Antibody for treating advanced rectal cancer and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses an antibody for treating advanced rectal cancer and application thereof.The antibody is a monoclonal antibody 3C1 of targeted carcino-embryonic antigen (CEA), the sequence of a heavy chain variable region (VH) of the antibody is as shown in SEQ ID NO: 1, the sequence of a light chain variable region (VL) of the antibody is as shown in SEQ ID NO: 5, and the indirect ELISA detection titer reaches 1: 512000; also provided is an antibody conjugate (ADC) in which the antibody and raltitrexed are coupled by a linker, the drug-antibody ratio (DAR) being 3.0-4.0, the monomer purity being greater than or equal to 95%; the antibody conjugate (ADC) can be used for treating CEA positive advanced rectal cancer, animal experiments show that the tumor inhibition rate of ADC is 86% (significantly higher than 35% of free raltitrexed), the weight loss rate of mice is low, the toxicity of small intestines is light, the problems that existing drugs are poor in targeting and high in toxicity are solved, and the antibody conjugate has the advantages of high efficiency and safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an antibody for treating advanced rectal cancer and its application; specifically, it includes a monoclonal antibody targeting carcinoembryonic antigen (CEA), its preparation method, an antibody-drug conjugate (ADC) comprising the antibody and the cytotoxic drug raltitrexed, and the application of the monoclonal antibody and ADC in the preparation of a drug for treating CEA-positive advanced rectal cancer, particularly suitable for patients with advanced rectal cancer who have poor tolerance to conventional chemotherapy and require precise targeted therapy. Background Technology

[0002] Advanced rectal cancer is a highly prevalent malignant tumor of the digestive tract worldwide, ranking among the top five malignant tumors in terms of incidence. In my country, over 500,000 new cases are diagnosed annually, with approximately 40% of patients already at an advanced stage at diagnosis, losing the opportunity for surgical cure. Chemotherapy has become the core means of prolonging survival. However, existing first-line chemotherapy drugs (such as fluorouracil, oxaliplatin, and irinotecan) have drawbacks including poor targeting and wide systemic distribution. After administration, they are prone to toxicity to normal tissues such as the gastrointestinal mucosa and bone marrow hematopoietic cells, leading to serious adverse reactions such as nausea, vomiting, diarrhea, and leukopenia. Approximately 30% of patients are forced to reduce their dosage or discontinue treatment due to intolerance to the toxicity, making it difficult to balance treatment efficacy with patients' quality of life.

[0003] Carcinoembryonic antigen (CEA) is a characteristic tumor marker of advanced rectal cancer, highly expressed on the surface of more than 90% of advanced rectal cancer cells, while lowly expressed or not expressed in normal gastrointestinal mucosa, liver, and other tissues. It possesses the advantage of "tumor-specific" targeting, making it an ideal target for precision treatment of advanced rectal cancer. However, existing anti-CEA monoclonal antibodies have significant technical shortcomings: most antibodies have insufficient affinity, with indirect ELISA titers often below 1:100,000, making it difficult to efficiently bind to CEA on the tumor surface; while some antibodies can bind to CEA, they cannot effectively mediate drug delivery, resulting in tumor inhibition rates of less than 30% when used alone, and requiring high-dose chemotherapy when combined with other drugs, failing to address toxicity issues and limiting clinical application.

[0004] Antibody-drug conjugates (ADCs), through the synergistic effect of "antibody targeted delivery + cytotoxic drug killing," can increase drug concentration at the tumor site and reduce exposure to normal tissues, representing a significant breakthrough in the treatment of advanced cancer. However, existing ADC drugs for advanced rectal cancer suffer from key process defects: poor linker stability, leading to premature drug release in the bloodstream and high levels of free drug residues; and uneven drug-antibody ratio (DAR) (often 1-2), resulting in large fluctuations in efficacy. Therefore, developing high-affinity anti-CEA antibodies and optimizing ADC conjugation processes are crucial to solving the problem of "low efficacy and high toxicity" in the treatment of advanced rectal cancer. Summary of the Invention

[0005] This invention first provides a monoclonal antibody targeting carcinoembryonic antigen (CEA), named 3C1, whose core features are as follows: Source and Screening: Secreted by hybridoma cells 3C1, these cells were obtained by immunizing Balb / c mice with GMP-grade human CEA antigen (Kaikai Biotechnology, catalog number CEA-HM201, purity ≥95%), fusing with SP2 / 0 myeloma cells at a ratio of 10:1, and undergoing three limiting dilution subcloning screenings. Indirect ELISA detection showed that its antibody titer reached 1:512000, which is significantly higher than existing anti-CEA antibodies. Sequence characteristics: The amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:1, and its complementarity-determining regions (CDRs) CDRH-1, CDRH-2, and CDRH-3 are SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO:5, and its CDRs CDRL-1, CDRL-2, and CDRL-3 are SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; the sequences were verified by comparison with the Kabat database, and the sequence identity of the three positive clones was ≥99%, excluding sequencing errors; Affinity: Using Biacore T200, the dissociation constant (KD) was calculated by fitting a 1:1 binding model with CM5 chip-conjugated antibody and gradient concentrations of CEA as target antigens, confirming that it has high specific binding ability with CEA.

[0006] This invention provides an ADC formed by the above-mentioned monoclonal antibody 3C1 and raltitrexed, with the following core features: Structural composition: It consists of monoclonal antibody 3C1, degradable linker SPDB, and cytotoxic drug raltitrexed, which are coupled to the maleimide group of SPDB through the free thiol group of the antibody hinge region; Quality control indicators: Drug-antibody ratio (DAR) of 3.0-4.0, homogeneity ≥90%; monomer purity ≥95% as determined by SEC-HPLC, aggregates ≤3%, free drug ≤0.5%; flow cytometry showed that its binding rate to CEA-positive rectal cancer cell line LS174T was ≥90%, and its activity retention was ≥85% (compared to the parent antibody). Stability: Accelerated stability test (stored at 40℃ and 75% humidity for 14 days) showed a DAR change of ≤0.3, a purity decrease of ≤2%, and no obvious aggregation, meeting the requirements for drug storage and clinical application.

[0007] This invention provides the application of the monoclonal antibody 3C1 and 3C1-raltitrexed ADC, specifically for the preparation of drugs for treating advanced rectal cancer, particularly suitable for CEA-positive advanced rectal cancer (xenograft model constructed from LS174T cell line expressing CEA). Animal experiments have demonstrated that, when administered at a raltitrexed equivalent of 5 mg / kg (twice a week for 3 weeks), the ADC tumor inhibition rate reached 86%, significantly higher than the 35% of free raltitrexed (P<0.01); moreover, the experimental group mice experienced a weight loss rate of only 5.2% and a small intestinal toxicity score of 1.2 (mild damage), significantly lower than the positive control group, demonstrating superior safety and efficacy compared to existing treatment regimens.

[0008] The advantages of this invention compared to the prior art are as follows: High affinity and specificity: The monoclonal antibody 3C1 has a titer of 1:512000 and strong binding specificity to CEA, laying the foundation for targeted delivery of ADCs; Significantly improved ADC efficacy: 3C1-raltitrexed ADC has a tumor inhibition rate of 86%, which is 143% higher than that of free raltitrexed, solving the problem of insufficient efficacy of traditional drugs; Safety optimization: ADCs reduce drug exposure in normal tissues through targeted delivery, resulting in low weight loss and mild small intestinal toxicity in mice, overcoming the high toxicity defects of existing chemotherapy and ADCs; Stable and controllable process: The ADC preparation process can achieve DAR uniformity ≥90%, purity ≥95%, and stability that meets clinical requirements, making it easy for industrial production. Attached Figure Description

[0009] Figure 1 Figure showing the results of indirect ELISA detection of serum titers in Balb / c mice after immunization.

[0010] Figure 2 The results of indirect ELISA detection of monoclonal antibody titers secreted by different hybridoma cell lines are shown in the figure.

[0011] Figure 3 A graph showing the binding kinetics of monoclonal antibody 3C1 to CEA detected by Biacore T200. Detailed Implementation

[0012] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0013] Example 1: Preparation of monoclonal antibodies against CEA (carcinoembryonic antigen) Six- to eight-week-old female Balb / c mice were intraperitoneally injected with an immunogen containing GMP-grade natural human carcinoembryonic antigen protein (Catalog No.: CEA-HM201, purity ≥95%) from Kaika Biotechnology (Shanghai). The immunogen was emulsified with an equal volume of Freund's complete adjuvant. Immunization was administered in three doses: 200 μg for the first immunization, 100 μg for the second immunization after two weeks, 100 μg for the third immunization after one week, and a 50 μg intraperitoneal pulse immunization without adjuvant after one week. Blood was collected from the tail vein three days after each immunization, and serum titers were measured using an indirect ELISA method. (See below for details.) Figure 1 .

[0014] Figure 1 The results showed that mouse number 3 had the highest and most stable titer, and mouse number 3 was used as the subject for subsequent experiments.

[0015] Three days after the initial immunization of mouse 3, the mice were euthanized, and the spleens were collected. Spleen cells were collected by washing with DMEM medium and grinding through a sieve. Spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 10:1. The mixture was centrifuged at 800 r·min⁻¹ for 10 min, and the supernatant was discarded. 1 mL of PEG fusion agent was added in a 37°C water bath, followed by the slow addition of 10 mL of preheated 37°C DMEM medium over 5 min. After adding 30 mL of medium, the mixture was centrifuged at 800 r·min⁻¹ for 10 min, and the supernatant was discarded. 40 mL of preheated 37°C HAT medium was added, and the mixture was thoroughly mixed before being evenly seeded into 96-well cell plates with feeder cells. The cells were then cultured in a 37°C, 5% CO₂ incubator. Single wells of cell clusters were selected for limiting dilution subcloning, with a total of three subcloning cycles performed. The concentration of HAT medium was gradually reduced, eventually being replaced with HT medium. The culture supernatant was analyzed by indirect ELISA. Finally, the hybridoma cell line that stably secreted anti-CEA antigen antibodies was expanded and cryopreserved. Figure 2 .

[0016] Figure 2 The results showed that, using the indirect ELISA method to detect the monoclonal antibody titers of different hybridoma cell lines, the antibody titer of 3C1 reached 1:512000, which was superior to the monoclonal antibodies of other hybridoma cell lines.

[0017] Prepare 5-6 week old female BALB / c mice. Sensitize the mice by intraperitoneal injection of 1 mL of liquid paraffin. About two weeks later, inject 1×10 63C1 hybridoma cells were used to observe the mice's condition daily. After about one week, the mice's abdomens became significantly distended and they had difficulty moving. Ascites fluid was collected using a 1.2 mm syringe. The collected ascites fluid was centrifuged at 2000 r·min⁻¹ for 10 min to separate blood cells, and then centrifuged again at 8000 r·min⁻¹ for 10 min. The supernatant was collected, and the monoclonal antibodies in the supernatant were purified using protein A and protein G. The eluted antibody solution was concentrated using an ultrafiltration concentrator.

[0018] Hybridoma cells secreting 3C1 antibody (logarithmic growth phase) were used to extract total RNA using Trizol reagent. RNA integrity was verified by 1% agarose gel electrophoresis (clear 28S / 18S bands). cDNA was synthesized using reverse transcriptase with Oligo(dT) primers as a PCR template. PCR amplification was performed using mouse IgG VH and κ-type VL specific primers (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles followed by a 72℃ extension for 10 min). The amplified product was recovered from the gel and ligated into the pMD19-T vector, transformed into DH5α competent cells, and at least three positive single clones were selected for colony PCR verification. Positive clones were sequenced using the dideoxy chain termination method. The obtained VH / VL sequences were uploaded to the Kabat database online alignment tool (http: / / www.kabatdatabase.com / ). The frame region (FR) and complementarity-determining region (CDR) were labeled according to the Kabat coding. The amino acid composition of CDR1 / 2 / 3 was confirmed by sequence alignment. Sequencing errors were eliminated (the consistency of the three clone sequences was ≥99%). Finally, the accurate sequence and structural information of the 3C1 antibody VH / VL were obtained, as shown in Table 1.

[0019] Table 1. Sequence analysis of the heavy and light chain variable regions of monoclonal antibody 3C1 A GE Healthcare CM5 carboxylated sensor chip (catalog number: BR100530) was loaded into a Biacore T200 and EDC / NHS mixture (1:1, v / v) was injected at a flow rate of 30 μL / min to activate the carboxyl groups on the chip surface for 420 s. The 3C1 monoclonal antibody was diluted to 20 μg / mL with 10 mM sodium acetate buffer (pH 4.5) and injected into the flow cell to couple it to the activated surface. A fixed volume control was applied, and the remaining active sites were blocked with ethanolamine for 420 s. A gradient concentration of the target antigen CEA was sequentially injected into the flow cell, and the binding signal was monitored. After each detection, glycine-HCl buffer was injected to remove the bound CEA antigen, and the chip surface was regenerated. A 1:1 binding model was fitted using Biacore Evaluation 3.0 software, and the dissociation constant (KD) between 3C1 and the target CEA was calculated. (See...) Figure 3 .

[0020] Table 2. Results of 3C1 Affinity Detection for Monoclonal Antibody Example 2: Preparation of antibody-drug conjugate (ADC) of monoclonal antibody 3C1 conjugated with raltitrexed Step 1: Pretreatment of anti-CEA antibody to generate conjugable thiol groups Antibody dialysis: Anti-CEA antibody 3C1 (100 mg, concentration 10 mg / mL) was dialyzed three times with DPBS (pH 7.4) for 4 hours each time to remove sodium azide and free impurities from the antibody storage solution and avoid interfering with the coupling reaction; Disulfide bond reduction: Add 0.5M DTT (molar ratio: DTT / antibody = 10:1) to the dialyzed antibody 3C1, and gently stir at 25℃ for 30 min to reduce the interchain disulfide bonds in the antibody hinge region (theoretically 4 free thiol groups are generated per antibody molecule). Desalting and purification: Using a PD-10 desalting column (pre-packed with Sephadex G-25), with DPBS (pH 7.4) containing 1 mM EDTA as the elution buffer, the reduced antibody was collected (peak shape was monitored by UV 280 nm), and the free thiol content was detected by the Ellman reagent method to ensure that each antibody molecule contains 3.5-4.0 -SH groups (to ensure subsequent conjugation efficiency).

[0021] Step 2: Synthesis of raltitrexed-linker (Drug-Linker) Linker activation: Dissolve 10 mg of degradable linker (SPDB) in anhydrous DMF (N,N-dimethylformamide), add N-hydroxysuccinimide (NHS, molar ratio: SPDB / NHS=1:1.2) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, molar ratio: SPDB / EDC=1:1.2), and react at 25 °C for 1 h to activate the carboxyl group of the linker; Drug-linker coupling: The activated linker solution was slowly added dropwise to raltitrexed solution (5 mg raltitrexed dissolved in 0.1 M citrate buffer, pH 5.0), with a molar ratio of raltitrexed to linker = 1:1.1. The mixture was stirred at 25 °C in the dark for 2 h. The reaction was monitored by reversed-phase HPLC (C18 column, mobile phase: acetonitrile / 0.1% trifluoroacetic acid water = 30:70), and the Drug-Linker peak (retention time approximately 8.5 min) was collected. Solvent removal: The collected Drug-Linker solution was evaporated using a rotary evaporator (35°C, vacuum) to remove DMF. The residual liquid was reconstituted with DPBS (pH 7.4) and the concentration was adjusted to 2 mg / mL for later use.

[0022] Step 3: Coupling reaction of anti-CEA antibody 3C1 with Drug-Linker Construction of the coupling reaction system: The reduced anti-CEA antibody 3C1 (concentration 8 mg / mL) from step 1 was mixed with the Drug-Linker solution from step 2 at a molar ratio of 1:8 (antibody: Drug-Linker), with a total reaction volume of 50 mL. The pH was adjusted to 6.5-7.0 (the optimal pH for the reaction of maleimide with thiol groups) using 1M HCl / NaOH. Mild coupling: Stir at 2-8℃ in the dark for 12 h, monitor the reaction progress by SEC-HPLC (Superdex 200 column, mobile phase: 0.02MPBS pH7.4), and terminate the reaction when the percentage of free Drug-Linker peak (retention time 15 min) is <5%; Quenching unreacted thiol groups: Add N-acetylcysteine ​​(molar ratio: NAC / Drug-Linker=2:1), stir at 2-8℃ for 30 min to quench unreacted free thiol groups and prevent antibody aggregation.

[0023] Step 4: Purification and refining of the ADC product Preliminary purification (removal of free drug): The conjugation reaction solution was loaded onto a Superdex 200 Increase 10 / 300GL gel column. 0.02M PBS (pH 7.4, containing 0.02% Tween 80) was used as the mobile phase at a flow rate of 0.5 mL / min. The ADC main peak was collected by dual-wavelength detection at 280 nm (antibody) and 254 nm (raltitrexed) (retention time 10-12 min). Free Drug-Linker (15 min) and antibody aggregates (8 min) were discarded. Fine purification (removal of unconjugated antibodies): The preliminarily purified ADC solution was loaded onto a HiTrap Q HP ion exchange column (5 mL), and eluted with a gradient of 0.02 M Tris-HCl buffer (pH 8.0) containing 0-0.5 M NaCl (elution time 30 min). The unconjugated antibodies were eluted first due to their higher isoelectric point (NaCl concentration 0.2 M). The ADC was eluted at a NaCl concentration of 0.3-0.4 M due to the charge change after drug binding. The ADC elution peak was collected. Aseptic processing: The finely purified ADC solution was filtered through a 0.22 μm polyethersulfone (PES) sterile filter membrane and dispensed into sterile vials (10 mg / 5 mL per vial) to obtain the ADC product, as shown in Table 3.

[0024] Table 3. Quality control and testing results of ADC finished products Example 3: Experiment on the inhibition of advanced rectal cancer by antibody-drug conjugate (ADC) of 3C1-raltitrexed. Laboratory animals: SPF-grade BALB / c nude mice, female, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; Tumor cells: human advanced rectal cancer cell line LS174T (CEA positive, purchased from Shanghai Fuheng Biotechnology Co., Ltd., catalog number: FH0014); Test drugs: 3C1-raltitrexed ADC (DAR=3.5, prepared in Example 2, raltitrexed equivalent concentration 1mg / mL), free raltitrexed (pharmaceutical grade, concentration 1mg / mL, positive control), physiological saline (blank control).

[0025] Establishment of xenograft tumor model: LS174T cells in logarithmic growth phase (1×10⁻⁶) were used. 7The tumor cells (10 mL / kg) were resuspended in physiological saline, and 0.2 mL was injected subcutaneously into the right side of each nude mouse. The mice were housed in an SPF environment. When the tumor volume reached 100-150 mm³, they were randomly divided into 4 groups (n=6 / group): model group (no drug administration), blank group (physiological saline, 10 mL / kg), positive control group (free raltitrexed, 5 mg / kg), and experimental group (3C1-raltitrexed ADC, at the equivalent of raltitrexed 5 mg / kg). Dosage regimen: Twice weekly via tail vein injection for 3 weeks, with a dosage of 10 mL / kg; Detection indicators: ① Twice weekly, the long diameter (L) and short diameter (W) of the tumor were measured using calipers, and the tumor volume V = 0.5 × L × W² was calculated. At the experimental endpoint (21 days after administration), mice were sacrificed, and the tumor weight was measured. The tumor inhibition rate was calculated as (average tumor weight of the model group - average tumor weight of the experimental group) / average tumor weight of the model group × 100%; ② Mouse body weight was measured weekly to assess toxicity; ③ Small intestinal tissue was taken for pathological sections, and gastrointestinal toxicity was assessed using a "0-4" scoring system (0 points for no damage, 4 points for severe damage). The results are shown in Table 4.

[0026] Table 4. Experimental results of antibody-drug conjugate (ADC) of 3C1-raltitrexed The results showed that the core indicators (mean tumor weight, tumor inhibition rate, and weight change rate) of the model group and the blank group were almost the same, proving that saline had no anti-tumor activity and did not interfere with tumor progression, thus excluding the interference of the "solvent effect" on the results, and the blank group setting was effective. The tumor inhibition rate of the positive control group was 35%, which was significantly higher than that of the model group / blank group, but lower than that of the experimental group (86%). This not only verified the positive control value of "raltitrexed itself has anti-rectal cancer activity", but also demonstrated the synergistic effect of the targeted conjugation of 3C1-raltitrexed ADC through the difference of "ADC efficacy > free drug". The positive control group experienced a decrease in weight due to the systemic distribution of free drug and the toxicity to normal tissues, while the experimental group only experienced a decrease of 5.2%, proving that 3C1-raltitrexed ADC reduced the damage of the drug to normal metabolic tissues. The positive control group scored 2.4 (severe damage), while the experimental group scored 1.0 (mild damage), verifying the logic that "free drug stimulates the gastrointestinal mucosa → ADC drug is only released in the tumor, reducing gastrointestinal exposure". The results showed that the 3C1-raltitrexed ADC had a significantly higher tumor inhibition rate against LS174T advanced rectal cancer xenografts than free raltitrexed (P<0.01); and the weight loss rate and gastrointestinal toxicity of the experimental group mice were significantly lower than those of the positive control group, proving that it has better safety while enhancing the anti-advanced rectal cancer effect.

[0027] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody for treating advanced rectal cancer, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region (VH) is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region (VL) is as shown in SEQ ID NO:

5.

2. The antibody according to claim 1, characterized in that, The amino acid sequences of the complementarity-determining regions CDRH-1, CDRH-2, and CDRH-3 of the VH are SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively, and the amino acid sequences of the complementarity-determining regions CDRL-1, CDRL-2, and CDRL-3 of the VL are SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

3. An antibody-drug conjugate (ADC), characterized in that, The ADC consists of the antibody as described in claim 1 or 2, the degradable linker SPDB, and raltitrexed.

4. The use of the antibody according to any one of claims 1-2 or the antibody conjugate according to claim 3 in the preparation of a drug for treating advanced rectal cancer, characterized in that, The advanced rectal cancer mentioned is carcinoembryonic antigen (CEA) positive advanced rectal cancer.

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