Bispecific antibody photoimmuno-suppressors, methods of making and use thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a bispecific antibody photoimmunoassay inhibitor, its preparation method, and its application. The bispecific antibody photoimmunoassay inhibitor is composed of a bispecific antibody against EGFR and HER3 linked to a photosensitizer. The photosensitizer includes dihydroporphyrin e6, IR700, verteporfin, and / or hematoporphyrin monomethyl ether. The near-infrared photosensitizer is coupled to the Fc segment or constant region of the bispecific antibody via hydrazone bonds or disulfide bonds. This bispecific antibody photoimmunoassay inhibitor can simultaneously target EGFR and HER3, precisely killing tumor cells through phototherapy, and has broad application prospects in tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bispecific antibody photoimmunoinhibitor, its preparation method, and its application. Background Technology

[0002] Gliomas are the most common type of primary intracranial tumor originating from glial cells in the central nervous system, accounting for approximately 30% of all brain tumors and 80% of all malignant tumors. According to the World Health Organization (WHO) classification of tumors of the central nervous system, gliomas are classified into grades I-IV based on malignancy. Glioblastoma (GBM) belongs to WHO grade IV, representing the most malignant, aggressive, and common subtype of adult malignant gliomas. Glioblastoma is extremely difficult to treat and has a very poor prognosis. The current standard treatment for glioblastoma is a comprehensive approach based on surgery, combined with radiotherapy and chemotherapy. This method is limited by key challenges such as complete tumor resection, blood-brain barrier obstruction, tumor heterogeneity and drug resistance, and the immunosuppressive microenvironment, resulting in very limited treatment efficacy. Therefore, there is an urgent need to develop novel treatment strategies and drugs to overcome the blood-brain barrier, improve tumor targeting, overcome drug resistance, modulate the immune microenvironment, or provide entirely new therapeutic mechanisms, thereby improving the treatment outcomes and survival prognosis of glioblastoma patients.

[0003] Akalux is the world's first approved photoimmunotherapy drug, composed of the anti-EGFR monoclonal antibody cetuximab and the near-infrared photosensitizer IR700 linked by a thioether bond. Its mechanism of action is based on dual targeting: cetuximab specifically binds to EGFR highly expressed in head and neck tumors (positive rate approximately 85%), achieving precise drug localization; IR700 undergoes a photophysical reaction under 690nm near-infrared light irradiation, producing a cell membrane perforation effect and releasing immunogenic death signals. The clinical treatment process consists of three phases: ① Intravenous infusion of Akalux (dose 21.5 mg / m²). 2 ); ② After 20-28 hours, use the Class III medical device BioBlade laser system (output power 30 J / cm²). 2 ① Irradiate the lesion through a fiber optic catheter; ② Photoactivation induces tumor cell membrane rupture and release of damage-associated molecular patterns (DAMPs), activates dendritic cells and enhances CD8+ T cell infiltration, forming a systemic anti-tumor immune response.

[0004] Akalux, the world's first photoimmunotherapy drug, offers new hope to patients with head and neck cancer. However, existing photoimmunotherapy drugs also have certain limitations: 1) Limited tissue penetration depth: The photosensitizer IR700 in Akalux absorbs near-infrared light. Although near-infrared light has enhanced tissue penetration compared to visible light, it is still limited. For some deep tumors, such as those located at the base of the skull or deep in the neck, closely adjacent to structures like large blood vessels, the light cannot effectively reach the tumor site, thus limiting the therapeutic effect of Akalux. 2) Antigen expression heterogeneity leading to poor targeting: In head and neck malignancies, tumor cells exhibit antigen expression heterogeneity, meaning that the EGFR expression levels on the surface of different tumor cells vary. Studies have shown that even within the same tumor, EGFR expression may be inconsistent. This heterogeneity affects the therapeutic effect of Akalux.

[0005] Currently, there are no photoimmunotherapy drugs that are effective in treating malignant tumors such as gliomas. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a bispecific antibody against EGFR and HER3, which can simultaneously target EGFR and HER3, providing technical support for the subsequent development of bispecific antibody photoimmunoassay inhibitors.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A bispecific antibody against EGFR and HER3, wherein the bispecific antibody against EGFR and HER3 is composed of an anti-EGFR antibody and an anti-HER3 antibody linked together; the anti-EGFR antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.9; the anti-HER3 antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.17 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.18.

[0009] Preferably, the bispecific antibody is composed of an anti-EGFR antibody and an anti-HER3 antibody linked by a linker, the amino acid sequence of which is shown in any one or more of SEQ ID NO.27 to SEQ ID NO.29.

[0010] Preferably, the amino acid sequence of the bispecific antibody is shown in any one or more of SEQ ID NO.19 to SEQ ID NO.26.

[0011] Preferably, the amino acid sequence of the bispecific antibody is shown in SEQ ID NO.20.

[0012] The second objective of this invention is to provide a bispecific antibody photoimmunoassay inhibitor with good targeting ability, which can simultaneously target EGFR and HER3, and precisely kill tumor cells through in vitro near-infrared light, showing broad application prospects in the treatment of glioblastoma.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] A bispecific antibody photoimmunosuppressant, wherein the bispecific antibody photoimmunosuppressant is formed by linking the aforementioned bispecific antibody with a near-infrared photosensitizer.

[0015] Preferably, the near-infrared photosensitizer is coupled to the Fc segment or constant region of the bispecific antibody via an hydrazone bond or a disulfide bond; the near-infrared photosensitizer includes one or more of dihydroporphyrin E6, IR700, verteporfen, and hematoporphyrin monomethyl ether.

[0016] The third objective of this invention is to provide a method for preparing the aforementioned bispecific antibody photoimmunoassay inhibitor.

[0017] To achieve the above objectives, the present invention adopts the following technical solution:

[0018] The preparation method of bispecific antibody photoimmunosuppressant includes the following steps:

[0019] 1) The aforementioned bispecific antibody was mixed with activators EDC and NHS and reacted to obtain an activated antibody solution;

[0020] 2) The activated antibody solution obtained in step 1) is mixed with the butyrate derivative solution to obtain the antibody-butyrate complex;

[0021] 3) The photosensitizer is mixed with EDC·HCl and NHS to react and obtain an activated photosensitizer;

[0022] 4) The activated photosensitizer obtained in step 3) is mixed and reacted with the antibody-butyrate complex to obtain the bispecific antibody photoimmunoinhibitor.

[0023] Preferably, in step 1), the reaction conditions include: stirring the reaction at room temperature for 1-2 hours.

[0024] Preferably, in step 1), before conjugation, the bispecific antibody is dissolved in PBS buffer solution at pH 7.4 to prepare an antibody solution of 5-10 mg / mL.

[0025] Preferably, in step 1), the final concentration of EDC is 5-10 mM; and the final concentration of NHS is 1-5 mM.

[0026] Preferably, in step 2), the reaction conditions include: stirring the reaction for 6-12 hours at room temperature to 37°C and pH 6-8.

[0027] Preferably, in step 2), samples can be taken periodically during the reaction process, and the reaction progress can be detected by methods such as HPLC to observe the formation of the antibody-butyrate derivative complex and the consumption of reactants.

[0028] Preferably, the reaction solution obtained in step 2) is subjected to dialysis or ultrafiltration centrifugation to obtain the antibody-butyrate complex.

[0029] Preferably, in step 3), the molar ratio of photosensitizer, EDC and NHS is 1:1.2:1.5.

[0030] Preferably, in step 3), the reaction conditions are: room temperature and light protection for 15-30 min.

[0031] Preferably, in step 4), the antibody-butyrate complex is dissolved in PBS at a concentration of 1-5 mg / mL.

[0032] Preferably, in step 4), the molar ratio of the antibody-butyrate complex to the photosensitizer is 1:5-20.

[0033] As a preferred option, the reaction conditions in step 4) are: stirring at 4°C in the dark for 4-6 h or reacting at room temperature for 2 h.

[0034] Preferably, in step 4), the concentration of the antibody-butyrate complex is 1-5 mg / mL.

[0035] Preferably, the reaction solution obtained in step 4) is subjected to ultrafiltration and centrifugation to obtain a bispecific antibody photoimmunoinhibitor.

[0036] More preferably, the ultrafiltration centrifugation includes: transferring the reaction solution to a 100 kDa ultrafiltration centrifuge tube, centrifuging at 4°C and 3000×g for 10-15 min, discarding the filtrate (containing free photosensitizer); adding PBS and repeating the centrifugation 3 times, retaining the retentate.

[0037] The fourth objective of this invention is to provide the application of the aforementioned bispecific antibody and / or the aforementioned bispecific antibody photoimmunoassay inhibitor in the preparation of antitumor drugs.

[0038] To achieve the above objectives, the present invention adopts the following technical solution:

[0039] The use of bispecific antibodies and / or bispecific antibody photoimmunosuppressants in the preparation of antitumor drugs, wherein the tumor includes glioma.

[0040] Preferably, the tumor is a glioblastoma.

[0041] Preferably, the antitumor drug is an antitumor drug for photoimmunotherapy; by intravenous injection of a bispecific antibody photoimmunoinhibitor, near-infrared light is irradiated onto the tumor site using an implanted optical fiber to activate the coupled photosensitizer and kill tumor cells.

[0042] Preferably, the wavelength of the near-infrared light is 650-700 nm, more preferably 689 nm or 664 nm.

[0043] Preferably, the bispecific antibody photoimmunosuppressant is used as adjuvant therapy after surgical resection of glioma, or for local in situ treatment of unresectable glioma sites, or in combination with chemotherapy and / or radiotherapy for antitumor treatment.

[0044] As a preferred option, deep tumors can be precisely irradiated by implanted ultra-fine optical fibers. Multimode fiber coupling technology is used to efficiently connect the laser source and the fiber end face. Combined with the microlens design at the end of the fiber, the penetration depth reaches 3-5cm, and it can be implanted in the body for continuous irradiation.

[0045] The beneficial effects of this invention are as follows:

[0046] 1. This invention proposes for the first time a photoimmunotherapy drug for treating deep tumors such as glioblastoma. This bispecific antibody photoimmunoinhibitor has good targeting ability and can simultaneously target EGFR and HER3. It can precisely kill tumor cells through in vitro near-infrared light and has broad application prospects in the treatment of glioblastoma.

[0047] 2. The bispecific antibody photoimmunosuppressant of the present invention can be used as an adjuvant therapy after surgical resection of glioma, or for local in situ treatment of unresectable glioma sites, and can also be combined with chemotherapy and radiotherapy to improve efficacy.

[0048] 3. The bispecific antibody photoimmunoassay inhibitor of this invention exhibits significant tumor-suppressing effects, which are superior to those of monoclonal antibody + photosensitizer. In vitro validation results show that, 6 days after the start of administration, the average tumor volume in the solvent control group of tumor-bearing mice reached 785 mmHg. 3 The tumor volume in the EGFR (10 mg / kg) + photosensitizer group was 529 mm. 3 The T / C percentage was 63.34%, and the TGI percentage was 36.66%; while the tumor volume in the EGFR+HER3 (10mg / kg)+photosensitizer treatment group of this invention was 447mm. 3 The T / C% was 53.62%, and the TGI% was 46.38%. Attached Figure Description

[0049] Figure 1The image shows the body weight of mice with CT2A subcutaneous tumors treated with photosensitizers and antibodies.

[0050] Figure 2 Day 6 tumor accumulation map of mice with CT2A subcutaneous tumor treated with photosensitizer and antibody;

[0051] Figure 3 The graph shows the tumor growth inhibition rate in mice with CT2A subcutaneous tumors treated with photosensitizers in combination with antibodies. Detailed Implementation

[0052] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0053] This invention's dual-antibody photoimmunoassay inhibitor specifically addresses the limitations of Akalux in tissue penetration and poor targeting due to antigen expression heterogeneity. The core value of the dual-antibody molecule design lies in its simultaneous recognition of two antigens, thereby overcoming tumor antigen expression heterogeneity and enhancing targeting. Target selection employs a primary target plus secondary target combination strategy. The primary target is a tumor cell-highly expressed and highly specific antigen, such as EGFR, ensuring targeting of most tumor cells. The secondary target is a complementary antigen to the primary target, such as tumor matrix-associated antigens like HER3, to cover tumor cells that escape due to antigen loss or low expression, reducing missed targets. Simultaneously, secondary targets can also be selected from components in the tumor microenvironment, such as CD13 of tumor vascular endothelial cells or CD44 of tumor-associated macrophages. This not only enhances antibody accumulation at the tumor site but also increases retention within tumor tissue through dual-target binding, reducing diffusion to normal tissues.

[0054] In this invention, regarding the optimization of the bispecific antibody structure, photosensitizers, such as IR700 or better near-infrared photosensitizers, are selected for conjugation to the Fc segment or constant region of the bispecific antibody to avoid affecting the affinity of the two antigen-binding domains. Simultaneously, the conjugation ratio is controlled to balance targeting and phototoxicity. Furthermore, a miniaturized bispecific antibody form is employed to reduce the molecular weight and improve tissue penetration, facilitating targeted delivery to deep tumors.

[0055] In this invention, to address the problem of insufficient penetration depth of near-infrared light, fiber optic implantation technology is used to deliver the light source directly to the vicinity of deep tumors, reducing the attenuation of light by tissues; a low-toxicity photosensitizer is injected locally at the tumor site, and the reactive oxygen species generation efficiency of the photosensitizer is enhanced through energy transfer, thereby reducing the required light intensity and minimizing damage to surrounding normal tissues.

[0056] In this invention, the antibody sequence information is shown in Table 1.

[0057] Table 1

[0058]

[0059] In this invention, the bispecific antibody is composed of an IgG molecule from an anti-EGFR antibody and a scFv domain from an anti-HER3 antibody. Both monoclonal antibody monomers exhibit efficacy exceeding that of commercially available products. The combined bispecific antibody structure effectively improves the stability of the entire fusion protein while retaining the affinity of the IgG molecule itself. Furthermore, the preparation of bispecific antibodies based on the IgG molecule effectively reduces uncertainties in stability and immunogenicity caused by excessive alterations to the molecular structure, offering significant advantages.

[0060] In this invention, the linker consists of a hydrazone (C=NN) bond acid-sensitive component (butyrate bond partially linked to the antibody) and a photosensitizer (disulfide bond (SS) linked to the antibody). When the drug is endocytosed by the target cells, the hydrazone bond breaks under low pH conditions, and the disulfide bond is reduced by cytoplasmic glutathione reductase, releasing the photosensitizer.

[0061] In this invention, a photosensitizer and implanted optical fiber are used: Verteporfin is the coupled payload, a second-generation porphyrin-based photosensitizer. Its maximum absorption wavelength is 689 nm (near-infrared region), and upon photoactivation, it generates reactive oxygen species (such as singlet oxygen), selectively damaging newly formed vascular endothelial cells or tumor cells through phototoxicity. A ceramic ferrule is used as the optical transmission interface, and a biocompatible ceramic ferrule (such as zirconia) is permanently implanted into the tumor target area via minimally invasive intervention, forming a stable anchoring point. The ferrule surface is treated to resist photoaging, has a built-in optical coupling interface, and can withstand long-term laser irradiation.

[0062] In this invention, a combined targeted synergistic effect is achieved: the implanted optical fiber can be combined with a dual-antibody photoimmunosuppressant. The antibody targets the photosensitizer to the tumor cell membrane, and then the fiber optic irradiation disrupts membrane integrity, enhancing anticancer activity. Furthermore, the fiber-guided near-infrared light can activate the photosensitizer-carrier complex, enabling responsive drug release within the tumor microenvironment.

[0063] In this invention, precise light energy delivery is achieved because deep tumors suffer from light scattering and absorption by the tissue, making it difficult for conventional surface irradiation to guarantee an effective light dose reaching the lesion. Implantable optical fibers can directly guide the laser into the tumor, achieving localized high-intensity light irradiation (e.g., direct irradiation of tumor tissue with 689 nm red light), ensuring that the photosensitizer is fully activated.

[0064] In this invention, the mechanism of action is as follows: After intravenous injection of a dual-antibody photoimmunoassay inhibitor, the antibody partially targets and binds to EGFR and HER3 on the surface of tumor cells. Subsequently, the lesion is irradiated with near-infrared light (689 nm) in vitro, and the implanted optical fiber activates the photosensitizer verteporfin to produce a phototoxic reaction, precisely killing tumor cells while protecting normal tissues. This design gives the dual-antibody photoimmunoassay inhibitor both targeted and photoselective properties, reducing systemic toxicity.

[0065] Example 1

[0066] 1. Protein expression of anti-EGFR / HER3 bispecific antibodies

[0067] (1) Harvesting the supernatant: The light chain cDNA and heavy chain cDNA of the artificially synthesized antibody were cloned into the pTT5 plasmid, and the correct construction of the plasmid was confirmed by sequencing. The sequenced plasmid was transfected, and fresh Freestyle293 Gibco medium was added after transfection. The final volume was 50 ml of medium, centrifuged at 1000 rpm for 10 min, and 0.5 ml of 2M Tris pH8.0 stock solution and 0.3 g of NaCl powder were added to the supernatant. The mixture was stirred evenly and filtered through a 0.22 μm filter membrane.

[0068] (2) Loading: Equilibrate 5-10 column volumes of Mabselect column with Buffer D and load the sample at a flow rate of 3 ml / min.

[0069] (3) Washing: After loading the sample, wash the column with 80ml Buffer D.

[0070] (4) Elution: Use 0.1M Glycine pH2.7 for direct elution. Collect the eluent in 15ml tubes. Add 800μl of 2M Tris pH8.0 stock solution and 900μl of 2M NaCl stock solution to the collection tube in advance, and mix the eluent quickly and evenly.

[0071] (5) Purity identification: SDS-PAGE gel electrophoresis (12.5% ​​SDS-Polyacrylamide gel) was used to identify the purity of the target protein.

[0072] (6) Concentrate the sample to 1-2 ml, add PBS, concentrate again, repeat 3 times, determine the concentration, dilute the EGFR / HER3 sample concentration, quick freeze in liquid nitrogen, and store at -80°C.

[0073] 2. Coupling reaction

[0074] (1) Conjugation preparation: Dissolve the antibody in an appropriate amount of PBS buffer solution (pH 7.4) to prepare an antibody solution of 5-10 mg / mL. Under stirring conditions, add appropriate amounts of activators EDC and NHS to the antibody solution to achieve final concentrations of approximately 5-10 mM and 1-5 mM, respectively. The role of EDC and NHS is to activate the carboxyl groups on the antibody, enabling them to react with butyrate derivatives. Stir the reaction at room temperature for 1-2 hours to fully activate the antibody. During the reaction, EDC and NHS will form an active ester intermediate with the carboxyl groups on the antibody, preparing for the subsequent hydrazone bond formation reaction.

[0075] (2) Preparation of butyrate derivatives: Dissolve butyrate hydrazine in ethanol or dimethyl sulfoxide (DMSO) to prepare a 20-50 mM solution.

[0076] (3) Hydrazone bond linkage reaction and termination: The activated antibody solution is mixed with the butyrate derivative solution and stirred for 6-12 hours at room temperature to 37°C and pH 6-8. This allows the hydrazine group of the butyrate derivative to react with the active ester intermediate on the activated antibody, forming a hydrazone bond, thereby linking the butyrate bond to the antibody moiety. During the reaction, samples can be taken periodically, and the reaction progress can be detected by methods such as HPLC to observe the formation of the antibody-butyrate derivative complex and the consumption of reactants.

[0077] (4) Product purification: Transfer the reaction mixture obtained in step (3) to a dialysis bag or ultrafiltration centrifuge tube, and perform dialysis or ultrafiltration purification using 0.01 mol / L PBS (pH 7.4). During dialysis, the buffer solution needs to be changed multiple times to remove unreacted butyrate derivatives, activators, and other small molecule impurities; ultrafiltration centrifugation should be performed according to the corresponding operating instructions to separate the product from impurities and retain the desired antibody-butyrate complex.

[0078] The specific structure and sequence of the bispecific antibody against EGFR and HER3 of the present invention are as follows:

[0079] The polypeptide chain from the N-terminus to the C-terminus contains: ①VL-HER3-linker1-VH-HER3-linker2-VH-EGFR-CH1-CH2-CH3, with the amino acid sequence shown in SEQ ID NO.19; or ②VH-HER3-linker1-VL-HER3-linker2-VH-EGFR-CH1-CH2-CH3, with the amino acid sequence shown in SEQ ID NO.20; or ③EGFR-CH1-CH2-CH3-linker2-VL-HER3-linker1-VH-HER3, with the amino acid sequence shown in SEQ ID NO.21; or ④EGFR-CH1-CH2-CH3-linker2-VH-HER3-linker1-VL-HER3, with the amino acid sequence shown in SEQ ID NO.22; while the light chain from the N-terminus to the C-terminus contains VL-EGFR-CL.

[0080] The polypeptide chain from the N-terminus to the C-terminus may contain: ⑤ VL-EGFR-linker1-VH-EGFR-linker2-VH-HER3-CH1-CH2-CH3, the amino acid sequence of which is shown in SEQ ID NO.23; ⑥ or VH-EGFR-linker1-VL-EGFR-linker2-VH-PDL1-CH1-CH2-CH3, the amino acid sequence of which is shown in SEQ ID NO.24; ⑦ or HER3-CH1-CH2-CH3-linker2-VL-EGFR-linker1-VH-EGFR, the amino acid sequence of which is shown in SEQ ID NO.25; ⑧ or HER3-CH1-CH2-CH3-linker2-VH-EGFR-linker1-VL-EGFR, the amino acid sequence of which is shown in SEQ ID NO.26; the light chain from the N-terminus to the C-terminus may contain VL-HER3-CL, wherein VL-HER3 is a light chain variable region that binds HER3.

[0081] In this invention, the amino acid sequence of linker1 is shown in SEQ ID NO.28 or SEQ ID NO.29, and the amino acid sequence of linker2 is shown in SEQ ID NO.27 or SEQ ID NO.28.

[0082] The combination of antibodies and photosensitizers (i.e. antibody-photosensitizer conjugates) is a common strategy in photodynamic therapy (PDT) to improve targeting. It utilizes the specific binding of antibodies to antigens on the surface of target cells to precisely deliver photosensitizers to the target site (such as tumor cells). Then, the photosensitizers are activated by light of a specific wavelength to generate reactive oxygen species (such as singlet oxygen), thereby achieving selective killing of target cells.

[0083] Example 2. Preparation and in vivo validation of tumor cell-targeting antibody-photosensitizer conjugates.

[0084] I. Experimental Materials and Reagents

[0085] Core antibody: Monoclonal antibodies against specific antigens on the surface of target cells (such as anti-EGFR and anti-HER3 antibodies, used for EGFR and HER3 positive tumor cells; purity >95% required, free from interfering substances such as sodium azide).

[0086] Photosensitizers: Select photosensitizers with modifiable groups (such as carboxyl, amino, and thiol groups) and stable photosensitizer activity, such as dihydroporphyrin e6 (Ce6), hematoporphyrin monomethyl ether (HMME), or commercial photosensitizers with active groups such as NHS esters and maleimide.

[0087] Coupling reagents:

[0088] If the antibody is coupled via an amino group (lysine residue): NHS activator (such as EDC・HCl / NHS, used to activate the carboxyl group of the photosensitizer).

[0089] If coupled via a thiol group (cysteine ​​residue): a maleimide-activated photosensitizer (or by adding Traut's reagent to convert the antibody amino group to a thiol group).

[0090] Buffer and consumable coupling buffer: 0.1M PBS (pH 7.2-7.4, free of amino or thiol groups to avoid interfering with the reaction); if using EDC, add 0.05M MES buffer (pH 5.0-6.0) to improve activation efficiency.

[0091] Purification consumables: ultrafiltration centrifuge tubes (molecular weight cutoff > antibody molecular weight, such as 100 kDa, used to separate conjugates from free photosensitizers), Sephadex G-25 chromatography column (desalting).

[0092] Cell-related: target cells (e.g., SK-BR-3, HER2 positive), negative control cells (e.g., MCF-7, HER2 negative), cell culture medium (e.g., RPMI-1640 + 10% FBS), CCK-8 kit, flow cytometry antibody (e.g., fluorescent secondary antibody).

[0093] II. Preparation of Antibody-Photosensitizer Conjugates

[0094] 1. Photosensitizer activation (carboxyl photosensitizer + EDC / NHS activation): Weigh the photosensitizer (such as Ce6) and dissolve it in a small amount of DMSO (final concentration <5%, to avoid affecting antibody activity). Add EDC·HCl and NHS according to the molar ratio of photosensitizer: EDC:NHS = 1:1.2:1.5. React at room temperature in the dark for 15-30 min (activating the carboxyl group results in an active ester).

[0095] 2. Coupling reaction: Slowly add the activated photosensitizer solution dropwise to the purified antibody-butyrate complex solution (dissolve the antibody-butyrate complex in PBS at a concentration of 1-5 mg / mL; the EGFR and HER3 bispecific antibodies are SEQ ID NO. 20), and mix at a molar ratio of antibody:photosensitizer = 1:5-20 (this ratio has been optimized through preliminary experiments; too high a ratio may affect antibody binding activity). Stir the reaction at 4°C in the dark for 4-6 h (or at room temperature for 2 h).

[0096] 3. Ultrafiltration purification of conjugate: Transfer the reaction solution to a 100 kDa ultrafiltration centrifuge tube, centrifuge at 3000×g for 10-15 min at 4℃, discard the filtrate (containing free photosensitizer); add PBS and repeat centrifugation 3 times, retain the retentate to obtain the antibody-photosensitizer conjugate.

[0097] 4. Verify the purification effect: Take the purified solution and measure the UV-Vis spectrum (antibody absorbs at 280 nm, photosensitizer has a peak at a specific wavelength, such as Ce6 at 664 nm). If there is only a peak at 280 nm, the purification is over-purified. If the peak at 664 nm is too strong, purification is required again.

[0098] III. In vivo validation

[0099] 0.2 mL (3 × 10) 5 CT2A cells were subcutaneously injected into the right posterior dorsal region of each BALB / c Nude mouse, resulting in an average tumor volume of approximately 100 mm². 3 Animals were randomly divided into 5 groups according to their body weight and tumor volume: solvent control group (PBS), EGFR (10 mg / kg), EGFR+HER3 (10 mg / g), EGFR (10 mg / kg)+photosensitizer, and EGFR+HER3 (10 mg / kg)+photosensitizer. Each group consisted of 3 animals. The drugs were administered once a day for a total of 6 days.

[0100] The results are as follows Figures 1-3 As shown, none of the single-drug test groups had a significant effect on mouse body weight, and no obvious abnormalities were observed in the mice's condition. As shown in the figure, 6 days after the start of administration, the average tumor volume of tumor-bearing mice in the solvent control group reached 785 mmHg. 3 The tumor volume in the EGFR (10 mg / kg) + photosensitizer group was 529 mm. 3The TGI% was 36.66%; the tumor volume in the EGFR+HER3 (10mg / kg)+photosensitizer treatment group was 447mm. 3 The TGI% was 46.38%. The bispecific antibody photoimmunosuppressant of the present invention (EGFR+HER3(10mg / kg)+photosensitizer treatment group) showed a significant tumor-suppressing effect compared with the control group, and the tumor-suppressing effect was superior to that of the monoclonal antibody+photosensitizer treatment group.

Claims

1. A bispecific antibody against EGFR and HER3, characterized in that, The bispecific antibody against EGFR and HER3 is composed of an anti-EGFR antibody and an anti-HER3 antibody linked together; the anti-EGFR antibody contains a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.9; the anti-HER3 antibody contains a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.17 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

18.

2. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody is composed of an anti-EGFR antibody and an anti-HER3 antibody linked by a linker, the amino acid sequence of which is shown in any one or more of SEQ ID NO.27 to SEQ ID NO.

29.

3. The bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the bispecific antibody is shown in any one or more of SEQ ID NO.19 to SEQ ID NO.

26.

4. A bispecific antibody photoimmunosuppressant, characterized in that, The bispecific antibody photoimmunosuppressant is formed by linking the bispecific antibody according to any one of claims 1 to 3 with a near-infrared photosensitizer.

5. The bispecific antibody photoimmunosuppressant according to claim 4, characterized in that, The near-infrared photosensitizer is coupled to the Fc segment or constant region of the bispecific antibody via an hydrazone bond or a disulfide bond; the near-infrared photosensitizer includes one or more of dihydroporphyrin E6, IR700, verteporfen, and hematoporphyrin monomethyl ether.

6. The method for preparing the bispecific antibody photoimmunoassay inhibitor according to any one of claims 4 to 5, characterized in that, Includes the following steps: 1) The bispecific antibody according to any one of claims 1 to 3 is mixed with activators EDC and NHS and reacted to obtain an activated antibody solution; 2) The activated antibody solution obtained in step 1) is mixed with the butyrate derivative solution to obtain the antibody-butyrate complex; 3) The photosensitizer is mixed with EDC·HCl and NHS to react and obtain an activated photosensitizer; 4) The activated photosensitizer obtained in step 3) is mixed and reacted with the antibody-butyrate complex to obtain the bispecific antibody photoimmunoinhibitor.

7. The preparation method according to claim 6, characterized in that, In step 4), the molar ratio of the antibody-butyrate complex to the photosensitizer is 1:5-20.

8. The use of the bispecific antibody according to any one of claims 1 to 3 and / or the bispecific antibody photoimmunoinhibitor according to any one of claims 4 to 5 in the preparation of antitumor drugs, characterized in that, The tumors include gliomas.

9. The application according to claim 8, characterized in that, The tumor is a glioblastoma.

10. The application according to claim 8, characterized in that, The anti-tumor drug is an anti-tumor drug for photoimmunotherapy; by intravenous injection of a bispecific antibody photoimmunoinhibitor, near-infrared light is irradiated onto the tumor site using an implanted optical fiber to activate the coupled photosensitizer to kill tumor cells.

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