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CN-121971646-A - Responsive antibody nano-drug conjugate and preparation method and application thereof

CN121971646ACN 121971646 ACN121971646 ACN 121971646ACN-121971646-A

Abstract

The invention discloses an ROS responsive antibody nano-drug conjugate, and a preparation method and application thereof, and belongs to the field of biological medicine. The conjugate comprises a nano-drug carrier encapsulated with a first chemotherapeutic drug and a second chemotherapeutic drug capable of cascade reaction, and a TIM-3 blocking antibody and a VISTA blocking antibody which are covalently connected on the surface of the carrier through an ROS responsive linker. After reaching the tumor site, the conjugate can respond to the high-level ROS releasing antibody in tumor microenvironment to block the immune check points of TIM-3 and VISTA synchronously and eliminate the immune inhibition, and the carried chemotherapeutic medicine has cascade reaction in tumor cell to induce immunogenic death and release tumor antigen and dangerous signal. The synergy of tumor cascade killing and effective blocking of the antibody can effectively remodel the immunosuppressive microenvironment, activate and enhance the anti-tumor immune response, and realize high-efficiency tumor killing. The conjugate has the advantages of accurate targeting, synergy and good biological safety, and provides a new strategy for tumor immunotherapy.

Inventors

  • LIANG JUNLONG
  • Qi Yongdan
  • SUN JIHONG

Assignees

  • 浙江大学医学院附属邵逸夫医院

Dates

Publication Date
20260505
Application Date
20260320

Claims (10)

  1. 1. An ROS-responsive antibody-nanopharmaceutical conjugate, comprising: a nano-drug carrier encapsulating a first chemotherapeutic drug and a second chemotherapeutic drug capable of undergoing a cascade reaction; And, TIM-3 blocking antibodies and VISTA blocking antibodies covalently linked to the surface of the nano-drug carrier through ROS-responsive linkers; wherein the ROS-responsive linker is cleavable by reactive oxygen species, thereby releasing the TIM-3 blocking antibody and the VISTA blocking antibody.
  2. 2. The ROS-responsive antibody-nanopharmaceutical conjugate of claim 1, wherein the nanopharmaceutical carrier material is poly (D, L-lactic-co-glycolic acid) (PLGA) or ferritin.
  3. 3. The ROS-responsive antibody nanodrug conjugate of claim 1, wherein the first chemotherapeutic is beta-lapachone and the second chemotherapeutic is tirapazamine.
  4. 4. ROS-responsive antibody nanopharmaceutical conjugate according to claim 1, wherein the ROS-responsive linker is preferably a linker comprising a ketal (TK) structural unit.
  5. 5. A method of preparing a ROS-responsive antibody-nanopharmaceutical conjugate, the method comprising: step S1, obtaining a nano-drug carrier encapsulated with a first chemotherapeutic drug and a second chemotherapeutic drug; S2, reacting the ROS responsive linker with the nano-drug carrier to obtain a linker modified nano-drug carrier; and S3, reacting the TIM-3 blocking antibody and the VISTA blocking antibody with the linker modified nano-drug carrier to obtain the antibody nano-drug conjugate.
  6. 6. The ROS-responsive antibody-nanopharmaceutical conjugate of claim 5, wherein step S1 comprises: Dissolving poly (D, L-lactic-co-glycolic acid) in an organic solvent to form a first solution; adding the first chemotherapeutic drug and the second chemotherapeutic drug solution dissolved in dimethyl sulfoxide into the first solution, and adding a bovine serum albumin aqueous solution to carry out ultrasonic emulsification treatment to form emulsion; And (3) dripping the emulsion into water, stirring, and centrifugally collecting to obtain the nano-drug carrier.
  7. 7. The ROS-responsive antibody nanopharmaceutical conjugate of claim 6, wherein the first chemotherapeutic is beta-lapachone, the second chemotherapeutic is tirapazamine, and the ratio of the mass of the beta-lapachone, the volume of the second chemotherapeutic solution, the volume of the first solution to the volume of the bovine serum albumin aqueous solution is (1.8-5.4) mg (0.1-0.3) mL (3-6) mL (5-10) mL.
  8. 8. The ROS-responsive antibody nano-drug conjugate of claim 5, wherein in step S2, the ROS-responsive linker is N-hydroxysuccinimide-ketal-N-hydroxysulfosuccinimide.
  9. 9. Use of the ROS-responsive antibody nano-drug conjugate of claims 1-4 for the manufacture of a medicament for tumor immunotherapy.
  10. 10. The use according to claim 9, wherein the medicament is for triggering immunogenic death of tumor cells and synergistically blocking TIM-3 and VISTA immune checkpoint pathways.

Description

Responsive antibody nano-drug conjugate and preparation method and application thereof Technical Field The invention belongs to the field of biological medicine, and in particular relates to a responsive antibody nano-drug conjugate, a preparation method and application thereof. Background Tumor immunotherapy has become an important strategy in the field of tumor therapy by mobilizing the patient's own immune system to recognize and eliminate tumor cells. Among them, immune checkpoint blocking therapies (e.g., anti-PD-1/PD-L1, anti-CTLA-4 therapies), adoptive cell immunotherapy (e.g., CAR-T), etc. have been significantly advanced and applied clinically. However, existing immunotherapy still faces many challenges, including poor immunogenicity, limited immune response rate, immunosuppressive Tumor Microenvironment (TME), and insufficient infiltration of immune cells in some patients, resulting in poor therapeutic effects or susceptibility to relapse. Induction of immunogenic death (ICD) of tumor cells is a promising strategy for enhancing anti-tumor immune responses. Dying tumor cells can release damage-associated molecular patterns (DAMPs) such as high mobility group box 1 (HMGB 1), calreticulin (CRT) and the like by means of chemotherapy, radiotherapy and the like, thereby activating antigen presenting cells and initiating adaptive immune responses. However, immune effects triggered by ICD are often strongly inhibited by multiple factors in the actual tumor microenvironment, rendering immune activation undesirable. Specifically, firstly, high-level Reactive Oxygen Species (ROS) existing in the tumor microenvironment can oxidize and neutralize a key immune signal molecule such as HMGB1 to weaken the stimulation capability of the HMGB1, secondly, an immune checkpoint molecule TIM-3 with high expression on the surface of tumor infiltration Dendritic Cells (DC) can be combined with the HMGB1, a specific structural domain of the immune checkpoint molecule TIM-3 can be 'isolated' on the surface of the DC to prevent the DC from endocytosis and subsequent antigen presentation processes, so that the activation and maturation of the DC are inhibited, and in addition, the activation and the function of T cells can be directly inhibited through the 'cis' or 'trans' inhibition effect of another immune checkpoint VISTA expressed on the T cells or antigen presentation cells. Therefore, although ICDs theoretically initiate immune responses, the high ROS levels inherent in tumor microenvironments and the coexistence of immunosuppressive pathways such as TIM-3 and VISTA form a powerful immunosuppressive network, severely limiting the practical efficacy of ICDs. At present, how to develop a novel therapeutic strategy capable of cooperatively overcoming the multiple immune suppression barriers and accurately relieving the micro-environmental suppression while inducing powerful ICD (ICD), so as to realize durable and efficient anti-tumor immune response is still a key technical problem to be solved in the field. Disclosure of Invention The invention aims to provide a responsive antibody nano-drug conjugate, a preparation method and application thereof. In order to solve the problems, the invention adopts the following technical scheme: in a first aspect of the invention, there is provided a ROS-responsive antibody nanopharmaceutical conjugate, the conjugate comprising: a nano-drug carrier encapsulating a first chemotherapeutic drug and a second chemotherapeutic drug capable of undergoing a cascade reaction; And, TIM-3 blocking antibodies and VISTA blocking antibodies covalently linked to the surface of the nano-drug carrier through ROS-responsive linkers; wherein the ROS-responsive linker is cleavable by reactive oxygen species, thereby releasing the TIM-3 blocking antibody and the VISTA blocking antibody. Further, the nano-drug carrier is made of poly (D, L-lactic-co-glycolic acid) (PLGA) or ferritin. Further, the first chemotherapeutic is beta-lapachone and the second chemotherapeutic is tirapazamine. Further, the linker is a stimuli-responsive linker comprising an ROS-responsive linker, preferably a linker comprising a ketal (TK) building block, or a Glutathione (GSH) -responsive linker, preferably a disulfide-containing linker. In a second aspect of the present invention, there is provided a method of preparing a ROS-responsive antibody-nanopharmaceutical conjugate, the method comprising: step S1, obtaining a nano-drug carrier encapsulated with a first chemotherapeutic drug and a second chemotherapeutic drug; S2, reacting the ROS responsive linker with the nano-drug carrier to obtain a linker modified nano-drug carrier; and S3, reacting the TIM-3 blocking antibody and the VISTA blocking antibody with the linker modified nano-drug carrier to obtain the antibody nano-drug conjugate. Further, the step S1 includes: Dissolving poly (D, L-lactic-co-glycolic acid) in an organic solvent to form a first solution; adding the first c