Human anti-transferrin receptor 1 antibodies and uses thereof

Abstract

The present application relates to the technical field of antibodies, in particular to human anti-transferrin receptor 1 antibody and application thereof. The present application provides a human anti-transferrin receptor 1 antibody, which widens the selectable range of antibodies, and the human anti-transferrin receptor 1 antibody of the present application can have cross-binding activity with TfR1 of different species of human, mouse and cynomolgus monkey, can penetrate the blood-brain barrier to deliver into the brain through TfR1-mediated endocytosis transport, can provide effective raw materials for preparing drugs crossing the blood-brain barrier, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular to human anti-transferrin receptor 1 antibodies and uses thereof. BACKGROUND

[0002] The blood-brain barrier (BBB) is a complex physiological barrier that isolates harmful substances from the peripheral circulation system, protects the central nervous system (CNS) from invasion and maintains normal homeostasis, and has multiple functions such as physical barrier, transport barrier, metabolic barrier, immune barrier and enzyme system barrier (Yan et al., Advanced Drug Delivery Reviews 2012, 64:640-665; Serlin et al., Seminars in Cell & Developmental Biology 2015, 38:2-6.). Generally, most small molecule drugs and almost all biological macromolecule drugs are difficult to break through the blood-brain barrier and reach the central nervous system (William et al., Current Opinion in Pharmacology 2006, 6:494-500; Per-Ola et al., Neuropharmacology 2017, 120:38e55). Breaking through the blood-brain barrier and achieving effective delivery across the blood-brain barrier has long been one of the key problems urgently needed to be solved in the field of central nervous system drug research and development.

[0003] Receptor-mediated transcytosis (RMT) is one of the ways for the body to achieve normal exchange of specific substances between the brain and the peripheral circulation through specific receptors on the surface of vascular endothelial cells constituting the blood-brain barrier. A large number of studies have shown that the strategy of achieving central nervous system drug delivery across the blood-brain barrier through receptor-mediated transcytosis has the advantages of high specificity, low toxicity and side effects, can carry multiple types of molecules, and does not damage the integrity of the blood-brain barrier, etc., and is a cross-blood-brain barrier drug delivery approach worthy of in-depth study (Yan et al., Advanced Drug Delivery Reviews 2012, 64:640-665; Serlin et al., Seminars in Cell & Developmental Biology 2015, 38:2-6.).

[0004] Among the RMT-associated transport receptors, transferrin receptor 1 (TfR1) is the most widely used and effective target (Zuchero et al., Neuron 2016, 89:70-82). TfR has two subtypes, TfR1 and TfR2; the two have 45% amino acid sequence identity and 66% sequence similarity. TfR2 is mainly highly expressed in hepatocytes and erythrocyte precursor cells, and is an iron ion sensor. TfR1 is highly expressed in hepatocytes, erythrocytes, the blood-brain barrier, and tumor cells, and is an endocytic transport receptor for iron ions. A large number of studies have confirmed that antibodies targeting TfR1 can preferentially accumulate on the peripheral side of the blood-brain barrier and enter the brain parenchyma through the RMT pathway, achieving drug delivery across the blood-brain barrier.

[0005] Currently, many international pharmaceutical companies have laid out research and development pipelines for antibody drugs that cross the blood-brain barrier. The core technology of Japan's JCR Pharmaceuticals is a blood-brain barrier crossing delivery system called "J-Brain Cargo ® ", which fuses and expresses relevant therapeutic protein / peptide drugs with antibodies targeting TfR1, and then delivers them to the brain through endocytosis to exert effector functions. Its representative product IZCARGO (development code JR141, TfR1 antibody fused with IDS) has been approved for marketing in Japan for the treatment of mucopolysaccharidosis type II (MPS II, also known as Hunter syndrome). Studies have shown that JR141 has good blood-brain barrier crossing drug delivery capacity; phase II / III clinical results show that JR141 has reached the primary clinical trial endpoint, with significant effectiveness, good safety and tolerability (Torayuki et al., Molecular Therapy 2019, 27(2): 456-464; Torayuki et al., Molecular Therapy 2021, 29: 671-679.). JCR Pharmaceuticals still has multiple similar products undergoing relevant preclinical or early clinical studies.

[0006] Therefore, it is an urgent technical problem in the art to develop more anti-transferrin receptor 1 antibodies for selection. SUMMARY

[0007] The present application develops a human anti-transferrin receptor 1 (TfR1) antibody, which has specific binding activity to TfR1, does not interfere with the binding of transferrin (Tf) to TfR1, can penetrate the blood-brain barrier by binding to TfR1, and can be used for blood-brain barrier crossing drug delivery. Based on this, the following technical solutions are proposed.

[0008] In a first aspect, the present application provides a human anti-transferrin receptor 1 antibody or an antigen binding fragment thereof, comprising the following complementarity determining regions:

[0009] CDR1-VL: the amino acid sequence is SGDALGDKYAS;

[0010] CDR2-VL: the amino acid sequence is EDSKRPS;

[0011] CDR3-VL: the amino acid sequence is QAYERGAV or QSHDGSYI or QAYDMGRI or QSYDSDIRS or QSRELLGP;

[0012] CDR1-VH: the amino acid sequence is TSYWIG;

[0013] CDR2-VH: the amino acid sequence is IIYPGDSDTRYSPSFQG;

[0014] CDR3-VH: the amino acid sequence is YNDLLSPMDY or YIDAFTPFDY or YSHFSRPFAD or YRILAHFDY or SHVFDLNMDD.

[0015] In some embodiments, the light chain variable region amino acid sequence is as set forth in SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 39, SEQ ID NO. 41, or SEQ ID NO. 43; and the heavy chain variable region amino acid sequence is as set forth in SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 40, SEQ ID NO. 42, or SEQ ID NO. 44.

[0016] In some embodiments, the humanized anti-transferrin receptor 1 antibody or antigen binding fragment thereof further comprises a constant region.

[0017] In some embodiments, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.

[0018] In some embodiments, the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, monkey, or human origin.

[0019] In some embodiments, the constant region comprises a light chain constant region and a heavy chain constant region; the amino acid sequence of the light chain constant region is as set forth in SEQ ID NO. 46, and the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID NO. 48.

[0020] In some embodiments, the humanized anti-transferrin receptor 1 antibody antigen binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, scFv.

[0021] In a second aspect, the present application provides a drug delivery carrier comprising the humanized anti-transferrin receptor 1 antibody or the antigen binding fragment thereof, or a nucleic acid fragment encoding the humanized anti-transferrin receptor 1 antibody or the antigen binding fragment thereof.

[0022] In a third aspect, the present application provides a drug comprising the humanized anti-transferrin receptor 1 antibody or the antigen binding fragment thereof, or a nucleic acid fragment encoding the humanized anti-transferrin receptor 1 antibody or the antigen binding fragment thereof, or the drug delivery carrier.

[0023] In a fourth aspect, the present application further provides a reagent or a kit for detecting transferrin receptor 1, comprising the humanized anti-transferrin receptor 1 antibody or the antigen binding fragment thereof.

[0024] In some embodiments, the antibody or the antigen binding fragment thereof in the reagent or the kit described above is labeled with a detectable label.

[0025] The detectable label refers to a kind of substance having a property such as luminescence, color development, radioactivity, etc. that can be directly observed by naked eyes or detected or probed by instruments, by which qualitative or quantitative detection of the corresponding target can be achieved.

[0026] In optional embodiments, the detectable label includes but is not limited to fluorescent dyes, enzymes that develop color of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0027] In actual use, the skilled in the art can select appropriate labels according to the detection conditions or actual needs, and no matter what kind of label is used, it all falls within the protection scope of the present application.

[0028] In optional embodiments, the fluorescent dye includes, but is not limited to, fluorescein-based dyes and their derivatives (such as including, but not limited to, fluorescein isothiocyanate (FITC), hydroxycoumarin (FAM), tetra-chloro-fluorescein (TET), and the like or analogs thereof), rhodamine-based dyes and their derivatives (such as including, but not limited to, red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like or analogs thereof), Cy series dyes and their derivatives (such as including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, and the like or analogs thereof), Alexa series dyes and their derivatives (such as including, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like or analogs thereof), and protein-based dyes and their derivatives (such as including, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), and the like).

[0029] In optional embodiments, the catalyzing-substrate-coloring enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0030] In optional embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lucigenin and its derivatives, and peroxymalate and its derivatives.

[0031] In optional embodiments, the nanoparticle-based label includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0032] In optional embodiments, the colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latex.

[0033] In optional embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0034] In a fifth aspect, the present application provides application of the humanized anti-transferrin receptor 1 antibody or antigen binding fragment thereof and the drug delivery carrier in vitro in detecting transferrin receptor 1 or preparing drugs crossing the blood-brain barrier.

[0035] On the basis of the amino acid sequence of the antibody or antigen-binding fragment thereof disclosed in the present application, it is easy for those skilled in the art to conceive that the antibody or antigen-binding fragment thereof is prepared by using genetic engineering technology or other technologies (chemical synthesis, hybridoma cells), for example, is isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody or antigen-binding fragment thereof according to any one of the above, which is easy for those skilled in the art to achieve, and based on this, no matter what technology is used to prepare the antibody or antigen-binding fragment thereof of the present application, it belongs to the protection scope of the present application.

[0036] Compared with the prior art, the present application has the beneficial effects that:

[0037] The present application provides a human anti-transferrin receptor 1 antibody, which widens the selection range of the antibody, and the human anti-transferrin receptor 1 antibody of the present application can have cross-binding activity with TfR1 of different species of humans, mice and cynomolgus monkeys, can penetrate the blood-brain barrier to deliver into the brain through TfR1-mediated endocytosis transport, provides an effective raw material for preparing a drug crossing the blood-brain barrier, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is an ELISA identification specific positive clone result graph.

[0039] Figure 2 is a vector structure map of antibody eukaryotic expression vectors pABG and pABL, wherein A is a vector structure map of pABG; B is a vector structure map of pABL.

[0040] Figure 3 is an antibody and human TfR1-His binding activity test result graph.

[0041] Figure 4 is an antibody and TfR1 binding activity test result graph under the condition of pre-coating transferrin.

[0042] Figure 5 is an antibody and TfR2 cross-binding reaction test result graph.

[0043] Figure 6 is a kinetic curve of AET1802 antibody and human TfR1-His interaction; wherein the thick line is the actual determination curve, and the black thin line is the fitting curve.

[0044] Figure 7 is a kinetic curve of AET1805 antibody and human TfR1-His interaction; wherein the thick line is the actual determination curve, and the black thin line is the fitting curve.

[0045] Figure 8is the kinetic curve of the interaction of AET1806 antibody with human TfR1-His; wherein, the thick line is the actual measured curve, and the black thin line is the fitted curve.

[0046] Figure 9 is the kinetic curve of the interaction of AET1807 antibody with human TfR1-His; wherein, the thick line is the actual measured curve, and the black thin line is the fitted curve.

[0047] Figure 10 is the kinetic curve of the interaction of AET1809 antibody with human TfR1-His; wherein, the thick line is the actual measured curve, and the black thin line is the fitted curve.

[0048] Figure 11 is the kinetic curve of the interaction of AET1811 antibody with human TfR1-His; wherein, the thick line is the actual measured curve, and the black thin line is the fitted curve.

[0049] Figure 12 is the evaluation result figure of antibody penetration rate based on the Transwell chamber monolayer hCMEC / D3 cell blood brain barrier model. DETAILED DESCRIPTION

[0050] In order to make the purposes, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. The materials, reagents and instruments used in the following embodiments can be obtained from commercial channels if not specifically stated. If not specifically stated, the technical means used in the embodiments is the conventional means known to those skilled in the art. In the following embodiments, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA or RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA or RNA if not specifically stated. The pABL and pABG in the following embodiments are recorded in “Du et al., A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-a subtypes: A candidate therapy for systemic lupus erythematosus, mAbs 2015, 7(5):969—980.” and “Dong Han et al., A novel human anti-TIGIT monoclonal antibody with excellent function in eliciting NK cell-mediated antitumor immunity, Biochemical and Biophysical Research Communications 2021, 534:134-140.” which are available from the applicant, and the material is only used for repeating the relevant experiments of the present application and cannot be used for other purposes.

[0051] Example 1: Generation of human anti-human TfR1 antibodies

[0052] I. Materials and methods

[0053] 1. Materials

[0054] The full human single-chain antibody phage display library was constructed and preserved by the Institute of Biological Engineering, Academy of Military Medical Sciences of Chinese People's Liberation Army (ZL200910091261.8), with a library capacity of 1.35×10 10Immuno tubes are MaxiSorp from Nunc (cat# 470319). 96-well ELISA plates are Costar products. Bacteriophage infected host strain is XL1-Blue (Stratagene, USA), plasmid amplification strain is Top 10 (Beijing Zixiao Biotech, China), helper phage is M13KO7 (Invitrogene, USA), horseradish peroxidase (HRP) labeled anti-M13KO7 antibody is a product from Beijing Yiqiao Biotech (cat# 11973-MM05T-H). Screening antigen is eukaryotic cell recombinant expressed hTfR1-His (amino terminal fusion histidine tagged human TfR1 extracellular domain, Beijing Yiqiao Biotech, cat# 11020-H07H). Plate washer is BioTeK ELx405, ELISA reader is Thermo Multiskan MK3. Nucleic acid sequencing service is provided by Mingchenzhixian Biotech (Beijing) Co., Ltd.

[0055] 2. Method

[0056] 2.1 Screening of phage antibody library

[0057] First round screening: Dilute recombinant protein hTfR1-His to 10 μg / mL x 1 mL with PBS (pH 7.2), add to immuno tubes, coat overnight at 4°C, discard liquid next day, add 1.5 mL PBS containing 2% BSA (blocking solution) to block at 37°C for 1 h; block phage antibody library (sub-library) with blocking solution containing 1‰ Tween20 at 37°C for 1 h; add blocked antibody library to immuno tubes, bind overnight at 4°C. Discard liquid and wash next day, washing conditions: PBST (PBS containing 1‰ Tween20) wash 15 times, PBS wash 5 times, 3 min / time. Elute with 0.1 mol / L Glycine-HCl (pH 2.2), act at room temperature for 10 min, immediately neutralize to pH 7.4 with 1 mol / L Tris. Collect eluted and neutralized solution, add 9 mL of logarithmic growth phase E. coli XL1-Blue to it, mix well, then incubate at room temperature for 30 min, then transfer to 37°C, 150 rpm shaker for 1 h. Take 1% of the infected bacteria from the bacteria solution and spread on 2xYT-CTG (2xYT medium containing final concentration of 50 μg / mL chloramphenicol, 10 μg / mL tetracycline and 0.5% glucose) agarose plates, incubate at 37°C overnight until clear colonies are formed, count the single colonies to determine the yield; at the same time, spread the remaining bacteria solution on 2xYT-CTG plates and incubate at 37°C overnight for expansion. Next day, scrape and collect the expanded bacteria, take an appropriate amount of bacteria solution and add to 150 mL 2xYT-CTG liquid medium to OD 600 =0.3~0.5, incubate at 37°C, 220 rpm until OD600 =0.7, helper phage M13KO7 was added at multiplicity of infection (MOI) of 50, and the mixture was incubated at room temperature for 30 min and then at 37 °C with shaking at 150 rpm for 1 h; kanamycin was added to a final concentration of 50 μg / mL, and IPTG was added to a final concentration of 0.15 mM, and the mixture was incubated at 30 °C with shaking at 200 rpm for 10-12 h; the culture supernatant was collected by centrifugation, and 1 / 4 volume of PEG8000 Buffer (containing 20% PEG8000 and 2.5 mol / L NaCl) was added, and the mixture was mixed and then placed on ice for at least 2 h; the mixture was centrifuged at 4 °C at 8000 rpm for 30-45 min, the supernatant was discarded, the precipitate was resuspended in 2-3 mL of PBS containing 2% BSA, and the mixture was filtered through a 0.45-μm filter membrane to remove bacteria, a small amount of the mixture was taken for titer determination, a certain volume of the mixture was used for the next round of screening, and the remaining mixture was stored at -80 °C for later use.

[0058] In the second round of screening, the antigen was coated at a concentration of 2.5 μg / mL x 1 mL, and 0.5% casein was used for blocking, and the washing intensity was increased as follows: PBST was used for washing for 25 times, PBS (containing 0.2 M NaCl) was used for washing for 5 times, and PBS was used for washing for 5 times, each for 3 min. In the third round of screening, the antigen was coated at a concentration of 1.0 μg / mL x 1 mL, and 2.5% skim milk was used for blocking, and the washing intensity was increased as follows: PBST was used for washing for >30 times, PBS (containing 0.2 M NaCl) was used for washing for 15 times, PBS was used for washing for 10 times, each for 5 min, and 0.1 M glycine-HCl (pH 4.0) was used for washing for 10 min. In the fourth round of screening, the antigen was coated at a concentration of 0.5 μg / mL x 1 mL, and 2.5% skim milk was used for blocking, and the washing intensity was increased as follows: PBST was used for washing for 20 times, PBS (containing 0.2 M NaCl) was used for washing for >30 times, PBS was used for washing for 10 times, each for 5 min, and 0.1 M glycine-HCl (pH 4.0) was used for washing for 10 min. After the last round of screening, the bacteria were not scraped and collected for use in picking single colonies for preliminary identification.

[0059] 2.2 Monoclonal identification

[0060] A sterile 96-deep-well plate was prepared, 2xYT-CTG medium was added at a volume of 200 μL / well, and single colonies were randomly picked and inoculated into the 96-well plate, which was incubated at 37 °C with shaking at 220 rpm overnight until saturation. The next day, a new sterile 96-deep-well plate was prepared, 2xYT-CT medium was added at a volume of 200 μL / well, and the saturated bacterial solution was transferred to the new 96-well plate at a volume of 40 μL / well, which was incubated at 37 °C with shaking at 220 rpm for about 1.5 h until the OD 6000.7. Helper phage M13KO7 was added at multiplicity of infection (MOI) of 50 in 200 μL / well, and incubated at room temperature for 45 min. 2× YT-CT medium containing kanamycin (final concentration of 50 μg / mL) and IPTG (final concentration of 0.15 mM) was added at 200 μL / well, and the phage was induced to express at 30 °C, 200 rpm for 12 h. The 96-well plate was centrifuged at 4000 rpm for 10 min, and the supernatant was collected in a new 96-well plate for ELISA identification. The 96-well enzyme-linked plate was coated with the target antigen and irrelevant control antigen at 0.5 μg / mL in 100 μL / well at 4 °C overnight. The enzyme-linked plate and phage supernatant were blocked with PBS containing 2.5% skim milk and PBST, respectively, at 37 °C for 1 h. After blocking, the phage supernatant was added to the enzyme-linked plate at 100 μL / well, and incubated at 37 °C for 1 h. The enzyme-linked plate was washed, and anti-M13-HRP secondary antibody was diluted 1 / 5000 with PBST containing 2.5% skim milk, and added to the enzyme-linked plate at 100 μL / well, and incubated at 37 °C for 1 h. The enzyme-linked plate was washed, and TMB color developing solution was added at 100 μL / well, and incubated at room temperature for 10-15 min before color development was stopped. The absorbance value at 450 nm (595 nm as control) was read by a microplate reader. The specific positive clones were sequenced, and the sequences were aligned to obtain the target antibody gene.

[0061] II. Results

[0062] Four rounds of screening with gradually increasing selection were performed on the antibody library, and the input and output of each round are shown in Table 1. 288 monoclonal antibodies were selected and cultured to produce phage antibodies. ELISA identified 96 specific positive clones, with a positive rate of 33.3%, and the results are shown in Table 1. Figure 1 Six antibody variable region genes with obvious enrichment were obtained by sequencing, and the antibody numbers and corresponding amino acid sequences of the light and heavy chain CDR regions are shown in Table 2.

[0063] Table 1. Input and output of each round of antibody library screening

[0064]

[0065] Table 2. Amino acid sequences of light and heavy chain CDR regions

[0066]

[0067] Example 2: Phage single-chain antibody is changed to whole antibody form

[0068] I. Materials and methods

[0069] 1. Materials

[0070] The vector structure maps of antibody eukaryotic expression vectors pABG and pABL are as follows:Figure 2 Restriction enzymes BsrG I, Hind III, Afl II, Nhe I and T4 ligase were products of NEB, Agarose Gel DNA Recovery Kit and DNA Purification Kit were products of TIANGEN, E. coli Top 10 competent cells were products of Jiangsu Kangwei Century Biotech Co., Ltd. FreeStyle TM 293-F mammalian cell expression system: cells FreeStyle TM 293-F were products of Invitrogene, serum-free 293 medium and Opti-MEM were products of Gibco, transfection reagent was product of PolyPlus trasfection. Protein purification system AKTA avant was product of GE, purification column HiTrap TM MabSelect SuRe, HiTrap TM Desalting Columns were provided by Cytiva. Spectrophotometer was Nanodrop ONE C . Other materials involved were from the same sources as in Example 1.

[0071] 2 Methods

[0072] Vectors pABG and pABL were used to construct antibody heavy chain and light chain expression plasmids, respectively. Using the phage plasmid returned by sequencing as a template, PCR was performed to amplify the antibody light chain variable region gene using L3F and LR as paired primers and to amplify the antibody heavy chain variable region gene using H5F and HR as paired primers. The primer sequences are listed in Table 3. The PCR products were recovered. The heavy chain variable region gene fragment and the pABG vector were respectively digested with Afl II and Nhe I, and the light chain variable region gene fragment and the pABL vector were respectively digested with BsrG I and Hind III. The fragments and vectors were recovered, respectively, and were connected with T4 enzyme. The recombinant vector plasmid was transformed into E. coli TOP10, plated, cultured at 37°C overnight, and single colonies were picked for sequencing. The correct clones were transferred and cultured to extract plasmids, and antibody light and heavy chain expression vectors were obtained. The light and heavy chain expression vectors were mixed at a molar ratio of 2:1 and were co-transfected into good FreeStyle TM 293-F cells, which were cultured at 37°C, 120 rpm, 5% CO2 horizontal shaker for 4 days, and the expression supernatant was collected by high-speed centrifugation. Based on the AKTA system, the HiTrap TM MabSelect SuRe pre-packed column was used for affinity purification, and the sample was eluted and collected with 0.1M sodium citrate buffer (pH 3.0). The HiTrap TMDesalting Columns The antibody was replaced into PBS buffer to obtain the antibody sample. The spectrophotometer was used to quantify the antibody concentration.

[0073] Table 3. Primers related to the construction of full antibody light and heavy chain expression plasmids

[0074]

[0075] II. Results

[0076] By molecular cloning, the variable region genes of each antibody were cloned into the corresponding expression vector to construct full human antibodies in the form of IgG1 λ The variable region genes and amino acid sequences of each antibody are listed in Table 4, and the light chain constant region gene and amino acid sequence are SEQ ID NO. 45 and SEQ ID NO. 46, respectively, and the heavy chain constant region gene and amino acid sequence are SEQ ID NO. 47 and SEQ ID NO. 48, respectively. The corresponding antibody samples were prepared by a mammalian cell expression system for subsequent evaluation.

[0077] Table 4. Antibody sequences

[0078]

[0079] Example 3: Binding activity of antibodies and antigen epitopes

[0080] I. Materials and methods

[0081] 1. Materials

[0082] Recombinant human transferrin (Tf) was a product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. (item number 11019-HNAH2). HRP-labeled goat anti-human IgG antibody was a product of Beijing Zhongshanjinqiao Biotechnology Co., Ltd.

[0083] 2. Methods

[0084] 2.1 Detection of the binding activity of antibodies

[0085] 96-well ELISA plates were coated with hTfR1-His diluted in PBS at 1 pg / mL x 100 pL / well at 4°C overnight. The ELISA plates were blocked with PBS containing 2.5% skim milk, and the purified antibody samples were diluted in PBST containing 2.5% skim milk and incubated at 37°C for 1 h. The antibodies were added to the ELISA plates at 100 pL / well, and incubated at 37°C for 1 h. The ELISA plates were washed, and HRP-labeled goat anti-human IgG antibody was added at a dilution of 1 / 2000 in PBST containing 2.5% skim milk at 100 pL / well, and incubated at 37°C for 1 h. The ELISA plates were washed, and TMB color developing solution was added at 100 pL / well, and incubated at room temperature for 10-15 min before stopping the color development. The absorbance values at 450 nm (with 595 nm as a reference) were read on a microplate reader, and the antibody concentration-absorbance value curve was plotted using Graphpad Prism Software 9.0.

[0086] 2.2 Detection of antibody binding epitope cross-reactivity with Tf binding epitope

[0087] The above steps were performed after pre-coating Tf in a 96-well plate at 1 pg / mL x 100 pL / well, and blocking, to detect whether the binding epitope of the antibody cross-reacted with the Tf epitope.

[0088] 2.3 Detection of antibody cross-reactivity with TfR2

[0089] 96-well ELISA plates were coated with hTfR1-His and hTfR2-His at 100 ng / well; wherein TfR2-his was prepared by recombinant expression in this experiment, and the amino acid sequence was SEQ ID NO. 49. The subsequent operation steps were the same as above, and the concentration of the antibody to be detected was 10 pg / mL.

[0090] II. Results

[0091] The ELISA identification results showed that the candidate antibodies had good binding activity with human TfR1-His ( Figure 3 ), and showed a concentration-dependent relationship. In the case of pre-coating transferrin (Tf), the candidate antibodies still showed the same trend of binding to TfR1 ( Figure 4 ), which could be preliminarily determined that the binding epitope of the candidate antibody did not cross-react with the Tf binding epitope, and there was no interference with the binding of Tf. At the same time, the candidate antibodies also had no cross-binding reaction with TfR2 ( Figure 5 ).

[0092] Example 4: Affinity of the antibody

[0093] I. Materials and methods

[0094] 1. Materials

[0095] The detection instrument is BIAcore TM T200 system, Sensor Chip CM5 (part number: BR100530), HBS-EP+ buffer (part number: BR-1006-69), Human Antibody Capture Kit (part number: BR-1008-39), Amino Coupling Kit (part number: BR-1000-50) are all products of Cytiva. Recombinant mouse TfR1 (mTfR1-His, part number: 50741-M07H) and cynomolgus monkey TfR1 (cTfR1-His, part number: 90253-C07H) are purchased from Beijing Yiqiao Godz Biological Technology Co., Ltd.

[0096] 2. Method

[0097] According to the instructions of the Human Antibody Capture Kit, dilute the prepared capture molecules (10 μl of capture molecule solution is added to 200 μl of immobilization buffer, mix well, and then aliquot into two EP tubes); run Immobilization (Wizard Template) in BIAcore T200 Control Software, set the flow rate to 10 μl / min, and the coupling time to 7 min. The capture molecules are immobilized on the CM5 chip 1, 2 channels by amino coupling.

[0098] The detection buffer system is HBS-EP+, and the detection temperature is 25°C; the key parameters such as the immobilization concentration of each antibody, the concentration gradient setting of the antigen hTfR-His, and the binding and dissociation time are determined by pre-experiment; a new Method is created in BIAcore T200 Control Software, and the antigen is set to 5 concentrations (as marked in the results); channel 2 is the detection channel, and channel 1 is the control; the capture flow rate is set to 10 μl / min, and the detection flow rate is set to 30 μl / min; the regeneration condition is 3M MgCl2 solution provided with the kit, 30 μl / min x 30 s.

[0099] The results are analyzed by Surface Bound / Kinetics / 1:1 binding fitting analysis in Biacore T200 Evaluation Software to obtain the kinetic parameters of the interaction.

[0100] II. Results

[0101] The response values of the human antibody capture molecules immobilized on the CM5 chip 1, 2 channels by amino coupling are 9052.3 RU and 8948.7 RU, respectively. Under the experimental conditions, the affinities of the antibodies determined to interact with different species of recombinant TfR1 are listed in Table 5, and the representative sensorgrams are shown inFigure 6~Figure 11 AET1809 and AET1802 have the highest affinity to human TfRl, with 2.09 nM and 5.66 nM, respectively, and the rest of the candidate antibodies have an affinity greater than 10 nM. Except for AET1806, all the other antibodies have cross-binding activity to mouse and cynomolgus TfRl.

[0102] Table 5. Results of SPR assay of antibody interaction affinity with different species TfRl

[0103]

[0104] Table: ka, association rate constant; kd, dissociation rate constant; Kd, dissociation equilibrium constant, i.e. affinity. NB indicates no binding. D , dissociation equilibrium constant, i.e. affinity. NB indicates no binding.

[0105] Example 5: Functional activity of antibodies on blood-brain barrier cell model

[0106] I. Materials and methods

[0107] 1. Materials

[0108] hCMEC / D3 cells were purchased from Quansheng Life Science (Shanghai) Co., Ltd. (Cat. No. CVCL U985). RPMI-1640 medium was an ATCC product (Cat. No. 30-2001), FBS was an AusGeneX product (Cat. No. FBS500-S), trypsin was a Gibco product (Cat. No. 25200-072), and penicillin-streptomycin (PS) was a HyClone product (Cat. No. SV30010). 6.5 mm Transwell chambers were a Corning product (Cat. No. 3413). The electrical resistivity meter EVOM3 was a WPI product. Goat anti-human IgG-Fc antibody was a Sigma-Aldrich product (Cat. No. I2136). ELISA-related reagents and consumables were the same as in Example 3.

[0109] 2. Methods

[0110] hCMEC / D3 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% PS at 37°C in a 5% CO2 incubator. Well-grown hCMEC / D3 cells were trypsinized and resuspended in complete medium to 8 x 10 5 cells / mL, and inoculated at 0.25 mL / well onto the upper layer of the transwell chamber, which was placed in a 24-well cell culture plate, and 1 mL of complete medium was added to the lower layer. Fresh medium was replaced daily, and the cells were continuously cultured for 6-7 days until the transwell electrical resistance (TEER value) reached 100 Ω·cm2. 2Left and right. 50 μg of the antibody to be evaluated was added to the upper chamber, and the upper and lower culture media were collected at 4 h and 8 h, respectively, for quantitative detection of the antibody. Three independent repeats were set for the antibody to be detected at different sampling time points. The control antibody JR-3 was a similar antibody disclosed by JCR Pharmaceutical in Japan (anti-hTfR antibody No. 3 described in the patent published as US2018 / 0171012A1); it was prepared by the method of Example 2 in the laboratory, and the light and heavy chain sequences were SEQ ID NO. 50 and SEQ ID NO. 51, respectively.

[0111] The antibody in the culture medium was quantitatively detected by ELISA. The goat anti-human IgG-Fc antibody was diluted with PBS (1:2000) and coated in a 96-well plate. The basic operation steps of ELISA were the same as in Example 3. The standard curve was drawn by detecting 9 gradients of the antibody to be evaluated with known concentrations, starting from 5 μg / mL, 2-fold gradient dilution. The upper and lower culture media collected were diluted according to the specific ratio for detection, and the corresponding antibody concentrations were calculated according to the standard curve. One repeat detection was set for each standard curve / culture medium sample. According to the detection results, the penetration rate of the antibody was calculated (the calculation formula is as follows), and the result graph was drawn by Graphpad Prism Software 9.0.

[0112]

[0113] II. Results

[0114] The functional activity of TfR1 antibody was evaluated in vitro by a single-layer hCMEC / D3 cell system based on Transwell chamber, and the results are shown in Figure 12 The results show that the five candidate antibodies AET1802, AET1805, AET1807, AET1809 and AET1811 can effectively penetrate the blood-brain barrier simulated by the single-layer cell, among which AET1809 is the best.

[0115] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A humanized anti-transferrin receptor 1 antibody or antigen binding fragment thereof, characterized in that, The CDRs include the following: CDR1-VL: the amino acid sequence is SGDALGDKYAS; CDR2-VL: the amino acid sequence is EDSKRPS; CDR3-VL: the amino acid sequence is QSYDSDIRS; CDR1-VH: the amino acid sequence is TSYWIG; CDR2-VH: the amino acid sequence is IIYPGDSDTRYSPSFQG; CDR3-VH: the amino acid sequence is YRILAHFDY.

2. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof of claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 41; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

42.

3. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof of claim 1, wherein, It also includes a constant region.

4. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof of claim 3, characterized in that, The constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.

5. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof of claim 3, wherein, The species origin of the constant region is bovine, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, monkey or human.

6. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof of claim 3, wherein, The constant region includes a light chain constant region and a heavy chain constant region; the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 46, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

48.

7. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof of claim 1, wherein, The humanized anti-transferrin receptor 1 antibody antigen binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, scFv.

8. A drug delivery vehicle, characterized in that, The humanized anti-transferrin receptor 1 antibody or antigen binding fragment thereof in any one of claims 1-7.

9. Use of the humanized anti-transferrin receptor 1 antibody or antigen binding fragment thereof in any one of claims 1-7 in the in vitro detection of transferrin receptor 1 for non-disease diagnosis and treatment purposes.

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