Humanized anti-transferrin receptor 1 antibody and application thereof

Abstract

The invention relates to the technical field of antibodies, in particular to a human anti-transferrin receptor 1 antibody and application thereof. The invention provides the humanized anti-transferrin receptor 1 antibody, the selectable range of the antibody is widened, meanwhile, the humanized anti-transferrin receptor 1 antibody can have cross-binding activity with TfR1 of different species of human, mice and cynomolgus monkeys, and can penetrate through a blood brain barrier to be delivered into the brain through the TfR1 mediated endocytosis transport effect, so that the human anti-transferrin receptor 1 antibody can be applied to the treatment of human and cynomolgus monkeys, and the human anti-transferrin receptor 1 antibody can be applied to the treatment of human and cynomolgus monkeys. Effective raw materials are provided for preparing blood-brain barrier-crossing medicines, and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to humanized anti-transferrin receptor 1 antibodies and applications thereof. Background Art

[0002] The blood-brain barrier (BBB) ​​is a complex physiological barrier that isolates harmful substances from the peripheral circulatory system, protects the central nervous system (CNS) from damage and maintains normal homeostasis. It 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.). Under normal circumstances, most small molecule drugs and almost all biological macromolecule drugs have difficulty breaking through the blood-brain barrier and reaching 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 issues that urgently need 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 the normal exchange of specific substances between the brain and the peripheral circulation through specific receptors on the surface of vascular endothelial cells that constitute the blood-brain barrier. A large number of studies have shown that the strategy of delivering central nervous system drugs across the blood-brain barrier through receptor-mediated transcytosis has the advantages of high specificity, low toxicity and side effects, the ability to carry a variety of molecules, and no damage to the integrity of the blood-brain barrier. It is a cross-blood-brain barrier drug delivery route 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-related 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 amino acid sequence identity between the two is 45% and the sequence similarity is 66%. TfR2 is mainly highly expressed in hepatocytes and erythrocyte precursor cells and is an iron ion sensor. TfR1 is highly expressed in hepatocytes, erythrocytes, 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 cross-blood-brain barrier delivery of drugs.

[0005] At present, many international pharmaceutical companies have laid out their R&D pipelines for antibody drugs that cross the blood-brain barrier. The core technology of Japan’s JCR Pharmaceuticals is called “J-Brain Cargo ® "The cross-blood-brain barrier delivery system of JCR Pharmaceuticals: the relevant therapeutic protein / peptide drugs are fused with antibodies targeting TfR1 and then delivered to the brain through endocytosis to exert their effector functions; its representative product IZCARGO (R&D code name 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 cross-blood-brain barrier delivery capabilities; Phase II / III clinical results showed that JR141 achieved the primary clinical trial endpoint, with significant efficacy, 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 a number of similar products undergoing relevant preclinical or early clinical studies.

[0006] Therefore, developing more optional anti-transferrin receptor 1 antibodies has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0007] The present invention develops a humanized 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 cross-blood-brain barrier drug delivery. Based on this, the following technical solution is proposed.

[0008] In a first aspect, the present invention provides a humanized anti-transferrin receptor 1 antibody or an antigen-binding fragment thereof, comprising the following complementary determining regions: CDR1-VL: amino acid sequence is SGDALGDKYAS; CDR2-VL: amino acid sequence is EDSKRPS; CDR3-VL: amino acid sequence is QAYERGAV or QSHDGSYI or QAYDMGRI or QSYDSDIRS or QSRELLGP; CDR1-VH: amino acid sequence is TSYWIG; CDR2-VH: amino acid sequence: IIYPGDSDTRYSPSFQG; CDR3-VH: the amino acid sequence is YNDLLSPMDY or YIDAFTPFDY or YSHFSRPFAD or YRILAHFDY or SHVFDLNMDD.

[0009] In some embodiments, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.39, SEQ ID NO.41 or SEQ ID NO.43; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.40, SEQ ID NO.42 or SEQ ID NO.44.

[0010] In some embodiments, the human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof further comprises a constant region.

[0011] In some embodiments, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0012] In some embodiments, the species of origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, monkey or human.

[0013] In some embodiments, 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.

[0014] In some embodiments, the antigen-binding fragment of the human anti-transferrin receptor 1 antibody is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv.

[0015] In a second aspect, the present invention provides a drug delivery carrier, which contains the human anti-transferrin receptor 1 antibody or its antigen-binding fragment, or contains a nucleic acid fragment encoding the human anti-transferrin receptor 1 antibody or its antigen-binding fragment.

[0016] In a third aspect, the present invention provides a drug, which contains the human anti-transferrin receptor 1 antibody or its antigen-binding fragment, or contains a nucleic acid fragment encoding the human anti-transferrin receptor 1 antibody or its antigen-binding fragment, or contains the drug delivery vector.

[0017] In a fourth aspect, the present invention also provides a reagent or kit for detecting transferrin receptor 1, which contains the human anti-transferrin receptor 1 antibody or its antigen-binding fragment.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof in the above reagent or kit is labeled with a detectable marker.

[0019] Detectable markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0020] In an optional embodiment, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent agents, and nanoparticle markers.

[0021] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0022] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0023] In an optional embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0024] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0025] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0026] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0027] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0028] In a fifth aspect, the present invention provides the use of the human anti-transferrin receptor 1 antibody or its antigen-binding fragment, and the drug delivery vector in in vitro detection of transferrin receptor 1 or in the preparation of drugs that cross the blood-brain barrier.

[0029] Based on the amino acid sequence of the antibody or its antigen-binding fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, hybridoma cells) to prepare the antibody or its antigen-binding fragment, for example, separating and purifying the antibody or its antigen-binding fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its antigen-binding fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its antigen-binding fragment of the present invention, it belongs to the protection scope of the present invention.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a humanized anti-transferrin receptor 1 antibody, which broadens the selectable range of antibodies. At the same time, the humanized anti-transferrin receptor 1 antibody of the present invention can have cross-binding activity with TfR1 of different species such as humans, mice and crab-eating macaques, and can penetrate the blood-brain barrier and be delivered into the brain through TfR1-mediated endocytosis, thereby providing an effective raw material for the preparation of drugs that cross the blood-brain barrier and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the result of ELISA identifying specific positive clones.

[0032] Figure 2 It is the vector structure map of the antibody eukaryotic expression vectors pABG and pABL, wherein A is the vector structure map of pABG; B is the vector structure map of pABL.

[0033] Figure 3 This is a graph showing the binding activity test results of the antibody and human TfR1-His.

[0034] Figure 4 This is a graph showing the binding activity test results of the antibody and TfR1 in the case of pre-coating with transferrin.

[0035] Figure 5 This is a graph showing the test results of the cross-binding reaction between the antibody and TfR2.

[0036] Figure 6 This is the kinetic curve of the interaction between AET1802 antibody and human TfR1-His; the thick line is the actual measurement curve, and the thin black line is the fitting curve.

[0037] Figure 7 This is the kinetic curve of the interaction between AET1805 antibody and human TfR1-His; the thick line is the actual measurement curve, and the thin black line is the fitting curve.

[0038] Figure 8This is the kinetic curve of the interaction between AET1806 antibody and human TfR1-His; the thick line is the actual measurement curve, and the thin black line is the fitting curve.

[0039] Fig. 9 This is the kinetic curve of the interaction between AET1807 antibody and human TfR1-His; the thick line is the actual measured curve, and the thin black line is the fitting curve.

[0040] Fig.10 This is the kinetic curve of the interaction between AET1809 antibody and human TfR1-His; the thick line is the actual measured curve, and the thin black line is the fitting curve.

[0041] Fig.11 This is the kinetic curve of the interaction between AET1811 antibody and human TfR1-His; the thick line is the actual measurement curve, and the thin black line is the fitting curve.

[0042] Fig.12 This is a graph showing the evaluation results of antibody penetration rate based on the Transwell chamber monolayer hCMEC / D3 cell blood-brain barrier model. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The materials, reagents, instruments, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. In the following embodiments, unless otherwise specified, the first position of each nucleotide sequence in the sequence table 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. The pABL and pABG in the following examples are both recorded in "Du et al., A fully human monoclonal antibody with novelbinding epitope and excellent neutralizing activity to multiple human IFN-asubtypes: 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 antitumorimmunity, Biochemical and Biophysical Research Communications 2021, 534:134-140.", which are available to the public from the applicant. The materials are only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0044] Example 1: Generation of human anti-human TfR1 antibodies

[0045] 1. Materials and Methods

[0046] 1. Materials

[0047] The fully synthetic human single-chain antibody phage display library was constructed and preserved by the Institute of Bioengineering, Military Medical Research Institute, Chinese Academy of Military Sciences (ZL200910091261.8), with a library capacity of 1.35×10 10. The immunotube was MaxiSorp (Cat. No. 470319) from Nunc. The 96-well ELISA plate was a Costar product. The host strain for phage infection was XL1-Blue (Stratagene, USA), the plasmid amplification strain was Top 10 (Beijing Quanshijin Biotechnology Co., Ltd.), the helper phage was M13KO7 (Invitrogene, USA), and the horseradish peroxidase (HRP)-labeled anti-M13KO7 antibody was a product of Beijing Sino Biological Technology Co., Ltd. (Cat. No. 11973-MM05T-H). The screening antigen was hTfR1-His (human TfR1 extracellular domain fused with histidine tag at the amino terminus, Beijing Sino Biological Technology Co., Ltd., Cat. No. 11020-H07H) recombinantly expressed in eukaryotic cells. The plate washer was BioTeK ELx405, and the microplate reader was Thermo Multiskan MK3. Nucleic acid sequencing services were provided by Mingchen Zhiyuan Biotechnology (Beijing) Co., Ltd.

[0048] 2. Methods

[0049] 2.1 Screening of phage antibody library

[0050] First round of screening: dilute the recombinant protein hTfR1-His to 10μg / mL×1mL with PBS (pH7.2), add it to the immunotube, coat it at 4℃ overnight, discard the solution the next day, add 1.5mL PBS (blocking solution) containing 2% BSA and block it at 37℃ for 1h; the phage antibody library (sub-library) is blocked with blocking solution containing 1‰ Tween20 at 37℃ for 1h; add the blocked antibody library to the immunotube and bind it at 4℃ overnight. Discard the solution and wash the next day. Washing conditions: wash 15 times with PBST (PBS containing 1‰ Tween20), wash 5 times with PBS, 3min / time. Add 0.1mol / L Glycine-HCl (pH2. 2) for elution, act at room temperature for 10min, and immediately neutralize to pH7.4 with 1mol / L Tris. Collect the neutralized eluate, add 9 mL of Escherichia coli XLl-Blue in the logarithmic growth phase, mix well, and infect at room temperature for 30 minutes, then transfer to a 37°C, 150 rpm shaker for 1 hour. Take 1% of the infected bacterial solution and spread it on a 2×YT-CTG (2×YT medium containing a final concentration of 50µg / mL chloramphenicol, 10µg / mL tetracycline, and 0.5% glucose) agar plate, culture at 37°C overnight until clear colonies are formed, count the single clones, and determine the output; at the same time, spread the remaining bacterial solution on a 2×YT-CTG plate and expand the culture at 37°C overnight. The next day, scrape and collect the expanded cultured bacteria, take an appropriate amount of bacterial solution and add it to 150 mL 2×YT-CTG liquid culture medium until the OD 600 =0.3~0.5, culture at 37℃, 220rpm until OD600 =0.7, add helper phage M13KO7 at MOI =50, let stand at room temperature for 30 minutes, and culture at 37℃, 150rpm for 1 hour; add kanamycin to a final concentration of 50μg / ml and IPTG to a final concentration of 0.15mM, and culture at 30℃, 200rpm for 10-12h; collect the culture supernatant by centrifugation, add 1 / 4 volume of PEG8000 Buffer (containing 20% ​​PEG8000 and 2.5mol / l NaCl), mix well and place on ice for at least 2h; centrifuge at 4℃, 8000rpm for 30-45min, discard the supernatant, resuspend the precipitate in 2-3mL PBS containing 2% BSA, filter and sterilize with 0.45μm filter membrane, take a small amount to determine the titer, take a certain volume for the next round of screening, and freeze the rest at -80℃ for later use.

[0051] Second round of screening: antigen coating amount was 2.5μg / mL×1mL, 0.5% casein blocking, washing intensity increased to: PBST washing 25 times, PBS (containing 0.2M NaCl) washing 5 times, PBS washing 5 times, 3min / time. Third round of screening antigen coating amount was 1.0μg / mL×1mL, 2.5% skim milk powder blocking, washing intensity increased to: PBST washing>30 times, PBS (containing 0.2M NaCl) washing 15 times, PBS washing 10 times, 5min / time; 0.1M Glycine-HCl (pH4.0) washing 10min. Fourth round of screening antigen coating amount was 0.5μg / mL×1mL, 2.5% skim milk powder blocking, washing intensity increased to: PBST washing 20 times, PBS (containing 0.2M NaCl) washing>30 times, PBS washing 10 times, 5min / time; 0.1M Glycine-HCl (pH4.0) washing 10min. After the last round of screening and expansion culture, the bacterial liquid is collected without scraping and used to pick single clones for preliminary identification.

[0052] 2.2 Monoclonal identification

[0053] Take a sterile 96-well plate, add 2×YT-CTG medium at 200µL / well, randomly pick monoclonal colonies and inoculate them into the 96-well plate, and culture them at 37°C and 220rpm overnight until saturation. The next day, take a new sterile 96-well plate, add 2×YT-CT medium at 200µL / well, transfer the saturated bacterial solution to a new 96-well plate at 40µL / well, and culture at 37°C and 220rpm for about 1.5h until OD 600~0.7. At the multiplicity of infection MOI=50, add helper phage M13KO7 at 200µL / well and let stand at room temperature for 45min. Add 2×YT-CT medium (final concentration 0.15mM) containing kanamycin (final concentration 50µg / mL) and IPTG at 200µL / well and culture at 30℃ and 200rpm for 12h to induce phage expression; centrifuge the 96-well plate at 4000rpm×10min, collect the supernatant in a new 96-well plate for ELISA identification. Take a 96-well ELISA plate, add the target antigen and irrelevant control antigen at 0.5μg / mL×100µL / well, and coat at 4℃ overnight. Block the ELISA plate and phage supernatant with PBS and PBST containing 2.5% skim milk powder, respectively, at 37℃ for 1h. After blocking, add phage supernatant to the ELISA plate at 100µL / well and combine at 37℃ for 1h. Wash the ELISA plate, dilute the anti-M13-HRP secondary antibody 1 / 5000 with PBST containing 2.5% skim milk powder, add 100µL / well to the ELISA plate, and bind at 37℃ for 1h. Wash the ELISA plate, add TMB colorimetric solution at 100µL / well, and let it stand at room temperature for 10-15min before stopping the color development. Read the absorbance value at 450nm wavelength (595nm as control) with an ELISA reader. Sequence the specific positive clones, align the sequences, and obtain the target antibody gene.

[0054] 2. Results

[0055] The antibody library was screened for four rounds with increasing degrees, and the input-output of each round is listed in Table 1. 288 monoclones were selected for culture, showing phage antibodies, and ELISA identified 96 specific positive clones with a positive rate of 33.3%. The results are as follows Figure 1 As shown; 6 antibody variable region genes with obvious enrichment were obtained by sequencing, and the output antibody numbers and the corresponding amino acid sequences of the light and heavy chain CDR regions are listed in Table 2.

[0056] Table 1. Input and output of each round of antibody library screening process

[0057] Table 2. Amino acid sequences of light and heavy chain CDR regions

[0058] Example 2: Phage single-chain antibody converted to full antibody format

[0059] 1. Materials and Methods

[0060] 1 Materials

[0061] The vector structure map of antibody eukaryotic expression vectors pABG and pABL is as follows Figure 2As shown. Restriction enzymes BsrG I, Hind III, Afl II, Nhel and T4 ligase are products of NEB, agarose gel DNA recovery kit and DNA product purification kit are products of Tiangen Biochemical Technology (Beijing) Co., Ltd., and Escherichia coli Top l0 competent cells are products of Jiangsu Kangwei Century Biotechnology Co., Ltd. FreeStyle TM 293-F mammalian cell expression system: Cell FreeStyle TM 293-F is a product of Invitrogene, serum-free 293 culture medium and Opti-MEM are products of Gibco, and transfection reagent is a product of PolyPlus trasfection. Protein purification system AKTA avant is a product of GE, and purification column HiTrap TM MabSelect SuRe, HiTrap TM Desalting columns were provided by Cytiva. Spectrophotometer was Nanodrop ONE C The sources of other materials are the same as those in Example 1.

[0062] 2 Methods

[0063] The 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 used to amplify the antibody light chain variable region gene with L3F and LR as paired primers, and the antibody heavy chain variable region gene with H5F and HR as paired primers. The primer sequences are listed in Table 3. Recover the PCR product. Afl II and Nhe I were used to digest the heavy chain variable region gene fragment and the pABG vector, and BsrG I and Hind III were used to digest the light chain variable region gene fragment and the pABL vector, respectively. The fragments and vectors were recovered, and the T4 enzyme was connected accordingly. The recombinant vector plasmid was transformed into Escherichia coli TOP10, plated, and cultured at 37°C overnight. Single clones were picked for sequencing. The clones with correct sequencing were transferred to the culture and extracted plasmids to obtain the light and heavy chain expression vectors of each antibody. The light and heavy chain expression vectors were mixed at a molar ratio of 2:1 and co-transfected with FreeStyle TM 293-F cells were cultured in a horizontal shaker at 37°C, 120 rpm, and 5% CO2 for 4 days, and the expression supernatant was collected by high-speed centrifugation. TM MabSelect SuRe prepacked column was used for affinity purification, and samples were eluted and collected with 0.1M sodium citrate buffer (pH 3.0). TMDesalting Columns replace the antibody with PBS buffer to obtain the antibody sample. The antibody concentration is quantified by spectrophotometer.

[0064] Table 3. Primers related to the construction of full antibody light and heavy chain expression plasmids

[0065] 2. Results: Through molecular cloning, the variable region genes of each antibody were cloned into the corresponding expression vector to construct IgG1 λ The fully human antibodies were named AET1802, AET1805, AET1806, AET1807, AET1809, and AET1811, respectively, and their variable region genes and amino acid sequences are listed in Table 4. 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.

[0066] Table 4. Antibody sequences

[0067] Example 3: Antibody Binding Activity and Antigenic Epitope

[0068] 1. Materials and Methods

[0069] 1 Materials

[0070] Recombinant human transferrin (Tf) was a product of Beijing Sino Biological Technology Co., Ltd. (Cat. No. 11019-HNAH2). HRP-labeled goat anti-human IgG antibody was a product of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0071] 2 Methods

[0072] 2.1 Antibody Binding Activity Detection

[0073] hTfR1-His diluted in PBS was added to the 96-well ELISA plate at 1μg / mL×100µL / well and coated overnight at 4℃. The ELISA plate was blocked with PBS containing 2.5% skim milk powder, and the purified antibody sample was diluted with PBST containing 2.5% skim milk powder and blocked at 37℃ for 1h. The corresponding antibodies were added to the ELISA plate at 100µL / well and bound at 37℃ for 1h. The ELISA plate was washed, and HRP-labeled goat anti-human IgG antibody was diluted 1 / 2000 with PBST containing 2.5% skim milk powder, and added to the ELISA plate at 100µL / well, and bound at 37℃ for 1h. The ELISA plate was washed, and TMB colorimetric solution was added at 100µL / well. The reaction was allowed to stand at room temperature for 10-15min before stopping the color development. The absorbance value at a wavelength of 450nm was read by an ELISA reader (595nm was used as a control), and the antibody concentration-absorbance value curve was plotted using Graphpad Prism Software 9.0.

[0074] 2.2 Detection of cross-binding epitopes between antibody and Tf

[0075] Pre-coat Tf in a 96-well plate at 1μg / mL×100µL / well, and perform the above steps after blocking to detect whether the binding epitope of the antibody crosses with the Tf epitope.

[0076] 2.3 Antibody cross-reactivity detection with TfR2

[0077] The 96-well ELISA plate was coated with hTfR1-His and hTfR2-His at 100 ng / well, wherein TfR2-his was prepared by recombinant expression in this experiment, and its amino acid sequence is SEQ ID NO. 49. The subsequent operation steps were the same as above, and the concentration of the antibody to be detected was 10 μg / mL.

[0078] 2. Results

[0079] ELISA results showed that the candidate antibodies had good binding activity with human TfR1-His ( Figure 3 ), and the relationship was concentration-dependent. In the case of pre-coating with transferrin (Tf), the candidate antibodies still showed the same trend of binding to TfR1 ( Figure 4 ), it can be preliminarily determined that the binding epitope of the candidate antibody does not cross with the Tf binding epitope, and there is no interference with the binding of Tf. At the same time, the candidate antibody also has no cross-binding reaction with TfR2 ( Figure 5 ).

[0080] Example 4: Antibody Affinity

[0081] 1. Materials and Methods

[0082] 1 Materials

[0083] The detection instrument is BIAcore TM T200 system, Sensor Chip CM5 (Cat. No. BR100530), HBS-EP+ buffer (Cat. No. BR-1006-69), Human Antibody Capture Kit (Cat. No. BR-1008-39), and Amino Coupling Kit (Cat. No. BR-1000-50) are all products of Cytiva. Recombinant mouse TfR1 (mTfR1-His, Cat. No. 50741-M07H) and cynomolgus monkey TfR1 (cTfR1-His, Cat. No. 90253-C07H) were purchased from Beijing Sino Biological Technology Co., Ltd.

[0084] 2 Methods

[0085] Prepare capture molecules by diluting according to the instructions of the human antibody capture kit (add 10 μl capture molecule solution to 200 μl fixation buffer, mix well and divide equally into two EP tubes); run Immobilization (Wizard Template) in BIAcore T200 Control Software, set the flow rate to 10 μl / min, the coupling time to 7 min, and fix the capture molecules to channels 1 and 2 of the CM5 chip by amino coupling.

[0086] The detection buffer system was HBS-EP+, and the detection temperature was 25°C. The preliminary experiment determined key parameters such as the fixed concentration of each antibody, the concentration gradient setting of the antigen hTfR-His, and the binding and dissociation time. A new Method was created in the BIAcore T200 Control Software, and 5 concentrations of the antigen were set (as indicated in the results). Channel 2 was the detection channel, and channel 1 was the control. The capture flow rate was set to 10 μl / min, and the detection flow rate was set to 30 μl / min. The regeneration condition was the 3M MgCl2 solution provided in the kit, 30 μl / min×30s.

[0087] Results Surface Bound / Kinetics / 1:1 binding fitting analysis was performed using Biacore T200 Evaluation Software to obtain the kinetic parameters of the interaction.

[0088] 2. Results

[0089] The response values ​​of human antibody capture molecules fixed to CM5 chip 1 and 2 channels by amino coupling were 9052.3 RU and 8948.7 RU, respectively. Under the experimental conditions, the affinity of the obtained antibodies for interaction with recombinant TfR1 of different species is listed in Table 5, and the representative sensor graphs are shown in Figure 6~Figure 11 As shown. AET1809 and AET1802 have the highest affinity to human TfR1, which are 2.09nM and 5.66nM respectively, and the affinities of the other candidate antibodies are all greater than 10nM. Except for AET1806, the other antibodies have cross-binding activity with mouse and cynomolgus monkey TfR1.

[0090] Table 5 Results of SPR determination of the affinity of antibodies for interaction with TfR1 of different species

[0091] In the table: ka, association rate constant; kd, dissociation rate constant; K D , the dissociation equilibrium constant, also known as affinity. NB means no binding.

[0092] Example 5: Functional activity of antibodies on blood-brain barrier cell model

[0093] 1. Materials and Methods

[0094] 1 Materials

[0095] hCMEC / D3 cells were purchased from Quanxin Quanyi (Shanghai) Company (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 double antibody (Penicillin-streptomycin, PS) was a HyClone product (Cat. No.: SV30010). 6.5mm Transwell chamber was a Corning product (Cat. No.: 3413). 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.

[0096] 2 Methods

[0097] hCMEC / D3 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% PS at 37°C in a 5% CO2 incubator. Well-growing hCMEC / D3 cells were trypsinized and resuspended in complete medium to 8×10 5 cells / mL, 0.25mL / well was inoculated into the upper layer of the transwell chamber, the chamber was placed in a 24-well cell culture plate, and 1mL of complete culture medium was added to the lower layer. Fresh culture medium was replaced daily, and culture was continued for 6-7 days until the single-layer cell resistivity (TEER value) reached 100Ω·cm 2About. 50 μg of the antibody to be evaluated was added to the upper chamber, and the upper and lower culture media were collected after 4 h and 8 h for quantitative detection of the antibody. Three independent replicates were set for the antibodies to be tested at different sampling time points. The control antibody JR-3 is a similar antibody disclosed by Japan JCR Pharmaceutical (anti-hTfR antibody No. 3 described in the patent with publication number US2018 / 0171012Al); this laboratory prepared it using the method of Example 2, and its light and heavy chain sequences are SEQ ID NO. 50 and SEQ ID NO. 51, respectively.

[0098] The ELISA method was used to quantitatively detect antibodies in the culture medium. Goat anti-human IgG-Fc antibody (1:2000) was diluted with PBS and coated on a 96-well plate. The basic ELISA operation steps are the same as in Example 3. The antibody to be evaluated with a known concentration was used as the standard, and the starting concentration was 5 μg / mL, and 9 gradients were continuously diluted by 2 times for detection, and a standard curve was drawn. The upper and lower culture media collected above were diluted at a specific ratio for detection, and the concentration of the corresponding antibody was calculated according to the corresponding standard curve. A repeated test was set for each standard curve / culture medium sample. According to the test results, the penetration rate of the antibody was calculated (the calculation formula is as follows), and Graphpad Prism Software 9.0 was used to draw the result graph.

[0099]

[0100] 2. Results

[0101] The functional activity of TfR1 antibody was evaluated in vitro by simulating the blood-brain barrier using a Transwell chamber-based monolayer hCMEC / D3 cell system. Fig.12 The results showed that the five candidate antibodies AET1802, AET1805, AET1807, AET1809, and AET1811 could effectively penetrate the blood-brain barrier simulated by monolayer cells, among which AET1809 was the best.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A human anti-transferrin receptor 1 antibody or an antigen-binding fragment thereof, characterized in that: The complementarity determining regions include: CDR1-VL: amino acid sequence is SGDALGDKYAS; CDR2-VL: amino acid sequence is EDSKRPS; CDR3-VL: amino acid sequence is QAYERGAV or QSHDGSYI or QAYDMGRI or QSYDSDIRS or QSRELLGP; CDR1-VH: amino acid sequence is TSYWIG; CDR2-VH: amino acid sequence: IIYPGDSDTRYSPSFQG; CDR3-VH: the amino acid sequence is YNDLLSPMDY or YIDAFTPFDY or YSHFSRPFAD or YRILAHFDY or SHVFDLNMDD.

2. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.39, SEQ ID NO.41 or SEQ ID NO.43; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.40, SEQ ID NO.42 or SEQ ID NO.

44.

3. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: Constant regions are also included.

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

5. The human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The species origin of the constant region is cow, 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 according to claim 3, characterized in that: 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 according to claim 1, characterized in that: The antigen-binding fragment of the human anti-transferrin receptor 1 antibody is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv.

8. A drug delivery carrier, characterized in that The present invention comprises the human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or comprises a nucleic acid fragment encoding the human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. A drug, characterized in that It contains the human anti-transferrin receptor 1 antibody or its antigen-binding fragment according to any one of claims 1 to 7, or contains a nucleic acid fragment encoding the human anti-transferrin receptor 1 antibody or its antigen-binding fragment according to any one of claims 1 to 7, or contains the drug delivery vector according to claim 8.

10. Use of the human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and the drug delivery vector according to claim 8 in in vitro detection of transferrin receptor 1 or preparation of a drug that crosses the blood-brain barrier.

Images