A ccr8 mutant and applications thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a CCR8 mutant and application thereof, an amino acid sequence of the CCR8 mutant is an amino acid sequence obtained by mutation on the basis of a sequence shown in SEQ ID No: 1, the mutation is I69A, Q277A and I287A, or the mutation is I69A, Q277A, I287A, C63Y, L168N, A236D and K306E. Through mutation of specific amino acid sites, the application produces a very strong space synergistic effect, and can be accurately recognized by a reference antibody for a conformational epitope while improving overall thermal stability.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology and relates to a CCR8 mutant and its application. Background Technology

[0002] CCR8 (CC chemokine receptor 8) belongs to the G protein-coupled receptor (GPCR) family. GPCRs are a class of membrane proteins with a seven-transmembrane structure, involved in various signal transduction processes, and are key specific targets of regulatory T cells (Tregs) in the tumor immune microenvironment. Clinical macromolecular antibodies screened against CCR8 are highly dependent on the extremely complex three-dimensional conformation formed by the receptor in the natural lipid bilayer. However, as a multi-transmembrane protein, wild-type CCR8 is highly hydrophobic, and in in vitro xenogeneic cell expression systems, it is prone to misfolding, resulting in extremely low upper membrane expression levels.

[0003] To improve the heterologous expression stability of GPCRs, researchers have attempted to introduce conventional stabilizing mutations based on the Ballesteros-Weinstein (BW) numbering system and homologous receptor mapping. However, simply superimposing these conventional mutations onto human CCR8, while barely maintaining the basic conformation, still results in expression abundance far below the levels required for industrial-grade antibody drug screening. The lack of existing recombinant protein tools that can achieve both high abundance display and perfect preservation of the receptor's natural antigenic epitopes severely hinders the development of anti-CCR8 targeted drugs. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this application is to provide a CCR8 mutant and its application.

[0005] To achieve the purpose of this application, the following technical solution is adopted:

[0006] In a first aspect, this application provides a CCR8 mutant, wherein the amino acid sequence of the CCR8 mutant is an amino acid sequence obtained by mutation based on the sequence shown in SEQ ID No:1, and the mutation is I69A, Q277A and I287A;

[0007] Alternatively, the mutations are I69A, Q277A, I287A, C63Y, L168N, A236D, and K306E.

[0008] This application involves amino acid mutations at specific sites (I69A, Q277A, I287A). The isoleucine residues (I69, I287) within the transmembrane helix (TM) have large side chains, easily causing physical repulsion due to inter-helix stacking. Glutamine (Q277), as a polar residue deeply embedded in the hydrophobic core, is prone to high-energy folding instability. Precisely replacing these sites with alanine, which has extremely high α-helical tendency and a very small side chain, effectively removes the steric hindrance between helices and eliminates local polar microenvironment defects, significantly reducing the receptor's folding free energy.

[0009] SEQ ID No:1:

[0010] MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASED GVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL.

[0011] In some embodiments, the nucleotide sequence encoding CCR8 includes the sequence shown in SEQ ID No:5.

[0012] SEQ ID No:5:

[0013]

[0014] Specific mutations are introduced into the hydrophobic core of the transmembrane region or the eighth helix (Helix 8) to enhance basic stability. Specifically, C63Y belongs to the aromatic ring space-filling strategy in the transmembrane region; L168N belongs to the polar network reconstruction strategy; A236D belongs to the helical conformation stabilization strategy; and K306E belongs to the intracellular side charge reversal anchoring lock.

[0015] The mutations in I69A, Q277A, and I287A, along with the mutations in C63Y, L168N, A236D, and K306E, produced an unexpected synergistic rearrangement of internal spatial structure, forming a perfect complementarity in spatial physical distribution. The mutations in C63Y, L168N, A236D, and K306E provide rigid support for the receptor globally, while I69A, Q277A, and I287A, like "microscopic wedges," precisely fill the hydrophobic stacking gaps within the chassis framework. This synergistic rearrangement of "rigid inside and flexible outside" not only allows the recombinant receptor to successfully pass through the host cell's folding quality control system, achieving an exponential leap in cell membrane surface abundance, but also ensures that the relative spatial position of the receptor's extracellular loop (ECL) region is absolutely fixed, thus perfectly preserving the natural complex conformation for recognition by the reference antibody Imzokitug.

[0016] Flow cytometry identification and comparative experiments confirmed that the above mutations significantly improved the display abundance (total expression level) of the target on the cell membrane compared with the wild-type baseline and the modified sequence containing only C63Y, L168N, A236D, and K306E mutations. This overcomes the industry bottleneck problems of easy misfolding, low abundance, and easy degradation of CCR8 and its common mutant sequences in in vitro heterologous expression systems in the prior art.

[0017] In some embodiments, the amino acid sequence of the CCR8 mutant is the sequence shown in SEQ ID No:3.

[0018] SEQ ID No:3:

[0019] MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSATDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASED GVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQALTYATHVTEAISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL.

[0020] In some embodiments, the nucleotide sequence encoding the CCR8 mutant includes the sequence shown in SEQ ID No:7.

[0021] SEQ ID No:7:

[0022]

[0023] In some embodiments, the amino acid sequence of the CCR8 mutant is the sequence shown in SEQ ID No:4.

[0024] SEQ ID No:4:

[0025] MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVYKKLRSATDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPNLVFYQVASED GVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKDIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQALTYATHVTEAISFTHCCVNPVIYAFVGEEFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL.

[0026] In some embodiments, the nucleotide sequence encoding the CCR8 mutant includes the sequence shown in SEQ ID No:8.

[0027] SEQ ID No:8:

[0028]

[0029] Secondly, this application provides a nucleic acid molecule that encodes the CCR8 mutant described in the first aspect.

[0030] Thirdly, this application provides a recombinant plasmid containing the nucleic acid molecule described in the second aspect.

[0031] In some embodiments, the vector for the recombinant plasmid is pcDNA3.1.

[0032] Fourthly, this application provides a host cell containing the nucleic acid molecule described in the second aspect or the recombinant plasmid described in the third aspect.

[0033] In some embodiments, the host cell includes the HEK293 cell line or the Expi293 cell line.

[0034] Fifthly, this application provides the application of the CCR8 mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant plasmid described in the third aspect, or the host cell described in the fourth aspect in the screening of drugs targeting CCR8, detection of antibody binding activity, and preparation of antigens.

[0035] Compared with the prior art, this application has the following advantages:

[0036] The mutant combination in this application is designed with rigorous structural principles. Based on C63Y, L168N, A236D, and K306E, three specific core mutation sites, I69A, Q277A, and I287A, are introduced to produce a strong spatial synergistic effect. This enhances protein expression abundance while perfectly preserving the natural complex conformation of the receptor's N-terminus and the second extracellular loop (ECL2). The second extracellular loop is a key conformational epitope region of the CCR8 receptor, enabling the CCR8 mutant described in this application to be accurately recognized by clinical-grade reference antibodies targeting the conformational epitope.

[0037] Specifically, experiments showed that while mutants containing only the three novel mutations could increase expression levels, their conformation was significantly impaired (Ratio value was only 0.07). However, further combination with the C63Y, L168N, A236D, and K306E mutations not only further increased expression levels but also restored the conformation ratio to a healthy level (Ratio 0.14). This indicates that the C63Y, L168N, A236D, and K306E mutations, along with the I69A, Q277A, and I287A mutations, produced a synergistic effect of "1+1>2," an effect that would not have been anticipated by those skilled in the art.

[0038] In the flow cytometry methodology for recombinant protein conformation identification, this application specifically establishes an evaluation system based on "absolute exemption from background in the negative control group" and "parallel detection in single-color fluorescent tubes," which completely eliminates the spatial steric hindrance and fluorescence compensation errors caused by conventional double staining, and achieves rigorous relative quantification of receptor conformation retention. Attached Figure Description

[0039] Figure 1 This is the result of detecting the total expression level of the negative control group on the Expi293 cell membrane.

[0040] Figure 2 This is the result of detecting the total expression level of control group A on the Expi293 cell membrane.

[0041] Figure 3 This is the result of detecting the total expression level of control group B on the Expi293 cell membrane.

[0042] Figure 4 This is the result of detecting the total expression level of experimental group C on the Expi293 cell membrane.

[0043] Figure 5 This is the result of detecting the total expression level of experimental group D on the Expi293 cell membrane.

[0044] Figure 6 This is the result of the conformation binding activity test in the negative control group.

[0045] Figure 7 This is the result of the conformation-dependent antibody binding activity test in control group A.

[0046] Figure 8 This is the result of the conformation-dependent antibody binding activity test in the control group B.

[0047] Figure 9 This is the result of the detection of conformation-dependent antibody binding activity in the experimental group C.

[0048] Figure 10 This is the result of the detection of conformation-dependent antibody binding activity in experimental group D. Detailed Implementation

[0049] The technical solution of this application will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of this application and should not be construed as specific limitations thereof.

[0050] Example 1

[0051] Construction and identification of eukaryotic expression vectors for CCR8 mutants.

[0052] 1. Target sequence design, tag fusion, and gene synthesis:

[0053] To standardize the quantitative detection criteria for flow cytometry and ensure the comparability of expression efficiencies among different constructs, this application standardized the N-terminus of all CCR8 construct sequences (including wild-type and all mutants): a natural signal peptide of influenza virus type A hemagglutinin (HA) and a FLAG tag sequence were fused downstream of the start codon of each sequence. The amino acid sequence of the fused peptide is shown in SEQ ID No:9.

[0054] SEQ ID No:9: MKTIIALSYIFCLVFADYKDDDDK.

[0055] In this fusion peptide, amino acids 1-18 are the HA signal peptide, and amino acids 19-24 are the FLAG tag.

[0056] The nucleotide sequence encoding the fusion peptide is shown in SEQ ID No:10.

[0057] SEQ ID No:10:

[0058] ATGAAGACCATCATCGCCCTGAGCTACATCTTCTGCCTGGTGTTCGCCGACTACAAGGACGACGACGACAAG.

[0059] Based on the above design, codon optimization and whole-genome synthesis were performed for the following four sequences respectively.

[0060] CCR8-Control Group A: Wild-type (WT) human CCR8 sequence, amino acid sequence as shown in SEQ ID No:1, nucleotide sequence as shown in SEQ ID No:5.

[0061] CCR8-Control Group B: Based on the wild-type human CCR8 sequence, mutations were made, including C63Y, L168N, A236D, and K306E. The amino acid sequence is shown in SEQ ID No:2, and the nucleotide sequence is shown in SEQ ID No:6.

[0062] CCR8-Experimental Group C: Based on the wild-type human CCR8 sequence, mutations were performed, including I69A, Q277A, and I287A. The amino acid sequence is shown in SEQ ID No:3, and the nucleotide sequence is shown in SEQ ID No:7.

[0063] CCR8-Experimental Group D: Based on the wild-type human CCR8 sequence, mutations were made, including C63Y, L168N, A236D, K306E, I69A, Q277A, and I287A. The amino acid sequence is shown in SEQ ID No:4, and the nucleotide sequence is shown in SEQ ID No:8.

[0064] SEQ ID No:2:

[0065] MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVYKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPNLVFYQVASED GVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKDIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEEFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL.

[0066] SEQ ID No:6:

[0067]

[0068] During gene synthesis, linearized vector homologous arms were added to the 5' and 3' ends of each sequence. The nucleotide sequence of the 5' homologous arm is shown in SEQ ID No:11, containing the HindIII site; the nucleotide sequence of the 3' homologous arm is shown in SEQ ID No:12, containing the EcoRI site.

[0069] SEQ ID No: 11: CAGATCTCGAGCTCAAGCTT.

[0070] SEQ ID No: 12: GCGGCCGCCTGCAGGAATT.

[0071] 2. Linearization of expression vectors:

[0072] The pcDNA3.1(+) vector was used as the basic backbone. This vector carries the ampicillin resistance gene and the neomycin G418 (geneticin) selection marker, contains the CMV promoter and a multiple cloning site (MCS) located in the positive (+) direction, and is suitable for high-efficiency expression in mammalian cells.

[0073] The enzyme digestion system is shown in Table 1.

[0074] Table 1

[0075]

[0076] Enzyme digestion procedure: Gently mix the above system and digest for 2 hours in a water bath or metal bath at 37℃. Verify the digestion product by 1.0% agarose gel electrophoresis. After confirming complete linearization of the vector (single clear band, approximately 5.4 kb in size), use an agarose gel DNA recovery kit (Tiangen DP209) to excise and purify the linearized vector. The elution volume is 30 μL. Measure the concentration of the purified product and the A260 / A280 ratio (between 1.8 and 2.0). Store at -20℃ for later use.

[0077] Note: HindIII and EcoRI both have a single recognition site in the pcDNA3.1(+) multiple cloning site (MCS). Double digestion produces linearized vectors with sticky ends, suitable for subsequent homologous recombination cloning. HindIII, EcoRI, and 10× rCutSmart buffer are all from New England Biolabs, with catalog numbers R0104V, R0101S, and B6004V, respectively.

[0078] 3. Homologous recombination (seamless cloning) ligation:

[0079] The linearized vectors were recombinated and ligated with the synthetic fragments of each CCR8 gene using the ClonExpress® II one-step seamless cloning kit (Nanjing Novizan Vazyme, commercial product number C112-01).

[0080] The homologous recombination reaction system is shown in Table 2 (20 μL system).

[0081] Table 2

[0082]

[0083] Enzyme ligation ratio calculation: The molar ratio of vector to insert fragment is 1:3. The amount of vector used is approximately 80 ng. The amount of insert fragment used is calculated based on the fragment length as (insert fragment length / vector length) × amount of vector used × 3 (the vector length is approximately 5.4 kb, and each CCR8 fragment is approximately 1.0-1.1 kb long). The final calculated amount to be added is approximately 44-49 ng.

[0084] Reaction procedure: Gently mix the above mixture, briefly centrifuge, and incubate at 37°C in a metal bath or PCR instrument for 30 minutes (following the recommended conditions for the ClonExpress® II kit). After the reaction, immediately cool on ice and use directly for transformation or store at -20°C.

[0085] 4. Transformation and colony screening:

[0086] Competent cells: Commercially available E. coli DH5α chemically competent cells (Tiangen product) were used.

[0087] Transformation procedure: Take 10 μL of ligation product and add it to 100 μL of DH5α competent cells thawed on ice. Gently mix and incubate on ice for 30 minutes. Then heat shock in a 42℃ water bath for 45 s, quickly transfer to ice and incubate for 2 minutes. Add 900 μL of preheated LB liquid medium (antibiotic-free) and revive the cells at 37℃ and 220 rpm for 1 hour.

[0088] Spreading and resistance screening: Take 200 μL of revived bacterial culture and spread it evenly on the surface of LB agar plates containing 100 μg / mL ampicillin. Incubate upside down in a 37℃ incubator for 16 h.

[0089] 5. Small-scale plasmid extraction and sequence validation:

[0090] Colony selection: Pick 3-5 plump and independent single colonies from the plate and inoculate them into 5 mL LB liquid medium (containing 100 μg / mL ampicillin) and incubate at 37℃ and 220 rpm for 16 hours with shaking.

[0091] Plasmid extraction: Plasmid DNA was extracted using a plasmid mini-extraction kit (Tiangen DP103) following the instructions. Finally, the plasmid was eluted with 50 μL of sterile deionized water.

[0092] Sequencing identification: 10 μL of plasmid sample was sent to a commercial sequencing company (Qingke) for bidirectional sequencing using universal primers T7 forward primer and BGH reverse primer.

[0093] The nucleotide sequence of the T7 forward primer is shown in SEQ ID No:13, and the nucleotide sequence of the BGH reverse primer is shown in SEQ ID No:14.

[0094] SEQ ID No: 13: TAATACGACTCACTATAGGG.

[0095] SEQ ID No: 14: TAGAAGGCACAGTCGAGG.

[0096] Validation criteria: The sequencing results were compared with the theoretically designed CCR8 mutation sequences to confirm 100% accuracy. The recombinant plasmids that were correctly identified by sequencing were named pcDNA3.1-CCR8-control group A, pcDNA3.1-CCR8-control group B, pcDNA3.1-CCR8-experimental group C, and pcDNA3.1-CCR8-experimental group D.

[0097] 6. Preparation and quality control of transfection-grade plasmids:

[0098] Expanded culture: Positive clones verified by sequencing were inoculated at a ratio of 1:1000 into 50 mL LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37°C with shaking at 220 rpm for 16 h (until OD). 600 The value reaches 2.0).

[0099] Endotoxin-free plasmid extraction: The endotoxin-free plasmid extraction kit (Tiangen endotoxin-free plasmid extraction kit) was used, and the operation was strictly carried out in accordance with the instructions. Finally, the plasmid DNA was eluted with sterile TE buffer (pH 8.0).

[0100] The determination was performed using a NanoDrop micro-spectrophotometer. The plasmid concentration was required to be no less than 1.0 μg / μL, the A260 / A280 ratio was between 1.8 and 2.0, and the A260 / A230 ratio was greater than 2.0.

[0101] Example 2

[0102] HEK293 cell suspension culture and transient transfection.

[0103] 1. Cell resuscitation and passage culture:

[0104] Cell line and culture medium: The host cell line used was the HEK293 suspension cell line (Expi293F). The culture medium used was a serum-free and animal-derived suspension medium (SMM 293-TII Expression Medium, purchased from SinoBiological Inc., catalog number M293TII).

[0105] Thawing procedure: Remove the cryovials of HEK293 cells from the liquid nitrogen container and thaw them rapidly by shaking in a 37°C water bath until a small amount of ice crystals remain. Transfer the cell suspension to a centrifuge tube containing 10 mL of preheated culture medium, mix gently, and centrifuge at 100×g for 5 minutes at room temperature. Discard the supernatant (to remove DMSO from the cryopreservation solution). Resuspend the cell pellet in 5 mL of fresh culture medium and transfer it to a 125 mL shake flask containing 45 mL of fresh culture medium. Incubate at 37°C, 8% CO2, and 120 rpm in a shaker.

[0106] Maintenance through passage: Passage every 2-3 days until the cell density reaches 4.0 × 10⁻⁶ cells / year. 6 When the cell count was 10 cells / mL, the cells were diluted to 1.0 × 10⁻⁶ cells / mL with fresh culture medium. 6 Cells / mL, continue culturing. During passage, take samples periodically and use trypan blue staining to count viable cells, ensuring that cell viability is always greater than 95% before using them for subsequent transfection experiments.

[0107] 2. Cell preparation before transfection:

[0108] 24 hours before transfection, HEK293 cells in the logarithmic growth phase (viability >95%) were harvested and the cell density was adjusted to 1.0 × 10⁶ cells / year using fresh SMM 293-TII medium. 6 Cells were inoculated at a density of 2.0 × 10⁶ / mL and cultured overnight in shake flasks. On the day of transfection, samples were taken for counting to confirm that the cell density had reached 2.0 × 10⁶ / mL. 6 Transfection can be performed when the number of cells / mL is greater than 95%.

[0109] 3. Preparation of the transfection complex:

[0110] Transient transfection was performed on CCR8-control group A, control group B, experimental group C, experimental group D, and negative control group (empty vector pcDNA3.1). Three biological replicates were set up for each group.

[0111] Transfection reagent: Linear polyethyleneimine (PEI, molecular weight 25 kDa, Polysciences, catalog number 23966-1) was used as the transfection reagent. PEI was prepared into a 1 mg / mL stock solution with sterile deionized water, filtered through a 0.22 μm filter for sterilization, aliquoted, and stored at -20℃ for later use.

[0112] Plasmid working solution: Each recombinant plasmid constructed in Example 1 was diluted with sterile TE buffer (pH 8.0) to a working concentration of 1.0 μg / μL.

[0113] Complex preparation (using 100 mL cell culture as an example): Take a sterile 1.5 mL centrifuge tube and add 5 mL of Opti-MEM™ I serum-depleted medium (Thermo Fisher, catalog number 31985062) as diluent. Add the corresponding plasmid and PEI according to the table below, and mix gently:

[0114] Table 3

[0115]

[0116] Incubation: Gently mix the above mixtures using a vortex mixer (avoiding the formation of air bubbles), and incubate at room temperature for 20 minutes to allow the plasmid and PEI to fully form a stable cationic polymer-plasmid DNA transfection complex.

[0117] Note: The final plasmid concentration was 1.0 μg / mL of cell culture, and the mass ratio of PEI to plasmid was strictly controlled at 3:1.

[0118] 4. Instantaneous transfection operation:

[0119] The incubated transfection complexes from each group were slowly added dropwise to the corresponding HEK293 cell culture in shake flasks (100 mL cell suspension per flask). During the addition, the flasks were gently shaken to ensure even distribution of the transfection complexes. After transfection, the flasks were returned to a shaker at 37°C, 8% CO2, and 120 rpm for further incubation.

[0120] 5. Post-transfection culture and quality control monitoring:

[0121] Culture time: Continue culture for 48 hours after transfection.

[0122] Viability monitoring: Samples were taken at 24 and 48 hours after transfection, and cell density and viability were counted using trypan blue staining to ensure that the viability was not less than 95%.

[0123] 6. Harvesting and Sample Collection:

[0124] After 48 hours of transfection and culture, all cell suspensions in each shake flask were collected and transferred to 50 mL centrifuge tubes. The tubes were centrifuged at 300×g for 5 minutes at room temperature, and the supernatant was gently discarded (a small amount of supernatant was retained for sample resuspending before flow cytometry). The collected cell pellet was then subjected to non-destructive collection and FACS staining as described in Example 3 (Flow Cytometry).

[0125] Note: Avoid using proteases such as trypsin to digest cells throughout the process to fully preserve the natural conformation of the recombinant CCR8 target site on the cell membrane surface (the core control point for conformation protection).

[0126] Example 3

[0127] CCR8 mutant conformational activity assessment based on monochromatic parallel flow cytometry.

[0128] 1. Experimental materials and reagent system:

[0129] 1.1 Host cell testing group:

[0130] HEK293 cells transfected and cultured for 48 hours in Example 2 were labeled as the following test groups (as shown in Table 4).

[0131] Table 4

[0132]

[0133] 1.2 Reference Scale Antibody:

[0134] The known reference antibody Imzokitug (also known as BMS-986340) was used as the assay tool for conformation binding activity. This antibody was purchased from MedChemExpress (MCE), catalog number HY-P991001. This antibody is a human IgG1 monoclonal antibody prepared using a mammalian expression system, which specifically recognizes the native conformational epitope of CCR8 and is used to assess whether each CCR8 mutant retains the correct three-dimensional conformation.

[0135] 1.3 Flow cytometry detection of antibodies:

[0136] Total expression level detection antibody: A direct labeling method was used, employing an anti-FLAG tag antibody with a PE fluorescent group (ABflo® PE Rabbit Anti-DYKDDDDK-tag mAb), brand: ABclonal, catalog number: A27399. This antibody directly recognizes the FLAG tag fused to the N-terminus of each CCR8 construct sequence, and its fluorescence intensity (PE channel) reflects the total expression level of each mutant on the cell membrane.

[0137] Conformation binding activity assay antibody: An indirect method was used, employing FITC-labeled anti-human IgG Fc fluorescent secondary antibody (BeyoFC™ FITC anti-human IgG Fc Antibody), brand: Beyotime, product number: AC0406. This secondary antibody recognizes and binds to the Fc fragment of the reference antibody Imzokitug (human IgG1) that has bound to the CCR8 mutant. Its fluorescence intensity (FITC channel) reflects the binding activity of the reference antibody to the native conformation of CCR8.

[0138] 1.4 Buffer solution preparation:

[0139] FACS buffer: 1×PBS (pH 7.4) containing 1% (w / v) bovine serum albumin (BSA), sterilized by filtration through a 0.22 μm filter, and stored at 4°C for later use.

[0140] 2. Non-destructive cell collection:

[0141] To avoid disrupting the natural extracellular conformation of CCR8 on the cell membrane surface through treatment with proteases such as trypsin, the use of trypsin was strictly avoided throughout the entire cell collection process.

[0142] After 48 hours of transfection and culture in Example 2, the cell suspensions from each group (negative control group, control group A, control group B, experimental group C, and experimental group D) were transferred to 50 mL centrifuge tubes and centrifuged at 300×g for 5 minutes at room temperature. The supernatant was gently discarded. The cell pellet was resuspended in pre-chilled FACS buffer and centrifuged again at 300×g for 5 minutes at room temperature, followed by washing once. The supernatant was discarded, and the cell pellet was resuspended in an appropriate amount of pre-chilled FACS buffer to adjust the cell density to 1.0×10⁶ cells / year. 6 Each tube is placed on ice for later use.

[0143] Gentle centrifugation (300×g, 5 minutes) and gentle pipetting resuspension were used throughout the process to avoid violent shaking that could cause cell membrane rupture or receptor detachment. All centrifugation steps were performed at room temperature (not 4°C) to reduce the potential impact of cell membrane lipid phase transitions caused by low temperature on receptor conformation.

[0144] 3. Quantitative system and control exemption logic:

[0145] A strategy of "single-color parallel detection" was established to eliminate the steric hindrance interference and fluorescence compensation error that may occur between the two fluorescent antibodies in the traditional double staining method.

[0146] 3.1 Single-color tube inspection design:

[0147] Table 5

[0148]

[0149] Branch A and branch B were stained independently and in parallel. Each group of cells was divided into two equal parts, and branch A and branch B were stained separately, without overlapping double staining. The two samples were analyzed independently, and the fluorescence intensity of the PE channel and FITC channel was recorded separately.

[0150] 3.2 Logic for Exemption from Negative Background Control:

[0151] We used "empty vector transfected cells (negative control group) + complete staining system" as the absolute negative background control for flow cytometry, exempting us from the conventional isotype control. The logic is as follows: the negative control group cells underwent the exact same transfection procedure (only the plasmid differed), the same culture conditions, and the same staining process as the experimental group, perfectly matching the background signals of the experimental group in terms of transfection disturbance, non-specific adsorption, and autofluorescence. By subtracting the background fluorescence value of the negative control group, we can accurately eliminate systemic background interference and obtain the true specific signal. This method is more rigorous than the conventional isotype control because the negative control group cells underwent the exact same transfection procedure as the experimental group cells (rather than untreated cells and isotype antibodies), resulting in a higher background matching degree.

[0152] 4. Flow cytometry staining detection procedure:

[0153] 4.1 Cell division:

[0154] Take cell suspensions of adjusted density from each group (1.0 × 10⁻⁶). 6 Each tube was divided into two equal portions (approximately 5.0 x 10⁻⁶ units each). 5 (1 cell), labeled as branch A tube and branch B tube.

[0155] 4.2 Branch A (Total Expression Detection, PE Direct Labeling Method):

[0156] Add PE-Anti-FLAG antibody (ABflo® PE Rabbit Anti-DYKDDDDK-tagmAb, catalog number A27399) to tube A, following the manufacturer's recommended dilution ratio (1:100, diluted with FACS buffer), adding 100 μL of the diluted antibody working solution to each tube. Gently mix and incubate at 4°C in the dark for 30 minutes. Wash the cells with 2 mL of pre-chilled FACS buffer, centrifuge at 300×g for 5 minutes at 4°C, and discard the supernatant. Repeat the washing twice (for a total of 3 washes) to thoroughly remove unbound free antibody. Finally, resuspend the cell pellet in 200 μL of pre-chilled FACS buffer and store on ice in the dark for later use in assays.

[0157] 4.3 Branch B (Conformation-binding activity assay, FITC indirect method):

[0158] Primary antibody incubation: Add the reference antibody Imzokitug (MCE, HY-P991001) to tube B of the branch line, and dilute with FACS buffer to a working concentration of 2 μg / mL (this concentration has been determined to be the saturation binding concentration in preliminary experiments). Add 100 μL of the diluted antibody working solution to each tube. Mix gently and incubate at 4°C in the dark for 45 minutes.

[0159] Primary antibody washing: Add 2 mL of pre-chilled FACS buffer to wash cells, centrifuge at 300×g for 5 minutes at 4°C, and discard the supernatant. Repeat washing twice (for a total of 3 washes).

[0160] Secondary antibody incubation: Add FITC-labeled anti-human IgG Fc fluorescent secondary antibody (BeyoFC™ FITC anti-human IgG Fc Antibody, product number AC0406) to tube B of the branch line, according to the manufacturer's recommended dilution ratio (usually 1:500, diluted with FACS buffer), adding 100 μL of the diluted secondary antibody working solution to each tube. Mix gently and incubate at 4°C in the dark for 30 minutes.

[0161] Secondary antibody washing: Add 2 mL of pre-chilled FACS buffer to wash cells, centrifuge at 300×g for 5 minutes at 4°C, and discard the supernatant. Repeat washing 3 times (4 times in total) to completely remove unbound free secondary antibody.

[0162] Finally, the cell pellet was resuspended in 200 μL of pre-cooled FACS buffer and stored on ice in the dark for later use in instrumental analysis.

[0163] Note: A negative control group (FITC secondary antibody added only, Imzokitug primary antibody not added) was set up in branch B to evaluate the non-specific binding of FITC secondary antibody and cell autofluorescence.

[0164] 4.4 Flow cytometry analysis:

[0165] Instrument preparation: Use a CytoFLEX flow cytometer (Beckman Coulter) or an equivalent model, power it on and warm it up, run quality control microspheres (such as Cytometer Setup and Tracking Beads) to verify the instrument's CV value and fluorescence channel sensitivity, and ensure that the instrument is stable.

[0166] Voltage settings: Using the negative control group cells as a baseline, the FSC (forward scattering) and SSC (side scattering) voltages were set so that the cell population was located in the central region of the FSC-SSC scatter plot; the voltages of the PE channel (excitation wavelength 488 nm, emission wavelength 575 nm) and FITC channel (excitation wavelength 488 nm, emission wavelength 525 nm) were set so that the fluorescence peak of the negative control group cells was located at the x-axis 10.1 -10 2 The interval (approximately the 1st to 1.5th logarithmic order of magnitude).

[0167] Gating strategy:

[0168] First, circle the main population of live cells (R1 gate) on the FSC-SSC scatter plot, and exclude cell debris and dead cells (dead cells are usually located in the FSC low / SSC high region).

[0169] Based on the R1 gate, fluorescence signals from the PE channel (branch A) and the FITC channel (branch B) were collected respectively.

[0170] Each tube collects 10,000-20,000 live cell events.

[0171] Data recording: The mean fluorescence intensity (MFI) of each group of cells in the PE channel and FITC channel was recorded separately.

[0172] The total expression levels of each group on the Expi293 cell membrane are as follows: Figures 1-5 As shown, the blue peak represents the cell population that failed to effectively express the FLAG-tagged fusion protein, i.e., the PE-negative (or low-expression) cell subpopulation. This group of cells may include: cells that were not successfully transfected with the plasmid, cells that were transfected with the plasmid but did not effectively express the exogenous CCR8 protein, or cells with extremely low expression levels that were not effectively labeled by the PE-Anti-FLAG antibody. In this application, the region located in the low fluorescence intensity area (approximately 10) 2 -10 3 The red peak represents the cell population that is FLAG negative (not expressed or low-expressing). The red peak represents the cell population that has been effectively labeled by the PE-Anti-FLAG antibody, i.e., the PE-positive (high-expressing) cell subpopulation. These cells successfully expressed the FLAG-tagged fused CCR8 protein, and the protein was efficiently transported to the cell membrane surface, where it could be specifically recognized and bound by the PE-Anti-FLAG antibody. In this application, the high fluorescence intensity region (approximately 10) 4 -10 5 The blue peak represents the FLAG-positive (highly expressed) cell population. The blue peak consistently represents the negative / unexpressed / low-expressed background population, while the red peak consistently represents the positive / high-expressed specific signal population. The degree to the right shift of the red peak relative to the blue peak reflects the expression abundance of the FLAG-tagged fusion protein on the cell membrane; the further to the right, the higher the expression level.

[0173] The results of conformation-dependent antibody binding activity assays for each group are as follows: Figures 6-10As shown, the blue peak represents the cell population that failed to be effectively labeled by the FITC fluorescent secondary antibody, i.e., the FITC-negative cell subpopulation. This subset of cells may include: cells that failed to successfully express exogenous CCR8 protein; or cells that, although expressing CCR8 protein, have a conformational error and cannot be recognized by the reference antibody Imzokitug. In this application, it is located in the low fluorescence intensity region (approximately 10). 2 The red peak (FITC-positive subset) represents the cell population that was effectively labeled with the FITC-labeled anti-human IgG Fc secondary antibody, i.e., the FITC-positive cell subset. These cells successfully expressed the CCR8 protein and maintained the correct native three-dimensional conformation, enabling them to be specifically recognized and bound by the reference antibody Imzokitug. In this application, it is located in the high fluorescence intensity region (approximately 10). 4 The blue peak represents the FITC-negative subset (conformation-negative / unbound cell population), while the red peak represents the FITC-positive subset (conformation-positive / specifically bound to the reference antibody cell population). The degree of rightward shift of the red peak relative to the blue peak reflects the binding activity of the reference antibody Imzokitug to the CCR8 mutant; the further to the right, the more cells successfully retain the correct native three-dimensional conformation and can be efficiently recognized by the conformation-dependent antibody.

[0174] 5. Data Judgment and Analysis Methods:

[0175] 5.1 Calculation of Net Mean Fluorescence Intensity (Net MFI):

[0176] To eliminate interference from cell autofluorescence and nonspecific binding, this application uses net mean fluorescence intensity (NetMFI) for precise quantification. All data have had the corresponding fluorescence values ​​of the negative control group subtracted.

[0177] Net average fluorescence intensity of PE = Average fluorescence intensity of PE (experimental group) - Average fluorescence intensity of PE (negative control group)

[0178] Net mean fluorescence intensity of FITC = Mean fluorescence intensity of FITC (experimental group) - Mean fluorescence intensity of FITC (negative control group)

[0179] 5.2 Calculation of Conformation Binding Ratio

[0180] To assess the degree of preservation of the native conformation while increasing expression levels in each mutant, this application establishes a "conformation binding ratio" evaluation index:

[0181] Conformation binding ratio = Net average fluorescence intensity of FITC / Net average fluorescence intensity of PE

[0182] This ratio reflects the conformation-binding activity per unit expression level. A higher conformation-binding ratio indicates better preservation of the receptor's native conformation at the same expression level.

[0183] 5.3 Two-dimensional evaluation model

[0184] This application establishes a two-dimensional evaluation model of "absolute expression abundance (PE Net MFI) and conformation ratio (Ratio)," with the following judgment criteria:

[0185] Table 6

[0186]

[0187] 6. Test Results:

[0188] The results of flow cytometry analysis for each group are shown in Table 7.

[0189] Table 7

[0190]

[0191] Ratio calculation is not applicable to the negative control group (denoted as N / A).

[0192] Results analysis:

[0193] (1) Absolute expression abundance (PE channel):

[0194] The net mean fluorescence intensity of PE in control group A (wild type) was 2329.2, indicating that wild type CCR8 was expressed at only a basal level in Expi293 cells.

[0195] The net mean fluorescence intensity of PE in control group B (4 mutation) was 9536.2, which was about 4.1 times higher than that in control group A, indicating that C63Y, L168N, A236D, and K306E can increase the expression level to some extent.

[0196] The net mean fluorescence intensity of PE in experimental group C (3 new mutations) was 8701.2, which was about 3.7 times higher than that in control group A, indicating that the three new mutations I69A, Q277A and I287A themselves have the ability to significantly increase expression levels.

[0197] The net average fluorescence intensity of PE in experimental group D (7 mutation) reached 19777.2, which was about 8.5 times that of control group A, about 2.1 times that of control group B, and about 2.3 times that of experimental group C. This achieved an exponential and significant increase in expression level, broke through the expression bottleneck of CCR8 in heterologous expression systems, and reached the expression abundance required for industrial-grade antibody screening.

[0198] (2) Conformation-binding activity (FITC channel):

[0199] The net mean fluorescence intensity of FITC in experimental group D was 2804.4, which was approximately 5.9 times that of control group A (474.4), approximately 2.0 times that of control group B (1426.4), and approximately 4.7 times that of experimental group C (596.4).

[0200] It is noteworthy that although the total expression level (net mean fluorescence intensity of PE = 8701.2) of experimental group C (3 mutations) was comparable to that of control group B (9536.2), its conformation binding activity (net mean fluorescence intensity of FITC = 596.4) was significantly lower than that of control group B (1426.4). This indicates that high expression with only 3 new mutations is accompanied by partial conformational collapse.

[0201] (3) Verification of conformation ratio and synergistic effect:

[0202] The conformation ratio of control group A was 0.20 (wild-type baseline).

[0203] The conformation ratio of control group B was 0.15, indicating that C63Y, L168N, A236D, and K306E maintained conformational stability well while increasing expression levels.

[0204] The conformation ratio of experimental group C was only 0.07, which was significantly lower than the wild-type baseline (0.20), indicating that the conformation was severely damaged when only 3 new mutations were present.

[0205] The conformational ratio of experimental group D was 0.14, which was not only significantly higher than that of experimental group C (0.07), but also within the same order of magnitude (0.10-0.40) as control groups A (0.20) and B (0.15). This demonstrates that the combination of mutations in I69A, Q277A, and I287A with mutations in C63Y, L168N, A236D, and K306E did not exacerbate conformational collapse; instead, it restored the conformational ratio from 0.07 to a healthy level of 0.14, while simultaneously maximizing expression levels.

[0206] (4) Quantitative proof of synergistic effect:

[0207] If it is only an additive effect, the expected value of experimental group D is PE≈9536.2+(8701.2-2329.2)=15908.2, FITC≈1426.4+(596.4-474.4)=1548.4;

[0208] The actual values ​​for experimental group D were PE=19777.2 (exceeding the sum of expectations by about 24.3%) and FITC=2804.4 (exceeding the sum of expectations by about 81.1%).

[0209] It is particularly noteworthy that the actual FITC value far exceeded the sum of expectations, indicating that the mutations of I69A, Q277A, and I287A, along with the mutations of C63Y, L168N, A236D, and K306E, produced a strong positive synergistic effect in conformational stability. The mutations of C63Y, L168N, A236D, and K306E "rescued" the conformational damage caused by I69A, Q277A, and I287A.

[0210] Conclusion: The above-mentioned "double high" data (highest PE signal and conformation binding ratio restored to healthy level) strongly demonstrate that this application, for the first time, combined three specific mutations, I69A, Q277A, and I287A, with C63Y, L168N, A236D, and K306E, producing an unexpectedly strong synergistic effect. That is, although experimental group C could increase expression level, the conformation was severely damaged (conformation ratio only 0.07), and although control group B had stable conformation, the expression level was insufficient (PE did not meet industrial-grade standards). Only experimental group D, which combined the two, achieved an exponential jump in expression level (net average fluorescence intensity of PE reached 19777.2) while restoring the conformation ratio to a healthy level (conformation ratio 0.14), perfectly locking the natural extracellular three-dimensional conformation of the CCR8 receptor.

[0211] The above description is only a specific embodiment of this application, but the protection scope of this application is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application fall within the protection and disclosure scope of this application.

Claims

1. A CCR8 mutant, characterized in that, The amino acid sequence of the CCR8 mutant is an amino acid sequence obtained by mutation based on the sequence shown in SEQ ID No:1, and the mutations are I69A, Q277A and I287A; Alternatively, the mutations are I69A, Q277A, I287A, C63Y, L168N, A236D, and K306E.

2. The CCR8 mutant according to claim 1, characterized in that, The amino acid sequence of the CCR8 mutant is the sequence shown in SEQ ID No:

3.

3. The CCR8 mutant according to claim 1, characterized in that, The amino acid sequence of the CCR8 mutant is the sequence shown in SEQ ID No:

4.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the CCR8 mutant as described in any one of claims 1-3.

5. A recombinant plasmid, characterized in that, The recombinant plasmid contains the nucleic acid molecule as described in claim 4.

6. The recombinant plasmid according to claim 5, characterized in that, The vector for the recombinant plasmid is pcDNA3.

1.

7. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 4 or the recombinant plasmid of claim 5 or 6.

8. The host cell according to claim 7, characterized in that, The host cells include the HEK293 cell line or the Expi293 cell line.

9. The application of the CCR8 mutant according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the recombinant plasmid according to claim 5 or 6, or the host cell according to claim 7 or 8 in the screening of drugs targeting CCR8, detection of antibody binding activity, and preparation of antigens.

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