB was also determined. Figure 8 The data in C further support the conclusion that the ability of VHH to cross the BBB depends on binding to transferrin, and also clearly show the relationship between the affinity of VHH for transferrin and its ability to mediate crossing the BBB. The higher the affinity of VHH for transferrin, the stronger the ability to cross the BBB.

[0300] Example 6 VHH binding to transferrin achieves intracellular delivery of its binding entity

[0301] The levels of transferrin receptors on the membrane surface of various types of cells (such as tumor cells) are also increased. Therefore, VHHs that bind to transferrin are beneficial for targeted drug delivery, especially intracellular delivery to tumor cells. To verify this hypothesis, recombinant VHH-PE38-His protein was produced in Escherichia coli and purified to homogeneity. The efficacy of intracellular delivery was tested on monolayer cultures of Hepa-G2 cells (continuously expressing TfR) and MDCK cells (ectopic TfR1 expression with or without transfected recombinant plasmid). Briefly, cells were seeded at the desired density into 96-well microplates containing 100 μL of culture medium and cultured overnight at 37°C. After washing the cells with fresh preheated culture medium, serially diluted VHH-PE38-His prepared in culture medium with or without human transferrin was added to the cell culture. At multiple time points after treatment, the cells were treated with 1% paraformaldehyde according to the instructions of the instructions. The luminescence signal was recorded with an integration time of 0.25-1 s per well and the data were analyzed using GraphPad.

[0302] Fig. 9 The data shown in show that transferrin-binding VHHs achieve intracellular delivery of binding entities into cells expressing transferrin receptors on the cell surface, and this function depends on the transferrin binding activity of the VHHs. VHHs with different binding epitopes (belonging to different bins or having different CDR sequences) also exhibit differential effects in intracellular delivery.

[0303] Example 7 VHH binding to transferrin achieves organ or tissue targeted delivery of binding entities

[0304] The biodistribution of intravenously injected recombinant proteins (e.g., VHH-OVA-His) has been studied in mice with different genetic backgrounds. At multiple time points after intravenous injection, after 30 mL PBS perfusion of blood vessels, samples were collected in liver, lung, bone marrow, heart, muscle, and blood, etc. The samples were weighed and homogenized, and soluble proteins were extracted and used for quantitative ELISA detection as described above. With reference to the standard curve formed by the purified recombinant protein, data were normalized with tissue weight or volume. According to the multiple changes compared with the control VHH not in conjunction with transferrin, the biodistribution of VHH-OVA-His protein was analyzed. Fig.10 The data shown in show that the selected transferrin-binding VHHs achieve targeted delivery to liver, brain, bone marrow, lung, muscle, kidney, eye and other organs or tissues expressing transferrin receptors. The transferrin-binding VHHs in the present invention have potential applications in drug delivery targeting different tissues, especially brain, liver and bone marrow.

[0305] Although preferred embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that such embodiments are provided only as examples. This does not mean that the present invention is limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not meant to be interpreted in a limiting sense. Without departing from the present invention, those skilled in the art will think of many variations, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that, when implementing the present invention, various alternatives to the embodiments of the present invention described herein may be adopted. Therefore, it is expected that the present invention will also cover any such substitutions, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for transporting molecules across a cell membrane by transferrin binding protein, wherein the transferrin binding protein comprises a single domain antibody fragment VHH, and the VHH comprises CDR1, CDR2 and CDR3, wherein the amino acid sequences of the CDR1, CDR2 and CDR3 are selected from any one of the following combinations: (1) CDR1: SEQ ID NO: 1, CDR2: SEQ ID NO: 22 and CDR3: SEQ ID NO: 45; (2) CDR1: SEQ ID NO: 1, CDR2: SEQ ID NO: 22 and CDR3: SEQ ID NO: 63; (3) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 23 and CDR3: SEQ ID NO: 46; (4) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 23 and CDR3: SEQ ID NO: 58; (5) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 40 and CDR3: SEQ ID NO: 58; (6) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 41 and CDR3: SEQ ID NO: 58; (7) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 23 and CDR3: SEQ ID NO: 66; (8) CDR1: SEQ ID NO: 3, CDR2: SEQ ID NO: 24 and CDR3: SEQ ID NO: 47; (9) CDR1: SEQ ID NO: 4, CDR2: SEQ ID NO: 25 and CDR3: SEQ ID NO: 48; (10) CDR1: SEQ ID NO: 5, CDR2: SEQ ID NO: 26 and CDR3: SEQ ID NO: 49; (11) CDR1: SEQ ID NO: 6, CDR2: SEQ ID NO: 27 and CDR3: SEQ ID NO: 50; (12) CDR1: SEQ ID NO: 6, CDR2: SEQ ID NO: 27 and CDR3: SEQ ID NO: 59; (13) CDR1: SEQ ID NO: 7, CDR2: SEQ ID NO: 28 and CDR3: SEQ ID NO: 51; (14) CDR1: SEQ ID NO: 8, CDR2: SEQ ID NO: 29 and CDR3: SEQ ID NO: 52; (15) CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 30 and CDR3: SEQ ID NO: 53; (16) CDR1: SEQ ID NO: 10, CDR2: SEQ ID NO: 31 and CDR3: SEQ ID NO: 54; (17) CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 32 and CDR3: SEQ ID NO: 55; (18) CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 33 and CDR3: SEQ ID NO: 56; (19) CDR1: SEQ ID NO: 13, CDR2: SEQ ID NO: 34 and CDR3: SEQ ID NO: 57; (20) CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 35 and CDR3: SEQ ID NO: 60; (21) CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 36 and CDR3: SEQ ID NO: 61; (22) CDR1: SEQ ID NO: 16, CDR2: SEQ ID NO: 37 and CDR3: SEQ ID NO: 62; (23) CDR1: SEQ ID NO: 17, CDR2: SEQ ID NO: 38 and CDR3: SEQ ID NO: 64; (24) CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 39 and CDR3: SEQ ID NO: 65; (25) CDR1: SEQ ID NO: 19, CDR2: SEQ ID NO: 42 and CDR3: SEQ ID NO: 67; (26) CDR1: SEQ ID NO: 20, CDR2: SEQ ID NO: 43 and CDR3: SEQ ID NO: 68; (27) CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 44 and CDR3: SEQ ID NO:

69.

2. The method according to claim 1, wherein the method is used to deliver a drug across the cell membrane of polarized and / or non-polarized cells.

3. The method according to claim 2, wherein the polarized and / or non-polarized cells express transferrin receptor on their cell membrane.

4. The method according to any one of claims 1-3, wherein the drug delivery across the cell membrane of unpolarized cells comprises intracellular delivery of the drug, and / or recycling of the endocytosed drug back into the blood circulation.

5. The method of any one of claims 1-4, wherein the drug delivery across the cell membrane of polarized cells comprises systemically administered drugs targeting the central nervous system (CNS) crossing the blood-brain barrier (BBB), orally administered drugs crossing the intestinal epithelium, and / or drugs penetrating multiple layers of cells in solid tissues.

6. The method of any one of claims 1-5, wherein the drug comprises a therapeutic or diagnostic substance.

7. The method according to any one of claims 1-6, wherein the drug comprises a small molecule compound, a synthetic peptide, a recombinant protein, an antibody or an antibody fragment, an enzyme, a nucleotide sequence fragment, a liposome, a lipid nanoparticle, a drug carrier, a modified virus and / or a gene therapy vector.

8. The method according to any one of claims 1-7, wherein the drug is bound to the transferrin binding protein by chemical coupling or gene fusion.

9. A transferrin binding protein, wherein the transferrin binding protein is a single domain antibody fragment VHH, which is capable of prolonging the circulation half-life of the entity to which it binds, and the VHH comprises CDR1, CDR2 and CDR3, wherein the amino acid sequences of the CDR1, CDR2 and CDR3 are selected from any one of the following combinations: (1) CDR1: SEQ ID NO: 1, CDR2: SEQ ID NO: 22 and CDR3: SEQ ID NO: 45; (2) CDR1: SEQ ID NO: 1, CDR2: SEQ ID NO: 22 and CDR3: SEQ ID NO: 63; (3) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 23 and CDR3: SEQ ID NO: 46; (4) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 23 and CDR3: SEQ ID NO: 58; (5) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 40 and CDR3: SEQ ID NO: 58; (6) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 41 and CDR3: SEQ ID NO: 58; (7) CDR1: SEQ ID NO: 2, CDR2: SEQ ID NO: 23 and CDR3: SEQ ID NO: 66; (8) CDR1: SEQ ID NO: 3, CDR2: SEQ ID NO: 24 and CDR3: SEQ ID NO: 47; (9) CDR1: SEQ ID NO: 4, CDR2: SEQ ID NO: 25 and CDR3: SEQ ID NO: 48; (10) CDR1: SEQ ID NO: 5, CDR2: SEQ ID NO: 26 and CDR3: SEQ ID NO: 49; (11) CDR1: SEQ ID NO: 6, CDR2: SEQ ID NO: 27 and CDR3: SEQ ID NO: 50; (12) CDR1: SEQ ID NO: 6, CDR2: SEQ ID NO: 27 and CDR3: SEQ ID NO: 59; (13) CDR1: SEQ ID NO: 7, CDR2: SEQ ID NO: 28 and CDR3: SEQ ID NO: 51; (14) CDR1: SEQ ID NO: 8, CDR2: SEQ ID NO: 29 and CDR3: SEQ ID NO: 52; (15) CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 30 and CDR3: SEQ ID NO: 53; (16) CDR1: SEQ ID NO: 10, CDR2: SEQ ID NO: 31 and CDR3: SEQ ID NO: 54; (17) CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 32 and CDR3: SEQ ID NO: 55; (18) CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 33 and CDR3: SEQ ID NO: 56; (19) CDR1: SEQ ID NO: 13, CDR2: SEQ ID NO: 34 and CDR3: SEQ ID NO: 57; (20) CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 35 and CDR3: SEQ ID NO: 60; (21) CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 36 and CDR3: SEQ ID NO: 61; (22) CDR1: SEQ ID NO: 16, CDR2: SEQ ID NO: 37 and CDR3: SEQ ID NO: 62; (23) CDR1: SEQ ID NO: 17, CDR2: SEQ ID NO: 38 and CDR3: SEQ ID NO: 64; (24) CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 39 and CDR3: SEQ ID NO: 65; (25) CDR1: SEQ ID NO: 19, CDR2: SEQ ID NO: 42 and CDR3: SEQ ID NO: 67; (26) CDR1: SEQ ID NO: 20, CDR2: SEQ ID NO: 43 and CDR3: SEQ ID NO: 68; (27) CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 44 and CDR3: SEQ ID NO:

69. 10 . The transferrin binding protein according to claim 9 , which enables a fusion protein comprising the transferrin binding protein to cross the blood-brain barrier (BBB).

11. The transferrin binding protein according to any one of claims 9-10, for use in oral delivery of drugs.

12. The transferrin binding protein according to any one of claims 9 to 11, which is capable of achieving intracellular delivery of entities bound thereto to cells expressing transferrin receptor 1. 13 . The transferrin binding protein according to claim 9 , which is capable of specifically binding to transferrin and does not interfere with the interaction between transferrin and transferrin receptor 1.

14. The transferrin binding protein according to any one of claims 9 to 13, wherein the transferrin is human transferrin.

15. The transferrin binding protein according to any one of claims 9 to 14, which has a higher affinity for iron-containing, iron-saturated transferrin than for iron-free, apotransferrin.

16. A polypeptide comprising the transferrin binding protein of any one of claims 9 to 15 and a therapeutic entity.

17. The polypeptide of claim 16, wherein the therapeutic entity is linked to the transferrin binding protein via a linker.

18. The polypeptide according to any one of claims 16-17, wherein the therapeutic entity is glucagon-like peptide-1 (GLP-1) or PE38.

19. One or more isolated nucleic acid molecules encoding the transferrin binding protein of any one of claims 9-15 or the polypeptide of any one of claims 16-18. A vector comprising the nucleic acid molecule of claim 19 .

21. A cell comprising the nucleic acid molecule of claim 19 or the vector of claim 20.

22. A method for preparing the transferrin binding protein according to any one of claims 9 to 15 or the polypeptide according to any one of claims 16 to 18, the method comprising culturing the cell according to claim 21 under conditions allowing expression of the transferrin binding protein or the polypeptide.

23. A pharmaceutical composition comprising the polypeptide of any one of claims 16-18.

24. A pharmaceutical composition comprising the transferrin binding protein according to any one of claims 9 to 15 and a therapeutic entity, wherein the therapeutic entity refers to a monomeric or multimeric protein or protein fragment having the function of treating or preventing a disease.

25. Use of the transferrin binding protein according to any one of claims 9 to 15 in combination with a therapeutic drug or a diagnostic agent in the preparation of a drug, wherein the transferrin binding protein is used to deliver the therapeutic drug or the diagnostic agent to cells or organs expressing transferrin receptor 1.

26. Use of the transferrin binding protein according to any one of claims 9 to 15 in combination with a targeted drug in the preparation of a drug, wherein the transferrin binding protein is used to deliver the targeted drug across the blood-brain barrier, intestinal epithelium and / or cell membranes expressing transferrin receptor 1.

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