PH-sensitive humanized anti-transferrin receptor 1 antibody

Abstract

The invention relates to the technical field of antibodies, in particular to a pH-sensitive humanized anti-transferrin receptor 1 antibody. The invention designs a pH-sensitive human anti-transferrin receptor 1 antibody or an antigen binding fragment thereof, the antibody has good binding activity to TfR1 under a neutral pH condition, the binding activity is obviously reduced or lost under a low pH condition, and the antibody can more efficiently penetrate through a blood-brain barrier to be delivered into the brain through a TfR1-mediated endocytosis transport effect by utilizing the pH-sensitive characteristic, so that the human anti-transferrin receptor 1 antibody or the antigen binding fragment thereof can be applied to the brain. The drug delivery system can be used as a delivery carrier of drugs for treating central nervous system diseases, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a pH-sensitive humanized anti-transferrin receptor 1 antibody. 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 from damage and maintains normal homeostasis. It 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 have difficulty breaking through the blood-brain barrier and reaching 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 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 not destroying the integrity of the blood-brain barrier. It is a cross-blood-brain barrier drug delivery route 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; 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 to achieve cross-blood-brain barrier delivery of drugs.

[0005] The endocytic transport mechanism based on TfR1 involves processes such as binding, endocytosis, dissociation, exocytosis, and release. By improving the molecular characteristics of antibodies in these processes, it is expected that the transport efficiency will be further improved. During the endocytic transport process, TfR1 will not enter the brain with the transported molecules, but will eventually return 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 to TfR1 under peripheral neutral pH conditions, and quickly dissociate from TfR1 when internalized in a low pH environment. This 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 that urgently needs to be solved in this field. Summary of the invention

[0007] The present invention provides a pH-sensitive 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; CDR1-VH: amino acid sequence is TSYWIG; CDR2-VH: amino acid sequence: IIYPGHSDTRYSPSFQG; CDR3-VH: The amino acid sequence is YNDLLSPMDY.

[0008] In some embodiments, the amino acid sequence of the above CDR2-VL is replaced with EDHKRPH, which still has pH sensitivity.

[0009] 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.

[0010] 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.

[0011] In some embodiments, the pH-sensitive human anti-transferrin receptor 1 antibody or antigen-binding fragment thereof further comprises a constant region.

[0012] In some embodiments, the constant region is any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0013] 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.

[0014] In some embodiments, 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.

[0015] In some embodiments, the antigen-binding fragment of the human anti-transferrin receptor 1 antibody is any one of F(ab')2, Fab', Fab, Fv, and scFv.

[0016] Furthermore, the present invention provides a reagent or a kit containing the pH-sensitive human anti-transferrin receptor 1 antibody or an antigen-binding fragment thereof.

[0017] In a specific implementation process, the antibody or antigen-binding fragment thereof in the reagent or kit is labeled with a detectable marker.

[0018] In the specific implementation process, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter what marker is used, it belongs to the protection scope of the present invention.

[0019] In some embodiments, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent agents, and nanoparticle labels.

[0020] In some embodiments, the fluorescent dyes include but are 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, Cy3.31 ...2, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.39, 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.).

[0021] In some embodiments, 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.

[0022] In some embodiments, the chemiluminescent reagents include, but are 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.

[0023] In some embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0024] Preferably, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres and latex.

[0025] Preferably, the colloidal metal includes but is not limited to colloidal gold, colloidal silver and colloidal selenium.

[0026] Furthermore, the present invention provides a drug containing the pH-sensitive human anti-transferrin receptor 1 antibody or an antigen-binding fragment thereof.

[0027] Preferably, the drug is a central nervous system drug and can cross the blood-brain barrier.

[0028] Furthermore, the present invention also provides the use of the pH-sensitive human anti-transferrin receptor 1 antibody or its antigen-binding fragment or the reagent or kit in in vitro detection of transferrin receptor 1 or preparation of drugs that cross the blood-brain barrier.

[0029] Compared with the prior art, the present invention has the following beneficial effects: The present invention designs a pH-sensitive humanized 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 the binding activity under low pH conditions. The pH-sensitive property can be utilized to more efficiently penetrate the blood-brain barrier and be delivered into the brain through TfR1-mediated endocytosis, thereby achieving purposes such as drug delivery. The antibody can be used as a delivery carrier for drugs for treating central nervous system diseases, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The information of TfR1 site in AET1802 / TfR1 interaction determined by HDX-MS, and the ordinate represents the difference in deuterium incorporation percentage (△%D).

[0031] Figure 2 The information of the AET1802 heavy chain site in the AET1802 / TfR1 interaction determined by HDX-MS is shown in the figure. The ordinate represents the difference in the percentage of deuterium incorporation (△%D).

[0032] Figure 3 The structural model of the AET1802 variable region and TfR1 complex was constructed.

[0033] Figure 4 The binding activity of the antibody was determined by ELISA under different pH washing conditions.

[0034] Figure 5 The results of antibody penetration evaluation based on the Transwell chamber monolayer hCMEC / D3 cell blood-brain barrier model; ns indicates no statistical difference.

[0035] Figure 6 The results of the functional activity evaluation of the antibody in TfR1 humanized mice; ns indicates no statistical difference. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels. 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 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 ineliciting NK cell-mediated antitumor immunity, 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.

[0037] Example 1: Determination of Antigen and Antibody Epitopes by Hydrogen-Deuterium Exchange Mass Spectrometry

[0038] 1. Materials and Methods

[0039] 1. Materials

[0040] hTfR1-His recombinant protein was purchased from Beijing Sino Biological Technology Co., Ltd. (Cat. No.: 11020-H07H). TM293-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. The antibody light chain expression vector pABL-1902L and heavy chain expression vector pABG-1902H were constructed and preserved by our laboratory. The spectrophotometer was Nanodrop ONE C The Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) Experimental Platform (LEAP PAL 3.0) includes the Orbitrap Fusion TM Tribrid TM Mass Spectrometer with Easy ETD (Thermo Fisher), HDX analysis software ProteomeDiscover2.2, and linear and conformational epitope quantitative analysis tool HDXWB; related services were provided by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0041] 2. Methods

[0042] 2.1 Antibody Preparation

[0043] AET1802 is an IgG1λ humanized antibody obtained by our laboratory from a fully synthetic phage display antibody library. The amino acid sequence of the light chain CDR1 of AET1802 is SEQ ID NO. 1, the amino acid sequence of CDR2 is SEQ ID NO. 2, the amino acid sequence of CDR3 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 amino acid sequence of the heavy chain CDR1 of AET1802 is SEQ ID NO. 8, the amino acid sequence of CDR2 is SEQ ID NO. 9, the amino acid sequence of CDR3 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.

[0044] 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 column was used for affinity purification, and samples were eluted and collected with 0.1M sodium citrate buffer (pH 3.0). TM Desalting Columns replace the antibody with PBS buffer to obtain the antibody sample. The antibody concentration is quantified by spectrophotometer.

[0045] 2.2 Determination of AET1802 / hTfR1 epitopes by hydrogen-deuterium exchange mass spectrometry

[0046] Antigen and antibody were added to 50mM HEPES, pH 7.4, 150mM NaCl, 4mM TCEP buffer to a final concentration of 5μM and placed at 4°C for 1 hour to form a stable complex. The individual antigens and antibodies were also diluted to 5μM. 5μL of sample was diluted to 20μL D20 (deuterium) and placed at different hydrogen deuterium exchange (HDX) time points (e.g. 0, 10, 60, 300, 900s), 25μL pre-cooled 4M guanidine hydrochloride and 1% trifluoroacetic acid were added to terminate the reaction, and then transferred to the LEAPPAL3.0 platform for mass spectrometry analysis. Samples without HDX were set up simultaneously. The sample passed through the fixed pepsin column at a flow rate of 120μL / min, and the enzymatic peptides were captured on the 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 the above processes were performed at 4 °C. Protein hydrolyzed peptides were identified on a mass spectrometer by secondary mass spectrometry (MS / MS) with a resolution of 65,000 (m / z 400). Each sample at each time point was measured three times. The MS / MS data files were processed by Proteome Discover software and the antigen-antibody two-dimensional peptide spectra were identified. The average m / z centroid value of the mass spectrum peak intensity of each hydrolyzed peptide was calculated by HDX Workbench software (10 ppm accuracy) and converted into deuterium incorporation percentage (%D); the difference in deuterium incorporation percentage between the two samples (∆%D) was calculated to analyze and determine the key amino acid residues involved in spatial interactions (the absolute value of ∆%D ≥5% was considered to be significantly different).

[0047] 2. Results

[0048] The amino acid residues of the AET1802 / TfR1 interaction interface were determined based on the HDX-MS method. Figure 1 and Figure 2 As shown. Figure 1 The results show that the EFKLSKVWRDQHF sequence on TfR1 is a potential epitope region, corresponding to sites 175-187. Figure 2 The results show that the YPGDSDTRYSPSF sequence on the AET1802 heavy chain is a potential epitope region, corresponding to the HCDR2 region of the antibody. Based on this method, no potential epitope region of the light chain could be detected.

[0049] Example 2: Histidine scanning of antibody CDR regions and identification of binding activity of mutants

[0050] 1. Materials and Methods

[0051] 1 Materials

[0052] DNA polymerase PrimerSTAR is a product of Takara, plasmid extraction kit is a product of Tiangen Biochemical Technology (Beijing) Co., Ltd., and Escherichia coli Top 10 competent cells are a product of Jiangsu Kangwei Century Biotechnology Co., Ltd. The 96-well ELISA plate is a product of Costar. HRP-labeled goat anti-human IgG antibody is a product of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. The plate washer is BioTeK ELx405, and the microplate reader is Thermo Multiskan MK3. Primer synthesis and DNA sequencing services are provided by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. The sources of other materials involved are the same as those in Example 1.

[0053] 2 Methods

[0054] Referring 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 light chain expression vectors pABL-1802L and pABG-1802H were used as templates to replace the codons of the corresponding sites in the CDR region with CAT or CAC (translated as histidine) to construct an expression vector with a single site mutated to histidine. Escherichia coli Top 10 competent cells were transfected, and the mutant plasmid was amplified and extracted. The light chain plasmid containing a single histidine mutation site was co-transfected with the original heavy chain plasmid into FreeStyle TM 293-F cells, and the antibody sample was obtained by affinity purification as described in Example 1; and so on, all histidine mutants of AET1802 were prepared.

[0055] 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 samples were gradiently diluted with PBST containing 2.5% skim milk powder (PBS containing 0.1% Tween20) (starting concentration 10μg / mL, 2-fold gradient serial dilution to 8 dilutions), and blocked at 37℃ for 1h. The antibodies were added to the ELISA plate at 100µL / well and bound at 37℃ for 1h. Washing the ELISA plate: Here, two parallel groups were set for each antibody sample, and washed with PBST at pH 7.4 and pH 6.0 respectively. 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. Wash the ELISA plate, add TMB colorimetric solution at 100 µL / well, and let it stand at room temperature for 10-15 min before stopping the color development. Read the absorbance at 450 nm (595 nm as a control) with an ELISA reader, and plot the antibody concentration-absorbance curve using Graphpad Prism Software 9.0 to compare the differences in binding activity between the antibody mutants and AET1802.

[0056] 2. Results

[0057] Compared with the parent antibody AET1802, the binding activity test results of the mutants with a single site mutation to histidine under pH7.4 and pH6.0 washing conditions are listed in Table 1. From the results, it can be seen that the completely inactivated sites include K31, Y32, Y91 of the light chain and Y27, W33, Y52, Y95 of the heavy chain), suggesting that they play an important role in the interaction and may be at the interface position; LCDR2 basically does not participate in the interaction. In particular, the mutation of D54H in the heavy chain CDR2 region showed weaker binding activity under low pH conditions, suggesting that it may be beneficial to the design of pH-sensitive TfR1 antibodies.

[0058] Table 1. Binding activity identification results of histidine mutants at each position in the CDR of the variable region of AET1802

[0059] Example 3: Construction of antigen-antibody complex structural model

[0060] 1. Materials and Methods

[0061] 1 Materials

[0062] The operating system was Windows 7 (64-bit), and the molecular simulation software was Accelrys ®Discover Studio 3.0 (DS 3.0 for short), structural graphics drawing tool PyMol. The templates for homology modeling (6A3W.pdb and 6MI2.pdb) and the hTfR1 structure (6WRV.pdb) used in molecular docking were both from the PDB database (http: / / www.rcsb.org).

[0063] 2 Methods

[0064] The comparative modeling (homologous modeling) method was used to construct the structural model of the variable region of AET1802. Blast searched the PDB database to determine the homologous sequences of the light and heavy chain variable regions of AET1802. Sequences and structures with high homology in both light and heavy chains were selected as homologous templates. The DS 3.0 Align Sequence Profiles (MODELER) module was used to align the target antibody sequence with the homologous template sequence. The Build Homology Models function in the DS 3.0 Homology Modeling module was used to construct the structural model of the antibody variable region. The probability density function (PDF), DOPE function (Discrete Optimized Protein Energy function) scoring and empirical analysis were used to preliminarily exclude obviously unreasonable models; Loop Refinement (MODELER) was used to optimize the CDR regions of the light and heavy chains of the antibody. The structural rationality of the model was further evaluated through the "Environment Matching Score of Residues (Profile-3D)", "Rarmchandran Plot" and empirical analysis, and the antibody variable region structural model was obtained. The hTfR1 structure was derived from 6WRV.pdb. Dock Proteins (ZDOCK) in the DS 3.0 Dock Proteins module was used to dock AET1802 and hTfR1. The interaction epitope range was limited according to the previous experimental results and antibody characteristics, and the complex structure model was constructed. The results were clustered and scored by ZDock and ZRank. Refine Dock Proteins (RDOCK) in the DS 3.0 Dock Proteins module was used to optimize the preferred clustering, and the final complex structure model was determined based on the ZDock Score and ZRank Score scores combined with empirical inspection.

[0065] 2. Results

[0066] According to the Blast search results, the antibody structures in 6A3W.pdb and 6MI2.pdb were selected as templates for AET1802 homology modeling. The antibody variable regions described in the two pdb structures have high similarity with the AET1802 light and heavy chain sequences. After structural modeling and optimization, the AET1802 variable region structure model 1802.M0001L0001.L0002.L0004 was obtained. Molecular docking generated 2000 complex structures (Pose), and analysis formed 100 clusters (Cluster). Based on the docking clustering situation, with reference to the relevant results of the aforementioned Examples 1 and 2, the top 20 clusters were specifically analyzed, as listed in Table 2, and finally Cluster 6 was selected for optimization. Refined Pose 9 is preferred as the AET1802 / TfR1 complex structure model, as shown in Table 2. Figure 3 shown.

[0067] Further, the variable region related sites of AET1802 were analyzed based on the complex structure model, and the results are listed in Table 3. At the same time, referring to the results of Example 2, it can be seen that the heavy chain D54H mutation is beneficial for designing pH-sensitive molecules. At the same time, it is also considered to introduce two mutations, S52H and S56H, in the light chain at the same time, in order to maintain the binding ability at neutral pH.

[0068] Table 2. Analysis of the docking results between the variable region structure model of AET1802 and TfR1 molecule

[0069] In the table, “+” indicates that the binding region contains the marked domain.

[0070] Table 3. Analysis results of the structural model of the AET1802 / TfR1 complex (Pose 9)

[0071] Example 4: Identification of pH-sensitive antibodies

[0072] 1. Materials and Methods

[0073] The preparation method of the mutants is the same as that described in Examples 1 and 2. The ELISA identification is the same as that described in Example 2.

[0074] 2. Results

[0075] According to the design of Example 3, two mutants were prepared, namely 1802-D54H (heavy chain D54H mutation) and 1802-DSS (heavy chain D54H, light chain S52H and S56H three mutation sites). 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 as follows Figure 4 As shown, the two antibodies showed obvious differences in binding activity under pH 7.4 and pH 6.0 washing conditions, which were pH sensitive.

[0076] Example 5: Functional activity of antibodies on blood-brain barrier cell model

[0077] 1. Materials and Methods

[0078] 1 Materials

[0079] 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). HRP-labeled goat anti-human IgG antibody was a product of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. The 96-well enzyme-linked plate was a Costar product. Other reagents and consumables were the same as in the previous embodiment.

[0080] 2 Methods

[0081] 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 4h and 8h 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 (patent publication number: US2018 / 0171012Al, CN107849555A), and its light and heavy chain amino acid sequences are SEQ ID NO.17 and SEQ ID NO. 18, respectively.

[0082] 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 2. 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 the result graph was drawn by Graphpad Prism Software 9.0. The t-test was used for inter-group difference analysis.

[0083]

[0084] 2. Results

[0085] 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. Figure 5 The pH-sensitive 1802-D54H showed significantly better penetration than AET802.

[0086] Example 6: Functional activity of antibodies in human TfR1 transgenic mice

[0087] 1. Materials and Methods

[0088] 1 Materials

[0089] TfR1 humanized mice (BALB / c-hTfR1, strain number: T055118) were purchased from Jicui Yaokang Biotechnology Co., Ltd. The fully automatic sample rapid grinding instrument 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 prepared in this laboratory against an irrelevant target, which has the same light and heavy chain constant regions as AET1802. Other reagents and consumables were the same as in Example 5.

[0090] 2 Methods

[0091] Ten-week-old male BALB / c-hTfR1 mice were injected with 200 μg of the antibody to be evaluated once through the tail vein, with 5 mice in each group. 12 hours later, the heart was perfused with PBS, and the brain tissue was weighed and ground with PBS at 500 μL / μg tissue, and the supernatant was centrifuged at 6000g×10min. The antibody concentration in the supernatant of the tissue homogenate was detected by the ELISA method described in Example 5.

[0092] 2. Results

[0093] ELISA quantitative test results Figure 6 As shown, the results showed that AET1802, 1802-D54H, and 1802-DSS can effectively cross the blood-brain barrier and enter the brain tissue, and the pH-sensitive 1802-D54H is significantly higher than AET802.

[0094] 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 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, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.11; 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, characterized in that: 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 . A medicament comprising the pH-sensitive human anti-transferrin receptor 1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 .

9. The drug according to claim 8, characterized in that It can cross the blood-brain barrier; preferably, the drug is a central nervous system drug.

10. 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 detecting transferrin receptor 1 in vitro or in preparing a drug that crosses the blood-brain barrier.

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