An antibody or antigen-binding fragment thereof against human transferrin receptor 1 and use thereof

Abstract

This invention relates to the field of biomedical technology, providing an antibody against human transferrin receptor 1 or its antigen-binding fragment and its applications. Specifically, it provides antibodies Ab116 and Ab274 against human transferrin receptor 1 and their antigen-binding fragments. Through fully human antibody transgenic animal immunoscreening technology, it overcomes the shortcomings of existing antibodies, such as poor cross-species reactivity, high immunogenicity, interference with iron metabolism, and low delivery efficiency. It exhibits specific binding activity to TfR1 in humans and cynomolgus monkeys, without blocking the physiological binding of Tf to TfR1. As a "brain delivery tool," this antibody can safely and efficiently mediate drug crossing the blood-brain barrier, achieving targeted brain delivery, significantly improving the exposure level of macromolecular drugs in the central nervous system. It provides a new tool with low immunogenicity and high-efficiency delivery for the treatment of Alzheimer's disease and other neurodegenerative diseases, and possesses good safety and tolerability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an antibody against human transferrin receptor 1 or its antigen-binding fragment and its applications. Background Technology

[0002] The blood-brain barrier is composed of specialized endothelial cells that interact with pericytes and astrocytes to maintain the optimal environment for neuronal function by clearing toxins and supplying the brain with nutrients and other metabolic needs. This barrier system has multiple mechanisms of action, including physical barriers, transport barriers, metabolic barriers, immune barriers, and enzyme system barriers, which synergistically block harmful factors from infiltrating brain tissue at multiple levels.

[0003] Antibody drugs have shown significant therapeutic potential in recent years in areas such as oncology, autoimmune diseases, and neurological disorders. However, in the treatment of central nervous system-related diseases, the blood-brain barrier has extremely low permeability to large molecule drugs, severely limiting their delivery to brain tissue. For example, in the treatment of Alzheimer's disease, although mainstream research strategies focus on antibodies targeting β-amyloid protein, studies have shown that only about 0.1%–0.2% of antibodies can enter brain tissue. Therefore, developing technical strategies that can effectively deliver large molecule drugs to the brain has become one of the core challenges in the field of biotherapy and is expected to promote the clinical translation of treatments for neurological and psychiatric diseases.

[0004] One current research direction is to identify and utilize targets selectively expressed on the surface of brain endothelial cells as a pathway for antibodies to cross the blood-brain barrier. Transferrin receptor (TfR1) is one of the most widely studied receptor-mediated transcellular transport targets. Expressed in brain endothelial cells, TfR1 is a crucial membrane protein regulating intracellular iron transport and can serve as a "transport gate" for antibody drugs to cross the blood-brain barrier. OX26 was the first antibody used to achieve drug crossing of the blood-brain barrier. Since then, numerous antibodies targeting TfR1 have been attempted for the brain delivery of various biomolecules.

[0005] Currently, many pharmaceutical companies are actively developing antibody drugs that cross the blood-brain barrier, generally employing a TfR1-mediated receptor-mediated transcellular transport strategy to improve the delivery efficiency of large molecule drugs in the central nervous system. Among them, Trotinemab (also known as RG6102) is a bifunctional fusion antibody developed by Roche, based on the existing Aβ antibody gantenerumab (originally developed by AC Immune), by fusing a TfR1-targeting Fab domain to the C-terminus of one of its heavy chains. In multiple preclinical animal models and early clinical studies, Trotinemab has shown higher brain tissue drug concentrations and more uniform brain distribution characteristics compared to its parent antibody gantenerumab, suggesting enhanced blood-brain barrier delivery capabilities. JCR Pharmaceuticals of Japan has developed a delivery platform called "J-Brain Cargo," which achieves effective drug crossing of the blood-brain barrier via cerebral vascular endothelial cells and delivery to brain tissue by fusing therapeutic proteins or peptides with TfR1-targeting antibodies. JR141 (trade name IZCARGO), a representative drug based on this platform, is a fusion protein of a TfR1 antibody and idurosidase, and has been approved for the treatment of mucopolysaccharidosis type II (Hunter's syndrome). Clinical studies have shown that the drug has good intracerebral delivery capabilities; Phase II / III trial results showed that it met the pre-specified primary endpoint and demonstrated good data support in terms of efficacy, safety, and tolerability.

[0006] Given the many limitations of existing anti-TfR1 antibodies in terms of their ability to cross the blood-brain barrier, target specificity, safety, and cross-species reactivity, there is an urgent need to develop more alternative anti-transferrin receptor 1 antibodies as drug delivery tools to cross the blood-brain barrier, thereby promoting the development and clinical translation of drugs for the treatment of neurological diseases.

[0007] This invention develops a human anti-TfR1 antibody derived from the immune screening of fully human antibody transgenic animals, requiring no subsequent humanization modification and exhibiting lower immunogenicity. This antibody demonstrates specific binding activity to TfR1 in both humans and cynomolgus monkeys, does not interfere with the binding of transferrin (Tf) to TfR1, and can penetrate the blood-brain barrier by binding to TfR1, making it suitable for cross-blood-brain barrier drug delivery. Summary of the Invention

[0008] Therefore, the purpose of this invention is to provide an antibody against human transferrin receptor 1 or its antigen-binding fragment and its applications. This invention develops a human anti-TfR1 antibody derived from fully human antibody transgenic animal immunoassay, requiring no subsequent humanization and exhibiting lower immunogenicity. This antibody has specific binding activity against TfR1 in humans and cynomolgus monkeys, does not interfere with the binding of transferrin (Tf) to TfR1, and can penetrate the blood-brain barrier by binding to TfR1, making it suitable for cross-blood-brain barrier drug delivery.

[0009] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides an antibody against human transferrin receptor 1 or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is selected from any one of the following: Antibody Ab116 is an antibody containing a light chain variable region and a heavy chain variable region. The light chain variable region contains a light chain CDR1 with the amino acid sequence QSISIY, a light chain CDR2 with the amino acid sequence AAS, and a light chain CDR3 with the amino acid sequence CQQTYSSPPWTF. The heavy chain variable region contains a heavy chain CDR1 with the amino acid sequence GFIVSSNY, a heavy chain CDR2 with the amino acid sequence IYSGGST, and a heavy chain CDR3 with the amino acid sequence CASHSFGYGNWFDPW. or, Antibody Ab274 is an antibody containing a light chain variable region and a heavy chain variable region. The light chain variable region contains light chain CDR1 with the amino acid sequence QDIYIY, light chain CDR2 with the amino acid sequence AAS, and light chain CDR3 with the amino acid sequence CQQSFSTPLTF. The heavy chain variable region contains heavy chain CDR1 with the amino acid sequence GFTFSRYG, heavy chain CDR2 with the amino acid sequence ISYDGSNK, and heavy chain CDR3 with the amino acid sequence CAKDRGDILYYMDVW.

[0010] The amino acid sequence of the heavy chain variable region of the antibody Ab116 is shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.13.

[0011] The amino acid sequence of the heavy chain variable region of the antibody Ab274 is shown in SEQ ID NO.14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.15.

[0012] The nucleotide sequence encoding the heavy chain variable region of antibody Ab116 is shown in SEQ ID NO.20, and the nucleotide sequence encoding the light chain variable region of antibody Ab116 is shown in SEQ ID NO.21.

[0013] The nucleotide sequence encoding the heavy chain variable region of antibody Ab274 is shown in SEQ ID NO.22, and the nucleotide sequence encoding the light chain variable region of antibody Ab274 is shown in SEQ ID NO.23.

[0014] The antibody also includes a light chain constant region and a heavy chain constant region; As one embodiment of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.16; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.18.

[0015] As another embodiment of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.24; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.18.

[0016] The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO.5.

[0017] Secondly, the present invention provides a nucleic acid molecule that encodes an antibody against human transferrin receptor 1 or an antigen-binding fragment thereof as described above.

[0018] Thirdly, the present invention provides a recombinant expression vector containing the nucleic acid molecule.

[0019] The recombinant expression vector is a transposon vector, a lentiviral vector, or an adeno-associated virus vector. Optionally, the transposon vector is a PiggyBac transposon donor vector.

[0020] Fourthly, the present invention provides a drug delivery carrier containing an antibody against human transferrin receptor 1 or an antigen-binding fragment thereof as described above.

[0021] As one embodiment of the present invention, the drug delivery carrier comprises: (a) An antibody against human transferrin receptor 1 or an antigen-binding fragment thereof as described above; and (b) Heterogeneous loads; The antibody or its antigen-binding fragment is connected to the heterologous payload via chemical coupling or gene fusion. Furthermore, the drug delivery carrier is used to deliver the heterologous payload to the brain across the blood-brain barrier.

[0022] The heterologous payload is selected from therapeutic proteins, peptides, nucleic acid drugs, enzymes, or nanoparticles; preferably, the therapeutic protein is an anti-Alzheimer's disease antibody or a neurotrophic factor.

[0023] Fifthly, the present invention provides a pharmaceutical composition comprising the antibody against human transferrin receptor 1 or an antigen-binding fragment thereof, or the drug delivery carrier as described above, and pharmaceutically acceptable excipients.

[0024] The application of the antibody against human transferrin receptor 1 or its antigen-binding fragment, or the drug delivery carrier, in the preparation of tools for delivering drugs or diagnostic reagents across the blood-brain barrier is also within the scope of protection of this invention.

[0025] The application of the antibody against human transferrin receptor 1 or its antigen-binding fragment in the preparation of a kit for detecting transferrin receptor 1 is also within the scope of protection of this invention.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. In this invention, the anti-human TfR1 monoclonal antibody is produced by immunizing fully humanized antibody mice. Using recombinantly expressed hTfR1-His (a near-shore protein of human TfR1 with an amino-terminal histidine fusion tag, catalog number CU75) in eukaryotic cells as the antigen, an immune library is used for screening to obtain a specific monoclonal antibody against the target antigen. This antibody can penetrate the blood-brain barrier by binding to TfR1 and can be used for cross-blood-brain barrier drug delivery.

[0027] 2. The antibody of the present invention has specific binding activity to hTfR1 and does not interfere with the binding of transferrin (Tf) to TfR1, and can be used as a drug delivery carrier.

[0028] 3. This invention has screened out a highly specific human anti-transferrin receptor 1 protein antibody, which has good affinity for human anti-transferrin receptor 1. Therefore, this antibody can be used to determine the content of human anti-transferrin receptor 1 in a detection system. Attached Figure Description

[0029] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The graph shows the results of the antibody binding activity assay with K562 cells; Figure 2 The image shows the results of the antibody blocking Tf-TfR1 binding activity assay. Figure 3 Antibody concentrations in the serum of hTfR1 mice after intravenous (iv) administration of different antibodies; Figure 4 Antibody concentrations in the whole brain of hTfR1 mice after intravenous (iv) administration of different antibodies; Figure 5The ratio of antibody concentration in total brain protein to serum antibody concentration in hTfR1 mice after intravenous (iv) administration of different antibodies; Figure 6 Figure showing the results of the binding activity assay of the modified antibody to K562 cells; Figure 7 Antibody concentrations in serum of hTfR1 / 5XFAD mice after intravenous (iv) administration of different antibodies; Figure 8 Antibody concentrations in the whole brain of hTfR1 / 5XFAD mice after intravenous (iv) administration of different antibodies; Figure 9 Antibody concentrations in the brain parenchyma of hTfR1 mice after intravenous (iv) administration of different antibodies; Figure 10 A map of the PiggyBac transposon donor vector; Figure 11 The image shows the results of the antibody binding activity assay with CHOK1-cynoCD71 cells. Detailed Implementation

[0030] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0031] Example 1: Generation of humanized anti-human TfR1 monoclonal antibody This embodiment aims to obtain a fully humanized antibody that specifically binds to hTfR1 (NM_001128148.3). The specific operation procedure includes: immunizing mice with the fully humanized antibody, screening and obtaining the antibody variable region sequence of antigen-specific B cells, fusing the variable region sequence with the human constant region gene to construct an expression vector, expressing and purifying the fully humanized antibody in a eukaryotic system, and optionally engineering the Fc region of the antibody to optimize its functional characteristics. The specific steps include the following: 1. Mouse immunization Six-week-old female fully humanized antibody mice (Smocmab, Nanmo Biotechnology) were used as immunization subjects, and hTfR1-His (a human TfR1 extracellular domain fused with histidine at the amino terminus, nearshore protein, catalog number CU75) recombinantly expressed in eukaryotic cells was used as the antigen.

[0032] First immunization: The antigen protein (50 µg) was fully emulsified with an equal volume of adjuvant using Freund's complete adjuvant before subcutaneous immunization. Booster immunization: On days 14 and 28 after the first immunization, the antigen protein (25 µg each time) was emulsified with an equal volume of adjuvant using Freund's incomplete adjuvant and then administered as a second and third immunization.

[0033] Before immunization, blood was collected from behind the eye socket as a negative control; 7 days after the third immunization, blood was collected to verify the immunization effect; the serum anti-human TfR1 antibody titer reached 10. 5 After completing the above, you can proceed to the next experimental step.

[0034] 2. Antigen-specific plasma B cell sorting and antibody sequence acquisition First, CD138 was enriched from mouse spleens using EasySep™ magnetic bead sorting technology. + Plasma B cells were used to obtain a high-purity antibody-secreting cell population. Subsequently, the target cells were automatically captured and arrayed using the Hypercell single-cell optofluidic platform, and then cultured in situ within a microchamber for antigen-specific screening. Positive cells were identified through real-time imaging and recovered non-contactly. Finally, the recovered target single cells were used in a 10x Genomics single-cell sequencing platform to construct a single-cell sequencing library containing V(D)J information using water-in-oil droplet technology, combined with 5′ gene expression sequencing, to simultaneously obtain the naturally paired antibody heavy and light chain variable region sequences of the target plasma B cells.

[0035] Sequencing data were analyzed, and the most promising antibody clones were screened based on sequence uniqueness, abundance, and CDR3 diversity, and named Ab116 and Ab274, respectively. Bioinformatics analysis determined the gene sequences and deduced amino acid sequences of their respective heavy chain variable regions (VH) and light chain variable regions (VL).

[0036] Table 1

[0037] Antibody Ab116: The amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 12.

[0038] The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO. 13.

[0039] Antibody Ab274: The amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 14.

[0040] The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO. 15.

[0041] 3. Construction and expression of fully humanized antibody recombinant expression vector The heavy chain and light chain sequences of the above antibodies were respectively linked to the human IgG1 constant region sequence to construct complete heavy chain and light chain genes.

[0042] The heavy chain uses the human IgG1 subtype constant region (amino acid sequence as shown in SEQ ID NO.16, and the codon-optimized gene sequence as shown in SEQ ID NO.17). The light chain uses the human κ light chain constant region (amino acid sequence as shown in SEQ ID NO.18, and the codon-optimized gene sequence as shown in SEQ ID NO.19).

[0043] The VH and VL amino acid sequences (SEQ ID NO. 12-15) of the above antibodies were codon-optimized to adapt them for efficient expression in human cells (such as HEK293); the optimized variable region DNA sequences are as follows: The VH nucleotide sequence of Ab116 is shown in SEQ ID NO.20, and the VL nucleotide sequence is shown in SEQ ID NO.21; The VH nucleotide sequence of Ab274 is shown in SEQ ID NO.22, and the VL nucleotide sequence is shown in SEQ ID NO.23.

[0044] The above VH sequence was fused within the framework to the human IgG1 heavy chain constant region gene (SEQ ID NO.17) to obtain the complete heavy chain gene: Ab116 heavy chain gene: SEQ ID NO.20 + SEQ ID NO.17 Ab274 heavy chain gene: SEQ ID NO.22 + SEQ ID NO.17; The above VL sequences were fused within the framework with the human κ light chain constant region gene (SEQ ID NO.19) to obtain the complete light chain gene: Ab116 light chain gene: SEQ ID NO.21 + SEQ ID NO.19; Ab274 light chain gene: SEQ ID NO.23 + SEQ ID NO.19.

[0045] Example 2: Construction, expression, and purification of recombinant antibodies This embodiment details the preparation process of recombinant antibodies against target antigens (Ab274 and Ab116), specifically including the construction of expression vectors, preparation of recombinant plasmids, transient transfection expression in mammalian cells, and affinity purification of antibodies.

[0046] 1. Construction of expression vectors and preparation of recombinant plasmids (1) Construction of Ab274 expression vector: The heavy chain gene of Ab274 (Ab274-hIgG1-H) was synthesized, and an Xba I restriction site was added to its 5′ end. This gene was then cloned into the expression vector Pcdna3.4-hIgG1 CH-293 (plasmid derived from Genewiz) (containing ampicillin resistance) using homologous recombination to construct the Ab274 heavy chain expression plasmid (named pAb274-H). The light chain gene of Ab274 (Ab274-LC) was cloned into the vector pUC-GW-Kan (plasmid derived from Genewiz) to construct the Ab274 light chain expression plasmid (named pAb274-L).

[0047] The heavy chain gene of Ab274 (Ab274-hIgG1-H) is formed by the fusion of SEQ ID NO. 22 (VH nucleotide sequence) and SEQ ID NO. 17 (human IgG1 heavy chain constant region nucleotide sequence).

[0048] The Ab274 light chain gene (Ab274-LC) is formed by the fusion of SEQ ID NO. 23 (VL nucleotide sequence) and SEQ ID NO. 19 (human κ light chain constant region nucleotide sequence).

[0049] (2) Construction of Ab116 expression vector: The heavy chain gene of Ab116 (Ab116-hIgG1-H) was synthesized, and an Xba I restriction site was added to its 5′ end. This gene was then cloned into the expression vector Pcdna3.4-hIgG1 CH-293 (containing ampicillin resistance) using homologous recombination to construct the Ab116 heavy chain expression plasmid (named pAb116-H). The light chain gene of Ab116 (Ab116-LC) was cloned into the vector pUC-GW-Kan to construct the Ab116 light chain expression plasmid (named pAb116-L).

[0050] Among them, the heavy chain gene of Ab116 (Ab116-hIgG1-H) is formed by fusing a codon-optimized heavy chain variable region nucleotide sequence (SEQ ID NO.20) with a human IgG1 heavy chain constant region sequence (SEQ ID NO.17).

[0051] The light chain gene (Ab116-LC) is formed by the fusion of SEQ ID NO. 21 (VL nucleotide sequence) and SEQ ID NO. 19 (human κ light chain constant region nucleotide sequence).

[0052] After construction, we transformed the recombinant plasmid into competent Escherichia coli (DH5α), screened for positive clones on LB plates containing ampicillin, and verified the correctness of the clones by colony PCR and Sanger sequencing. Finally, we prepared a sufficient amount of mini-scale recombinant plasmid DNA, which was then used to transfect host cells.

[0053] 2. Transient expression and purification of recombinant antibodies in mammalian cells The corresponding heavy chain expression plasmids and light chain expression plasmids were respectively suspended in Opti-MEM medium, and the transfection reagent polyethyleneimine (PEI, Sigma) was added. The specific transfection combinations are as follows: For the Ab116 antibody, the Ab116 heavy chain expression plasmid (pAb116-H) and the Ab116 light chain expression plasmid (Ab116-L) were mixed in an equimolar ratio and then co-transfected. For the Ab274 antibody, the Ab274 heavy chain expression plasmid (pAb274-H) and the Ab274 light chain expression plasmid (pAb274-L) were mixed in an equimolar ratio and then co-transfected.

[0054] Each transfection mixture was co-transfected into the logarithmic growth phase human embryonic kidney cell line HEK293. The cells were cultured for 7 days post-transfection. The culture supernatants were collected and purified by affinity chromatography using Protein A resin to obtain high-purity recombinant antibodies Ab116 and Ab274 for subsequent biological function assays.

[0055] Example 3: Detection of binding affinity of anti-TfR1 antibody 1. ELISA test The binding affinity of anti-TfR1 antibody to His-labeled human TfR1 protein (hTfR1-His, ACROBiosystems, catalog number: CD1-H5243) was verified using ELISA. PBS-diluted hTfR1-His was added to each well of a 96-well ELISA plate at a concentration of 1 μg / mL × 100 µL / well and incubated overnight at 4°C. The ELISA plate was blocked with PBS containing 3% BSA, and the purified antibody sample was diluted with PBST containing 1% BSA and blocked at 37°C for 2 h. Antibody was added to each well at a concentration of 100 µL / well and incubated at 37°C for 1 h. The ELISA plate was washed, and HRP-labeled goat anti-human IgG antibody was diluted 1 / 10000 with PBST containing 2.5% skim milk and added to each well at a concentration of 100 µL / well. The plate was incubated at 37°C for 1 h. Wash the ELISA plate, add 100 µL of TMB chromogenic solution to each well, and incubate at room temperature for 10–15 min before stopping the reaction. Read the absorbance at 450 nm using a microplate reader (595 nm as a control), and plot the antibody concentration-absorbance curve using Graphpad Prism Software 9.0. Monoclonal antibodies with affinity levels in the nanomolar (nM) range were obtained by affinity ELISA. Figure 1 As shown in the figure, the results indicate that the screened antibodies have good specific recognition and binding ability to human TfR1 protein.

[0056] 2. Flow cytometry detection Flow cytometry was used to detect the binding activity of anti-TfR1 antibody to the human myeloid leukemia cell line K562 (which highly expresses human TfR1 on its surface). K562 cells were cultured at 1 × 10⁶ cells per well. 6 Cells were resuspended at a density of 1,000 mcg in PBS containing 2% fetal bovine serum and aliquoted into 96-well U-shaped cell culture plates. After centrifugation and discarding the supernatant, serially diluted test antibodies were added, and the cells were incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with PBS containing 2% fetal bovine serum to remove unbound antibodies. Fluorescein-labeled goat anti-human IgG secondary antibody was added, and the cells were incubated at 4°C in the dark for 20 minutes. After washing again, the cells were resuspended in fixation buffer and analyzed using flow cytometry. The antibody binding activity was assessed by analyzing the relationship between antibody concentration and mean fluorescence intensity using GraphPadPrism 9.0 software to fit a curve. Figure 1 The results showed that the antibody could specifically bind to the native human TfR1 on the surface of K562 cells with nanomolar affinity, confirming its good biological activity.

[0057] Example 4: Detection of crossover between antibody-binding epitopes and human transferrin-binding epitopes To investigate the competitive binding relationship between the target anti-human transferrin receptor 1 monoclonal antibody (Anti-hTfR1 mAb) and its endogenous ligand human transferrin (hTf) to hTfR1 on the surface of K562 cells, a flow cytometry-based competitive binding assay was performed. K562 cells were collected and adjusted to a concentration of 2 × 10⁻⁶ cells. 6 / mL, take 1×10 per dose 5 Cells were collected in flow cytometry tubes. First, cells were co-incubated with a series of 5-fold serially diluted Anti-hTfR1 mAb at 4°C for 30 minutes. Then, without washing, pre-optimized concentrations of biotin-labeled hTf (Biotin-hTf) were added, and incubation continued at 4°C in the dark for another 30 minutes. After incubation, cells were washed twice with PBS containing 1% BSA, and then stained with fluorescein-labeled streptavidin at 4°C in the dark for 30 minutes. After a final wash, flow cytometry was used to analyze the average fluorescence intensity of the corresponding fluorescence channel of Biotin-hTf to assess the inhibitory effect of the antibody on hTf binding. The positive control was JR-141 antibody, a humanized anti-human transferrin receptor (CD71; TfR) antibody. Figure 2 The results showed that the candidate antibody at any concentration did not interfere with the binding of Tf to TfR1, and the candidate antibody could be identified as a non-blocking antibody, and its binding epitope did not overlap with the Tf binding epitope.

[0058] Example 5: Functional activity of antibodies penetrating the blood-brain barrier A humanized TfR1 mouse model (hTfR1 mouse, NM-HU-215059, Nanmo Biotechnology) was engineered to express chimeric TfR1 protein (NM_001128148.3), in which the extracellular region of the mouse TfR1 protein was replaced by the corresponding human TfR1 extracellular region. This experiment aimed to detect the drug exposure levels in serum and brain tissue of hTfR1 gene-humanized mice after a single tail vein administration of the test antibody. Ten-week-old hTfR1 mice were used in the experiment and housed in an AAALAC-certified SPF facility. Animals were randomly divided into three groups according to body weight and received a single intravenous injection of 10 mg / kg of Ab274, JR141 antibody, and isotype control antibody, respectively. Serum was collected at 4, 8, and 24 hours after administration; 24 hours after administration, the left brain tissue was dissected after cardiac perfusion with physiological saline. The brain tissue was weighed and directly homogenized for the determination of whole-brain drug concentration. The concentrations of the target antibody drug in serum and brain tissue homogenates were quantitatively analyzed using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).

[0059] The specific method is as follows: Goat anti-human IgG (H+L) capture antibody was diluted to 2 μg / mL using carbonate buffer and coated onto an ELISA plate, then incubated overnight at 4°C. After washing, the plate was blocked at 37°C for 2 hours with 2% BSA / PBST. Serum standards and control samples were serially diluted with blank mixed serum; brain tissue samples and corresponding standards were prepared using 1% BSA / PBS as the matrix. After washing, the test samples and standard curve samples were added separately and incubated at 37°C for 1 hour. After washing, horseradish peroxidase-labeled goat anti-human IgG detection antibody diluted 1:50000 with 1% BSA / PBS was added and incubated at 37°C for 1 hour. After thorough washing, TMB substrate was added for color development, and the reaction was terminated with stop solution. Absorbance values ​​were read at 450 nm and 630 nm. Finally, the actual concentration of the test antibody in each sample was calculated based on a four-parameter logarithmic fitting model of the standard curve. Figure 3 The antibody concentrations in the serum of hTfR1 mice after intravenous (iv) administration of different antibodies were measured. Figure 4 Antibody concentrations in the whole brain of hTfR1 mice after intravenous (iv) administration of different antibodies; Figure 5 The ratio of antibody concentration in total brain protein to serum antibody concentration in hTfR1 mice after intravenous (iv) administration of different antibodies was studied. The results showed that, following a single tail vein administration, antibodies capable of binding to hTfR were all detectably and stably exposed in serum, exhibiting typical kinetic characteristics over time. More importantly, these antibodies also demonstrated significant distribution ability in brain tissue, with significantly higher intrabrain drug concentrations and brain tissue / serum distribution ratios compared to control antibodies. These data demonstrate that antibodies targeting hTfR possess highly efficient blood-brain barrier penetration capabilities.

[0060] Example 6: Construction, expression, and binding affinity verification of the Fc mutant antibody (N297A) To further reduce the Fc effector function of the antibody, site-directed mutagenesis was performed on the obtained antibody in the Fc region. The asparagine (N) codon at position 297 of the CH2 domain of the human IgG1 heavy chain constant region was mutated to an alanine codon (N297A mutation). The amino acid sequence of the mutated heavy chain constant region is shown in SEQ ID NO.24, and the optimized gene sequence of the mutated heavy chain constant region codon is shown in SEQ ID NO.5, to eliminate its binding ability to the Fcγ receptor and complement protein C1q. The specific steps are as follows: 1. Construction and expression of mutant plasmids: The codon (AAC) of asparagine (N) at position 297 of the CH2 domain of the human IgG1 heavy chain constant region was mutated to the codon (GCC) of alanine (A), and the N297A mutation was introduced to obtain heavy chain gene fragments containing the N297A mutation (NM-G3202-Ab116-H-N297A; NM-G3202-Ab274-H-N297A).

[0061] The mutated heavy chain gene fragment (NM-G3202-Ab116-H-N297A or NM-G3202-Ab274-H-N297A) and the parental antibody heavy chain plasmids pAb116-H and pAb274-H constructed in Example 2 were digested with restriction endonuclease Xba I. After purification by agarose gel electrophoresis, they were directionally ligated using T4 DNA ligase to construct heavy chain expression plasmids pAb116-H(N297A) and pAb274-H(N297A) containing the N297A mutation, respectively.

[0062] After construction, the recombinant plasmid was transformed into competent E. coli, such as DH5α. Positive clones were screened on LB agar plates containing ampicillin, and the correctness of the mutation sites was further verified by colony PCR and Sanger sequencing. After confirmation, sufficient plasmid DNA was prepared for subsequent transfection.

[0063] 2. Transient expression and purification of mutant antibodies in mammalian cells The verified mutant heavy chain plasmid was mixed with the corresponding wild-type light chain plasmid in an equimolar ratio; the wild-type light chain expression plasmids were the recombinant plasmids pAb116-L and pAb274-L constructed in Example 2; the specific combination is as follows: For Ab116-N297A antibody: mix pAb116-H(N297A) and pAb116-L in an equimolar ratio; For Ab274-N297A antibody: mix pAb274-H(N297A) and pAb274-L in an equimolar ratio.

[0064] The mixed plasmids were suspended in Opti-MEM medium, and polyethyleneimine (PEI) transfection reagent was added for co-transfection at a mass ratio (e.g., PEI:DNA = 3:1). The transfection system was then added to HEK293 cells pre-cultured to the logarithmic growth phase and cultured for another 7 days. The supernatant was collected, and cell debris was removed by centrifugation. The cells were then purified by affinity chromatography using Protein A agarose resin. The purified antibodies were desalted by dialysis or ultrafiltration to obtain high-purity N297A mutant antibodies Ab116-N297A and Ab274-N297A.

[0065] Activity verification: The flow cytometry method described in Example 3 was used to detect the binding activity of the mutant antibody with K562 cells.

[0066] like Figure 6 As shown, the antibody modified with the N297A mutation still maintains a high binding affinity for human TfR1.

[0067] Example 7: Functional activity of Fc mutant antibody (N297A) in penetrating the blood-brain barrier This embodiment aims to detect the drug exposure levels in serum and brain tissue of hTfR1 / 5XFAD gene-humanized mice (NM-HU-234838, Nanmo Biotechnology) after a single tail vein administration of Fc mutant antibody (N297A).

[0068] Ten-week-old hTfR1 / 5XFAD mice were used in the experiment and housed in an AAALAC-certified SPF facility. Animals were randomly divided into three groups according to body weight, receiving a single intravenous injection of 10 mg / kg of Ab274-N297A, Ab116-N297A, Trotinemab (a known anti-hTfR1 antibody capable of penetrating the blood-brain barrier, used as a positive control), and an isotype control antibody (human IgG1 isotype control, which does not recognize any target antigen, used as a negative control). Serum was collected at 4, 8, and 24 hours after administration. Twenty-four hours after administration, the left brain tissue was dissected after cardiac perfusion with saline. The brain tissue was weighed and directly homogenized for determining the whole-brain drug concentration. 30% dextran solution and the remaining brain homogenate (volume ratio 1:1) were added sequentially to 2 ml centrifuge tubes, mixed, and centrifuged (4℃, 5400g, 15 min). The upper and middle layers were collected and mixed again. Take 450 µL of the mixed liquid, add 50 µL of 5% Triton X-100, mix well, and lyse on ice for 15 min. Centrifuge (4℃, 10000g, 10 min), and collect the supernatant. One portion is used for BCA detection of total protein, and the remaining supernatant is divided into two tubes and stored at -80℃ for subsequent detection of brain parenchymal drug concentration. The concentration of the target antibody drug in serum, whole brain, and brain parenchymal samples was detected by double-antibody sandwich ELISA. The concentration of the target antibody drug in serum and brain tissue homogenate was quantitatively analyzed using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: Dilute goat anti-human IgG (H+L) capture antibody to 2 μg / mL with carbonate buffer, coat it onto an ELISA plate, and incubate overnight at 4℃. After washing, block with 2% BSA / PBST at 37℃ for 2 hours. Serum standards and control samples were serially diluted with blank mixed serum; brain tissue samples (whole brain or devascularized brain parenchyma homogenate) and corresponding standards were prepared using 1% BSA / PBS as the matrix. After washing the plates, the test samples and standard curve samples were added separately and incubated at 37°C for 1 hour. After washing the plates, horseradish peroxidase-labeled goat anti-human IgG detection antibody diluted 1:50000 with 1% BSA / PBS was added and incubated at 37°C for 1 hour. After thorough washing, TMB substrate was added for color development, and the reaction was terminated with stop solution. Absorbance values ​​were read at 450 nm (detection wavelength) and 630 nm (reference wavelength). Finally, the actual concentration of the test antibody in each sample was calculated based on a four-parameter logarithmic fitting model of the standard curve.

[0069] Figure 7 Antibody concentrations in serum of hTfR1 / 5XFAD mice after intravenous (iv) administration of different antibodies; Figure 8 Antibody concentrations in the whole brain of hTfR1 / 5XFAD mice after intravenous (iv) administration of different antibodies; Figure 9The antibody concentrations in the brain parenchyma of hTfR1 mice after intravenous (iv) administration of different antibodies were measured. Results showed that, following a single tail vein administration, antibodies binding to the human transferrin receptor (hTfR) exhibited stable exposure in serum and displayed typical kinetic characteristics over time. More importantly, these antibodies also demonstrated significant distribution ability in brain tissue, with both whole-brain and brain parenchymal drug concentrations superior to the control antibody. These data demonstrate that both candidate antibodies, Ab116-N297A and Ab274-N297A, can effectively penetrate the blood-brain barrier.

[0070] Example 8: Cross-species binding ability of antibodies This embodiment aims to detect the cross-species binding ability of anti-TfR1 antibodies (Ab116-N297A and Ab274-N297A) to the transferrin receptor (cynoCD71) derived from cynomolgus monkeys. First, a CHOK1 cell line stably expressing cynoCD71 was constructed, and then flow cytometry was used to detect the binding activity of the antibodies to this cell line.

[0071] 1. Construction of a stable CHOK1-cynoCD71 expression cell line Following the methodology of Rajendra et al. (2017) (RAJENDRA Y, PEERY RB, BARNARD GC. Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g / L using the piggyBac transposon system[J]. Biotechnology Progress, 2016, 32(5): 1301-1307. DOI: 10.1002 / btpr.2307.), we constructed a stable CHOK1-cynoCD71 expression cell line using the PiggyBac transposon system. The specific procedure is as follows: 1.1 Construction and Transfection of the PiggyBac Expression Vector First, the transferrin receptor gene (cynoCD71, NP_001244232.1) derived from cynomolgus monkeys was cloned into the PiggyBac transposon donor vector (see diagram). Figure 10In this study, a recombinant transposon donor plasmid was constructed. Subsequently, this donor plasmid and an auxiliary plasmid encoding the PiggyBac transposase (pCMV-hyPBase) were co-transfected into CHOK1 cells at a 9:1 mass ratio using electroporation. After electroporation, the cells were placed in antibiotic-free cell culture medium and incubated at 37°C. Forty-eight hours post-transfection, the medium was replaced with puromycin-containing cell culture medium. After transient expression of the transposase in the cells, it recognized the inverted terminal repeats (ITRs) at both ends of the donor plasmid and efficiently integrated the cynoCD71 expression cassette into the CHOK1 cell genome via a "cut-paste" mechanism.

[0072] 1.2 Selection of stably transfected cells Forty-eight hours after transfection, puromycin was added to the culture medium for positive selection, which continued for about 10–14 days. The selection medium was changed every 3–4 days until the cell viability recovered to more than 90% to remove cells that had not integrated the foreign gene and obtain a stable transduced mixed cell population.

[0073] 1.3 Screening and Identification of Monoclonal Cells The mixed cell population was isolated into single clones using a limiting dilution method. Single cells were seeded into 96-well plates and cultured to expand into single-clonal cell lines. Flow cytometry (FACS) was used to screen and identify the single-clonal cell lines, and the expression level of cynoCD71 protein was detected. Finally, a stable single-clonal cell line with high cynoCD71 protein expression and stable growth was obtained and named CHOK1-cynoCD71.

[0074] 1.4 Flow cytometry detection of antibody binding activity to CHOK1-cynoCD71 The binding activity of anti-TfR1 antibody to CHOK1-cynoCD71 was detected by flow cytometry; the specific steps are as follows: CHOK1-cynoCD71 cells were added at a rate of 1 × 10⁻⁶ per well. 6Cells were resuspended at a density of 1,000 mcg in PBS containing 2% fetal bovine serum (FBS) and aliquoted into 96-well U-shaped cell culture plates. After centrifugation and discarding the supernatant, serially diluted working solutions of the test antibodies (Ab116-N297A and Ab274-N297A) were added, and the cells were incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with PBS containing 2% FBS to remove unbound antibodies. Fluorescein-labeled goat anti-human IgG secondary antibody was added, and the cells were incubated at 4°C in the dark for 20 minutes. After washing again, the cells were resuspended in fixation buffer (Biolegend, 424401) and analyzed by flow cytometry. The antibody binding activity was assessed by analyzing the relationship between antibody concentration and mean fluorescence intensity using GraphPad Prism 9.0 software to fit a curve. Figure 11 The results showed that the Ab116-N297A and Ab274-N297A antibodies could specifically bind to the native conformation of monkey TfR1 on the surface of CHOK1-cynoCD71 cells with nanomolar affinity, confirming their good biological activity.

[0075] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An antibody against human transferrin receptor 1 or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment is selected from any of the following: Antibody Ab116 is an antibody containing a light chain variable region and a heavy chain variable region. The light chain variable region contains a light chain CDR1 with the amino acid sequence QSISIY, a light chain CDR2 with the amino acid sequence AAS, and a light chain CDR3 with the amino acid sequence CQQTYSSPPWTF. The heavy chain variable region contains a heavy chain CDR1 with the amino acid sequence GFIVSSNY, a heavy chain CDR2 with the amino acid sequence IYSGGST, and a heavy chain CDR3 with the amino acid sequence CASHSFGYGNWFDPW. or, Antibody Ab274 is an antibody containing a light chain variable region and a heavy chain variable region. The light chain variable region contains light chain CDR1 with the amino acid sequence QDIYIY, light chain CDR2 with the amino acid sequence AAS, and light chain CDR3 with the amino acid sequence CQQSFSTPLTF. The heavy chain variable region contains heavy chain CDR1 with the amino acid sequence GFTFSRYG, heavy chain CDR2 with the amino acid sequence ISYDGSNK, and heavy chain CDR3 with the amino acid sequence CAKDRGDILYYMDVW.

2. The antibody against human transferrin receptor 1 or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody Ab116 is shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

13. And / or, the amino acid sequence of the heavy chain variable region of the antibody Ab274 is shown in SEQ ID NO.14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

15.

3. The antibody against human transferrin receptor 1 or its antigen-binding fragment according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of antibody Ab116 is shown in SEQ ID NO.20, and the nucleotide sequence encoding the light chain variable region of antibody Ab116 is shown in SEQ ID NO.

21. And / or, the nucleotide sequence encoding the heavy chain variable region of antibody Ab274 is shown in SEQ ID NO.22, and the nucleotide sequence encoding the light chain variable region of antibody Ab274 is shown in SEQ ID NO.

23.

4. The antibody against human transferrin receptor 1 or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody also includes a light chain constant region and a heavy chain constant region; The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.16, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

18.

5. The antibody against human transferrin receptor 1 or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody also includes a light chain constant region and a heavy chain constant region; The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.24, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

18.

6. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody against human transferrin receptor 1 as described in any one of claims 1 to 5, or its antigen-binding fragment.

7. A recombinant expression vector comprising the nucleic acid molecule of claim 6.

8. A drug delivery carrier, characterized in that, An antibody or antigen-binding fragment thereof containing the anti-human transferrin receptor 1 as described in any one of claims 1 to 5.

9. A pharmaceutical composition, characterized in that, The drug contains an antibody against human transferrin receptor 1 as described in any one of claims 1 to 5 or an antigen-binding fragment thereof, or a drug delivery carrier as described in claim 8, and pharmaceutically acceptable excipients.

10. The use of the antibody against human transferrin receptor 1 as described in any one of claims 1 to 5 or its antigen-binding fragment, or the drug delivery carrier as described in claim 8, in the preparation of a tool for delivering drugs or diagnostic reagents across the blood-brain barrier.

11. The use of the antibody against human transferrin receptor 1 as described in any one of claims 1 to 5, or the antigen-binding fragment thereof, in the preparation of a kit for detecting transferrin receptor 1.

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