A nerve growth factor, its preparation method and application

Abstract

This invention discloses a nerve growth factor, its preparation method, and its applications. The nerve growth factor is a heterodimer, with the amino acid sequence of its A chain as shown in SEQ ID NO: 56 and the amino acid sequence of its B chain as shown in SEQ ID NO: 61. It possesses analgesic, long-lasting, and blood-brain barrier-penetrating properties.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nerve growth factor, its preparation method and application, and more specifically, to a painless, long-acting, blood-brain barrier-penetrating nerve growth factor, its preparation method and application. Background Technology

[0002] Nerve growth factor (β-NGF / NGF) was the first neurotrophic factor discovered by Rita Levi-Montalcini in mouse sarcoma cells in 1953. β-NGF can maintain and promote the survival, differentiation, maturation, and executive function of sympathetic nerves and sensory nerve cells derived from the neural crest, and is an important factor involved in the regeneration and functional repair of damaged nerves. β-NGF can also promote the growth, differentiation, and activity of cholinergic neurons in the central nervous system. β-NGF is first synthesized in the cell into a 241-amino acid precursor molecule (preproNGF). The amino acid sequence homology of preproNGF is as high as 78% in humans, rats, mice, guinea pigs, chimpanzees, and squirrel monkeys. The last three amino acids at the C-terminus are RRA / G. In the endoplasmic reticulum, the signal peptide (pre region) of preproNGF is cleaved by signal peptidase to form proNGF (composed of 223 amino acids). It is then transported to the Golgi apparatus, where it is cleaved by Furin enzyme to remove the leader peptide (pro region) and produce mature NGF protein (composed of 120 amino acids), denoted as NGF(120). When expressing β-NGF in vitro using mammalian cells, there are often two amino acid (RA / G) or three amino acid (RRA / G) residues missing at the C-terminus. The former is denoted as NGF(118), and the latter as NGF(117). NGF(120), NGF(118) and NGF(117) can be regarded as functional variants of a type of β-NGF, and their stability and in vitro recombinant expression efficiency are different.

[0003] Currently, commercially available β-NGF is generally isolated and purified from the submandibular glands of adult male mice. It is widely used to treat peripheral nerve injuries such as Guillain-Barré syndrome, facial neuritis, toxic peripheral nerve injury, peripheral nerve injury caused by radiotherapy and chemotherapy, brachial plexus injury, various nerve transections, optic nerve injury, auditory nerve injury, and diabetic peripheral neuropathy. The molecular weight of β-NGF monomer is approximately 13.5 kDa. As a protein drug, it is easily filtered by the glomeruli during metabolism in vivo, resulting in a short half-life. Studies have shown that the elimination half-life (T1 / 2) of murine β-NGF administered intramuscularly in mice is approximately 2–3 hours; the elimination half-life (T1 / 2) of a single intravenous injection of concentrated human β-NGF in rats is approximately 4 hours. Furthermore, because β-NGF is a biological macromolecule, it is difficult to cross the blood-brain barrier (BBB), thus hindering its therapeutic effects on central nervous system injuries or neurodegenerative diseases such as traumatic brain injury, spinal cord injury, traumatic brain injury, stroke, neurological injuries caused by multiple sclerosis or acute cerebrovascular disease, hypoxic-ischemic encephalopathy in newborns, cerebral palsy in children, and Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and vascular dementia. Moreover, intramuscular injection of β-NGF commonly causes adverse reactions such as injection site pain or pain in the lower limb on the injection side. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of β-NGF having a short half-life, making it difficult to cross the blood-brain barrier, and often causing painful side effects after intramuscular injection in the prior art.

[0005] Therefore, the present invention provides a nerve growth factor, wherein the amino acid sequence of the nerve growth factor, from the N-terminus to the C-terminus, consists of NGF... mut It is composed of Linker1, Fc, Linker2 and TfR1BS connected together;

[0006] The NGF mut It is a human nerve growth factor mutant containing amino acid residue mutations, and its binding selectivity with TrkA receptor and p75NTR receptor is not less than 1.5 times that of the wild-type human nerve growth factor mature peptide shown in SEQ ID NO: 1 with TrkA receptor and p75NTR receptor.

[0007] The Fc is an Fc fragment of IgG or a variant thereof, consisting of an IgG hinge region or a fragment thereof, a CH2 region and a CH3 region;

[0008] The TfR1BS is defined as having an affinity constant KD value of 1×10⁻⁶. -9 - 5×10 -7M binds to the binding sequence of the TfR1 receptor with binding affinity.

[0009] Specifically, the aforementioned nerve growth factor is a homodimer, and the TfR1BS is in a bivalent form.

[0010] Specifically, the aforementioned nerve growth factor is a heterodimer, and the TfR1BS is in monovalent form.

[0011] Specifically, the aforementioned NGF mut The NGF mutant having a K32A mutation and / or an R114I mutation on the amino acid residues relative to SEQ ID NO: 1; or the NGF mutant having a K88L mutation on the amino acid residues relative to SEQ ID NO: 1.

[0012] Specifically, the aforementioned TfR1BS is a VHH molecule; the VHH molecule includes complementarity-determining regions CDR1, CDR2, and CDR3; CDR1, CDR2, and CDR3 are respectively composed of the following sequences: SEQ ID NO: 2, 3, and 4; or SEQ ID NO: 6, 7, and 8; or SEQ ID NO: 10, 11, and 12; or SEQ ID NO: 14, 15, and 16; or SEQ ID NO: 18, 19, and 20; or SEQ ID NO: 22, 19, and 23.

[0013] Specifically, the above TfR1BS is the amino acid sequence shown in SEQ ID NO: 5, 9, 13, 17, 21, 24, 25 or 26.

[0014] The present invention also provides a nucleic acid that encodes any of the nerve growth factors described above.

[0015] The present invention also provides an expression vector comprising the above-mentioned nucleic acid.

[0016] Specifically, the aforementioned expression vector also contains nucleic acid encoding Furin enzyme.

[0017] The present invention also provides a recombinant host cell containing the above-mentioned nucleic acid or containing any of the above-mentioned expression vectors.

[0018] The present invention also provides a method for preparing nerve growth factor, comprising the following steps: expressing the above-mentioned nucleic acid to obtain the nerve growth factor; or expressing the expression vector described in any one of the above-mentioned methods to obtain the nerve growth factor; or culturing the above-mentioned recombinant host cells and separating and purifying the nerve growth factor.

[0019] Specifically, the above preparation method includes the following steps:

[0020] (1) Transfect the expression vector into mammalian cells;

[0021] (2) Screening for cell lines containing the expression vector or integrated with the nucleic acid encoded thereon;

[0022] (3) The obtained cell line was cultured, the culture medium was collected and the nerve growth factor was separated and purified.

[0023] The present invention also provides a pharmaceutical composition comprising any of the above-described nerve growth factors, or the above-described nucleic acids, or any of the above-described expression vectors, or the above-described recombinant host cells, and pharmaceutically acceptable carriers and / or excipients.

[0024] The present invention also provides the use of any of the above-described nerve growth factors, or the above-described nucleic acids, or the above-described expression vectors, or the above-described recombinant host cells, or the above-described pharmaceutical compositions in the preparation of a medicament for treating nerve injury diseases.

[0025] Specifically, the aforementioned nerve injury diseases are at least one of peripheral nerve injury diseases, central nervous system injury diseases, and central nervous system degenerative diseases.

[0026] Specifically, the aforementioned peripheral nerve injury diseases include Guillain-Barré syndrome, facial neuritis, toxic peripheral neuropathy, peripheral nerve injury caused by radiotherapy and chemotherapy, brachial plexus injury, nerve transection injury, optic nerve injury, auditory nerve injury, and diabetic peripheral neuropathy.

[0027] Specifically, the aforementioned central nervous system injury diseases include craniocerebral injury, spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, nerve injury caused by acute cerebrovascular disease, neonatal hypoxic-ischemic encephalopathy, and cerebral palsy in children.

[0028] Specifically, the aforementioned central nervous system degenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and vascular dementia.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] The nerve growth factor provided by this invention is modified by amino acid mutation of NGF and fused with human IgG Fc region and TfR1BS sequence. In vivo, it can reduce the pain side effects caused by the natural NGF mechanism and prolong its plasma half-life. Through the transcytosis mechanism mediated by the binding of TfR1BS sequence to TfR1 receptor, it can efficiently cross the blood-brain barrier and play a role in treating nerve damage diseases.

[0031] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0032] Figure 1 The NGF in Embodiment 1 of this invention mut -Fc-VHHA(2+) recombinant expression vector map.

[0033] Figure 2 This is a graph showing the change in pain threshold 1 hour after a single subcutaneous injection into the hind leg paw pads of mice in Example 3 of the present invention.

[0034] Figure 3 The NGF in Embodiment 5 of this invention K32A Figure 1 shows the results of drug concentration detection in the cerebral cortex of mice in an in vivo brain uptake assay using -Fc-TfR1BS.

[0035] Figure 4 The NGF in Embodiment 6 of this invention K32A Figure 1 shows the results of drug concentration detection in the cerebral cortex of B6-hTFRC (CDS) transgenic mice in an in vivo brain uptake assay of -Fc-TfR1BS.

[0036] Figure 5 The NGF in Embodiment 7 of this invention K32A -Fc-VHHQ(1+) TBI model mouse pharmacodynamic test results; A, Time axis of TBI model mouse pharmacodynamic test, the day of modeling is recorded as D0, the drug is administered once every 3 days for 4 consecutive weeks, and the first administration is 6 hours after modeling; B, Trend statistics of neurological deficit score (mNSS) of each group of mice during the administration period; C, Statistics of mNSS score of each group of mice on the third day; D, Statistics of mNSS score of each group of mice on the seventh day; E, Results of spontaneous alternation rate of each group in the mouse Y maze test.

[0037] Figure 6 The NGF in Embodiment 7 of this invention K32A Figure 1 shows the efficacy test results of the -Fc-VHHQ(1+) TBI model mouse; A, representative image of NeuN immunohistochemical staining in the brain tissue of each group of mice; B, statistical graph of NeuN positive cell count in the brain tissue of each group of mice.

[0038] Figure 7 The NGF in Embodiment 8 of this invention K32A -Fc-VHHQ(1+) MCAO / R model mouse efficacy test results; A, MCAO / R model mouse efficacy test time axis, grouped one day before modeling, denoted as D0, administered once every 3 days, for 4 consecutive weeks, with the first administration 6 hours after reperfusion; B, mouse weight statistics for each group during administration; C, Longa score statistics for each group during administration.

[0039] Figure 8 The NGF in Embodiment 8 of this invention K32AFigure 1 shows the efficacy test results of the -Fc-VHHQ(1+) MCAO / R model mice; A, representative image of NeuN immunohistochemical staining in the brain tissue of each group of mice; B, statistical graph of NeuN positive cell count per unit area in the brain tissue of each group of mice. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0041] This invention provides a nerve growth factor, wherein the amino acid sequence of the nerve growth factor, from the N-terminus to the C-terminus, consists of NGF... mut It is composed of Linker1, Fc, Linker2 and TfR1BS.

[0042] Wherein, the NGF mut The mutant is a human nerve growth factor containing one or two amino acid residue mutations, and its binding selectivity with the TrkA receptor and p75NTR receptor is not less than 1.5 times that of the wild-type human nerve growth factor mature peptide shown in SEQ ID NO: 1 with the TrkA receptor and p75NTR receptor; more preferably not less than 5 times; and most preferably not less than 10 times. In one embodiment, NGF mut The NGF mutant is defined as follows: the amino acid residue at position 32 of the amino acid residue shown in SEQ ID NO: 1 is mutated from lysine (Lys, K) to alanine (Ala, A) (denoted as K32A, the same below), or / and the amino acid residue at position 114 is mutated from arginine (Arg, R) to isoleucine (Ile, I) (R114I); or the amino acid residue at position 88 is mutated from lysine (Lys, K) to leucine (Leu, L) (K88L).

[0043] The Fc is the Fc fragment of IgG or a variant thereof, composed of the IgG hinge region or a fragment thereof, the CH2 region, and the CH3 region; the Fc variant refers to a functional mutant that enhances, weakens, or eliminates the effector function of the Fc region, including but not limited to complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and functional mutants that increase affinity for the neonatal Fc receptor (FcRn) to prolong its half-life. Examples well-known to those skilled in the art include mutations such as N297A, L234A / L235A / P331S, and L234A / L235A / P329G that weaken or eliminate the effector function of the Fc region. The IgG hinge region, CH2 region, and CH3 region can originate from the same subtype of IgG, such as IgG1, IgG2, IgG3, or IgG4; or they can originate from different subtypes of IgG, for example, an Fc fragment or a variant thereof consisting of the human IgG3 hinge region or a fragment thereof, the human IgG1 CH2 region, and the human IgG1 CH3 region; or, for example, an Fc fragment or a variant thereof consisting of the human IgG2 hinge region or a fragment thereof, the human IgG4 CH2 region, and the human IgG4 CH4 region.

[0044] The TfR1BS is a transferrin receptor 1 (TfR1) binding sequence with an affinity constant KD value of 1 × 10⁻⁶. -9 - 5×10 -7 M binds to a protein or polypeptide sequence of TfR1 with an affinity including, but not limited to, antibodies, antibody fragments, polypeptides, or polypeptide fragments; preferably, the TfR1 receptor binding sequence has an affinity constant KD value of 1 × 10⁻⁶. -9 - 5×10 -7 The binding affinity of M is to bind to the variable domain (VHH) of a single-chain antibody (scFv), Fab, or a heavy-chain-only alpaca antibody containing the TfR1 receptor; more preferably, the TfR1BS is a VHH, and its complementarity-determining regions CDR1, CDR2, and CDR3 are composed of the following sequences: SEQ ID NO: 2, 3, and 4; or SEQ ID NO: 6, 7, and 8; or SEQ ID NO: 10, 11, and 12; or SEQ ID NO: 14, 15, and 16; or SEQ ID NO: 18, 19, and 20; or SEQ ID NO: 22, 19, and 23; most preferably, the TfR1BS is any amino acid sequence shown in SEQ ID NO: 5, 9, 13, 17, 21, 24, 25, and 26.

[0045] Linker1 and Linker2 are peptide linker sequences containing at least one amino acid selected from glycine (G), serine (S), alanine (A), and threonine (T), with a sequence length of no more than 30 amino acids. In some preferred embodiments, the peptide linker sequence is (GGG)n or (GGGGS)n, where n is 0 or at least an integer of 1. For example, (GGG)1 is equivalent to GGG; another example is (GGGGS)3, which is equivalent to GGGGSGGGGSGGGGS.

[0046] Those skilled in the art will understand that Linker1 and Linker2, acting as spacers, should possess a certain degree of flexibility to prevent NGF (Neural GF) damage. mut The Fc and TfR1 receptor binding sequences interfere with each other or affect each other's structure or function. Those skilled in the art will understand that the sequences of Linker1 and Linker2 may be identical or different.

[0047] Those skilled in the art will understand that the nerve growth factor forms a dimer through covalent cross-linking between the two polypeptide chains in the Fc region. This dimer can be a homodimer, where TfR1BS is bivalent, meaning two TfR1BS sequences are respectively linked to the two polypeptide chains of the Fc region; or it can be a heterodimer, where TfR1BS is monovalent, with only one TfR1BS sequence linked to one polypeptide chain of the Fc region. In the homodimer, each monomer sequence is completely identical, representing NGF. mut -Linker1-Fc-Linker2-TfR1BS; the heterodimer, in which one monomer sequence is NGF mut -Linker1-Fc, the other monomeric sequence is NGF mut -Linker1-Fc -Linker2-TfR1BS. The heterodimer can be prepared using techniques well-known and widely used by those skilled in the art, such as KiH, KiHs-s, DD-KK, EW-RVT, and EW-RVTs-s.

[0048] The present invention also provides a nucleic acid that encodes any of the nerve growth factors described above.

[0049] The present invention also provides an expression vector comprising the above-mentioned nucleic acid and an expression control element.

[0050] The expression control elements are control sequences required for the effective replication and expression of the nucleic acid, such as origin of replication, promoter, and enhancer; essential processing information sites, such as ribosome binding sites, KOZAK sequences, RNA splicing sites, intron sequences, polyadenylation sites, and transcription terminator sequences; and selection marker genes, such as dihydrofolate reductase (DHFR) genes, glutamine synthase (GS) genes, neomycin resistance genes, puromycin resistance genes, bleomycin resistance genes, hygromycin resistance genes, and polychromycin resistance genes.

[0051] Furthermore, the above expression vector also includes nucleic acid encoding Furin enzyme, which improves the processing efficiency of enzymatic cleavage to remove the leader peptide (pro region) when preproNGF is expressed in cells.

[0052] The present invention also provides a recombinant host cell containing the above-mentioned nucleic acid or containing any of the above-mentioned expression vectors.

[0053] Optionally, the recombinant host cell is a mammalian cell, and its original cells are Chinese hamster ovary cells (CHO), human embryonic kidney 293 cells, COS cells, HeLa cells, or MDCK cells. Original cells refer to cells that have not been transfected with the expression vector.

[0054] The present invention also provides a method for preparing nerve growth factor, comprising the following steps: expressing the above-mentioned nucleic acid to obtain the nerve growth factor; or expressing the expression vector described in any one of the above-mentioned methods to obtain the nerve growth factor; or culturing the above-mentioned recombinant host cells and separating and purifying the nerve growth factor.

[0055] Specifically, the above preparation method includes the following steps:

[0056] (1) Transfect the expression vector into mammalian cells;

[0057] (2) Screening for cell lines containing the expression vector or integrated with the nucleic acid encoded thereon;

[0058] (3) The obtained cell line was cultured, the culture medium was collected and the nerve growth factor was separated and purified.

[0059] Transfection is the process of introducing an expression vector into a host cell, such as a mammalian cell. Transfection methods include calcium phosphate precipitation, electroporation, liposomes, and polymers (such as PEI). Based on whether the target gene is integrated into the host cell genome, transfection can be divided into stable transfection and transient transfection. The specific method used depends on the experimental purpose and economic and technical factors. For example, stable transfection is often used to develop stable and efficient expression cell lines for clinical and commercial applications to express and prepare proteins required by the pharmaceutical, food, and animal husbandry industries; while transient transfection is effectively used for high-throughput drug screening and early product analysis and evaluation.

[0060] Typically, selectable marker genes on expression vectors are used to screen for cell lines that express the target nerve growth factor stably and efficiently. These marker genes can induce drug resistance; examples of such drugs include neomycin (G418), puromycin, bleomycin, hygromycin, and polymycin. Through drug screening (e.g., cells integrating the marker gene survive while those without die), cell lines stably transfected with the expression vector or whose genomes integrate nucleic acids encoding the nerve growth factor can be obtained. Furthermore, these marker genes not only induce drug resistance but also increase the copy number of themselves and neighboring target genes with increasing drug concentration (i.e., selection pressure), thereby increasing the expression level of the target protein. Examples of such marker genes include dihydrofolate reductase (DHFR) and glutamine synthase (GS), with corresponding screening drugs being methotrexate (MTX) and methionine iminosulfone (MSX). Therefore, those skilled in the art will understand that by combining the above-mentioned antibiotic drug screening with a DHFR-MTX or GS-MSX screening system, stable and efficient cell lines can be obtained.

[0061] The methods for isolating and purifying nerve growth factor are well known in the art. Based on the physicochemical properties of nerve growth factor, common purification techniques such as ammonium sulfate precipitation, ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography can be selected. For example, since nerve growth factor contains Fc, affinity chromatography techniques, such as Protein A or Protein G affinity chromatography packing materials, can be conveniently used for purification. Another example is utilizing the presence of the TfR1BS sequence in nerve growth factor, using MabSelect... TM VL or MabSelect TM Purification was performed using VH3 affinity chromatography packing material.

[0062] The present invention also provides a pharmaceutical composition comprising any of the above-described nerve growth factors, or the above-described nucleic acids, or any of the above-described expression vectors, or the above-described recombinant host cells, and pharmaceutically acceptable carriers and / or excipients.

[0063] The pharmaceutically acceptable carriers and / or excipients refer to stabilizers, protectants, excipients, antioxidants, surfactants, and buffer salts that are harmless to the subjects at the doses and concentrations used, including but not limited to hydroxypropyl methylcellulose, polyvinylpyrrolidone, human serum albumin, gelatin, glutamic acid, histidine, arginine, glycine, mannitol, dextran 40, sucrose, trehalose, methionine, phosphate, citrate, acetate, sodium chloride, polyethylene glycol 6000 (PEG6000), polyvinyl alcohol, polysorbate 80 (Tween-80), carboxymethyl cellulose, and phosphate, citrate, acetate, and sodium chloride.

[0064] The pharmaceutical composition may be administered by any suitable formulation and via any suitable route, including but not limited to parenteral routes, such as in the form of an injectable preparation that can be injected subcutaneously, intravenously, or intramuscularly; or intranasal routes, such as in the form of nasal drops, aerosols, or sprays; or intraocular or ocular surface routes, such as in the form of eye drops.

[0065] The pharmaceutical composition typically comprises an effective dose of the painless, long-acting, blood-brain barrier-penetrating nerve growth factor of the present invention. The effective dose refers to a dose of the drug or pharmaceutical composition sufficient to eliminate, improve, alleviate, reduce, and / or prevent or delay one or more symptoms of a particular disorder, symptom, or disease. Those skilled in the art will understand that, for a given individual, the effective dose depends on various factors, including the bioactivity / efficacy or toxicity of the nerve growth factor, the route of administration, the frequency of administration, the severity of the disease itself, and the individual's functional status.

[0066] The present invention also provides the use of any of the above-described nerve growth factors, or the above-described nucleic acids, or the above-described expression vectors, or the above-described recombinant host cells, or the above-described pharmaceutical compositions in the preparation of a medicament for treating nerve injury diseases.

[0067] Specifically, the aforementioned nerve injury diseases are selected from at least one of peripheral nerve injury diseases, central nervous system injury diseases, and central nervous system degenerative diseases.

[0068] The peripheral nerve injury diseases mentioned include Guillain-Barré syndrome, facial neuritis, toxic peripheral neuropathy, peripheral nerve injury caused by radiotherapy and chemotherapy, brachial plexus injury, nerve transection injury, optic nerve injury, auditory nerve injury, and diabetic peripheral neuropathy.

[0069] The central nervous system injury diseases mentioned include craniocerebral injury, spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, nerve injury caused by acute cerebrovascular disease, neonatal hypoxic-ischemic encephalopathy, and cerebral palsy in children.

[0070] The central neurodegenerative diseases mentioned include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and vascular dementia.

[0071] The effects of the nerve growth factor of the present invention, its preparation method, and its application are studied through specific embodiments below. In the embodiments of the present invention, unless otherwise specified, the technical terms used are those commonly used by those skilled in the art; experimental methods without specific conditions refer to conventional experimental methods; the experimental materials used are all commercially available products unless otherwise specified, and the composition and preparation methods of various reagents and culture media can be found in conventional experimental manuals.

[0072] Example 1: NGF mut Preparation of -Fc-VHHA(2+)

[0073] In this embodiment, different pain-free or pain-reducing mutations of NGF(117) are used to construct different NGFs with the same Fc and TfR1BS sequences. mut -Fc-TfR1BS homodimer molecule: The TfR1BS sequence is the VHHA sequence shown in SEQ ID NO: 27; Fc is the Fc sequence of the natural variant of human IgG1, IGHG1*03, which has a D356E / L359M double mutation (nG1m1 heteroallelic mutation); three amino acid residues of L234A / L235A / P331S are introduced into the Fc region to eliminate the Fc region effector function, and the N-terminal 5 amino acid residues are deleted (EPKSC) to reduce potential structural perturbation of the hinge region; (SEQ ID NO: 28) different NGFs were prepared mut -Fc-TfR1BS homodimer molecule, in which TfR1BS is in a bivalent form, denoted as NGF. mut -Fc-VHHA(2+). To facilitate comparison of different NGFs... mut The analgesic effect of the -Fc-VHHA(2+) molecule was observed, and a wild-type NGF(117)-Fc fusion protein, denoted as NGF, was prepared. wt -Fc. To ensure NGF mut / NGF wt The Fc and VHHA sequences are independent of each other, and their integrity, structure, and function do not interfere with each other, in NGF mut The peptide linker sequence Linker1 between Fc and VHHA was ligated using (GGGGS)3, and the peptide linker sequence Linker2 between Fc and VHHA was ligated using (GGGGS)4. Different NGFs mutThe structure of the -Fc-VHHA(2+) molecule is shown in Table 1. Each molecular construct contains the natural human NGF signal peptide and the pro region. Those skilled in the art will understand that the signal peptide and pro region sequences will be cleaved during expression in the host cell.

[0074] Table 1 NGF mut -Fc-VHHA(2+) molecular structure

[0075]

[0076] In this embodiment, different NGF (117) mutations were observed in NGF. mut -Fc-VHHA(2+) molecule and control NGF wt All -Fc molecules were prepared using the same method. The NGF F12E mutation was the point mutation described in CN109153709B.

[0077] (1) Obtaining DNA encoded by molecular constructs

[0078] The above NGFs are respectively wt -Fc、NGF K32A -Fc-VHHA(2+), NGF R114I -Fc-VHHA(2+), NGF K88L -Fc-VHHA(2+), NGF F12E -Fc-VHHA(2+) and NGF K32A / R114I The amino acid sequence of -Fc-VHHA (2+) was reverse-translated into a DNA sequence. Simultaneously, referencing the CHO cell preferred codon table and considering factors such as codon degeneracy, GC content, transcribed mRNA structure, and stability, OptimumGene was used to determine the optimal DNA sequence. TM The software optimizes the DNA sequence to design DNA sequences suitable for encoding various molecular constructs. Specifically, as shown in SEQ ID NO: 35-40, the 5' end contains an Avr II restriction site (CCTAGG) and a KOZAK sequence (GCCACC), and the 3' end contains a Bstz17 I restriction site (GTATAC) and a stop codon (TGA).

[0079] The encoding DNA of the above molecular constructs was synthesized through chemical synthesis.

[0080] (2) Construction of recombinant expression vectors and acquisition of mixed clonal populations

[0081] The expression vector in this embodiment was constructed using the Freedom pCHO 1.0 vector from Life Technologies.

[0082] This vector contains two exogenous gene expression units (SU1 and SU2). To improve the processing efficiency of preproNGF during intracellular expression by removing the leader peptide (pro region) through enzymatic cleavage, the entire SU2 expression unit of this vector was replaced with a Furin enzyme expression unit via seamless cloning. This unit contains a weak PGK promoter, a Furin-encoding DNA sequence (SEQ ID NO: 75, encoding the mouse Furin enzyme shown in SEQ ID NO: 74), and an SV40 polyadenylation signal sequence. This modified vector is designated pCHO-Furin. The exogenous gene promoter in the SU1 expression unit of this vector is a hybrid sequence derived from the EF1 and CMV promoters, exhibiting 5–10 times higher transcriptional activity than the commonly used CMV promoter. Furthermore, this vector contains two selection markers: a puromycin resistance gene and a DHFR gene, corresponding to puromycin and methotrexate (MTX), respectively. Therefore, stable and highly efficient cell lines can be obtained through puromycin resistance screening combined with the DHFR-MTX selection system.

[0083] The artificially synthesized NGF mentioned above were respectively wt -Fc、NGF K32A -Fc-VHHA(2+), NGF R114I -Fc-VHHA(2+), NGF K88L -Fc- VHHA(2+), NGF F12E -Fc-VHHA(2+) and NGF K32A / R114I The coding DNAs for -Fc-VHHA(2+) were double-digested with Avr II and Bstz17 I, then ligated into the double-digested pCHO-Furin expression vector using T4 DNA ligase to construct recombinant expression vectors for each molecular construct. These vectors were transformed into *E. coli* clone strain XL10 and cultured at 37°C on LB medium containing 50 μg / ml kanamycin for recombinant selection. After enzyme digestion confirmation, further sequencing confirmed sequence agreement with the designed sequence, confirming successful construction of each recombinant expression vector. The recombinant expression vector map is shown below. Figure 1 .

[0084] In this embodiment, the host cell used is Freedom CHO-S cell from Life Technologies, which is derived from CHO-K1 cell. Both growth and passage were carried out in serum-free culture medium.

[0085] The above recombinant expression vector plasmids were digested with NruI and linearized. (Refer to Freestyle) TM The MAX kit instructions specify the use of liposomes (Freestyle) TMCHO-S cells were transfected using the MAX method. Following the Freedom CHO-S kit manual, the cells underwent two rounds of drug-induced screening: a first round of screening with 10 μg / ml puromycin and 100 nM MTX (abbreviated as 10P / 100M), and a second round of screening with 50 μg / ml puromycin and 1000 nM MTX (abbreviated as 50P / 1000M). After these two rounds of drug-induced screening, mixed clonal populations of each molecular construct were obtained.

[0086] (3) Culture of mixed clonal populations of various molecular constructs and purification of target proteins

[0087] The frozen molecular constructs were revived and cultured in mixed clonal populations, and the culture volume was expanded to 600-800 ml. After culturing in a carbon dioxide shaker at 37°C, 8% CO2, and 106 rpm for 10 days, the supernatant of each molecular construct was collected by centrifugation.

[0088] Each molecular construct was first purified by Protein A affinity chromatography, followed by removal of residual polymers and degradation fragments using Superdex 200 Prep Grade gel filtration chromatography, yielding pure protein products for each construct. SDS-PAGE and SEC-HPLC analysis confirmed that the protein purity of each construct was above 95%.

[0089] Example 2: NGF mut Activity identification of -Fc-VHHA(2+)

[0090] The NGFs with different NGF (117) mutations prepared in Example 1 were used to prepare various NGFs. mut -Fc-VHHA(2+) molecules were analyzed for protein content using the Lowry method. Using the mouse nerve growth factor activity reference standard distributed by the National Institutes for Food and Drug Control as a reference, biological activity was detected using the "TF-1 cell / MTS colorimetric method" (General Chapter 3530, Volume III, 2020 edition of the Chinese Pharmacopoeia) to calculate specific activity. Furthermore, the surface plasmon resonance (SPR) method (General Chapter 1105, United States Pharmacopeia-National Formulary, effective December 1, 2020) was used to detect the activity of each NGF using a Cytiva Biacore 8K molecular interaction analyzer. mut-Fc-VHHA(2+) molecules and their affinity for human TrKA receptor (33-417) (with a mouse IgG2a Fc tag, ACRO product, catalog number: TRA-H5259) or human NGFR receptor (p75NTR) (with an Fc tag, ACRO product, catalog number: NGR-H5254) pre-coated and immobilized on a CM5 chip (Cytiva product, catalog number: BR100530): For the high-affinity TrKA receptor, the KD value was calculated using a 1:1 Langmuir binding model for global fitting; for the low-affinity p75NTR receptor, the KD value was calculated using steady-state affinity analysis.

[0091] The specific activity and KD values ​​of the affinity for TrkA and p75NTR receptors of each molecular construct are shown in Table 2. This indicates that compared to the wild-type NGF (117) control molecule NGF... wt Compared to -Fc, different NGF (117) mutations in NGF mut -Fc-VHHA(2+) molecules were well preserved in terms of specific activity; different NGF(117) mutations in NGF were also observed. mut The affinity of the -Fc-VHHA(2+) molecule for the TrkA receptor remained essentially unchanged, while its affinity for the p75NTR receptor decreased by approximately one to two orders of magnitude. Simultaneously, the binding selectivity of both the TrkA and p75NTR receptors (i.e., the ratio of the p75NTR receptor affinity KD value to the TrkA receptor affinity KD value) was the same as that of the control molecule NGF. wt -Fc binding selectivity is more than 1.5 times higher. Among them, NGF(117)K32A mutation NGF K32A -Fc-VHHA(2+) molecules exhibit superior specific activity and receptor binding selectivity compared to F12E-mutant NGF. F12E -Fc-VHHA(2+) molecules.

[0092] Those skilled in the art will understand that by increasing the binding selectivity of NGF to TrkA receptors and p75NTR receptors, i.e., reducing the affinity of NGF for p75NTR receptors while maintaining the affinity of NGF for TrkA receptors, the activation of p75NTR-mediated nociceptive pathways can be eliminated, and the TrkA-mediated neuronal survival, differentiation, and protective effects can be maintained, thus achieving the goal of NGF being "painless but neurotrophic."

[0093] Table 2 NGF mut -Fc-VHHA(2+) molecular activity identification results

[0094]

[0095] Example 3: NGFmut -Fc-VHHA(2+) Mouse Short-Term Pain Induction Test

[0096] In this embodiment, different NGF (117) mutations prepared in Example 1 were obtained by subcutaneous single injection into the paw pads of the left and right hind legs of mice. mut -Fc-VHHA(2+) molecules, and set up a saline negative control and NGF wt -Fc positive control group, pain threshold was measured 1 hour after drug administration, and the levels of each NGFmut-Fc-VHHA(2+) molecule, saline and NGF were compared. wt - Pain threshold changes induced by Fc (difference from baseline, ΔPWT, unit: g). Detailed experimental description is as follows: Male BALB / c mice weighing 25-30g were randomly divided into groups (n=4). After environmental acclimatization, the baseline pain threshold of the mice was measured using a Von Frey tactile meter (UGO product, catalog number: 38450). After measurement, insulin was administered subcutaneously to the left and right hind legs of the mice, with a single-sided administration volume of 25 μL, for a total of 50 μL per mouse. One hour after administration, the pain threshold of the left and right hind legs was measured again. The pain threshold for each mouse was the average of the left and right hind legs. Results are shown in Table 3. Figure 2 As shown, after a single injection of 3.74 μg / mouse into the footpads of mice, the wild-type positive control NGF levels decreased by 1 hour. wt -Fc caused a 2.9g decrease in the pain threshold, while each NGF mut The pain threshold reduction induced by -Fc-VHHA(2+) molecules was less than 2g, especially NGF. K32A -Fc-VHHA(2+) molecules and NGF R114I The pain threshold induced by the -Fc-VHHA(2+) molecule decreased to about 1g, which is similar to that of the saline group.

[0097] Table 3 NGF mut -Fc-VHHA(2+) molecule pain threshold after a single subcutaneous injection into the left and right hind paw pads of mice

[0098]

[0099] Note: NS is the saline control group.

[0100] Example 4: NGF K32A Preparation of -Fc-TfR1BS

[0101] In this embodiment, the K32A mutation of NGF(117) is used as an example to construct and prepare NGF containing different TfR1 binding sequences as shown in Table 4. K32A-Fc-TfR1BS molecules. The affinity constants (KD) of each TfR1BS sequence with recombinant proteins of the extracellular domain of human, monkey, and mouse TfR1 receptors and the apical domain of the human TfR1 receptor, as detected by biomembrane interference (BLI), are shown in Table 5. In parentheses at the end of each molecular construct name, "2+" indicates that the molecule is a homodimer, and its TfR1BS exhibits a bivalent form; "1+" indicates that the molecule is a heterodimer, and its TfR1BS exhibits a monovalent form. The TfR1BS construct 8D3 scFv was used as a positive control. This 8D3 scFv is a single-chain antibody (SEQ ID NO: 41) spliced ​​from the VH and VL of a rat anti-mouse TfR1 monoclonal antibody in the prior art.

[0102] Table 4 NGF K32A -Fc-TfR1BS molecular structure

[0103]

[0104] Table 5. Affinity constant KD values ​​of TfR1BS and TfR1 receptor (BLI method, unit nM)

[0105]

[0106] Note: NB indicates no binding; ND indicates not detected; α, NGF K32A - Affinity constant KD value of Fc-8D3 scFv(2+) with mouse TfR1; b, VHHA affinity constant data are the apparent Kd values ​​of Fc-VHHA (bivalent) binding to CHO cells expressing human TfR or mouse TfR as described in WO2020144233A1 by flow cytometry.

[0107] (1) Preparation of TfR1BS bivalent molecules (homodimers)

[0108] In this embodiment, NGF is used. K32A -Fc-8D3 scFv(2+) (SEQ ID NO: 44) and NGF K32A -Fc-VHHC(2+) (SEQ ID NO: 45) is used as a representative to illustrate NGF K32A The preparation method of -Fc-TfR1BS(2+) homodimer molecules is basically the same as that of NGF described in Example 1. mut The preparation method for -Fc-VHHA(2+) is the same, still using pCHO-Furin as the expression vector to achieve co-expression with Furin enzyme, thereby improving the processing efficiency of preproNGF in Freedom CHO-S cells to remove the leader peptide (pro region) by enzymatic cleavage. The difference is that: NGF K32AThe -Fc-8D3 scFv(2+) molecular signal peptide uses the Gis sequence (GNT-induced secretion sequence) described in CN108610398B, combined with the natural NGF signal peptide, to enhance the secretory expression of the target protein. An Fc variant (SEQ ID NO: 42) composed of the human IgG3 hinge region, human IgG1 CH2 region, and CH3 region is selected as the Fc. This human IgG3 hinge region, possessing three EPKSCDTPPPCPRCP tandem repeat sequences, effectively avoids potential structural and functional interference between NGF and the Fc region. Its Linker1 uses a short polypeptide linker sequence (GGG).

[0109] NGF K32A The Fc of the -Fc-VHHC(2+) molecule also uses the Fc sequence of the natural variant of human IgG1, IGHG1*03, and performs LALAPG mutation to eliminate the effect function of the Fc region. At the same time, it deletes 5 amino acid residues at its N-terminus (EPKSC) to reduce potential structural perturbation of the hinge region; (SEQ ID NO: 43) its Linker1 uses the peptide linker sequence (GGGGS)3.

[0110] Same as in Example 1, the above-mentioned NGF K32A -Fc-8D3 scFv(2+) and NGF K32A The amino acid sequences of -Fc-VHHC(2+) were reverse-translated into DNA sequences, and after sequence optimization, an Avr II restriction site (CCTAGG) and a KOZAK sequence (GCCACC) were added to the 5' end, and a Bstz17 I restriction site (GTATAC) and a stop codon (TGA) were added to the 3' end, resulting in the corresponding coding DNA sequences, SEQ ID NO: 46 and 47, respectively.

[0111] Same as in Example 1, the above-mentioned NGF K32A -Fc-8D3 scFv(2+) and NGF K32A The encoding DNA sequence of -Fc-VHHC(2+) was digested with Avr II and Bstz17 I, and then ligated into the pCHO-Furin expression vector, which had also been digested with T4 DNA ligase, to construct NGF. K32A -Fc-8D3 scFv(2+) and NGF K32A The recombinant expression vector of -Fc-VHHC(2+) was linearized by digestion with NruI and then transfected into CHO-S cells as described in Example 1. Two rounds of drug pressure screening were performed at 10P / 100M and 50P / 1000M to obtain NGF. K32A -Fc-8D3 scFv(2+) and NGFK32A A mixed clonal population of -Fc-VHHC(2+).

[0112] Using the same method as in Example 1, NGF was cultured. K32A -Fc-8D3 scFv(2+) and NGF K32A A mixed clonal population of -Fc-VHHC(2+) cells was collected, and the culture supernatant was purified according to the method in Example 1 to obtain NGF. K32A -Fc-8D3 scFv(2+) and NGF K32A The purity of Fc-VHHC(2+) was determined to be above 95% by SDS-PAGE and SEC-HPLC.

[0113] (2) Preparation of TfR1BS monovalent molecules (heterodimers)

[0114] In this embodiment, the NGF was constructed using KiH or KiHs-s technology, which is well-known and widely used by those skilled in the art. K32A The -Fc-TfR1BS(1+) heterodimer molecule exhibits a monovalent form where the TfR1BS sequence is linked to only one polypeptide chain of Fc. KiH technology utilizes steric hindrance and spatial complementarity to induce Fc dimers to form heterodimers, reducing the proportion of homologous mismatch dimers. One chain's CH3 region introduces a larger amino acid residue to form a "bulge" (T366W mutation, denoted as the Knob chain), while the other chain introduces smaller amino acid residues to form a "hole" (T366S / L368A / Y407V mutation, denoted as the Hole chain), thus promoting heterodimer formation. KiHs-s technology further introduces disulfide bonds based on KiH, specifically by introducing the S354C mutation into the Knob chain and the Y349C mutation into the Hole chain, forming interchain disulfide bonds and further improving the stability and yield of the heterodimer. Those skilled in the art will understand that the TfR1BS sequence can be placed at the C-end of the Knob chain or at the C-end of the Hole chain.

[0115] For example, in this embodiment, 8D3 scFv, VHHA, VHHC, VHHG, VHHN, VHHQ, VHHX, VHHY, VHHX1, and VHHX2 were selected as TfR1BS sequences to construct different NGF sequences as shown in Table 4. K32A -Fc-TfR1BS(1+) heterodimer molecules. Among them, TfR1BS is a construct molecule of 8D3 scFv or VHHA as a control molecule; the full amino acid sequences and CDR region sequences of VHHC, VHHG, VHHN, VHHQ, VHHX, VHHY, VHHX1 and VHHX2 are shown in Table 6.

[0116] Table 6. Amino acid sequence list of VHH and its CDR1, CDR2, and CDR3

[0117]

[0118]

[0119] For ease of explanation, NGF will be used. K32A The polypeptide chain carrying the TfR1BS sequence in the -Fc-TfR1BS(1+) heterodimer molecule is denoted as chain A, which possesses "NGF" K32A The structure is “-Linker1-Fc-Linker2-TfR1BS”; the other polypeptide chain without the TfR1BS sequence is designated as chain B, which has the structure “NGF”. K32A The structure is "-Linker1-Fc".

[0120] For example, TfR1BS are heterodimeric constructs of 8D3 scFv and VHHA, respectively. Both use the natural variant of human IgG1, IGHG1*03, as the Fc sequence and undergo LALAPS mutation to eliminate the Fc region effector function. Simultaneously, the N-terminal five amino acid residues (EPKSC) are deleted to reduce potential structural perturbation of the hinge region. (Knob chain: SEQ ID NO: 48; Hole chain: SEQ ID NO: 49) Both heterodimers are constructed using KiH technology, with TfR1BS positioned at the C-terminus of the Hole chain. The B-chain amino acid sequences of these two constructs are identical, both being SEQ ID NO: 52.

[0121] For example, TfR1BS are heterodimeric constructs of VHHC, VHHG, VHHN, VHHQ, VHHX, VHHY, VHHX1, and VHHX2, respectively. All constructs use human IgG1 as the Fc sequence and undergo LALAPG mutation to eliminate the Fc region effector function. Simultaneously, the N-terminal five amino acid residues (EPKSC) are deleted to reduce potential structural perturbation of the hinge region. (Knob chain: SEQ ID NO: 76; Hole chain: SEQ ID NO: 77) All heterodimers are constructed using KiHS-S technology, with TfR1BS positioned at the C-terminus of the Knob chain. The B-chain amino acid sequences of these eight constructs are identical, all being SEQ ID NO: 61.

[0122] Same as in Example 1, the above-mentioned NGF K32AThe amino acid sequences of the A and B chains of the -Fc-TfR1BS(1+) heterodimer molecule (SEQ ID NO: 50-61) were reverse-translated into DNA sequences. After sequence optimization, an Avr II restriction site (CCTAGG) and a KOZAK sequence (GCCACC) were added to the 5' end, and a Bstz17 I restriction site (GTATAC) and a stop codon (TGA) were added to the 3' end to obtain the corresponding coding DNA sequences (SEQ ID NO: 62-73).

[0123] Same as in Example 1, the above-mentioned NGF K32A The encoding DNA sequences of the B chain of the -Fc-TfR1BS(1+) heterodimer molecule (SEQ ID NO: 64, 73) were digested with Avr II and Bstz17 I, respectively, and then ligated into the pCHO-Furin expression vector, which was also digested with T4 DNA ligase, to construct B chain recombinant expression vectors.

[0124] At the same time, the above-mentioned NGF K32A The encoding DNA sequence of the A chain of the -Fc-TfR1BS(1+) heterodimer molecule (SEQ ID NO: 62-63, 65-72) was double-digested with Avr II and Bstz17 I, and then ligated into the pCHO-SU1 expression vector, which had also been double-digested, using T4 DNA ligase to construct recombinant expression vectors of the A chain. The pCHO-SU1 expression vector was prepared by digesting Freedom pCHO 1.0 with restriction endonuclease SfiⅠ and religating it, removing the expression unit SU2 and containing only the expression unit SU1. Like the pCHO-Furin vector, the pCHO-SU1 vector contains a heterozygous strong promoter derived from the EF1 and CMV promoters, and also contains two selection markers: a puromycin resistance gene and a DHFR gene.

[0125] Then the above NGF K32A The recombinant expression vectors of the A and B chains of the -Fc-TfR1BS(1+) heterodimer molecule were linearized by NruI restriction enzyme digestion, mixed at a 1:2 ratio, and transfected into CHO-S cells as described in Example 1. Two rounds of drug pressure screening were performed at 10P / 100M and 50P / 1000M to obtain the various NGFs. K32A A mixed clonal group of -Fc-TfR1BS(1+) heterodimer molecules.

[0126] Following the method in Example 1, mixed clonal populations of cells containing each of the above-mentioned NGFK32A-Fc-TfR1BS(1+) heterodimer molecules were cultured, and the culture supernatant was collected. The individual NGF molecules could then be obtained through the following separation and purification methods. K32A-Fc-TfR1BS(1+) protein: First, it was purified by Protein A affinity chromatography, and then purified by MabSelect. TM VH3 affinity chromatography packing material (NGF) K32A Except for -Fc-8D3 scFv(1+), which uses MabSelect TM VL affinity chromatography packing material (all Cytiva products) was used for affinity chromatography. Finally, Superdex 200 Prep Grade gel filtration chromatography was used to remove residual polymers and degradation fragments, yielding NGFs with SDS-PAGE and SEC-HPLC purity both exceeding 95%. K32A -Fc-TfR1BS(1+) protein purified.

[0127] Example 5: NGF K32A -Fc-TfR1BS mice in vivo cross-BBB brain uptake effect test

[0128] In this embodiment, to evaluate the NGF of the present invention mut -Fc-TfR1BS transBBB brain entry effect, exemplarily, the NGF prepared in Example 1 wt -Fc、NGF K32A -Fc-VHHA(2+) and NGF prepared in Example 4 K32A -Fc-8D3 scFv(2+), NGF K32A -Fc-VHHA(1+), NGF K32A -Fc-8D3 scFv(1+), NGF K32A -Fc-VHHC(2+), NGF K32A -Fc-VHHG(1+) and NGF K32A -Fc- VHHQ(1+) was injected into BALB / c mice via tail vein injection, and the concentration of the target protein in the brain and plasma was measured 24 hours later. The experimental procedure is detailed below:

[0129] BALB / c mice weighing approximately 30g were injected intravenously via the tail vein at a dose of 0.1 μmol / kg, with an administration volume of approximately 150 μL. Blood was collected 24 hours later by enucleation, and anticoagulated plasma was collected. The heart was perfused with physiological saline (0.9% sodium chloride solution), and brain tissue was dissected. RIPA tissue lysis buffer was added at a ratio of 5 μL / mg wet weight of brain tissue, and the brain tissue was homogenized. The supernatant was collected by centrifugation. The target protein—NGF—in the plasma and brain tissue homogenate supernatant was detected using ELISA. K32A -Fc-TfR1BS protein content.

[0130] (1) Select male BALB / c mice weighing approximately 30g;

[0131] (2) The drug was administered via tail vein injection at a dose of 0.1 μmol / kg (dose volume 150 μL);

[0132] (3) Blood was collected from the eyeballs 24 hours after injection. Whole blood of mice was collected using anticoagulant tubes containing EDTA-K2 (BKMAN product, catalog number: 110403014). The blood was centrifuged at 5000 rpm and 4℃ for 15 min, and the supernatant was collected as plasma sample.

[0133] (4) Immediately after blood collection, the heart was perfused with physiological saline (0.9% sodium chloride solution). After perfusion, the whole brain was dissected and the cerebral cortex tissue was separated and weighed. RIPA tissue lysis buffer (Beyotime product, catalog number: ST507) was added at a ratio of 5 μL of lysis buffer per 1 mg of tissue. Then, the cerebral cortex tissue was homogenized on ice using a handheld tissue homogenizer (Tuohe Technology product, model: MY-20).

[0134] (5) After homogenization, place on ice and pyrolyze on a horizontal shaker (1000 rpm) for 2 hours;

[0135] (6) After pyrolysis, centrifuge at 12,000 rpm and 4°C for 20 min;

[0136] (7) After centrifugation, collect the supernatant, which is the tissue lysis sample of the cerebral cortex.

[0137] In this embodiment, NGF in the plasma sample and the tissue lysate sample of the cerebral cortex were detected by ELISA. K32A The content of -Fc-TfR1BS was determined. The experimental procedure is as follows:

[0138] (1) Dilute the coating antibody anti-hFc (Sigma product, catalog number: I2136) to 2 μg / mL with ELISA coating buffer (1.59 g Na2CO3, 2.93 g NaHCO3, dissolved in ultrapure water and brought to a final volume of 1 L);

[0139] (2) Add 100 μL of the diluted coating antibody solution from step (1) to each well of the microplate and incubate at 4°C overnight;

[0140] (3) After overnight incubation, aspirate the liquid from the wells and wash three times with PBST;

[0141] (4) Prepare 2.5% skim milk powder using PBST, adding 300 μL to each well;

[0142] (5) Incubate at 37°C for 2 hours, then wash 3 times with PBST;

[0143] (6) The corresponding protein stock solution injected in the mouse brain uptake experiment was used as the standard for ELISA detection and diluted with PBST to 1500 pM, 500 pM, 166.7 pM, 55.6 pM, 18.5 pM, 6.2 pM, 2.1 pM and 0 pM respectively;

[0144] (7) Dilute the samples to be tested (the above-mentioned plasma samples and tissue lysate samples from the cerebral cortex) to an appropriate multiple using PBST;

[0145] (8) Take 100 μL of the diluted standard and sample and add them to the wells of the enzyme-labeled plate coated with anti-hFc antibody after washing with PBST in step (5). Perform two replicates for both the standard and the sample and incubate at 37°C for 1 hour.

[0146] (9) Dilute the HRP-labeled secondary antibody (HRP-labeled anti-hFc antibody, ABclonal product, catalog number: AS035) with PBST at a ratio of 1:2000;

[0147] (10) Aspirate the sample from the wells of the ELISA plate after incubation in step (8), wash 3 times with PBST, add 100 μL of the diluted HRP-labeled secondary antibody from step (9) to each well, and incubate at 37°C for 1 hour;

[0148] (11) Aspirate all HRP-labeled secondary antibody solution from the wells and wash 3 times with PBST;

[0149] (12) Add 100 μL of substrate solution (R&D Systems product, catalog number: DY999B) to each well (operate in the dark) and incubate at 37°C in the dark for 15 min;

[0150] (13) Add 50 μL of stop solution (2M H2SO4) to each well to stop the reaction, and read OD450 using an enzyme-linked immunosorbent assay (ELISA) reader.

[0151] A standard curve was fitted based on the OD450 readings of the standards, and then the concentration of the target protein in each sample was calculated. The results are as follows: Figure 3 As shown in Table 7, 24 hours after tail vein injection, compared with NGF without TfR1BS sequence... wt -Fc, each NGF K32A The average concentration of -Fc-TfR1BS molecules in the cerebral cortex is approximately NGF. wt -Fc was 4 to 23 times higher; among them, the monovalent heterodimer of TfR1BS had a higher average concentration in the cerebral cortex, which was 1.6 to 1.7 times higher than that of the corresponding divalent homodimer of TfR1BS; among the tested heterodimers, NGF K32A-Fc-VHHG(1+) and NGF K32A -Fc-VHHQ(1+) exhibits high brain penetration efficiency, with its average concentration in the cerebral cortex being approximately equal to that of the control molecule NGF. K32A 2.2 times and 2.5 times that of -Fc-VHHA(1+).

[0152] The TfR1BS sequence is a positive control molecule of 8D3 scFv. Regardless of whether it is monovalent or bivalent, it has the highest average concentration in the cerebral cortex 24 hours after tail vein injection. However, it is prone to aggregation and has poor stability during purification.

[0153] The results of plasma protein analysis 24 hours after tail vein injection showed that the construct NGF was present in the TfR1BS monovalent heterodimer. K32A -Fc-VHHQ(1+) had the highest plasma concentration, with a mean value approximately equal to that of the control molecule NGF. K32A -Fc-8D3scFv(1+) and NGF K32A -Fc-VHHA(1+) 8 times and 18 times.

[0154] Table 7 NGF K32A -Fc-TfR1BS in vivo brain uptake assay: cerebral cortex and plasma drug concentrations in mice (24h, the results shown are the average)

[0155]

[0156] Example 6: NGF K32A -Fc-TfR1BS hTfR1 transgenic mice in vivo cross-BBB brain uptake effect test

[0157] In this embodiment, NGF was evaluated. mut The effect of -Fc-TfR1BS on brain entry across the BBB in hTfR1 transgenic mice. Exemplarily, the NGF prepared in Example 1... wt -Fc and NGF prepared in Example 4 K32A -Fc-VHHA(1+), NGF K32A -Fc-VHHC(2+), NGF K32A -Fc-VHHC(1+), NGF K32A -Fc-VHHN(1+), NGF K32A -Fc- VHHQ(1+), NGF K32A -Fc-VHHY(1+) and NGF K32A-Fc-VHHX2(1+) was injected via tail vein into male 8-12 week old B6-hTFRC(CDS) mice (product of Suzhou Cyagen Biotechnology Co., Ltd., catalog number: C001584) using the same method as in Example 5. The content of the target protein in plasma and cerebral cortex tissue lysate samples was detected 24 hours later using the method in Example 5. Results are as follows... Figure 4 As shown in Table 8, the results also demonstrate that, compared to NGF without the TfR1BS sequence, wt -Fc, 24 h after tail vein injection in hTfR1 transgenic mice, each NGF K32A The average concentration of -Fc-TfR1BS molecules in the cerebral cortex is approximately NGF. wt -Fc 4 to 36 times; TfR1BS monovalent heterodimer NGF in hTfR1 transgenic mice K32A -Fc-VHHC(1+) also has better brain penetration efficiency than the TfR1BS bivalent homodimer NGF. K32A -Fc-VHHC(2+), the mean concentration of the former in the cerebral cortex 24 hours after injection was approximately 2.4 times that of the latter; among the tested heterodimers, NGF K32A -Fc-VHHX2(1+), NGF K32A -Fc-VHHY(1+) and NGF K32A -Fc-VHHQ(1+) exhibits high brain penetration efficiency, with its average concentration in the cerebral cortex being approximately equal to that of the control molecule NGF. K32A -Fc-VHHA(1+) was 1.6, 1.7, and 2.7 times more potent than other drugs.

[0158] Table 8 NGF K32A -Fc-TfR1BS in B6-hTFRC (CDS) transgenic mice brain uptake assay: cerebral cortex and plasma drug concentrations (24h, results shown are mean values)

[0159]

[0160] Example 7: NGF K32A The therapeutic effect of -Fc-VHHQ(1+) on traumatic brain injury (TBI)

[0161] In this embodiment, NGF is used. K32A Taking -Fc-VHHQ(1+) as an example, NGF was evaluated. mut The therapeutic effect of -Fc-TfR1BS on a mouse model of traumatic brain injury prepared by controlled cortical impaction (CCI).

[0162] In this embodiment, a traumatic brain injury mouse model was established by inducing brain tissue injury in 8-10 week old male C57BL / 6 mice using a PCI3000 precision craniocerebral injury device (Hatteras product). After an adaptation period, the mice were randomly divided into a model control group and an NGF group according to their body weight. K32A Six mice were administered NGF (1+) at low dose (0.35 mg / kg) and high dose (1.75 mg / kg). The first administration of NGF was initiated 6 hours after the traumatic brain injury. K32A -Fc-VHHQ(1+) was administered via tail vein injection, while the model control group received an equal volume of physiological saline; administration was repeated every 3 days for 4 consecutive weeks. The day of model establishment was recorded as D0. Neurological deficit scores (mNSS) were assessed in all mice on days 1, 3, 7, 14, and 21 post-modeling, and Y-maze behavior was tested on day 22 post-administration. At the experimental endpoint, all mice were euthanized, and brain tissue was collected for GFAP, Iba1, and NeuN staining to analyze changes in glial cell proliferation and neuron number. The Unpaired T Test was used for statistical analysis.

[0163] The results are as follows Figure 5 , Figure 6 As shown in Tables 9 and 10: On the first day after modeling, NGF K32A The mNSS scores of the -Fc-VHHQ(1+) 0.35 mg / kg and 1.75 mg / kg groups were not significantly different from those of the model control group; however, on days 3 and 7, the total mNSS score of the 1.75 mg / kg group was significantly lower than that of the model control group (P<0.05). On day 22, the Y-maze test results showed that the spontaneous alternation rate of mice in the 1.75 mg / kg group was significantly higher than that of the model control group (P<0.05). Furthermore, brain tissue pathological staining also showed NGF... K32A The percentage of GFAP and Iba1 positive areas in the cortical and hippocampal regions of mice in the -Fc-VHHQ(1+) 0.35mg / kg and 1.75mg / kg dose groups was reduced to some extent compared with the model control group, while the number of NeuN positive cells per unit area of ​​brain tissue was increased to some extent compared with the model control group.

[0164] Table 9. Results of GFAP immunostaining in brain tissue of TBI mice

[0165]

[0166] Note: All data in the table are expressed as mean ± standard deviation (SD). The statistical method used is the Unpaired T-test. * P<0.05 indicates the statistical analysis results of the model control group and the low or high dose group.

[0167] Table 10 Results of Iba1 immunostaining in brain tissue of TBI mice

[0168]

[0169] Note: All data in the table are expressed as mean ± standard deviation (SD). The statistical method used is the Unpaired T-test. * P<0.05 indicates the statistical analysis results of the model control group and the low or high dose group.

[0170] The results showed that NGF K32A -Fc-VHHQ(1+) at a dose of 1.75 mg / kg can improve neurological function in mice with traumatic brain injury to a certain extent, reduce neuroinflammation, and improve motor ability and spatial cognition in mice with traumatic brain injury.

[0171] Example 8: NGF K32A The therapeutic effect of -Fc-VHHQ(1+) on ischemic brain injury (MCAO / R)

[0172] In this embodiment, NGF is used. K32A Taking -Fc-VHHQ(1+) as an example, NGF was evaluated. mut The therapeutic effect of -Fc-TfR1BS on a mouse model of stroke prepared by cerebral ischemia-reperfusion (MCAO / R).

[0173] In this embodiment, a cerebral ischemia-reperfusion (MCAO / R) mouse model was established using 8-10 week old male C57BL / 6 mice. After an adaptation period, the mice were randomly divided into a model control group and an NGF group according to their body weight. K32A -Fc-VHHQ(1+) low-dose group (0.35mg / kg) and high-dose group (1.75mg / kg), 6 mice in each group.

[0174] The modeling process is as follows: After weighing, the mice were anesthetized by intraperitoneal injection of 2% tribromoethanol at a dose of 20 ml / kg. After deep anesthesia, the neck hair was removed with an animal shaver, and the surgical area was disinfected with iodine and alcohol. Then, a longitudinal incision was made on the right side of the neck about 5 mm lateral to the midline. The superficial fascia was cut to expose the right sternocleidomastoid muscle. Blunt dissection was performed between the sternocleidomastoid muscle and the anterior neck muscles to expose the carotid sheath. The common carotid artery (CCA) and vagus nerve were carefully dissected until the bifurcation of the CCA. The medial external carotid artery (ECA) and the lateral posterior internal carotid artery (ICA) were bluntly dissected. 6-0 silk sutures were used at the proximal end of the common carotid artery, 1 mm from the bifurcation of the common carotid artery. A 6-0 silk suture is passed under the common carotid artery and a slipknot is tied near the bifurcation of the common carotid artery. The internal carotid artery is clamped with an arterial clamp, and a small V-shaped incision is made at the common carotid artery with ophthalmic scissors. A pre-prepared suture plug (product of Guangzhou Jialing Biotechnology Co., Ltd., item number: L2000) is then inserted along the common carotid artery into the internal carotid artery until the suture plug marker is at the bifurcation of the common carotid artery. The slipknot is tightened to secure the suture plug. The muscle and skin are sutured layer by layer (the suture plug head must be exposed during suturing to facilitate removal of the suture plug for reperfusion), and the wound is disinfected. After 90 minutes of ischemia, the suture plug marker is removed from the common carotid artery, and excess suture plug is cut off to complete reperfusion (this operation must be performed under mouse anesthesia). After the operation, the mouse is placed on a heating pad and returned to its cage for normal feeding after it regains consciousness.

[0175] Six hours after reperfusion, mice were first administered the test drug NGFK32A-Fc-VHHQ(1+) via tail vein injection, while the model control group received an equal volume of physiological saline. Administration was repeated every 3 days for 4 weeks. Mice were grouped the day before modeling (D0). All mice were weighed and scored using the Longa scale on D0, D4, D11, D18, and D25. At the experimental endpoint, all mice were euthanized, and brain tissue was collected for NeuN staining to analyze changes in neuron count. The Unpaired T Test was used for statistical analysis.

[0176] The results are as follows Figure 7 and Figure 8 As shown: On days 0 and 4, there was no significant difference in body weight between groups. On days 11, 18, and 25, NGF... K32A The body weight of the -Fc-VHHQ(1+) 0.35mg / kg dose group was not significantly different from that of the model control group. NGF K32A The body weight of the -Fc-VHHQ(1+) 1.75 mg / kg dose group was significantly higher than that of the model control group (P<0.05). NGF K32AThe Longa score in the -Fc-VHHQ(1+) 1.75 mg / kg dose group was lower than that in the model control group on days 11 and 18. Furthermore, immunohistochemical staining of brain tissue also showed NGF... K32A The number of NeuN-positive cells per unit area in the brain tissue of mice in the -Fc-VHHQ(1+) 0.35mg / kg and 1.75mg / kg dose groups was significantly increased compared with the model control group (P<0.05).

[0177] The results showed that NGF K32A -Fc-VHHQ(1+) significantly reduced neuronal death in the acute phase of stroke at both doses of 0.35 mg / kg and 1.75 mg / kg; at the high dose of 1.75 mg / kg, it significantly reduced weight loss in stroke mice during the acute phase and improved neurological function in stroke mice to some extent.

[0178] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A nerve growth factor, characterized in that: The nerve growth factor is a heterodimer, and the amino acid sequence of its A chain is shown in SEQ ID NO: 56, and the amino acid sequence of its B chain is shown in SEQ ID NO:

61.

2. A nucleic acid, characterized in that: The nucleic acid encodes the nerve growth factor as described in claim 1.

3. An expression vector, characterized in that: The expression vector comprises the nucleic acid as described in claim 2.

4. The expression vector as described in claim 3, characterized in that: It also contains nucleic acids that encode Furin enzymes.

5. A recombinant host cell, characterized in that: The recombinant host cell contains the nucleic acid as described in claim 2, or contains the expression vector as described in any one of claims 3-4.

6. A method for preparing nerve growth factor, characterized in that, The method includes the following steps: expressing the nucleic acid of claim 2 to obtain the nerve growth factor; or expressing the expression vector of any one of claims 3-4 to obtain the nerve growth factor; or culturing the recombinant host cell of claim 5 and separating and purifying the nerve growth factor.

7. The method for preparing nerve growth factor as described in claim 6, characterized in that, Includes the following steps: (1) Transfect the expression vector into mammalian cells; (2) Screening for cell lines containing the expression vector or integrated with the nucleic acid encoded thereon; (3) The obtained cell line was cultured, the culture medium was collected and the nerve growth factor was separated and purified.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the nerve growth factor of claim 1, or the nucleic acid of claim 2, or the expression vector of any one of claims 3-4, or the recombinant host cell of claim 5, and pharmaceutically acceptable carriers and / or excipients.

9. The use of the nerve growth factor of claim 1, or the nucleic acid of claim 2, or the expression vector of any one of claims 3-4, or the recombinant host cell of claim 5, or the pharmaceutical composition of claim 8 in the preparation of a medicament for treating neurological injury diseases; wherein the neurological injury disease is traumatic brain injury or ischemic brain injury.

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