A group of humanized anti-respiratory syncytial virus fusion protein antibodies and their applications

Abstract

This invention discloses a set of humanized anti-respiratory syncytial virus (RSV) fusion protein antibody pairs and their applications, relating to the field of antibody preparation technology. The pair includes a first antibody and a second antibody. The amino acid sequence of the first heavy chain variable region is selected from one of SEQ ID NO:1 to SEQ ID NO:81; the amino acid sequence of the first light chain variable region is selected from one of SEQ ID NO:82 to SEQ ID NO:162; the amino acid sequence of the second heavy chain variable region is selected from one of SEQ ID NO:163 to SEQ ID NO:243; and the amino acid sequence of the second light chain variable region is selected from one of SEQ ID NO:244 to SEQ ID NO:270. This invention solves the problems of existing humanized antibody expression systems being inefficient and unstable, difficult to scale up, and having poor performance of murine antibodies, failing to meet the requirements of RSV detection for highly sensitive, highly specific, and low-cost antibody reagents. By employing a dual-promoter lentiviral vector system and a stable cell line screening strategy, it overcomes the shortcomings of traditional dual-plasmid transient transfection, such as low efficiency and large batch-to-batch variability. Its humanized design reduces the risk of HAMA interference.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation technology, specifically to a set of humanized anti-respiratory syncytial virus fusion protein antibody pairs and their applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is a common respiratory pathogen. RSV is a single-stranded, negative-sense RNA virus with an envelope, primarily existing in two subtypes, A and B. Its F protein is a key target mediating infection and neutralizing antibodies, and it mutates rapidly due to the lack of proofreading function in its RNA polymerase. It is the leading viral pathogen causing acute lower respiratory tract infections (such as bronchiolitis and pneumonia) in children under 5 years old, especially infants under 2 years old. Infection may have long-term effects on children's respiratory health, such as increasing the risk of persistent wheezing or asthma. Currently, there are no specific antiviral drugs or vaccines available for the general population; early identification and accurate diagnosis are crucial for guiding clinical medication.

[0003] Currently, the detection of respiratory syncytial virus (RSV) mainly relies on three types of technologies: molecular detection, antigen detection (such as immunochromatographic test strips), and serological detection. For infants and young children, antigen detection is promising because it is rapid, convenient, and well-tolerated by infants and young children, better meeting their physiological characteristics and diagnostic and treatment needs.

[0004] However, the core raw material for viral antigen detection—monoclonal antibodies—is currently mainly murine antibodies, produced through mouse ascites or hybridoma technology. These antibodies have several inherent drawbacks: First, murine antibodies easily trigger human anti-mouse antibody (HAMA) reactions, potentially leading to false positives or false negatives. Existing solutions rely on large amounts of immunoblockers, which are not only costly but may also introduce batch-to-batch variability, affecting stability. Second, various interfering substances present in mucosal samples may non-specifically bind to the Fc fragment of non-humanized antibodies, thus interfering with the detection signal. Furthermore, batch-to-batch variations in activity and specificity of antibodies produced using hybridoma technology may occur, directly affecting the uniformity and reliability of the kit quality. In addition, current commercially available respiratory syncytial virus (RSV) chromatography detection generally faces a shortage of high-affinity antibodies, while murine antibodies themselves often have limited affinity, resulting in insufficient sensitivity when detecting samples with low viral loads, easily leading to false negatives.

[0005] To address these issues, humanized antibodies have become an important research and development direction. The basic modification approach involves obtaining the complementarity-determining regions (CDRs) of the heavy and light chains of murine antibodies and transplanting them into the framework of human antibodies and the Fc fragment of IgG, thereby significantly reducing immunogenicity and enhancing anti-interference capabilities. However, current methods in this field mostly employ transient expression via dual plasmid co-transfection, lacking efficient and stable expression systems, which poses a key constraint to achieving large-scale production and cost control.

[0006] RSV is one of the leading pathogens causing severe lower respiratory tract infections in infants, the elderly, and immunocompromised individuals worldwide. Its seasonal prevalence and localized outbreaks create a continuous and substantial demand for clinical testing. Therefore, there is an urgent market need to develop highly sensitive, highly specific, and low-cost humanized antibodies to overcome the limitations of murine antibodies and further obtain superior diagnostic reagents.

[0007] Therefore, this application is hereby submitted. Summary of the Invention

[0008] The technical problem to be solved by this invention is that existing humanized antibody expression systems are inefficient and unstable, difficult to mass-produce, and mouse antibodies have poor performance, which cannot meet the requirements of RSV detection for highly sensitive, highly specific, and low-cost antibody reagents. The purpose is to provide a set of humanized anti-respiratory syncytial virus fusion protein antibody pairs and their applications.

[0009] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a pair of humanized anti-respiratory syncytial virus (RSV-N) fusion protein antibodies, comprising a first antibody (Clone-A) and a second antibody (Clone-B). The first antibody comprises a first heavy chain variable region and a second light chain variable region. The amino acid sequence of the first heavy chain variable region is selected from one of SEQ ID NO: 1 to SEQ ID NO: 81. The amino acid sequence of the first light chain variable region is selected from one of SEQ ID NO: 82 to SEQ ID NO: 162. The second antibody comprises a second heavy chain variable region and a second light chain variable region. The amino acid sequence of the second heavy chain variable region is selected from one of SEQ ID NO: 163 to SEQ ID NO: 243. The amino acid sequence of the second light chain variable region is selected from one of SEQ ID NO: 244 to SEQ ID NO: 270.

[0010] In one specific embodiment, the first heavy chain variable region comprises ICDR1, ICDR2, and ICDR3, whose amino acid sequences are shown at positions 26-34, 52-58, and 97-102 of SEQ ID NO: 1 to SEQ ID NO: 81, respectively; the first light chain variable region comprises CDR1′, CDR2′, and CDR3′, whose amino acid sequences are shown at positions 24-38, 54-60, and 93-101 of SEQ ID NO: 82 to SEQ ID NO: 162, respectively, having at least 80%, 85%, 90%, or 95% sequence identity, and the first antibody is capable of specifically binding to RSV-N.

[0011] Specifically, the amino acid sequence of ICDR1 can be GHSITRDYV, GHTITRDYV, or GHAITRDYV.

[0012] The amino acid sequence of ICDR2 can be ISYIGIT, ISYLGIT, or ISYVGIT.

[0013] The amino acid sequence of ICDR3 can be ADGYIA, ADGYIA, ADGYIA, AEGYLA, AEGYLA, AEGYLA, ANGYVA, ANGYVA and ANGYVA.

[0014] The amino acid sequence of CDR1′ can be RASESVDNYGFSLIN, RASESVDNYGFTLIN, RASESVDNYGFALIN, RASETVDNYGFSLIN, RASETVDNYGFTLIN, RASETVDNYGFALIN, RASEAVDNYGFSLIN, RASEAVDNYGFTLIN, RASEAVDNYGFALIN.

[0015] The amino acid sequence of CDR2′ can be AASNIGS, AASSNLGS, or AASNVGS.

[0016] The amino acid sequence of CDR3′ can be QQTKEVPRT, QQTKELPRT, or QQTKEIPRT.

[0017] In one specific embodiment, the second heavy chain variable region comprises IICDR1, IICDR2, and IICDR3, whose amino acid sequences are shown at positions 26-33, 51-58, and 97-103 of SEQ ID NO: 163 to SEQ ID NO: 243, respectively; the second light chain variable region comprises CDR1″, CDR2″, and CDR3″, whose amino acid sequences are shown at positions 27-32, 50-52, and 89-94 of SEQ ID NO: 244 to SEQ ID NO: 270, respectively, having at least 80%, 85%, 90%, or 95% sequence identity, and wherein the second antibody is capable of specifically binding RSV-N.

[0018] Specifically, the amino acid sequence of IICDR1 can be GYTFSDYE, GYTFTDYE, or GYTFADYE.

[0019] The amino acid sequence of IICDR2 can be FRPGSYST, FRPGTYST, or FRPGAYST.

[0020] The amino acid sequence of IICDR3 can be SRRDFAY, SRREFAY, SRRNFAY, TRRDFAY, TRREFAY, TRRNFAY, ARRDFAY, ARREFAY, and ARRNFAY.

[0021] The amino acid sequence of CDR1″ can be QDVTNY, QDLTNY, or QDITNY.

[0022] The amino acid sequence of CDR2″ can be RDN, REN, or RNN.

[0023] The amino acid sequence of CDR3″ can be IQYDEF, LQYDEF, or VQYDEF.

[0024] In one specific embodiment, both the first antibody and the second antibody further comprise a heavy chain constant region and a light chain constant region; wherein the heavy chain constant region is a human IgG1 constant region and the light chain constant region is a human kappa light chain constant region.

[0025] The humanized monoclonal antibody used in this protocol has been validated to have good binding ability and target recognition efficiency. Competitive ELISA testing showed that the key binding parameters IC50 for the antibody and the target antigen were 2.62 ng / mL and 1.553 ng / mL, respectively, indicating high affinity. In cross-reactivity tests, the antibody showed no cross-reactivity with dozens of other common respiratory pathogens (such as influenza virus, adenovirus, and rhinovirus), demonstrating high specificity and effectively avoiding false positives. Through accelerated degradation experiments (such as under high temperature and repeated freeze-thaw cycles), the antibody maintained its activity and structural integrity, exhibiting good stability, which is beneficial for batch-to-batch consistency and long-term storage.

[0026] Secondly, this invention provides a biomaterial selected from: (1) An isolated polynucleotide encoding a first heavy chain variable region and / or a first light chain variable region and a second heavy chain variable region and / or a second light chain variable region as described in any one of claims 2-3; (2) An expression vector comprising the above-mentioned polynucleotides; (3) A host cell containing the above expression vector.

[0027] Thirdly, the present invention provides a method for preparing the above-mentioned humanized anti-respiratory syncytial virus fusion protein antibody pair, comprising the following steps: a) Single B-cell technology for sorting B lymphocytes specifically recognized by RSV-N protein; b) Gene cloning and construction of dual-promoter expression vectors; c) Screening of stable transfected cell lines.

[0028] Specifically, the inventors designed a highly efficient and stable method for producing target humanized monoclonal antibodies. The core of this method lies in obtaining highly specific antibody genes from the source of innate immune memory and achieving their stable expression using genetic engineering and cell engineering techniques. The specific process is as follows: First, B cells were enriched from peripheral blood of donor mice with an immune response background. Memory B lymphocytes capable of specifically recognizing the target antigen were labeled and sorted by flow cytometry using a fluorescently labeled RSV-N protein-specific probe, and then unicellularized. Subsequently, single-cell reverse transcription PCR was used to specifically amplify gene fragments encoding the variable regions (V regions) of the antibody light and heavy chains, thereby preserving their inherent high affinity and specificity at the gene level.

[0029] In the crucial step of constructing stable expression cell lines, this method underwent systematic optimization: Using pCDH-CMV-MCS-EF1-copGFP-T2A-Puro as the backbone, an antibody light chain expression cassette (containing a human IgG signal peptide) was inserted downstream of the CMV promoter, and an antibody heavy chain expression cassette was inserted downstream of the EF1α promoter. A cHS4 insulator sequence was added between the two cassettes to prevent promoter crosstalk. The vector contained a WPRE element to enhance mRNA stability, a cPPT sequence to increase viral titer, and a GFP tag and puromycin resistance selection marker driven by a bicistronic system. The constructed vector was then transfected into 293T cells, and the supernatant was collected after 48 hours. ELISA analysis showed that the antibody specifically reacted with the target antigen. Lentiviral vectors were further packaged and used to infect HEK293F cells, and puromycin was added for selection to obtain stable expression lines. Compared with single-promoter vectors, the dual-promoter design resulted in a light-to-heavy chain expression ratio closer to 1:1, and improved antibody assembly efficiency by approximately 30%. The inducible Tet-On system showed a 200-fold fold induction after the addition of Dox, with extremely low background expression.

[0030] Subsequently, mammalian expression host cells (HEK293 cells) were infected using the aforementioned lentivirus. To screen for cell clones capable of stable, high-level antibody expression, cells were subjected to stress selection in a culture medium containing specific resistance drugs after transfection. This selection strategy is based on the resistance marker gene carried by the vector; only cells that successfully integrated the antibody expression cassette could survive and proliferate. After approximately 2-3 weeks of continuous drug screening and limiting dilution monoclonalization, multiple monoclonal cell lines were obtained. By detecting the antibody expression levels and binding activity in the supernatant of each clone, a master cell bank with good growth status, stable expression, and high antibody activity was finally selected, thus providing a reliable source of seed cells for subsequent large-scale, reproducible production.

[0031] Furthermore, this application provides a strategy for the stable and efficient in vitro expression of this group of humanized antibodies in mammalian cells. To achieve large-scale antibody production, this scheme designs and optimizes a batch / feed culture-based fermentation process. By precisely controlling culture parameters at key points, introducing specific additives, and dynamically adjusting nutrient supply, the final antibody yield and long-term stability are significantly improved.

[0032] The core process of fermentation is as follows: Suitable host cells with high expression are selected, and inoculated and amplified using an optimized serum-free, chemically defined culture medium. During fermentation, efficient expression is achieved through the synergistic control of multiple parameters, including culture temperature, pH, dissolved oxygen level, and additives.

[0033] Fourthly, the present invention provides an immunoassay kit, wherein the kit is an immunochromatographic test strip; the first antibody is labeled with a detectable marker; and the second antibody is immobilized on a detection line of a nitrocellulose membrane.

[0034] In one specific embodiment, the detectable marker is colloidal gold, fluorescent microspheres, or latex microspheres.

[0035] Fifthly, the present invention provides the use of the humanized antibody pair as described above in the preparation of a diagnostic agent for detecting respiratory syncytial virus.

[0036] This application provides specific applications of the humanized antibody combination in in vitro diagnostics. The core of this approach lies in the successful application of the antibody in, but not limited to, the development of colloidal gold immunochromatographic test strips, establishing a rapid and accurate point-of-care testing (POCT) method for detecting respiratory syncytial virus. Clinical performance validation data confirms the advantages of the humanized monoclonal antibody in terms of sensitivity and anti-interference ability during the detection of mucosal tissue samples.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention provides a set of humanized RSV-N specific monoclonal antibodies, which, while retaining high affinity and specificity, reduce the risk of HAMA interference through humanization and improve the anti-interference ability in complex mucosal tissue samples.

[0038] 2. The humanized monoclonal antibody production method provided by this invention overcomes the shortcomings of traditional dual-plasmid transient transfection, such as low efficiency and large batch-to-batch variability, by employing a dual-promoter lentiviral vector system and a stable cell line screening strategy. This method enables stable and continuous antibody expression, laying the foundation for large-scale production.

[0039] 3. The humanized monoclonal antibody provided by this invention is applied to immunochromatographic test strips, which show higher detection sensitivity and lower nonspecific background in comparative tests with clinical samples. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 This is a flow cytometry diagram of single B cell screening in Example 1 of the present invention; Figure 2 This is an agarose gel electrophoresis image of the antibody V region sequence amplification in this invention; Figure 3 This is a schematic diagram of the recombinant antibody vector of the present invention; Figure 4 The following is a performance test of the humanized antibody in Example 4 of the present invention, wherein 4.1: affinity test results; 4.2: specificity test results; 4.3: stability test results; Figure 5 This invention relates to the application of humanized antibodies in immunochromatographic test strips in Example 5, wherein 5.1: sensitivity test; 5.2 anti-interference test; 5.3 different sampling method test. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0042] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0043] Example 1: Obtaining and humanizing mouse monoclonal antibodies 1. Preparation of single-cell suspension from mouse spleen Recombinant protein RSV-N was diluted with physiological saline and emulsified with Freund's complete adjuvant. Balb / c mice were immunized intraperitoneally. On day 14, mice were immunized again using recombinant protein and Freund's incomplete adjuvant in the same manner, for a total of 2-3 immunizations. Seven days after the last immunization, blood was collected from the tail of the mice. Serum was separated and serum titer was detected using an indirect ELISA method. Mice with titers meeting the requirements were given a booster immunization of 100 μg of recombinant protein for two days. The mice were then euthanized, and a single-cell suspension of the spleen was prepared.

[0044] 2. B lymphocyte enrichment 1) Washing the sample: Transfer the mouse spleen single cell suspension to a 15ml centrifuge tube, add an appropriate amount of sterile PBS (containing 2% fetal bovine serum), centrifuge at 1800rpm for 5min, discard the supernatant, and repeat twice; 2) Microscopic counting: After the last wash, each sample was resuspended in 300 μL of PBS (containing 2% fetal bovine serum) and the cells were counted under an optical microscope to avoid excessively high cell suspension concentration; 3) Antibody incubation: Add anti-CD4 and anti-CD8 flow cytometry antibodies at a ratio of 1:50, mix the sample well, and incubate on ice in the dark for 30 minutes. 4) Washing the sample: After incubation, add an appropriate amount of PBS (containing 2% fetal bovine serum), centrifuge at 1800 rpm for 5 min, discard the supernatant, and repeat 3 times; 5) Magnetic bead incubation: Prepare a 200 μL system of biotin-labeled MicroBeads at a ratio of 1:10. Gently pipette to resuspend the sample, mix well, and stain on ice in the dark for 30 min. 6) Washing the sample: After incubation, add an appropriate amount of PBS (containing 2% fetal bovine serum), centrifuge at 1800 rpm for 5 min, discard the supernatant, repeat 3 times, and then resuspend the sample in 500 μL of PBS (containing 2% fetal bovine serum) in a sterile 1.5 mL centrifuge tube. 7) Cell negative selection: Attach the centrifuge tube containing the sample to the magnetic rack, let it stand for 2-3 minutes, aspirate the cell suspension along the other side of the tube wall, and place it in a sterile 15mL centrifuge tube.

[0045] 3. Flow cytometry sorting of individual RSV-N protein-specific memory B lymphocytes (e.g.) Figure 1 (As shown) 1) Blocking: After the B lymphocyte enrichment sample (including non-immunized mice as a control), centrifuge at 1800 rpm for 5 min, discard the supernatant, add 100 μL of PBS containing 5% mouse serum, mix well, and incubate on ice in the dark for 10 min. 2) Add 1 μL of biotin-labeled RSV-N protein directly, mix gently, and incubate at 4°C in the dark for 25 min. 3) Wash the sample: Add an appropriate amount of PBS (containing 2% fetal bovine serum), centrifuge at 1800 rpm for 5 min, discard the supernatant, and repeat 3 times; 4) Surface staining: Cell surface staining was performed using 200 μL of PBS (containing 2% fetal bovine serum). Antibodies were prepared at a ratio of 1:100. The antibody mixture was used to resuspend the samples from step 3. After mixing, the mixture was incubated on ice in the dark for 30 min. The antibodies and fluorescence channels used are as follows:

[0046] 5) Washing the sample: Add PBS (containing 2% fetal bovine serum) to wash the cells, centrifuge at 1800 rpm for 5 min, discard the supernatant, repeat 3 times, then wash once with sterile PBS, and finally resuspend the sample in 500 μL of sterile PBS and store it on ice in the dark for sorting. 6) Prepare cell collection plates: Add cell capture solution to each well of a 96-well plate at a rate of 10 μL, seal with sealing film, briefly detach, and place on ice for later use; 7) Single cell sorting: Debug the flow cytometer to sort live / dead cells. - CD19 + B220 + IgG + CD27 + Streptavidin + A single living cell; 8) Sample preservation: After each 96-well plate is filled, seal it with sealing film, centrifuge it briefly in a horizontal centrifuge, and then store it at -80℃.

[0047] Example 2 Preparation of humanized antibodies 1. Antibody variable region cloning First, total RNA was extracted from antigen-specific memory B cells obtained through sorting, and complementary DNA (cDNA) was synthesized using this RNA as a template via reverse transcription polymerase chain reaction (RT-PCR). Subsequently, using this cDNA as a template, a set of specific primers covering multiple immunoglobulin variable region (V region) families was used to initially amplify the variable region genes of the antibody light and heavy chains, respectively. The amplification products were then analyzed by electrophoresis (e.g.,...). Figure 2 As shown in the figure, primer combinations with single bands and correct size are selected, and then large-scale, high-fidelity PCR amplification is performed to obtain sufficient target gene fragments.

[0048] To facilitate subsequent cloning, the purified PCR product needs to be tailed: 2 U of Taq DNA polymerase is added to the product, and it is incubated at 72°C for 20 minutes to form a prominent adenine (A) tail at the 3' end, thus completing the poly(A) tailing. Subsequently, using TA cloning technology, the tailed product is efficiently ligated to a linearized T vector containing a complementary thymine (T) protrusion using ligase. The ligation product is then transformed into *E. coli* DH5α competent cells, and plasmid introduction is promoted by heat shock.

[0049] To rapidly screen positive clones, the transformed colonies were initially screened by PCR using universal M13 primers. Single colonies with positive PCR results were selected for small-scale culture in a shaking atmosphere, and plasmids were extracted. Finally, the recombinant plasmids were subjected to Sanger double-stranded sequencing to accurately confirm the correctness and integrity of the cloned variable region gene sequence, laying the foundation for subsequent construction of humanized antibody vectors.

[0050] 2. Antibody variable region analysis First, the sequenced nucleotide sequences and their deduced amino acid sequences were submitted to internationally recognized authoritative databases and analytical tools for comparison and annotation. The specific process is as follows: Preliminary analysis was performed using the IgBlast tool from the National Center for Biotechnology Information (NCBI) to identify variable (V), diversity (D), and linker (J) gene fragments in the sequence, clarify their germline gene origin, and determine the location and extent of somatic high-frequency mutations (SHMs). Subsequently, the sequences were imported into the IMGT / V-QUEST database, specifically designed for immunoglobulins and T-cell receptors, for refined analysis. The IMGT database provides a standardized numbering system and detailed domain definitions.

[0051] Through the above analysis, the complete amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) were accurately identified. Specifically, the boundaries and sequences of the three hypervariable loops—CDR1, CDR2, and CDR3—that determine the antibody-antigen binding specificity were accurately delineated in VH and VL (see sequence listing for details); the sequences of the four relatively conserved backbone regions (FR1-FR4) surrounding the CDR were determined. Simultaneously, using the human antibody sequence with the highest homology to the V region sequence of the parental mouse antibody as a candidate template, the CDR region of the mouse antibody was directly transplanted onto the selected FR region of the human antibody to synthesize the target sequence.

[0052] 3. Construction of humanized dual-plasmid vectors (e.g.) Figure 3 (As shown) Primers were designed based on sequence analysis results, and the target fragments were amplified using high-fidelity enzymes. Linear vectors were prepared from pAbvec-hIgG plasmids using double digestion with AgeI and SalI, and from pAbvec-hIgK plasmids using double digestion with AgeI and BsiWI. The fragments and vectors were recovered separately and in-fusion cloning was performed according to the kit. The cells were transformed into DH5α competent cells, and correct colonies were verified and sent for testing. Successfully validated plasmids were named pAbvec-hIgK-Clone-A(L), pAbvec-hIgG-Clone-A(H), pAbvec-hIgK-Clone-B(L), and pAbvec-hIgG-Clone-B(H).

[0053] 4. Construction of humanized dual-promoter stable transfection plasmid 1) Design and synthesize specific primers to obtain linearized vector fragments from the vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro via reverse PCR.

[0054] 2) Design and synthesize specific PCR primers respectively. Using pAbvec-hIgK-Clone-A(H), pAbvec-hIgG-Clone-A(L), pAbvec-hIgK-Clone-B(H), and pAbvec-hIgG-Clone-B(L) as templates, high-fidelity DNA polymerase was used to amplify antibody light and heavy chain expression cassettes containing the kozak sequence-signal peptide-V region sequence-C region sequence.

[0055] 3) After agarose gel electrophoresis of the enzyme digestion products, the linearized vector fragments of the target size were recovered by gel excision. Using a multi-fragment recombination kit, an antibody light chain expression cassette (containing an IgG signal peptide and a His tag) was inserted downstream of the CMV promoter, and an antibody heavy chain expression cassette was inserted downstream of the EF1α promoter. A cHS4 insulator sequence was added between the two expression cassettes to prevent promoter crosstalk. The light chain / heavy chain genes carrying clone Clone-A were constructed and named pCDH-CMV-Clone-A(L)-EF1-Clone-A(H)-PGK-copGFP-T2A-Puro vector; and the light chain / heavy chain genes carrying clone Clone-B were named pCDH-CMV-Clone-B(L)-EF1-Clone-B(H)-PGK-copGFP-T2A-Puro vector.

[0056] 5. Validation of transient transfection with humanized dual-plasmid vectors 1) Cell Culture and Preparation: After rapidly thawing the frozen 293F cells in a 37°C water bath, centrifuge at 1000 rpm for 5 minutes to remove the cryopreservation supernatant containing the cryoprotectant. Gently resuspend the cells in pre-warmed FreeStyle™ 293 expression medium and seed them in shake flasks, precisely controlling the initial seeding density at 0.5-0.6 × 10⁶ cells / year. 6 Cells / mL, and cell viability as detected by trypan blue staining, must be higher than 90%. Place the shake flasks in a constant-temperature shaker at 37°C, 5% CO2, and saturated humidity, and culture at 112 rpm. Monitor cell density and viability daily; when cell density reaches approximately 3.0 × 10⁶ cells / mL... 6 When the cell count reaches 0.5 × 10⁶ cells / mL, promptly dilute and passage the cells to maintain them in the vigorous logarithmic growth phase. Repeat this process several times until the cell growth curve stabilizes, ensuring that the cells used for transfection are in optimal growth condition. 24 hours before transfection, dilute the cells to 0.5 × 10⁶ cells / mL. 6 Density of cells / mL.

[0057] 2) Preparation of transfection complex and transfection: Linear polyethyleneimine (PEI) was used as the transfection reagent. First, 2 μg / 10 6 Calculate the required total amount of plasmid DNA using the cell ratio. Dilute in Opti-MEM™ at a DNA:PEI ratio of 1:3 (μg:μL). Allow to stand at room temperature for 15 minutes to form a stable DNA-PEI complex.

[0058] 3) Cell transfection and culture: Add the transfection complex dropwise and evenly to the cell culture medium, gently shaking the flask during the addition process to ensure rapid dispersion of the complex and avoid excessive local concentrations that could cause cell toxicity. After transfection, adjust the culture conditions to 37℃, 8% CO2, and 110-130 rpm for incubation.

[0059] 4) Post-transfection enhancement and feeding: To improve antibody expression, a stepwise feeding strategy was adopted. On Day 1 post-transfection, a final concentration of 0.5% of the dedicated transfection enhancer and 7% of FM02AB nutrient supplement were added to the culture system. On Day 4 post-transfection, 0.3% of the transfection enhancer and 7% of FM02AB were added again to continuously support cell metabolism and efficient synthesis of recombinant proteins.

[0060] 5) Expression detection: Expression monitoring can begin on the second day after transfection (Day 2). Collect a small amount of cell supernatant and centrifuge to remove cell debris. Preliminary detection is performed using an indirect ELISA: an RSV-N protein-coated ELISA plate is used to capture the secreted antibody in the culture supernatant. Then, the expression level of the humanized monoclonal antibody is qualitatively and semi-quantitatively assessed by reacting the substrate with HRP-labeled anti-human IgG Fc antibody.

[0061] 6. High-efficiency production of humanized dual-promoter stable-transfer plasmids 1) Lentiviral packaging HEK-293T and HEK-293F cells were revived separately and passaged at least three times to ensure stable growth. One day before transfection, HEK-293T cells in logarithmic growth phase with a density of approximately 80% were digested and seeded at an appropriate density into 6-well plates. When the cell density reached approximately 60%, half the medium was replaced.

[0062] Prepare the transfection mixture in sterile tubes as follows (per well): Take 250 μL of Opti-MEM. ® In the culture medium, add packaging plasmids PspAX2 (1 μg), PMD2.G (1 μg), and either pCDH-CMV-Clone-A(L)-EF1-Clone-A(H)-PGK-copGFP-T2A-Puro or pCDH-CMV-Clone-B(L)-EF1-Clone-B(H)-PGK-copGFP-T2A-Puro plasmid (2 μg) sequentially. Mix gently and incubate at room temperature for 5 minutes. Then add 10 μL of Mirus transfection reagent, mix gently again, and incubate at room temperature for 20 minutes to form the transfection reagent complex. Add the complex dropwise and evenly to the 293T cell culture medium in a 6-well plate. Then incubate the cells at 37°C with 5% CO2.

[0063] 2) Viral transduction Forty-eight hours after transfection, cell supernatant containing lentiviral particles was collected and centrifuged at 2000 rpm for 8 minutes to remove cell debris. The viral supernatant was carefully collected, and polybrene was added to a final concentration of 8 μg / mL to enhance viral infection efficiency. Cells in good condition with a density of 2–4 × 10⁻⁴ cells were collected. 6 HEK-293F suspension cells were resuspended in the viral supernatant containing polybrene and incubated in a shaker at 37°C and 5% CO2 for 2 hours. After incubation, the cells were centrifuged to remove the viral solution, resuspended in fresh serum-free medium, and cultured in a shaker.

[0064] On days 3–4 after transduction, an appropriate concentration of puromycin was added to the culture medium to begin screening for stable cell lines. Thereafter, the medium was replaced with fresh puromycin at the same concentration every 2–4 days, and screening continued until all untransduced cells died and the positive cell population proliferated stably. Finally, monoclonal cell lines capable of stably expressing the target antibody were obtained using the limiting dilution method and cryopreserved to establish a master cell bank.

[0065] Example 3: Large-scale in vitro production of humanized antibodies Based on established, stably expressing cell lines, this invention achieves efficient and stable production of humanized antibodies through precise time-segmented control. Specific process parameters and operations are as follows, in sequence according to culture time: Days 1-2: Environmental control: Temperature is strictly maintained at 37.0 ± 0.5°C; pH is controlled within the range of 7.0 – 7.2; dissolved oxygen (DO) is set to 40% air saturation and maintained by adjusting the intake ratio and speed.

[0066] Nutrition and Monitoring: After inoculation, cells primarily rely on the nutrients in the basal culture medium. Daily offline sampling is performed to monitor cell density, viability (target >95%), and glucose concentration. When the glucose concentration falls below 4 g / L, a 400 g / L glucose solution is added to maintain the concentration within the range of 4-6 g / L. Simultaneously, lactate production is monitored, and its concentration is controlled at a low level of <2 g / L.

[0067] Days 3-5: Environmental control: The culture temperature was gradually reduced to 33.5±0.5°C, and the pH control range was adjustable to 6.8-7.2. As cell density increased, the dissolved oxygen setpoint was increased to ≥50%, and agitation and aeration were increased accordingly to ensure sufficient oxygen supply.

[0068] Nutrition and Additives: Initiate a dynamic fed-batch strategy. In addition to glucose, begin feeding glutamine or a more stable dipeptide alternative (such as L-alanyl-L-glutamine) to maintain ammonia concentration at a low level (<2 mM). To cope with the high-density culture stress, add a caspase inhibitor (final concentration 1-10 μM) on day 3 to inhibit programmed cell death. Simultaneously, add proline (final concentration 5-20 mM) to improve the endoplasmic reticulum folding environment.

[0069] Process monitoring: Monitor key parameters twice daily: cell density (controlled at 5-8 × 10⁻⁶). 6 The parameters included cells / mL, viability (>90%), glucose / lactic acid concentration, and ammonium ion (NH4⁺) accumulation (<5 mM).

[0070] Days 6-10: Environmental control: Temperature should be further adjusted and stabilized at 32-33°C. pH must be strictly controlled within a narrow range of 6.8-7.0. Dissolved oxygen needs to be further increased to ≥60% to meet the oxygen demand under high cell density.

[0071] Nutrition and Additives: The nutrient feed was switched to a supportive mode, with a focus on adding key amino acids (including but not limited to tyrosine and phenylalanine). To protect the secreted antibodies, protective additives such as trehalose were introduced (final concentration 0.5-2% w / v). Caspase inhibitors (final concentration 1-10 μM) and proline (final concentration 5-20 mM) were continuously supplemented.

[0072] Fermentation termination: Cultivation is terminated when the viability rate continues to decline to <70%.

[0073] Example 4: Performance testing of humanized antibodies (e.g.) Figure 4 (As shown) 1. Affinity: KD value measured by competitive ELISA. 1) Coating: The RSV-N protein was coated at 100 ng / well in the wells of the microplate and coated overnight at 4°C. Then the liquid was discarded, and 300 μL of PBST (PBS containing 0.05% Tween-20) was added to wash twice. After discarding the washing solution, the microplate was patted dry on absorbent paper.

[0074] 2) Blocking: Add 300 μL of blocking solution (PBST containing 2% BSA) to the above ELISA plate, incubate at 37°C for 3 hours, then discard the blocking solution and wash the plate 3 times in the same way.

[0075] 3) Competitive Binding Reaction: Prepare a series of free antigen solutions with varying concentrations. Add a fixed concentration of monoclonal antibody to each free antigen solution. Add the mixture to the wells of the plate coated with the antigen. Include a negative control (without free antigen) and a blank control. Incubate at 37°C to allow the competitive reaction to reach equilibrium. Discard the liquid in the wells and wash the plate four times as before.

[0076] 4) Detection: Dilute the enzyme-labeled secondary antibody (HRP-labeled mouse anti-human IgG) with antibody dilution buffer, add 100 μL to each well, and incubate at 37°C for 1 h; discard the liquid in the wells and wash the plate 4 times as before.

[0077] 5) Color development and reading: Under light-protected conditions, add 100 μL of 3,3',5,5'-tetramethylbenzidine solution to each well and incubate at 37°C for 5 min. Add 50 μL of stop solution to each well and measure the absorbance of each well at 450 nm using a microplate reader. Calculate IC50 using nonlinear fitting. 50 ≈KD value.

[0078] 2. Specificity The humanized antibody Clone-B was prepared into a solution with a concentration of 1.0–2.0 mg / mL using 0.02 M PBS buffer at pH 7.4, and accurately streaked onto the detection line (T line) of the nitrocellulose membrane at a spray volume of 1.0–1.2 μL / cm. The colloidal gold-labeled humanized antibody Clone-A was uniformly atomized onto the sample pad and dried at 37°C for later use. The anti-chicken IgY antibody was prepared into a solution with a concentration of 0.5–1.0 mg / mL using 0.02 M PBS buffer at pH 7.4, and streaked onto the control line (C line) at a spray volume of 0.8–1.0 μL / cm.

[0079] We used antigens or inactivated virus samples from a variety of common respiratory pathogens, including influenza A virus, influenza B virus, rhinovirus, parainfluenza virus, SARS-CoV-2, and respiratory adenovirus, for cross-reactivity testing. All tests were performed at room temperature, and results were read 15 minutes after sample addition. The results showed that, within the set observation time, except for respiratory syncytial virus, no visible color development was observed on the test lines (T lines) for all other tested pathogen samples, and the control lines (C lines) showed normal color development. This fully demonstrates that the test strip does not cross-react with the above-mentioned respiratory pathogens, has high specificity, and can effectively avoid false positive results caused by cross-reactivity.

[0080] 3. Stability Testing: 1) Accelerate storage stability testing Paired antibodies (as coating antibodies and colloidal gold-labeled antibodies, respectively) were coated onto the nitrocellulose membrane detection line (T line) and labeled onto the gold pad according to the process requirements. The test strips were then stored in constant temperature incubators at room temperature (25℃) and 37℃, respectively. Samples were taken on days 0, 3, and 7 of storage, and tested using RSV positive clinical samples and negative control samples with known titers. Results were read 15 minutes after sample addition.

[0081] 2) Repeated freeze-thaw stability test The antibody solution was placed at -20°C and subjected to multiple freeze-thaw cycles. After each freeze-thaw cycle, 10-20 µL of the antibody solution was taken, re-labeled or coated with colloidal gold, and prepared into test strips. The same positive / negative samples were used for testing, and the results were recorded after 15 minutes.

[0082] In all stability tests, the NC membrane background of the test strips was clean, with no nonspecific color development or residue. The test results showed: The colorimetric intensity of the test lines for all negative samples was below grade G1. All positive samples showed a color intensity of G7 or higher on the test lines, and the signal intensity did not decrease significantly with storage time or number of freeze-thaw cycles.

[0083] The detection sensitivity of the test strip remained stable throughout, meeting the preset detection threshold requirements.

[0084] Example 5: Application and performance verification of humanized antibodies in immunochromatographic test strips (e.g.) Figure 5 (As shown) 1. Assembly and preparation of colloidal gold test strips 1) Preparation of colloidal gold and antibody labeling Colloidal gold solutions with uniform particle sizes (40–60 nm) were prepared using the trisodium citrate reduction method. Humanized antibody Clone-A was dialyzed under mild conditions to displace the antibody into a low ionic strength buffer. Under optimal pH conditions (pH 8.2–8.5), the dialyzed antibody was slowly added to the colloidal gold solution, forming a stable gold-labeled antibody complex via electrostatic adsorption. Subsequently, a 1% BSA solution was added for blocking to reduce non-specific adsorption. The labeled complex was purified by centrifugation and resuspended in a 1% trehalose buffer to prepare the working solution for the gold-labeled pad.

[0085] 2) Preparation of test line (T line) and quality control line (C line) Humanized antibody Clone-B was diluted to a concentration of 1.0-2.0 mg / mL with 0.02 M PBS buffer (pH 7.4). Using a gold sprayer, it was precisely coated onto the test line (T line) of a nitrocellulose membrane (NC membrane) at a spray volume of 1.0-1.2 μL / cm. The control line (C line) was coated using anti-chicken IgY antibody, diluted to 0.5-1.0 mg / mL with the same buffer solution at a spray volume of 0.8-1.0 μL / cm. The coated NC membranes were then dried at 37°C for later use.

[0086] 3) Assembly of test strips The sample pad, gold label pad, coated NC membrane, and absorbent pad are sequentially stacked and pasted onto a PVC base plate, cut into test strips of specified width, and placed into a plastic cartridge to produce the finished test kit.

[0087] 2. Sensitivity Verification Sensitivity testing was performed using RT-PCR-confirmed clinical positive samples (n=84), covering a CT value range of 18-32. Results showed that the overall detection sensitivity of this test strip for all positive samples was 98.0% (100 / 102). Particularly for samples with low viral load (CT value >30, n=18), the detection rate reached 88.8% (16 / 18), significantly better than the detection rate of the reference commercially available murine antibody reagent (55.5%, 10 / 18). Further serial dilution experiments determined the limit of detection (LoD) for RSV antigen of this test strip to be 1.25 × 10⁻⁶.3 The TCID50 / mL indicates that the humanized antibody in this invention has a good ability to capture trace amounts of viral particles.

[0088] 3. Anti-interference capability verification Next, the test strip's ability to resist interference with the nasal mucosal matrix was evaluated. Real nasal matrix samples from healthy individuals were used for testing without adding any immune blocking agents, with a sample size of n=40.

[0089]

[0090] The results showed that the fully humanized antibody, by completely avoiding the non-specific interaction between the mouse Fc fragment and human matrix components, maintained an extremely low false positive rate even without the use of blocking agents, which was significantly better than the combination of fully mouse and human-mouse antibodies, demonstrating its good anti-interference performance in real clinical samples.

[0091] Example 6: Performance and Advantages of Humanized Antibodies in Pediatric Clinical Samples To evaluate the performance of the humanized antibody provided by this invention in actual pediatric clinical diagnosis and to expand its application scenarios, this embodiment specifically conducted a comparative study on nasal and oral pharyngeal swab samples from children. This study aims to verify the feasibility of using oral pharyngeal swabs as an alternative sampling method to address the issues of poor tolerance and low cooperation among young children regarding nasopharyngeal swab collection, thereby providing a better subject experience.

[0092] Paired samples were collected from pediatric patients diagnosed with respiratory syncytial virus (RSV) infection by RT-PCR, specifically nasal and pharyngeal swab samples collected at the same time point. Samples were categorized into three groups based on RT-PCR CT values: strongly positive (CT < 25, n = 9), moderately positive (CT 25-30, n = 17), and weakly positive (CT > 30, n = 11). Under each viral load gradient (strong, moderate, and weak), the test results for nasal and pharyngeal swab samples from the same patient remained consistent (37 / 37). This demonstrates that the test strip using the humanized antibody of this invention maintains the same detection sensitivity when using pharyngeal swab samples as it does for nasal swab samples.

[0093] Simultaneously, nasal and pharyngeal swab samples were collected from healthy volunteer children and tested using the same test strip without the addition of any immunoblockers to assess nonspecific reactions in real, complex mucosal samples. The sample size was n=25. Results showed that neither nasal nor pharyngeal swab samples elicited significant nonspecific color development (0 / 25). This indicates that humanized antibodies can effectively overcome interference from complex matrix samples (such as mucus, symbiotic bacteria, and food residue) in children's nasopharynx, achieving low background and high specificity detection without relying on additional immunoblockers.

[0094] Where X1 is S, T, or A; X2 is I, L, or V; X3 is D, E, or N; and X4 is V, L, or I. The sequence shown under the horizontal line is the FR sequence, and the sequence circled in the box is the CDR sequence.

[0095] This invention relates to the following sequences: The amino acid sequence of the first heavy chain variable region (SEQ ID NO: 1~SEQ ID NO: 81):

[0096] The amino acid sequence of the first light chain variable region (SEQ ID NO: 82~SEQ ID NO: 162):

[0097] The amino acid sequence of the variable region of the second heavy chain (SEQ ID NO: 163~SEQ ID NO:243):

[0098] The amino acid sequence of the variable region of the second light chain (SEQ ID NO: 244~SEQ ID NO: 270):

[0099] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pair of humanized anti-respiratory syncytial virus fusion protein antibodies, characterized in that, The antibody includes a first antibody and a second antibody. The first antibody includes a first heavy chain variable region and a second light chain variable region. The amino acid sequence of the first heavy chain variable region is selected from one of SEQ ID NO: 1 to SEQ ID NO:

81. The amino acid sequence of the first light chain variable region is selected from one of SEQ ID NO: 82 to SEQ ID NO:

162. The second antibody includes a second heavy chain variable region and a second light chain variable region. The amino acid sequence of the second heavy chain variable region is selected from one of SEQ ID NO: 163 to SEQ ID NO:

243. The amino acid sequence of the second light chain variable region is selected from one of SEQ ID NO: 244 to SEQ ID NO:

270.

2. The humanized anti-respiratory syncytial virus fusion protein antibody pair according to claim 1, characterized in that, The first heavy chain variable region comprises ICDR1, ICDR2, and ICDR3, whose amino acid sequences are shown at positions 26-34, 52-58, and 97-102 in SEQ ID NO:1 to SEQ ID NO:81, respectively; the first light chain variable region comprises CDR1′, CDR2′, and CDR3′, whose amino acid sequences are shown at positions 24-38, 54-60, and 93-101 in SEQ ID NO:82 to SEQ ID NO:162, respectively, having at least 80%, 85%, 90%, or 95% sequence identity, and the first antibody is capable of specifically binding to RSV-N.

3. The humanized anti-respiratory syncytial virus fusion protein antibody pair according to claim 1, characterized in that, The second heavy chain variable region comprises IICDR1, IICDR2, and IICDR3, whose amino acid sequences are shown at positions 26-33, 51-58, and 97-103 of SEQ ID NO: 163 to SEQ ID NO: 243, respectively; the second light chain variable region comprises CDR1″, CDR2″, and CDR3″, whose amino acid sequences are shown at positions 27-32, 50-52, and 89-94 of SEQ ID NO: 244 to SEQ ID NO: 270, respectively, having at least 80%, 85%, 90%, or 95% sequence identity, and wherein the second antibody is capable of specifically binding RSV-N.

4. The humanized anti-respiratory syncytial virus fusion protein antibody pair according to claim 1, characterized in that, Both the first antibody and the second antibody further comprise a heavy chain constant region and a light chain constant region; wherein the heavy chain constant region is a human IgG1 constant region and the light chain constant region is a human kappa light chain constant region.

5. A biomaterial, characterized in that, Selected from: (1) An isolated polynucleotide encoding a first heavy chain variable region and / or a first light chain variable region and a second heavy chain variable region and / or a second light chain variable region as described in any one of claims 2-3; (2) An expression vector comprising the above-mentioned polynucleotides; (3) A host cell containing the above expression vector.

6. A method for preparing a humanized anti-respiratory syncytial virus fusion protein antibody pair as described in any one of claims 2-5, characterized in that, Includes the following steps: a) Single B-cell technology for sorting B lymphocytes specifically recognized by RSV-N protein; b) Gene cloning and construction of dual-promoter expression vectors; c) Screening of stable transfected cell lines.

7. An immunoassay kit, characterized in that, It comprises a first antibody and a second antibody as described in any one of claims 1-4; the first antibody is used as a detection antibody, and the second antibody is used as a capture antibody.

8. The immunoassay kit according to claim 7, characterized in that, The kit is an immunochromatographic test strip; the first antibody is labeled with a detectable marker; the second antibody is immobilized on the detection line of a nitrocellulose membrane.

9. The immunoassay kit according to claim 7, characterized in that, The detectable markers are colloidal gold, fluorescent microspheres, or latex microspheres.

10. The use of a humanized antibody pair as described in any one of claims 1-4 in the preparation of a diagnostic reagent for detecting respiratory syncytial virus.

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