A pH-sensitive human anti-transferrin receptor 1 antibody

Abstract

The present invention relates to the field of antibody technology, and in particular to a pH-sensitive human anti-transferrin receptor 1 antibody. The present invention designs a pH-sensitive human anti-transferrin receptor 1 antibody or an antigen-binding fragment thereof, which has good binding activity to TfR1 under neutral pH conditions, and significantly reduces or loses its binding activity under low pH conditions. Utilizing its pH-sensitive properties, it can more efficiently penetrate the blood-brain barrier and enter the brain through TfR1-mediated endocytosis, thereby achieving purposes such as drug delivery. It can be used as a delivery vehicle for drugs used to treat central nervous system diseases and has broad application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, and particularly relates to a pH-sensitive human anti-transferrin receptor 1 antibody. BACKGROUND

[0002] The blood-brain barrier (BBB) is a complex physiological barrier that isolates harmful substances in the peripheral circulation system, protects the central nervous system from invasion and maintains normal homeostasis, and has multiple functions such as physical barrier, transport barrier, metabolic barrier, immune barrier and enzyme system barrier. Under normal circumstances, 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. 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. It is a cross-blood-brain barrier drug delivery pathway worthy of in-depth study.

[0004] Among the RMT-related transport receptors, transferrin receptor 1 (TfR1) is the most widely used and effective target. TfR has two subtypes, TfR1 and TfR2, with 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, 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] The endocytosis transport mechanism based on TfR1 involves processes such as binding, endocytosis, dissociation, exocytosis and release. By improving the molecular properties of the antibody in these processes, it is expected to further improve the transport efficiency. In the endocytosis transport process, TfR1 does not enter the brain with the transported molecules, but eventually returns to the peripheral side of the BBB; at the same time, the pH value of the internalization compartment environment is generally 5.5-6.0. By designing a pH-sensitive TfR1 antibody, it can efficiently recognize and bind TfR1 under peripheral neutral pH conditions, and quickly dissociate from TfR1 in the low-pH environment of internalization. In this way, it can avoid being degraded after endocytosis or being taken back to the periphery by TfR1 after entering the brain, thereby improving the transport efficiency.

[0006] Therefore, how to develop a pH-sensitive TfR1 antibody has become a technical problem urgently to be solved in the field. SUMMARY

[0007] The present application provides a pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof, comprising the following complementarity determining regions:

[0008] CDR1-VL: the amino acid sequence is SGDALGDKYAS;

[0009] CDR2-VL: the amino acid sequence is EDSKRPS;

[0010] CDR3-VL: the amino acid sequence is QAYERGAV;

[0011] CDR1-VH: the amino acid sequence is TSYWIG;

[0012] CDR2-VH: the amino acid sequence is IIYPGHSDTRYSPSFQG;

[0013] CDR3-VH: the amino acid sequence is YNDLLSPMDY.

[0014] In some embodiments, the amino acid sequence of CDR2-VL is replaced by EDSKRPH, which still has pH sensitivity.

[0015] In some embodiments, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 11; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 15.

[0016] In some embodiments, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 15.

[0017] In some embodiments, the pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof further comprises a constant region.

[0018] In some embodiments, the constant region is any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.

[0019] 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.

[0020] In some embodiments, the constant region comprises a light chain constant region as set forth in SEQ ID NO. 6 and a heavy chain constant region as set forth in SEQ ID NO. 13.

[0021] In some embodiments, the human anti-transferrin receptor 1 antibody antigen-binding fragment is any one of F(ab’)2, Fab’, Fab, Fv, scFv.

[0022] Further, the present application provides a reagent or kit comprising the pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof.

[0023] In specific embodiments, the antibody or antigen-binding fragment thereof in the reagent or kit is labeled with a detectable label.

[0024] In specific embodiments, the skilled person in the art can select a suitable label according to the detection conditions or actual needs, and no matter what label is used, it falls within the protection scope of the present application.

[0025] In some embodiments, the detectable label includes but is not limited to fluorescent dyes, enzymes that develop color with substrates, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

[0026] In some embodiments, the fluorescent dye includes, but is not limited to, fluorescein-based dyes and their derivatives (e.g., 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 (e.g., 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 (e.g., 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 (e.g., 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 (e.g., including, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), and the like).

[0027] In some embodiments, the catalyzing-substrate-color-developing enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0028] In some embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacyanin, ruthenium bispyridyl and its derivatives, acridan ester and its derivatives, dioxetane and its derivatives, lucigenin and its derivatives, and peroxyoxalate and its derivatives.

[0029] In some 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.

[0030] Preferably, the colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latex.

[0031] Preferably, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0032] Further, the present application provides a medicament containing the pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof.

[0033] Preferably, the medicament is a central nervous system drug. The medicament is capable of crossing the blood-brain barrier.

[0034] Further, the application also provides application of the pH-sensitive human anti-transferrin receptor 1 antibody or antigen binding fragment thereof or the reagent or kit in vitro detection of transferrin receptor 1 or preparation of a drug crossing the blood-brain barrier.

[0035] Compared with the prior art, the application has the following beneficial effects:

[0036] The application designs a pH-sensitive human anti-transferrin receptor 1 antibody or antigen binding fragment thereof, which has good binding activity to TfR1 under neutral pH conditions and significantly reduced or lost binding activity under low pH conditions. The pH-sensitive property can be used to more efficiently penetrate the blood-brain barrier and deliver into the brain through TfR1-mediated endocytosis, so as to achieve the purpose of drug delivery, and can be used as a delivery carrier for central nervous system disease treatment drugs, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 For determination of TfR1 site information in AET1802 / TfR1 interaction by HDX-MS, the ordinate represents the difference in deuterium incorporation percentage (△%D).

[0038] Figure 2 For determination of AET1802 heavy chain site information in AET1802 / TfR1 interaction by HDX-MS, the ordinate represents the difference in deuterium incorporation percentage (△%D).

[0039] Figure 3 For the structure model of the AET1802 variable region and TfR1 complex constructed.

[0040] Figure 4 For ELISA identification of the binding activity of the antibody under different pH washing conditions.

[0041] Figure 5 For the antibody penetration rate evaluation results based on the Transwell chamber single-layer hCMEC / D3 cell blood-brain barrier model; ns indicates no statistical difference.

[0042] Figure 6 For the functional activity evaluation results of the antibody in TfR1 humanized mice; ns indicates no statistical difference. DETAILED DESCRIPTION

[0043] To make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are some 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 work fall within the protection scope of the present application. In the following examples, the experimental methods are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. In the following examples, the materials, reagents, instruments, etc. can be obtained from commercial channels, unless otherwise specified. In the following examples, 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, unless otherwise specified. In the following examples, pABL and pABG 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 materials are only used for repeating the relevant experiments of the present application, and cannot be used for other purposes.

[0044] Example 1: Hydrogen-deuterium exchange mass spectrometry determination of antigen-antibody epitope

[0045] I. Materials and methods

[0046] 1. Materials

[0047] hTfR1-His recombinant protein was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. (Item No.: 11020-H07H). FreeStyle TM293-F mammalian cell expression system: cells FreeStyle TM 293-F is a product of Invitrogene, serum-free 293 medium and Opti-MEM are products of Gibco, transfection reagent is a product of PolyPlus trasfection. Protein purification system AKTA avant is a product of GE, purification column HiTrap TM MabSelect SuRe, HiTrap TM Desalting Columns are provided by Cytiva. Antibody light chain expression vector pABL-1902L and heavy chain expression vector pABG-1902H are constructed and preserved by the laboratory. Spectrophotometer is Nanodrop ONE C The hydrogen-deuterium exchange mass spectrometry (HDX-MS) experimental platform (LEAP PAL 3.0) includes Orbitrap Fusion TM Tribrid TM MassSpectrometer with Easy ETD (Thermo Fisher), HDX analysis software Proteome Discover 2.2, and linear and conformational epitope quantification tool HDXWB; related services are provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0048] 2、Method

[0049] 2.1 Preparation of antibodies

[0050] AET1802 is an IgG1λ human antibody screened from a fully synthetic phage display antibody library in the laboratory. The CDR1 amino acid sequence of the light chain of AET1802 is SEQ ID NO. 1, the CDR2 amino acid sequence is SEQ ID NO. 2, the CDR3 amino acid sequence is SEQ ID NO. 3, the amino acid sequence of the light chain variable region is SEQ ID NO. 4, the nucleic acid sequence encoding the light chain variable region is SEQ ID NO. 5, and the amino acid and nucleic acid sequences of the light chain constant region are SEQ ID NO. 6 and SEQ ID NO. 7, respectively. The CDR1 amino acid sequence of the heavy chain of AET1802 is SEQ ID NO. 8, the CDR2 amino acid sequence is SEQ ID NO. 9, the CDR3 amino acid sequence is SEQ ID NO. 10, the amino acid sequence of the heavy chain variable region is SEQ ID NO. 11, the nucleic acid sequence encoding the heavy chain variable region is SEQ ID NO. 12, and the amino acid and nucleic acid sequences of the heavy chain constant region are SEQ ID NO. 13 and SEQ ID NO. 14, respectively.

[0051] The light chain expression vector pABL-1902L and the heavy chain expression vector pABG-1902H were mixed at a molar ratio of 2:1 and co-transfected into 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 columns were used for affinity purification, and samples were eluted and collected using 0.1 M sodium citrate buffer (pH 3.0). TM Desalting columns are used to replace the antibody with PBS buffer to obtain the antibody sample. Antibody concentration is quantified by spectrophotometry.

[0052] 2.2 Determination of AET1802 / hTfR1 epitopes by hydrogen-deuterium exchange mass spectrometry

[0053] Antigen and antibody were added to a 50 mM HEPES, pH 7.4, 150 mM NaCl, and 4 mM TCEP buffer to a final concentration of 5 μM each. The mixture was incubated at 4°C for 1 hour to allow for the formation of a stable complex. Separate antigen and antibody were also diluted to 5 μM. A 5 μL sample was diluted into 20 μL D20 (deuterium) and subjected to hydrogen-deuterium exchange (HDX) at various time points (e.g., 0, 10, 60, 300, and 900 s). The reaction was terminated by the addition of 25 μL of pre-cooled 4 M guanidine hydrochloride and 1% trifluoroacetic acid. The sample was then transferred to the LEAPPAL 3.0 platform for mass spectrometry analysis. A sample not subjected to HDX was also set up simultaneously. The sample was passed through an immobilized pepsin column at a flow rate of 120 μL / min. The enzymatically digested peptides were captured on a C18 capture column and desalted. Desalted peptides were separated using a 2.1 mm × 5 cm C18 column (1.9 μm Hypersil Gold, Thermo Fisher) with a linear gradient of 4–40% acetonitrile and 0.3% formic acid. All procedures were performed at 4°C. Protein-digested peptides were identified by MS / MS at a resolution of 65,000 (m / z 400). Each sample at each time point was analyzed in triplicate. Proteome Discover software was used to process the MS / MS data files and identify the antigen-antibody two-dimensional peptide spectra. HDX Workbench software was used to calculate the average m / z centroid value of the mass spectral peak intensity for each digested peptide (with 10 ppm accuracy) and convert it to percent deuterium incorporation (%D). The difference in deuterium incorporation between the two samples (∆%D) was calculated to identify key amino acid residues involved in steric interactions (an absolute ∆%D value ≥5% was considered significant).

[0054] II. Results

[0055] Amino acid residues of the AET1802 / TfRl interaction interface were determined based on HDX-MS method, and the results are shown in Table 1 and Table 2. Figure 1 and Figure 2 As can be seen from the results, the sequence of EFKLSKVWRDQHF on TfRl is a potential epitope region, and the corresponding site is 175-187. Figure 1 As can be seen from the results, the sequence of YPGDSDTRYSPSF on the heavy chain of AET1802 is a potential epitope region, which corresponds to the HCDR2 region of the antibody. Based on this method, no potential epitope region of the light chain was detected. Figure 2 As can be seen from the results, the sequence of YPGDSDTRYSPSF on the heavy chain of AET1802 is a potential epitope region, which corresponds to the HCDR2 region of the antibody. Based on this method, no potential epitope region of the light chain was detected.

[0056] Example 2: Antibody CDR region histidine scanning and mutant binding activity identification

[0057] I. Materials and methods

[0058] 1. Materials

[0059] DNA polymerase PrimerSTAR is a product of Takara Company, plasmid extraction kit is a product of Tiangen Biochemical Technology (Beijing) Co., Ltd., E. coli Top 10 competent cells are products of Jiangsu Kangwei Century Biological Technology Co., Ltd. 96-well enzyme-linked plate is a product of Costar. HRP-labeled goat anti-human IgG antibody is a product of Beijing Zhongshanjinqiao Biotechnology Co., Ltd. Plate washing machine is BioTeK ELx405, and enzyme marker is Thermo Multiskan MK3. Primer synthesis and DNA sequencing service are provided by Beijing Tianyihuiyuan Biotechnology Co., Ltd. Other materials are from the same source as in Example 1.

[0060] 2. Methods

[0061] According to the site-directed mutagenesis PCR method described in the literature (Wang Ronghao et al., Journal of Xiamen University, 2008, 47(sup 2):282-285), the codons of the corresponding sites in the CDR region were replaced with CAT or CAC (translated as histidine) respectively, and expression vectors with single histidine mutation sites were constructed. E. coli Top 10 competent cells were transfected, and mutant plasmids were amplified and extracted. The light chain plasmid containing a single histidine mutation site was co-transfected with the original heavy chain plasmid into FreeStyle 293-F cells, and the antibody sample was obtained by affinity purification as described in Example 1; in the same way, all histidine mutants of AET1802 were prepared. TM 293-F cells, and the antibody sample was obtained by affinity purification as described in Example 1; in the same way, all histidine mutants of AET1802 were prepared.

[0062] 96-well ELISA plates were coated with hTfR1-His diluted in PBS at 1 μg / mL x 100 μL / 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 (PBS containing 0.1% Tween 20) with a gradient of 2.5% skim milk at a starting concentration of 10 μg / mL, 2-fold serial dilution for 8 dilutions, and incubated at 37°C for 1 h. The antibody samples were added to the ELISA plates at 100 μL / well, and incubated at 37°C for 1 h. The ELISA plates were washed: for each antibody sample, two sets were set up in parallel, and washed with PBST at pH 7.4 and pH 6.0, respectively. HRP-labeled goat anti-human IgG antibody was diluted 1 / 2000 in PBST containing 2.5% skim milk, and added to the ELISA plates at 100 μL / well, and incubated at 37°C for 1 h. The ELISA plates were washed, and TMB color developing solution was added at 100 μL / well. After incubation at room temperature for 10-15 min, the color development was stopped. The absorbance values at 450 nm (with 595 nm as a control) were read by a microplate reader, and the antibody concentration-absorbance value curves were plotted by Graphpad Prism Software 9.0 to compare the binding activity differences between the antibody mutants and AET1802.

[0063] II. Results

[0064] The results of the binding activity detection of the mutants with single-site mutation to histidine compared to the parent antibody AET1802 under pH 7.4 and pH 6.0 washing conditions are listed in Table 1. As can be seen from the results, the completely inactivated sites include light chain K31, Y32, Y91 and heavy chain Y27, W33, Y52, Y95, suggesting that they play an important role in the interaction and may be located at the interface position; LCDR2 is basically not involved in the interaction. In particular, the mutation of D54H in the heavy chain CDR2 region shows weaker binding activity under low pH conditions, suggesting that it may be beneficial for the design of pH-sensitive TfR1 antibodies.

[0065] Table 1. Identification results of binding activity of AET1802 variable region CDR site histidine mutants

[0066]

[0067] Example 3: Construction of antigen-antibody complex structure model

[0068] I. Materials and methods

[0069] 1. Materials

[0070] The operating system was Windows 7 (64-bit), and the molecular simulation software was Accelrys ®Discover Studio 3.0 (DS 3.0), the structural graphics drawing tool PyMol. The template for homology modeling (6A3W.pdb and 6MI2.pdb) and the structure of hTfR1 used in molecular docking (6WRV.pdb) were all from the PDB database (http: / / www.rcsb.org).

[0071] 2. Methods

[0072] The comparative modeling (homology modeling) method was used to construct the structural model of the variable region of AET1802. The PDB database was searched by Blast to determine the homologous sequences of the light and heavy chain variable regions of AET1802, and the sequences and structures with high homology of light and heavy chains were selected as homology templates. The DS 3.0 Align Sequence Profiles (MODELER) module was used to align the target antibody sequence with the homology template sequence. The Build Homology Models function in the DS 3.0 Homology Modeling module was used to construct the structure model of the antibody variable region. The probability density function (PDF), DOPE function (Discrete Optimized Protein Energy function) score and empirical analysis were used to preliminarily exclude obviously unreasonable models. The Loop Refinement (MODELER) was used to optimize the light and heavy chain CDR regions of the antibody, and the structure rationality of the model was further evaluated by "residual environment matching score (Profile-3D)", "Rarmchandran Plot" and empirical analysis, and the antibody variable region structure model was obtained by optimization. The structure of hTfR1 was derived from 6WRV.pdb, and the Dock Proteins (ZDOCK) in the Dock Proteins module of DS 3.0 was used to dock AET1802 and hTfR1, and the range of the interacting epitope was limited according to the previous experimental results and the characteristics of the antibody to construct the complex structure model. The results were analyzed by clustering and ZDock, ZRank scoring. The Refine Dock Proteins (RDOCK) in the Dock Proteins module of DS 3.0 was used to optimize the preferred cluster, and the final complex structure model was determined according to the ZDock Score and ZRank Score scoring values, combined with empirical inspection.

[0073] II. Results

[0074] Based on the Blast search results, the antibody structures in 6A3W.pdb and 6MI2.pdb were selected as the templates for homology modeling of AET1802, both of which have high sequence similarity to the variable regions of AET1802 light and heavy chains. After structure modeling and optimization, the AET1802 variable region structure model 1802.M0001L0001.L0002.L0004 was obtained. Molecular docking produced 2000 complex structures (Pose), and 100 clusters were analyzed. Based on the docking cluster results, referring to the results of the previous Examples 1 and 2, the top 20 clusters were analyzed in detail, as shown in Table 2, and Cluster 6 was finally selected for optimization. The Refined Pose 9 was selected as the AET1802 / TfR1 complex structure model, as shown in Figure 3

[0075] Further, based on the complex structure model, the variable region related sites of AET1802 were analyzed, and the results are shown in Table 3. At the same time, referring to the results of Example 2, it can be seen that the mutation of heavy chain D54H is beneficial for designing pH-sensitive molecules. At the same time, it is also considered to introduce two mutations of light chain S52H and S56H at the same time, in order to maintain the binding ability at neutral pH.

[0076] Table 2. Analysis of AET1802 variable region structure model and TfR1 molecular docking results

[0077]

[0078] In the table, "+" indicates that the binding region contains the indicated domain.

[0079] Table 3. Analysis results of AET1802 / TfR1 complex structure model (Pose 9)

[0080]

[0081] Example 4: Identification of pH-sensitive antibodies

[0082] I. Materials and methods

[0083] The preparation method of the mutants is the same as described in Examples 1 and 2. The ELISA identification is the same as described in Example 2.

[0084] II. Results

[0085] ​According to the design of Example 3, two mutants, 1802-D54H (heavy chain D54H mutation) and 1802-DSS (heavy chain D54H, light chain S52H and S56H three mutation sites) were prepared. The light chain variable region sequence of H-D54H is SEQ ID NO. 11, and the heavy chain variable region sequence is SEQ ID NO. 15. The light chain variable region sequence of 54H52H56H is SEQ ID NO. 16, and the heavy chain variable region sequence is SEQ ID NO. 15. The ELISA identification results are shown in Figure 4 As shown in the table, the two antibodies showed obvious differences in binding activity under pH 7.4 and pH 6.0 washing conditions, and had pH sensitivity.

[0086] Example 5: Functional activity of the antibody on the blood-brain barrier cell model

[0087] I. Materials and methods

[0088] 1. Materials

[0089] hCMEC / D3 cells were purchased from Quansheng Quanyi (Shanghai) Co., Ltd. (catalog number: CVCL U985). RPMI-1640 medium is an ATCC product (catalog number: 30-2001), FBS is an AusGeneX product (catalog number: FBS500-S), trypsin is a Gibco product (catalog number: 25200-072), and penicillin-streptomycin (PS) is a HyClone product (catalog number: SV30010). 6.5mm Transwell chamber is a Corning product (catalog number: 3413). The electrical resistivity tester EVOM3 is a WPI product. Goat anti-human IgG-Fc antibody is a Sigma-Aldrich product (catalog number: I2136). HRP-labeled goat anti-human IgG antibody is a Beijing Zhongshanjingqiao Biotechnology Co., Ltd. product. 96-well enzyme-linked plate is a Costar product. Other reagents and consumables are as described in the previous examples.

[0090] 2. Methods

[0091] 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, resuspended in complete medium to 8x10 5 cells / mL, inoculated at 0.25mL / well onto the upper layer of the transwell chamber, and the chamber was placed in a 24-well cell culture plate, and 1mL of complete medium was added to the lower layer. Fresh medium was replaced daily, and the cells were cultured continuously for 6-7 days until the monolayer cell electrical resistivity (TEER value) reached 100Ω·cm 2The left and right chambers were added with 50 μg of the antibody to be evaluated, 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 of Japan (Patent Publication No. US2018 / 0171012A1, CN107849555A), and the amino acid sequences of the light and heavy chains thereof were SEQ ID NO. 17 and SEQ ID NO. 18, respectively.

[0092] 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 2. The standard curve was drawn by detecting 9 gradients of the antibody to be evaluated at a known concentration, with a starting concentration of 5 μg / mL and a 2-fold gradient. The upper and lower culture media collected were diluted according to a specific ratio for detection, and the concentration of the corresponding antibody was calculated according to the corresponding 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 (calculation formula as follows), and the result graph was drawn by Graphpad Prism Software 9.0, and the difference between groups was analyzed by t-test.

[0093]

[0094] II. Results

[0095] The functional activity of the TfR1 antibody was evaluated in vitro by simulating the blood-brain barrier based on the Transwell chamber monolayer hCMEC / D3 cell system, and the results are shown in Figure 5 The pH-sensitive 1802-D54H showed a significantly better penetration rate than AET802.

[0096] Example 6: Functional activity of the antibody in vivo in human TfR1 transgenic mice

[0097] I. Materials and methods

[0098] 1. Materials

[0099] TfR1 humanized mice (BALB / c-hTfR1, strain number: T055118) were purchased from Jiesai Yekang Biotechnology Co., Ltd. The full-automatic sample rapid grinder was a product of Shanghai Jingxin Industrial Development Co., Ltd. (Model: JXFSTPRP-24L). The electronic balance was Sartorius BSA124S-CW. The centrifuge was Eppendorf 5417R. The isotype-independent control antibody was an antibody against an unrelated target prepared in the laboratory, which had the same light and heavy chain constant regions as AET1802. Other reagents and consumables were the same as in Example 5.

[0100] 2 Method

[0101] 10-week-old male BALB / c-hTfR1 mice, single tail vein injection of 200 μg of the antibody to be evaluated, 5 in each group. 12h after heart perfusion with PBS, brain tissue was weighed, 500 μL / μg tissue was added to PBS and ground, and the supernatant was obtained by centrifugation at 6000g for 10 min. The detection of antibody concentration in the supernatant of tissue homogenate used the ELISA method described in Example 5.

[0102] II. Results

[0103] The results of ELISA quantitative detection are shown in Table 1. Figure 6 As shown in Table 1, the results show that AET1802, 1802-D54H and 1802-DSS can effectively cross the blood-brain barrier into the brain tissue, and the pH-sensitive 1802-D54H is significantly higher than AET802.

[0104] 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 it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements 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 pH-sensitive 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; CDR1-VH: amino acid sequence is TSYWIG; CDR2-VH: amino acid sequence: IIYPGHSDTRYSPSFQG; CDR3-VH: amino acid sequence is YNDLLSPMDY; or comprising the following complementarity determining regions: CDR1-VL: amino acid sequence is SGDALGDKYAS; CDR2-VL: amino acid sequence is EDHKRPH; CDR3-VL: amino acid sequence is QAYERGAV; CDR1-VH: amino acid sequence is TSYWIG; CDR2-VH: amino acid sequence: IIYPGHSDTRYSPSFQG; CDR3-VH: The amino acid sequence is YNDLLSPMDY.

2. The pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

15.

3. The pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to claim 1, wherein: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.16; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

15.

4. The pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that Constant regions are also included.

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

6. The pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The constant region includes the light chain constant region shown in SEQ ID NO.6 and the heavy chain constant region shown in SEQ ID NO.

13.

7. The pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antigen-binding fragment of the human anti-transferrin receptor 1 antibody is any one of F(ab')2, Fab', Fab, Fv, and scFv.

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

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