Acidic pH-dependent modified antibody specifically binding to v5 exon of CD44 and application thereof

Abstract

The invention relates to the field of biomedical antibodies, in particular to an acid pH-dependent modified antibody specifically binding to a v5 exon of CD44 and application of the antibody. The invention provides an acid pH-dependent modified antibody specifically binding to a v5 exon of CD44 or a fragment thereof. The antibody or the fragment thereof comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 2; the light chain variable region is obtained by replacing at least one amino acid residue in the light chain variable region of the parent antibody with a histidine residue. According to the present invention, the monoclonal antibody is modified so as to provide the high affinity under the acidic pH (pH 6.0), and the acidic pH dependent monoclonal antibody is provided, and can specifically bind to human CD44v5 under the pH 6.0 condition.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical antibodies, specifically to an acidic pH-dependent modified antibody that specifically binds to exon 5 of CD44 and its applications. Background Technology

[0002] Antibodies, as important targeting molecules in the biomedical field and core components of CAR immunotherapy, provide immune cells with the ability to specifically target specific targets. The complex and variable characteristics of the tumor microenvironment (TME) are a key factor limiting traditional antibodies and antibody-based immunotherapies. Recent studies have shown that the microenvironment of solid tumors exhibits unique acidic characteristics, typically with a pH range of 6.0-6.8, while the extracellular environment of normal tissues maintains a physiological pH of 7.3-7.4. This acidic environment mainly stems from factors such as vigorous glycolytic metabolism in tumor cells, regional hypoxia, and poor vascular perfusion. Under acidic conditions, the affinity between antibodies and antigens is generally reduced, leading to a significant weakening of the function of immunotherapies that rely on antigen-antibody interactions. The acidic characteristics of the tumor microenvironment provide new insights for antibody engineering. Traditional antibodies often exhibit decreased binding capacity under acidic conditions, a phenomenon that may lead to reduced efficacy in tumor treatment. However, through structural biology and computer-aided design, researchers have found that this "disadvantage" can be transformed into an "advantage" for targeted therapy. Histidine, with its imidazole side chain having a pKa of approximately 6.4, allows its protonation state to change with pH, ​​forming a positively charged state near the pH of the tumor microenvironment. This property makes histidine an ideal "molecular switch" for pH-dependent antibody design.

[0003] Molecular dynamics simulations and computational-aided design provide powerful tools for antibody engineering. Through computational methods such as homology modeling, molecular docking, and virtual mutation, the structure and interactions of antigen-antibody complexes can be predicted, key amino acid residues can be identified, and binding states under different pH conditions can be simulated. These methods not only significantly reduce the workload of experimental screening but also provide a theoretical foundation at the molecular level, contributing to a better understanding of antibody-antigen recognition mechanisms.

[0004] CAR-M technology, as a cell immunotherapy strategy that has emerged in recent years, combines the natural phagocytic function of macrophages with the specific targeting ability of CAR molecules. However, similar to traditional CAR-T therapy, CAR-M faces the challenge of functional inhibition in the tumor microenvironment (TME) of solid tumors, with the acidic environment having a particularly significant impact on the recognition domain (usually a single-chain antibody ScFv). To address this bottleneck, this invention focuses on developing pH-dependent antibodies. Through structural biology and computational simulation techniques, the antibody binding sites are precisely predicted and modified to maintain high affinity under acidic conditions. The modified antibodies are then integrated into CAR molecules to construct CAR-M adapted to the tumor microenvironment, and their anti-tumor function under acidic conditions is systematically evaluated, aiming to provide an innovative solution to overcome the inhibition of immunotherapy by the TME. Summary of the Invention

[0005] Objective of the Invention: The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing an acidic pH-dependent modified antibody that specifically binds to exon 5 of CD44 and its applications. This invention provides an acidic pH-dependent monoclonal antibody that specifically binds to human CD44v5 at pH 6.0 by modifying a monoclonal antibody to exhibit high affinity at an acidic pH (pH 6.0).

[0006] To address the aforementioned technical problems, this invention discloses an acid-pH-dependent modified antibody that specifically binds to exon 5 of CD44 and its applications. The specific technical solution is as follows:

[0007] In a first aspect, the present invention provides an acidic pH-dependent modified antibody or fragment thereof that specifically binds to exon v5 of CD44, the antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region;

[0008] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; the light chain variable region is obtained by replacing at least one amino acid residue of the parent antibody light chain variable region with a histidine residue; the parent antibody is an anti-CD44v5 antibody, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.1; the histidine residue replacement includes replacing the 97th threonine residue of the light chain variable region sequence shown in SEQ ID NO.1 with a histidine residue.

[0009] The antibody or its fragment comprises a heavy chain and a light chain; the amino acid sequence of the heavy chain is shown in SEQ ID NO. 4, and the amino acid sequence of the light chain is shown in SEQ ID NO. 17. The light chain is a variant of the light chain of the parent antibody, and the amino acid sequence of the light chain of the parent antibody is shown in SEQ ID NO. 3. This variant involves the substitution of histidine residues. Replacing the 101st threonine residue in the variable region sequence of the light chain shown in SEQ ID NO. 1 with a histidine residue corresponds to replacing the 117th threonine residue in the light chain shown in SEQ ID NO. 3 with a histidine residue.

[0010] In a second aspect, the present invention provides a gene encoding the antibody or a fragment thereof described in the first aspect.

[0011] Thirdly, the present invention provides a recombinant vector containing the gene described in the second aspect.

[0012] Fourthly, the present invention provides a chimeric antigen receptor comprising the antibody or a fragment thereof described in the first aspect. Preferably, the light chain variable region and the heavy chain variable region of the antibody or fragment are linked by a linker peptide to form an ScFv fragment; preferably, the linker peptide is GGGGSGGGGSGGGGS. The ScFv fragment, from the N-terminus to the C-terminus, consists of a heavy chain variable region, a linker, and a light chain variable region.

[0013] The chimeric antigen receptor comprises, from N-terminus to C-terminus, a signal peptide, an ScFv fragment, a hinge region, a transmembrane segment, and an intracellular segment; the signal peptide is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO. 8; the hinge region is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO. 9; the transmembrane segment is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO. 10; and the intracellular segment is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO. 11.

[0014] Fifthly, the present invention provides a gene encoding the chimeric antigen receptor described in the fourth aspect.

[0015] In a sixth aspect, the present invention provides a chimeric antigen receptor macrophage, wherein the chimeric antigen receptor macrophage expresses the chimeric antigen receptor described in the fourth aspect.

[0016] In a seventh aspect, the present invention provides the use of the antibody or fragment thereof described in the first aspect or the chimeric antigen receptor macrophage described in the sixth aspect in the preparation of a tumor therapeutic drug. The tumor includes breast cancer.

[0017] Beneficial effects:

[0018] This invention provides an acidic pH-dependent monoclonal antibody that specifically binds to human CD44v5 at pH 6.0 by modifying a monoclonal antibody to give it high affinity at an acidic pH (pH 6.0). Attached Figure Description

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0020] Figure 1 Predict the three-dimensional structure of the anti-CD44v5 antibody.

[0021] Figure 2 This section assesses the quality of the anti-CD44v5 antibody three-dimensional structure prediction model. A represents the Ramachandran Plot, and B represents the Profile-3D assessment of the anti-CD44v5 antibody prediction model's quality.

[0022] Figure 3 For predicting the three-dimensional structure of antigens.

[0023] Figure 4 This section assesses the quality of antigen three-dimensional structure prediction models. A represents the Ramachandran Plot, and B represents the Profile-3D assessment of the antigen prediction model's quality.

[0024] Figure 5 For antigen-antibody molecule docking.

[0025] Figure 6 This is a histidine pH-dependent scan, showing only some sites.

[0026] Figure 7 The purified antibody was analyzed by SDS-PAGE. The middle column is the marker, the left side of the marker is the non-reduction loading, and the right side is the reduction loading.

[0027] Figure 8 This section describes antibody affinity assays. A shows the ELISA curves for WT and T578H at pH 7.4 and pH 6.0, respectively. B compares antibody affinity at an antigen concentration of 0.05 μg / mL and an antibody concentration of 0.5 ng / mL, and performs significance analysis. C presents statistical data on the affinity of WT and all point-mutated antibodies at pH 7.4 and pH 6.0.

[0028] Figure 9 To detect the affinity of T578H antibody for BLI.

[0029] Figure 10 This is a schematic diagram of a CAR sequence.

[0030] Figure 11 Comparison of phagocytic capacity of CAR-M and UTD under normal pH and acidic pH conditions.

[0031] Figure 12 Comparison of the killing abilities of CAR-M and UTD under normal pH and acidic pH conditions.

[0032] Figure 13 To evaluate the phagocytic and killing capacity of pH-dependent CAR-M under normal and acidic pH conditions, where A represents the phagocytic rate of pH-dependent CAR-M against MDA-MB-231 / CD44v at 12 and 24 hours under normal and acidic pH conditions; and B represents the killing rate of pH-dependent CAR-M against MDA-MB-231 / CD44v under normal and acidic pH conditions, with effective-to-target ratios set at 10:1, 3:1, 1:1, 1:3, and 1:10. Detailed Implementation

[0033] In the following examples, unless otherwise specified, the PBS contained 135mM NaCl, 4.7mM KCl, 10mM Na2HPO4, 2mM NaH2PO4, and had a pH of 7.3±0.1. The D-PBS contained 0.90mM CaCl2, 0.49mM MgCl2, 2.67mM KCl, 1.47mM KH2PO4, 137.93mM NaCl, 8.06mM Na2HPO4, and had a pH of 7.4±0.1.

[0034] Example 1 Antibody and Antigen Modeling

[0035] Due to the lack of 3D structure data for anti-CD44v5 antibodies, this invention predicts the 3D structure of proteins based on their amino acid sequences. To accurately predict protein structure, this invention uses the online tool tFold (https: / / drug.ai.tencent.com / cn) to perform de novo structure modeling of the CD44v5 scFv antibody. The specific process is as follows: 1. Antibody Modeling Process

[0036] First, the amino acid sequences (SEQ ID NO.1 and SEQ ID NO.2, respectively) of the light chain (VL) and heavy chain (VH) variable regions of the humanized anti-CD44v5 antibody were obtained by sequencing and input into the tFold platform in FASTA format. To improve the modeling accuracy of the antibody complementarity-determining region (CDR), the "AntibodyMode" workflow of tFold was selected. This mode optimizes the conformational sampling of the CDR region through a pre-trained antibody-specific language model and geometric constraints. During the modeling process, the number of iterations was set to 3, generating 5 candidate models. The optimal structure was selected based on the model local confidence (pLDDT) and global conformational energy score, resulting in a high-precision three-dimensional conformation of the anti-CD44v5 antibody. Figure 1 ).

[0037] 2. Antibody model quality assessment

[0038] The generated model was imported into Discovery Studio (2019 version) in PDB file format. The antibody model was evaluated using both Ramachandran plot and Profile-3D. The Ramachandran plot evaluates the plausibility of the skeletal structure of two adjacent peptide units based on the minimum contact distance between non-bonded atoms in the protein. For a model to be considered of acceptable quality, the number of amino acids located in the disallowed region should be less than 5% of the total number of amino acids. Profile-3D uses a 3D-1D scoring function to detect the matching degree between the predicted model and the amino acid sequence; a higher score indicates greater confidence in the homology model. The results show that the Verify Score of the model in this invention is 260.64, significantly higher than the Verify Expected Low Score (143.066) and very close to the Verify Expected High Score (317.924). This indicates that the antibody model is of very high quality and can be used for antigen-antibody affinity analysis. Figure 2 ).

[0039] 3. Antigen modeling process

[0040] Following antibody modeling, this invention employs GDFold for antigen prediction modeling. The amino acid sequence of CD44 (SEQ ID NO.14) is exported to the GDfold2 website using FASTA, with the following key parameters set: use_physics, enabling AMBER force field constraints (default weight 0.3); flexible_region, initiating adaptive enhanced sampling for a specified residue range (format: chain ID: start-end); num_models: number of models generated (default 5). Gradient descent optimization is performed on the generated PDB file until energy convergence (energy threshold ΔE < 0.1 kcal / mol). The optimal structure is selected based on the global energy score (total_score) and local confidence (pLDDT). Figure 3 ).

[0041] 4. Antigen model quality assessment

[0042] After model training was completed, Laplace plots and Profile-3D were used to evaluate model quality. The results showed that the CD44v antigen model had a Verify Score of 63.01, significantly higher than the Verify Expected Low Score (40.6152), indicating high model quality suitable for antigen-antibody affinity analysis. Figure 4 ).

[0043] 5. Antibody-antigen interaction analysis

[0044] After antigen and antibody modeling, antibody-antigen molecular docking was performed using the Zdock module in Discovery Studio. First, the antibody and antigen proteins (PDB files) were imported for preprocessing. Hydrogenation and protonation were performed: hydrogen atoms were added using the PrepareProtein tool to optimize the protonation state (e.g., the protonation state of His, salt bridge formation, etc.); energy minimization was achieved by optimizing the local energy of the complex using the CHARMm force field to eliminate atomic conflicts; solvent treatment was performed to remove water of crystallization molecules or retain key water molecules (e.g., water of crystallization participating in the hydrogen bond network). Next, the binding interface was defined, and the molecular docking pocket was defined based on the antibody complementarity-determining region (CDR) to limit the docking search space. The molecular docking program was executed, and finally, each docking configuration was scored using the ZDockScore (combining shape complementarity, electrostatics, and desolvation energy) to select the optimal binding mode. The antibody binding pocket and corresponding amino acid sequence were then analyzed. Figure 5 ).

[0045] Example 2: Construction and affinity analysis of mutant antibodies

[0046] 1. Amino acid saturation mutation

[0047] Virtual amino acid saturation mutagenesis was performed on the antibody-antigen complex obtained in Example 1 using Discovery Studio to provide data for modifying antibodies with high binding affinity. Protein pretreatment was performed first. The BuildMutants module was used to saturate-mutate target residues in the full-length VL sequence of the light chain (amino acid sequence as shown in SEQ ID NO.3) and the full-length VH sequence of the heavy chain (amino acid sequence as shown in SEQ ID NO.4), replacing them with all 20 amino acids. The mutated residues are as follows:

[0048] VH: GLY45, PHE46, THR47, PHE48, SER49, GLY50, PHE51, TRP52, ILE70, ASN71, SER72, ASP73, GLY74, SER75, ALA76, MET77, LEU116, VAL117, LYS118, GLY119, PHE120.

[0049] VL: GLN508, SER509, LEU510, LEU511, ASP512, SER513, ASN514, GLY515, LYS516, THR517, TYR518, LE U536, VAL537, SER538, TRP575, GLN576, GLY577, THR578, HIS579, PHE580, PRO581, GLN582, THR583.

[0050] The above sorting method for amino acid mutation sites is as follows: amino acids 1-461 of the heavy chain full sequence are sorted as 1-461, followed by amino acids 1-239 of the light chain full sequence being sorted as 462-700.

[0051] The mutants were ranked and analyzed based on the Mutation Energy values, specifically using the formula: ΔG mut =ΔΔG bind (mutant)-ΔΔGb ind (wildtype).

[0052] If ΔΔG mut <-0.5 kcal / mol (stable) indicates that this mutation leads to an increase in affinity;

[0053] If -0.5 kcal / mol < ΔΔG mut <0.5 kcal / mol (no effect), meaning this mutation has no effect on affinity;

[0054] If ΔΔG mut>0.5 kcal / mol (unstable), meaning that this mutation will lead to a decrease in affinity.

[0055] Finally, mutants were screened based on ΔΔG values, interaction analysis, and structural rationality. (Based on ΔΔG...) mut Evaluation of candidate amino acid sequences showed that almost every amino acid site was either "stable" or "unstable," indicating that mutations at candidate amino acid sites have a significant impact on affinity and are key amino acids for antigen-antibody binding.

[0056] 2. Histidine scan

[0057] The protonation state of histidine (pKa ~ 6.4) affects antibody-antigen interactions with pH changes and can act as a molecular switch. This invention performs a histidine pH-dependent scan on the aforementioned key amino acids and calculates their binding free energy difference, i.e., the mutation difference ΔΔG. mut Focus on ΔΔG under acidic conditions (pH = 6.0). mut Mutation sites with a concentration less than -0.5 kcal / mol were selected as candidate amino acids. Based on this, nine key amino acids were predicted for further screening. Figure 6 ).

[0058] 3. Construction of vectors for mutant antibodies

[0059] Nine amino acid mutations (S72H, D73H, F120H, D512H, S513H, K516H, Y518H, T578H, and T583H) were obtained through molecular docking and virtual mutation screening, and mutation vectors were constructed. Mutation primers (as shown in Table 1) were designed, and point mutations were performed on the WT antibody vectors pCDNA3.4-TOPO-CD44v5Ab-L and pCDNA3.4-TOPO-CD44v5Ab-H using the MutExpress II Fast Mutagenesis Kit V2. pCDNA3.4-TOPO-CD44v5Ab-L and pCDNA3.4-TOPO-CD44v5Ab-H were synthesized by Sangon Biotech. They were obtained by inserting the full-length VL sequence (amino acid sequence as shown in SEQ ID NO.3, coding gene sequence as shown in SEQ ID NO.5) and the full-length VH sequence (amino acid sequence as shown in SEQ ID NO.4, coding gene sequence as shown in SEQ ID NO.6) into the plasmid vector pCDNA3.4-TOPO, respectively. The double restriction enzyme sites for insertion were BamHI and XhoHI.

[0060] The target mutant plasmid was amplified using Phanta Max Super-Fidelity DNA Polymerase. After thawing and rehydration, all components of the reaction system were thoroughly mixed (shaking well) to construct a 50 μL reaction system. The specific components were as follows: 25 μL 2×Max Buffer, 1 μL dNTP Mix (10 mM), 1 μL template DNA, 2 μL upstream mutant primer, 2 μL downstream mutant primer, 1 μL Phanta Max Super-Fidelity DNA Polymerase, and ddH2O to a final volume of 50 μL. The PCR reaction program was as follows: 98℃ pre-denaturation for 30 s, followed by 35 cycles: 98℃ pre-denaturation for 15 s, 65℃ annealing for 15 s, 72℃ extension for 30-60 s / 1 kb, and a final extension at 72℃ for 5 min, followed by a 4℃ forever. 9 μL of the PCR mixture was added to 1 μL of 10× loading buffer (Beyotime, R0211) and mixed thoroughly. The PCR products were then separated and identified using 1% agarose gel electrophoresis. Since the remaining 41 μL of PCR amplification product contained the original template plasmid, Dpn I digestion was performed before recombination to remove the methylated template plasmid. The reaction mixture consisted of 41 μL of PCR amplification product and 1 μL of Dpn I. The mixture was gently pipetted and mixed, then briefly centrifuged to the bottom of the tube and incubated at 37°C for 2 hours. The product was purified using a gel extraction kit (Qiagen). The concentration of the purified product was determined using a NanoDrop spectrophotometer and used as the Dpn I digested product.

[0061] Prepare the recombinant reaction system on ice: 200 ng Dpn I digest, 4 μL 5×CE II buffer, 2 μL Exnase II, and ddH2O to a final volume of 20 μL. Gently pipette the mixture to mix, briefly centrifuge to collect the contents at the bottom of the tube, and incubate at 37°C for 30 minutes to obtain the recombinant product. Then, place the tube on ice. Add 5 μL of the recombinant product to 100 μL of Lstbl3 competent cells (Thermo Scientific) and incubate on ice for 30 minutes. Next, heat shock the cells at 42°C for 30 seconds, then quickly transfer them back to ice and incubate for 2 minutes. Add 1 mL of LB medium and incubate at 37°C with shaking at 180 rpm for 1 hour. Collect the cells by centrifugation (2000×g, 2 minutes), resuspend, and evenly spread on LB agar plates containing 50 μg / mL kanamycin. Incubate overnight for approximately 15 hours. The following day, 3-5 morphologically sound single colonies were randomly selected and transferred to 5 mL of LB broth containing kanamycin (50 μg / mL). The cultures were incubated at 37°C and 160 rpm with shaking for 10 hours. After incubation, bacterial plasmids were extracted using the Qiagen plasmid mini-prep kit, and their concentration was determined using a NanoDrop spectrophotometer (>200 ng / μL). Qualified samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing. The obtained sequencing results were compared with the expected reference sequence. Based on this, point mutation plasmids were constructed.

[0062] Table 1. Mutant primer sequences

[0063]

[0064]

[0065] 4. Expression and purification of mutant antibodies

[0066] (1) 293FT cells were cultured in DMEM high-glucose medium with 10% v / v FBS, placed on a shaker, and seeded at 5 × 10⁶ cells / year. 5 Cells / well, culture until cells reach 70%-80% confluence.

[0067] (2) Dissolve the plasmid mixture in 100 μL of Opti-MEM medium (Gibco) according to the amount of plasmid DNA of 2 μg (light chain: heavy chain = 1:1 m / m) to obtain the first mixture; dissolve 4 μL of Lipofectamine 2000 (Invitrogen) in 100 μL of Opti-MEM medium, let stand for 5 minutes to obtain the second mixture, mix the first mixture and the second mixture to prepare a liposome solution, and let stand for 20 minutes again after mixing to form a transfection complex.

[0068] (3) Add the transfection complex described in step (2) to the 293FT cells with 70%-80% confluence obtained in step (1), mix gently, and then place on a shaker for culture. After 6 hours, replace with DMEM high-glucose medium containing 2% v / v FBS and maintain the culture temperature at 37°C.

[0069] (4) Collect the supernatant for the first time at 48h, and centrifuge at 3000rpm for 10min to remove cell debris. Filter the supernatant further using a 0.45μm filter membrane, aliquot and store at -80℃, taking care to avoid repeated freeze-thaw cycles. Collect again at 24-hour intervals, repeating the collection process three times, and combine the supernatants to increase yield.

[0070] (5) Equilibrate the Protein A+G column with 6 column volumes of PBS (pH 7.4).

[0071] (6) Add the filtered cell supernatant obtained in step (4), and wash away non-specific binding with 10 column volumes of PBS. Elute the antibody with 0.1M glycine (pH 3.0), and immediately neutralize the eluted antibody to pH 7.0 with 1M Tris-HCl (pH 8.5) to protect the antibody. SDS-PAGE results showed successful antibody expression, indicating that the expression system prepared in this invention is stable. Figure 7 ).

[0072] 5. Construction of antigen plasmid pET-28a CD44v2-v10

[0073] (1) RNA extraction and reverse transcription

[0074] Total RNA isolation and extraction: HCC 1937 cells were washed with PBS, and total RNA was extracted using the TRIzol method. The cell pellet was lysed with 0.5 mL of TRIzol (room temperature, 5 min), and 0.2 mL of chloroform was added, mixed, and allowed to stand. After centrifugation at 12000 g for 10 min at 4 °C, the supernatant was transferred to a new tube and mixed with 0.25 mL of isopropanol to precipitate RNA (room temperature, 10 min). The precipitate was recovered by centrifugation, washed with 75% ethanol, dried, and finally dissolved in 50 μL of DEPC H2O at 65 °C for 10 min. RNA concentration was detected using a NanoDrop instrument.

[0075] cDNA synthesis: Using the Abm 5X All-In-One RT MasterMix kit, 2 μg of total RNA was reverse transcribed in a 20 μL system. The reaction program was: incubation at 25°C for 10 min, extension at 42°C for 50 min, and heat inactivation at 85°C for 5 min. The resulting cDNA was used directly for downstream experiments or stored at -20°C.

[0076] (2) Amplification of the target fragment CD44v2-v10

[0077] Specific primers were designed for CD44v2-v10 (SEQ ID NO.15), and BamHI and XhoI restriction enzyme sites were introduced at the 5' end, as follows:

[0078] Upstream primer:

[0079] 5'-CAGCAAATGGGTCGCGGATCCTTGATGAGCACTAGTGCTACAGCA-3';

[0080] Downstream primer:

[0081] 5'-GTGGTGGTGGTGGTGCTCGAGTTATGATAAGGAACGATTGACATTAGAG-3'.

[0082] Using Yisheng Bio 2×Hieff PCR amplification was performed using the Gold PCR Master Mix premixed reagent. The PCR amplification product was mixed with 10× loading buffer (Beyotime, R0211) and separated by 1% agarose gel electrophoresis. The target band of about 1200 bp was excised under UV light and purified using the Omega gel extraction kit according to the manufacturer's instructions.

[0083] (3) Construction of pET-28a-CD44v2-v10 recombinant plasmid (double enzyme digestion method)

[0084] The pET-28a vector and the CD44v2-v10 PCR product prepared in step (2) were digested with BamHI / XhoHI. After digestion, 2 μL of 10× loading buffer was added to each reaction system. After separation by 1% agarose gel electrophoresis, the target band was excised and purified using a gel extraction kit (Omega). The concentration was detected by a Nanodrop instrument. The ligation system was prepared according to the vector:insertion = 1:4 molar ratio: 0.02 pmol of linearized pET-28a vector, 0.08 pmol of CD44v2-v10 fragment, 1 μL of T4 DNA ligase, 2 μL of 10× ligase reaction buffer, and ddH2O to a final volume of 20 μL. The prepared ligation system was thoroughly mixed and briefly centrifuged. The cells were incubated at 16℃ for 18 h for competent transformation. E. coli DH5α competent cells were thawed on ice. Add all ligation products, gently mix, incubate on ice for 30 min, heat shock in a 42°C water bath for 90 s, and quickly transfer to ice and incubate for 3-5 min. Add 200 μL of antibiotic-free LB medium and incubate at 37°C with shaking at 120 rpm for 1 h. Spread the bacterial culture evenly on LB agar plates containing kanamycin (50 μg / mL) and incubate upside down at 37°C for 16 h. Pick 3 single colonies and inoculate them into kanamycin LB liquid medium. Incubate at 37°C with shaking at 120 rpm until the logarithmic growth phase (OD200). 600 ≈0.6). The sample was sent to Genewiz for Sanger sequencing. After sequence alignment confirmed its correctness, the bacterial strain was amplified and plasmid was extracted to obtain the antigen plasmid pET-28a CD44v2-v10.

[0085] 6. Antigen expression and purification

[0086] (1) The prepared antigen plasmid pET28a-CD44v2-v10 was transformed into BL21(DE3) competent cells and cultured overnight at 37°C inverted. Single clones were then picked for small-scale amplification (5 mL). The cells were then inoculated into 500 mL of LB medium at a ratio of 1:100 v / v and shaken at 37°C for 4-5 hours until the bacterial culture reached OD. 600 =0.6-0.8, add 1mM IPTG, and induce protein expression at 16℃ for 12 hours. After centrifugation at 10000rpm for 10min at 4℃, discard the supernatant, collect the precipitate, and freeze at -20℃ for later use or for further experiments.

[0087] (2) Take the fresh or thawed bacterial pellet obtained in step (1) and resuspend the bacterial cells thoroughly with lysis buffer. Add the protease inhibitor mixture (PMSF final concentration 1mM) and sonicate on ice (power 650W, 55% amplitude, sonication 3s / 6s interval, duration 35min) until the bacterial solution is clear to obtain the lysis buffer. Take 20μL of the lysis buffer and mix it with an equal volume of ddH2O and 40μL of 2×SDS-PAGE loading buffer, and label it as the whole bacterial lysis buffer sample.

[0088] (3) Centrifuge the lysis buffer at 4℃ and 12000rpm for 30min, and take the clear supernatant (if turbid, centrifuge again). Take 20μL of supernatant and mix it with 20μL of 2×SDS-PAGE loading buffer, and label it as supernatant sample.

[0089] (4) Take 5 mL of premixed Ni-NTABeads 6FF packing material (loading capacity 40 mg / mL) and pack it into the chromatography column. After removing the protective solution, equilibrate the packing material with 50 mL of lysis buffer (25 mL × 2 times) to complete the column equilibration.

[0090] (5) Load the clarified lysis supernatant from step (3) onto the equilibrated chromatography column and collect the flow-through under gravity. Repeat the loading once, and mix 20 μL of the flow-through with 20 μL of 2×SDS-PAGE loading buffer, labeling it as the flow-through sample.

[0091] (6) Wash away non-specifically bound proteins sequentially with 60 mL of lysis buffer and 50 mL of wash buffer (containing 25-50 mM imidazole), and collect the wash buffer sample. Wash buffer formulation: 25-50 mM imidazole, 50 mM NaH2PO4 and 500 mM NaCl dissolved in 1 liter of water, adjust the pH to 8.0 with NaOH solution, and sterilize with a 0.22 or 0.45 μM filter membrane.

[0092] (7) The target protein was eluted using a gradient of 40 mL elution buffer (containing 250 mM imidazole), and the eluent was collected in fractions. 20 μL of the eluent was used to prepare a sample, and the protein concentration was determined by A280 absorbance (using the elution buffer as a blank control). Elution buffer formulation: 7.8 g NaH2PO4·2H2O, 29.2 g NaCl, and 17 g imidazole were dissolved in 1 liter of water. The pH was adjusted to 8.0 using NaOH solution, and the solution was sterilized using a 0.22 or 0.45 μM filter membrane.

[0093] (8) After obtaining the target protein through SDS-PAGE and Western blotting analysis, ultrafiltration and concentration were performed using a Millipore 30K Ultra-15mL centrifuge ultrafiltration tube. The tube was centrifuged at 4000×g for 15-45 min at 4℃, and concentrated to the target volume of 200μL. Then, 1×PBS was added, and the solution was centrifuged again to concentrate it to 200μL. The PBS replacement process was repeated, and the solution in the ultrafiltration tube was continuously collected until the concentrated protein solution was obtained. The protein concentration was confirmed using the BCA method and a Nano Drop instrument, and the protein was aliquoted and stored at -20℃.

[0094] The lysis buffer formulation used above is as follows: 78g NaH2PO4·2H2O and 292.3g NaCl dissolved in 1 liter of water, pH adjusted to 8.0 using NaOH solution, and sterilized using a 0.22 or 0.45 μM filter membrane.

[0095] 7. Antibody affinity analysis

[0096] The CD44v antigen prepared in "6. Antigen Expression and Purification" above was used for indirect ELISA detection to evaluate the antibody affinity at pH 7.4 and pH 6.0 (the pH of the reaction environment was controlled by adjusting the pH of the coating buffer and PBS, with the pH adjusted to 6.0 by adding hydrochloric acid). First, three concentrations of antigen (0.25 μg / mL, 0.5 μg / mL, and 1 μg / mL) were prepared using coating buffer (0.71 g Na₂CO₃ and 3.64 g NaHCO₃ dissolved in 1 L of water) and coated onto ELISA plates, respectively, and incubated overnight at 4°C. Blocking was then performed. After blocking, the monoclonal antibody to be tested was serially diluted with PBS from a starting concentration of 1 μg / mL, setting 11 gradient concentrations. 100 μL of the diluted antibody solution was added to each well, and the plates were incubated at 37°C for 1 hour. After washing the plate, add enzyme-labeled secondary antibody (horseradish peroxidase-labeled goat anti-human IgG / H+L, A0201, Beyotime) and incubate at 37°C for 1 hour. After washing the plate again, add 200 μL of TMB chromogenic solution (TMB chromogenic solution is prepared by mixing TMB chromogenic solution A and TMB chromogenic solution B in a 1:1 volume ratio. TMB chromogenic solution A: 13.6 g CH3COONa, 1.6 g citric acid, and 0.3 mL 30% H2O2, and bring the volume to 500 mL with ultrapure water; TMB chromogenic solution B: 0.2 g EDTA-2Na, 0.95 g citric acid, 50 mL glycerol, and 0.2 g TMB, and bring the volume to 500 mL with ultrapure water) to perform the colorimetric reaction, and promptly add 50 μL of 2M sulfuric acid to stop the reaction. Read the absorbance value (OD). 450 / 570 ). Calculate the maximum OD value (OD) at 50% for each antigen concentration. 450 / 570 The corresponding monoclonal antibody concentration (EC)50 EC obtained at three antigen concentrations 50 The values ​​are combined pairwise, and the affinity constant is calculated using the formula Ka = (n-1) / [2(n[Ab']t-[Ab]t)]. In the formula, n represents the ratio of the two antigen concentrations (high / low), and [Ab']t and [Ab]t represent the EC values ​​corresponding to the higher and lower antigen concentrations, respectively. 50 The Ka value is calculated as the average of the multiple Ka values, which is the affinity constant of the monoclonal antibody. This is compared to the non-mutant wild-type (WT, i.e., Figure 8 Compared with WT (1D8 in A), the results showed that only T578H exhibited a significantly enhanced affinity at pH=6.0 compared to WT. Figure 8 Among them, T578H has an affinity of up to 4.07 × 10⁻⁶ at pH 6.0. 9 M -1 This is far higher than the affinity of WT at pH=6.0 (5.13×10⁻⁶). 8 M -1 The full-length sequence of the light chain amino acids obtained from the T578H mutation site is shown in SEQ ID NO.17.

[0097] Bio-layer interferometry (BLI) is a label-free technique for real-time monitoring of intermolecular interactions (such as antibody-antigen binding). By detecting changes in the interference mode of light reflected from the tip of a biosensor, BLI can track the dynamics of molecular binding and dissociation in real time, thereby calculating antibody affinity. Compared to indirect ELISA, BLI is more accurate and direct; therefore, this invention continues to use BLI to accurately detect the affinity of the T578H antibody at pH 7.4 and pH 6.0. The results show that the affinity of T578H at pH 7.4 and pH 6.0 is 4.65 × 10⁻⁶. -9 M and 3.85×10 -9 M( Figure 9 This result is largely consistent with the ELISA results. These results indicate that we have successfully obtained an antibody that maintains high affinity even under acidic pH conditions (pH=6.0) in the tumor microenvironment.

[0098] Example 3: CAR-M Construction and Analysis

[0099] Considering that antibody affinity generally decreases under acidic conditions, and that the recognition principle of CAR-M relies on the recognition of target antigens by ScFv single-chain antibodies, depending on antigen-antibody interactions, this invention hypothesizes that the phagocytic and killing function of CAR-M will decrease under acidic conditions. To verify this hypothesis, this invention adjusts the pH of the cell culture medium to 6.0 using hydrochloric acid (HCl) to simulate an acidic environment.

[0100] 1. CAR-M construction:

[0101] Based on the CD44v5 antibody sequence, a linker (GGGGSGGGGSGGGGS) was used to connect the antibody heavy chain and light chain variable regions (from N-terminus to C-terminus: heavy chain variable region - linker - light chain variable region) to obtain the sequence of the ScFv fragment of the anti-CD44v5 single-chain antibody (SEQ ID NO.7). Then, the CD8α signal peptide (SEQ ID NO.8), ScFv sequence, IgG hinge region (SEQ ID NO.9), transmembrane segment of internal signal FcεRI (SEQ ID NO.10), and intracellular signal domain (also known as intracellular segment, SEQ ID NO.11) were sequentially linked from N-terminus to C-terminus to obtain a CAR element. The T2A element (SEQ ID NO.12, with an additional GSG motif to improve the ribosome jumping efficiency of the 2A peptide) and the RQR8 suicide gene (SEQ ID NO.13, with an added stop codon TAA) were further assembled with the C-terminus of the CAR element to obtain the final CAR sequence (e.g., T2A element (SEQ ID NO.12, with an additional GSG motif to improve the ribosome jumping efficiency of the 2A peptide) and the RQR8 suicide gene (SEQ ID NO.13, with an additional stop codon TAA) were then used to further assemble the CAR element into a final CAR sequence (e.g., ... Figure 10 As shown in the figure, the plasmid was artificially synthesized by Sangon Biotech and integrated into the lentiviral plasmid vector pLenti6 / v5 through two restriction enzyme sites, BamHI and XhoI, to obtain the pLenti6 / v5-CAR plasmid.

[0102] pLenti6 / v5-CAR lentiviral plasmid was co-transfected into 293T cells with lentiviral packaging plasmids psPAX2 and pMD2.G at a ratio of 6:3:1. After 48 hours, the supernatant was collected, centrifuged at 2000g for 15 minutes, and filtered through a 0.45μm filter to remove cell debris. 5× virus concentrate (Lentivirus Concentration Solution kit, Yisheng) was mixed with the filtered virus supernatant at a volume ratio of 1:4. After mixing and allowing to settle at 4℃ for 12 hours, the virus mixture was centrifuged at 2000g for 30 minutes. The supernatant was discarded, leaving a white precipitate at the bottom of the centrifuge tube. The precipitate was resuspended in 1 / 10 volume of DMEM medium to obtain the target lentiviral suspension, which was used immediately or aliquoted and stored at -80℃. THP1 cells were then transfected using the centrifugation transfection method. The specific procedure was as follows: THP-1 cells in logarithmic growth phase (1×10⁶ cells) were transfected into 293T cells. 6 Cells were seeded into 6-well plates (1 mL medium / well). 1 mL of medium containing the target lentivirus (1 / 10 volume) and polybrene (final concentration 8 μg / mL) was slowly added to each well, and the mixture was gently stirred. The plates were centrifuged at 32°C and 1900 rpm for 60 minutes. After centrifugation, the cells were incubated in an incubator (37°C, 5% CO2) for 1 hour. The centrifugation and transfection process was repeated once. After 24 hours, the medium was changed, and the cells were incubated statically for 48 hours to obtain CAR-THP1 (CAR-M). CAR-M cells were screened with 5 μg / mL blastidin. After 48 hours, the cells were centrifuged at 700g and resuspended in clean, complete medium containing 10% serum. Subsequently, the CAR-M was sorted by flow cytometry using anti-CD34 antibody (CD34 Monoclonal Antibody, QBEND / 10, PE, Invitrogen), and finally anti-CD44v5 CAR-M with transfection efficiency and membrane loading efficiency of over 90% was obtained.

[0103] To further refine the evaluation of the biological characteristics of anti-CD44v5 CAR-M, this embodiment constructed a CD44v overexpressing cell line based on the commonly used triple-negative breast cancer cell line MDA-MB-231, named MDA-MB-231 / CD44v (abbreviated as CD44v-OE). The amino acid sequence and encoding nucleotide sequence of human CD44v are shown in SEQ ID NO.14 and SEQ ID NO.16. PCR primers (CD44v-F and CD44v-R) for amplification were designed and synthesized, and XhoⅠ and BamHI restriction sites were added. PCR amplification yielded the PCR product. The PCR primer sequences are as follows:

[0104] CD44v-F: 5'-CTACCGGACTCAGATCTCGAGATGGACAAGTTTTGGTGGCACG-3'; CD44v-R: 5'-CGTCATCCTGTAGTCGGATCCTTACACCCCAATCTTCATGTCCA-3'.

[0105] Next, the PLVX-flag-puro expression vector and PCR product were subjected to double digestion with XhoⅠ and BamHI, respectively. After the reaction, 2 μL of 10× loading buffer was added to each and mixed well. After separation by 0.8% agarose gel electrophoresis, the corresponding bands were excised and purified using a gel extraction kit (Qiagen). The concentration of the purified product was determined by NanoDrop spectrophotometer and used for later use. Subsequently, single-fragment recombination ligation was performed using ClonExpress Ultra One Step Cloning Kit V3. 5 μL of the recombinant product was added to 100 μL of Stbl3 competent cells (Thermo Scientific) and incubated on ice for 30 minutes. Then, the cells were heat-shocked in a 42°C water bath for 30 seconds and quickly transferred back to ice and incubated for 2 minutes. After adding 1 mL of LSOC medium, the cells were cultured at 37°C with shaking at 180 rpm for 1 hour. The culture was centrifuged (2000g, 2 minutes) to collect bacterial cells, resuspended, and evenly spread on SOC agar plates containing 50 μg / mL ampicillin (Amp). Incubation was carried out overnight for approximately 15 hours. The next day, 3-5 morphologically sound single colonies were randomly selected and transferred to 5 mL of SOC liquid medium containing ampicillin (50 μg / mL). Incubation was carried out at 37°C and 160 rpm with shaking for 10 hours. After incubation, bacterial plasmids were extracted using a Qiagen plasmid mini-prep kit, and their concentration was determined using a NanoDrop spectrophotometer (>200 ng / μL). Qualified samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing. Only after confirming the sequence accuracy was subsequent cell transfection performed. Cell transfection was performed using Lipofectamine. TM Transfection with the 2000 transfection reagent was performed strictly according to the product instructions. Twenty-four hours after transfection, the medium was replaced with puromycin-containing selective medium for stable clone selection. One week after selection, cells were collected and total protein was extracted. Western blotting (WB) was used to verify whether the cells stably overexpressed CD44v protein.

[0106] Anti-CD44v5 CAR-M, after stimulation with 100 nM PMA (phorbol ester, stimulation for 24 hours), was co-cultured 1:1 with target antigen-positive cells MDA-MB-231 / CD44v in 6-well plates, with a cell mass of 1 × 10⁶ cells / well. 6 Tumor cells / well. This was used to assess its phagocytic capacity. Stimulated CAR-M cells were co-cultured with stably luciferase-expressing MDA-MB-231 / CD44v cells at different effector-to-target ratios (macrophage number: tumor cell number) in 96-well packs. Each well contained 5000 tumor cells, and the number of macrophages was adjusted according to the set effector-to-target ratio. Bioluminescence of the co-cultured cells was measured after 24 hours. The survival rate and killing rate were calculated using the bioluminescence values: survival rate = (sample well reading - individual culture medium well reading) / (average of individual tumor culture well readings - individual culture medium well readings) × 100%; killing rate = (sample well reading - average of individual tumor culture well readings) / (average of individual culture medium well readings - average of individual tumor culture well readings) × 100%. This assessed its killing ability. Results showed that compared to normal pH, the phagocytic and killing abilities of CAR-M cells were significantly reduced in an acidic environment. Figure 11 and Figure 12 In contrast, the phagocytic and killing abilities of the UTD group were not significantly weakened under acidic conditions. This indicates that acidic environments primarily interfere with CAR-M phagocytosis and killing by affecting antibody recognition rather than the cell's state and phenotype.

[0107] Because CAR-M molecules struggle to exert their efficient phagocytic and killing capabilities in acidic environments, and the tumor microenvironment is often acidic (pH 6.0-6.8) due to lactic acid secretion and accumulation, combined with the previous conclusion that "acidic environments primarily interfere with CAR-M phagocytosis and killing by affecting antibody recognition rather than the cell's state and phenotype," modifying CAR molecules to enable them to efficiently recognize target antigens in acidic environments is an effective way for CAR-M to overcome tumor microenvironment inhibition. Based on the previous pH-dependent modification of the anti-CD44v5 antibody, it is natural to consider replacing the ScFv on the CAR molecule with the heavy chain variable region and light chain variable region of the T578H mutant antibody, that is, mutating the corresponding CDR site threonine (Thr) to histidine (His).

[0108] Based on this approach, point mutations were performed on the ScFv of the CAR molecule to construct anti-CD44v5 CAR-THP1 (referred to as pH-dependent CAR-M, abbreviated as pH CAR-M). Point mutations were then performed on the pLenti6 / v5-CAR plasmid, with pH CAR-F and pH CAR-R primers for the upstream and downstream sides, respectively. The specific point mutation process is the same as in "3. Construction of the mutant antibody vector" in Example 2, resulting in the pLenti6 / v5-pH CAR-M plasmid. The specific pH CAR-M construction process is the same as in "CAR-M construction," ultimately yielding pH CAR-M. The specific primer sequences are as follows: pH CAR-F: 5'-CCACCACTTCCCACAGACATTCGGCGGGGGGA-3';

[0109] pH CAR-R: 5'-TCTGTGGGAAGTGGTGGCCCTGCCAGCAGTAGTACACG-3'.

[0110] The phagocytic and cytotoxic functions of pH CAR-M were evaluated, comparing the differences between normal conditions (pH 7.4) and acidic conditions (pH 6.0). The results showed that, unlike unmodified CAR-M, which exhibited a significant decline in phagocytic and cytotoxic abilities under acidic conditions, pH CAR-M maintained strong phagocytic and cytotoxic abilities under acidic conditions. Both abilities were stronger than under normal physiological conditions (pH 7.4), suggesting that the pH CAR-M designed in this invention still possesses the potential for highly efficient recognition, phagocytosis, and killing of tumor cells in the acidic tumor microenvironment, and does not cause off-target toxicity to normal physiological tissues at physiological pH, demonstrating its advantage in acidic pH response. Figure 13 ).

[0111] This invention provides an acid-pH-dependent modified antibody that specifically binds to exon 5 of CD44, along with its application and related ideas and methods. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. An acid-pH-dependent modified antibody or fragment thereof that specifically binds to exon 5 of CD44, characterized in that, The antibody or its fragment includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; The light chain variable region is obtained by replacing one amino acid residue of the parent antibody light chain variable region with a histidine residue; the parent antibody is an anti-CD44v5 antibody, and the amino acid sequence of the parent antibody light chain variable region is shown in SEQ ID NO.1; the histidine residue replacement is to replace the 97th threonine residue of the light chain variable region sequence shown in SEQ ID NO.1 with a histidine residue.

2. The antibody or fragment thereof according to claim 1, characterized in that, The antibody or its fragments include heavy chains and light chains; The amino acid sequence of the heavy chain is shown in SEQ ID NO.4; The amino acid sequence of the light chain is shown in SEQ ID NO.

17.

3. A gene encoding the antibody or a fragment thereof as described in claim 1 or 2.

4. A recombinant vector containing the gene of claim 3.

5. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor includes the antibody or a fragment thereof as described in claim 1 or 2.

6. The chimeric antigen receptor according to claim 5, characterized in that, The light chain variable region and heavy chain variable region of the antibody and its fragment are linked by linker peptides to form ScFv fragments; The chimeric antigen receptor, from N-terminus to C-terminus, comprises a signal peptide, a ScFv fragment, a hinge region, a transmembrane segment, and an intracellular segment. The signal peptide is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO. 8; The hinge region is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.9; The transmembrane segment is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.10; The intracellular segment is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.

11.

7. The gene encoding the chimeric antigen receptor as described in claim 5 or 6.

8. A chimeric antigen receptor macrophage, characterized in that, The chimeric antigen receptor macrophages express the chimeric antigen receptor as described in claim 5.

9. The use of the antibody or fragment thereof as described in claim 1 or the chimeric antigen receptor macrophage as described in claim 8 in the preparation of a drug for treating tumors.

10. The application according to claim 9, characterized in that, The tumors mentioned include breast cancer.

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