WO2022027828A1 - Nano-aptamère pour l'administration d'anticorps multi-spécifiques, application de celui-ci et procédé de construction associé - Google Patents

Nano-aptamère pour l'administration d'anticorps multi-spécifiques, application de celui-ci et procédé de construction associé Download PDF

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WO2022027828A1
WO2022027828A1 PCT/CN2020/122360 CN2020122360W WO2022027828A1 WO 2022027828 A1 WO2022027828 A1 WO 2022027828A1 CN 2020122360 W CN2020122360 W CN 2020122360W WO 2022027828 A1 WO2022027828 A1 WO 2022027828A1
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antibody
segment
nano
aptamer
αpd1
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Chinese (zh)
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沈松
王均
江澄涛
许从飞
杨显珠
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华南理工大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies

Definitions

  • the present invention relates to the technical field of medicine, in particular to a nanometer aptamer for multispecific antibody delivery and its application and construction method.
  • Immune checkpoint blocking antibodies have become a new hot spot in the global biopharmaceutical field. Clinical results show that immune checkpoint antibody therapy can activate anti-tumor immune responses to varying degrees in some tumor patients, and produce specific anti-tumor memory effects for several years. Although a variety of immune checkpoint blocking antibodies have been successively approved for the treatment of various types of tumors and various indications, showing great commercial value and clinical application prospects, different types of tumors and different patients with the same type of tumor are not immune to immune checkpoints.
  • bispecific antibodies and even multispecific antibodies, have attracted increasing attention as an effective strategy, developed to overcome the problem of insufficient drug potency of monoclonal antibodies.
  • Traditional monoclonal antibodies are composed of two identical heavy and light chains, while bispecific antibodies contain two different H and L chains that can specifically target two different antigens or two of one antigen at the same time. different epitopes.
  • researchers have developed more than 100 bispecific antibody construction models, and more than 85 bispecific antibodies are in clinical development.
  • bispecific/multispecific antibodies can greatly improve the titer and disease treatment effect of antibodies through dual or multiple recognition, their structural design complexity is high, and the complexity of design, preparation, purification and other processes is compared with that of monoclonal antibodies.
  • bispecific/multispecific antibodies can be used to develop new and simple strategies to achieve "multivalent”, “multispecific” and “multifunctional” of monoclonal antibodies, it is expected to greatly improve The clinical efficacy of monoclonal antibodies, applying more monoclonal antibodies in development or clinically approved to the treatment of solid tumors.
  • the purpose of the present invention is to provide a universal nanobody delivery platform (named as nanoaptamer) that can greatly improve the efficacy of antibodies, which can quickly, efficiently and controllably bind multiple One or more types of specific antibodies, so as to significantly enhance the disease treatment effect of antibody drugs.
  • a nanometer aptamer for multispecific antibody delivery which is formed by linking a nanocarrier with an anti-Fc segment antibody or an anti-Fc segment antibody fragment through a chemical bond;
  • the Fab domain of the anti-Fc segment antibody or anti-Fc segment antibody fragment can non-covalently bind to the Fc domain of the specific antibody delivered by the nano-aptamer; the specificity delivered by the nano-aptamer
  • the antibody has the same species origin as the Fc segment recognized by the anti-Fc segment antibody or the anti-Fc segment antibody fragment.
  • Another object of the present invention is to provide an application of the above-mentioned nano-aptamers in the preparation of immunotherapy drugs.
  • Another object of the present invention is to provide an application of the above-mentioned nano-aptamer in the preparation of a multispecific antibody delivery system.
  • Another object of the present invention is to provide a specific antibody delivery system, including the above-mentioned nano-aptamers, and specific antibodies.
  • Another object of the present invention is to provide an application of the above-mentioned multispecific antibody delivery system in the preparation of immunotherapy drugs.
  • Another object of the present invention is to provide a method for constructing a nano-aptamer, the nano-aptamer is formed by linking a nano-carrier with an anti-Fc segment antibody or an anti-Fc segment antibody fragment part through a chemical bond; the anti-Fc segment The chemical bond is formed by one-step or multi-step reaction of the segment antibody or the anti-Fc segment antibody fragment part with the nanoparticle;
  • the Fab domain of the anti-Fc segment antibody or anti-Fc segment antibody fragment can non-covalently bind to the Fc domain of the delivered specific antibody; the delivered specific antibody is bound to the anti-Fc segment antibody or anti-Fc segment.
  • the Fc segments recognized by the antibody fragments are of the same species origin.
  • the present invention has the following beneficial effects:
  • the inventors of the present invention have constructed a universal nanobody delivery platform (named as nanoaptamers, ⁇ Fc-NPs or imNAs) that can greatly improve the efficacy of antibodies based on rich experience accumulation and a large number of creative experiments.
  • Nano aptamer anti-Fc antibody or anti-Fc antibody fragment and delivered specific antibody can quickly, efficiently and controllably bind to one or more types of therapeutic monoclonal antibodies through simple physical mixing , so as to easily realize the "multivalent” and "multispecific” of the antibody.
  • the present invention creatively applies the constructed nanobody delivery platform to the preparation of immunotherapy drugs or therapeutic drugs for tumors or autoimmune diseases for the first time.
  • the anti-Fc-segment antibody or anti-Fc-segment antibody fragment of the nano-aptamer of the present invention binds to the antibody by means of antibody-antigen specific recognition, which does not destroy the structure of the antibody, and overcomes the damage of the antibody by traditional chemical bonding and fixation.
  • the structure of the drug, the closure of its antibody recognition region, the significant impact on the function of the antibody drug, the high complexity and the high difficulty, provide a new idea for the development of combined antibody therapy and a simple structure design.
  • the nano-aptamer of the present invention can also expose the Fab segment of the antibody to the outside, so that the function of the antibody can be retained to the greatest extent.
  • the present invention has been proved by a large number of in vitro and in vivo pharmacological tests that the multispecific antibody delivery system imNA ⁇ PD1 & ⁇ PDL1 obtained by combining nano aptamers with specific antibodies has significant advantages compared with the combined treatment of free monoclonal antibodies, and can significantly promote the effect- The interaction of target cells to enhance the anti-tumor ability mediated by T cells has good clinical translation prospects and great practical significance.
  • Figure 1 is a schematic diagram of the preparation of nano-aptamer ⁇ Fc-NP
  • Fig. 2 is an aldehyde group detection diagram after oxidation of anti-IgG Fc antibody ( ⁇ Fc);
  • Figure 3 is an SDS-PAGE picture of the binding mode of anti-Fc antibody to nanoparticles
  • Fig. 4 is the particle size of nano-aptamer ⁇ Fc-NP
  • Fig. 5 is the scanning electron microscope picture after nanometer aptamer and binding antibody
  • Figure 6 is a graph showing the binding efficiency of ⁇ Fc measured by ELISA
  • Figure 7 is an ultra-high-resolution micrograph of nano-aptamers binding to therapeutic monoclonal antibodies
  • Figure 8 shows the ability of NanoFCM to detect the simultaneous binding of ⁇ Fc-NP to two monoclonal antibodies
  • Figure 9 is the efficiency of nano-aptamer binding to therapeutic monoclonal antibody as a function of time
  • Figure 10 shows the proportion of antibodies bound to nano-aptamers when different ratios of ⁇ PD1 and ⁇ PDL1 are fed;
  • Figure 11 is the detection of the ability of the therapeutic monoclonal antibody bound to the nano-aptamer to bind the corresponding antigen
  • Figure 12 is a basic characterization of ⁇ Fc-NP ⁇ PD1 ;
  • Figure 13 shows the expression of PDL1 and PD1 in B16-F10 melanoma cells and CD8 + T cells stimulated in vitro;
  • Figure 14 shows the binding of imNA ⁇ PD1 & ⁇ PDL1 to B16-F10 melanoma cells
  • A the curve of extracellular fluorescence intensity with time
  • B CLSM image of the binding of B16-F10 cells to imNA ⁇ PD1 & ⁇ PDL1 , the scale bar is 5 ⁇ m
  • C Flow histogram of the change of fluorescence intensity with time before and after trypan blue quenching: trypan blue can quench extracellular fluorescence, so the fluorescence detected by flow cytometry after quenching is considered as intracellular fluorescence; FITC fluorescence marked on NP);
  • Figure 15 shows the binding of imNA ⁇ PD1 & ⁇ PDL1 to CD8 + T cells
  • Figure 16 shows the binding of NP ⁇ PD1 or NP ⁇ PD1 to B16-F10 melanoma cells (A histogram, B mean fluorescence intensity statistics, ****P ⁇ 0.0001; FITC fluorescently labeled on NP);
  • Figure 17 shows the binding of NP ⁇ PD1 or NP ⁇ PD1 to CD8 + T cells (A histogram, B statistics of mean fluorescence intensity, ***P ⁇ 0.001; FITC fluorescently labeled on NP);
  • Figure 18 shows the interaction between tumor cells and CD8 + T cells mediated by bispecific Nanobodies observed by laser confocal;
  • Figure 19 shows the cytokine release of imNA ⁇ PD1 & ⁇ PDL1 in vitro to promote the killing of B16-F10 cells by CD8 + T cells (ELISA kit detects IFN- ⁇ (A), granzyme B (B) and perforation in cell culture supernatant Concentration of oxalate (C); **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001);
  • Figure 20 is the image of the high-content imaging analysis system continuously observing the killing of T cells on tumor cells
  • Figure 21 is the H33342 release assay to determine the viability of B16-F10-OVA melanoma cells (the ratio of OVA-specific CD8 + T cells to B16-F10-OVA tumor cells is A) 5:1 and B) 10:1; **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001);
  • Figure 22 shows the particle size distribution of ⁇ Fc(H)-NP and imNA Keytruda & Tecentiq (A and B): ⁇ Fc(H)-NP is 141.7 ⁇ 4.3 nm, imNA Keytruda & Tecentiq is 158.7 ⁇ 7.0 nm; CD133 determined by H33342 release experiment + Human colorectal cancer cell viability: The ratio of tumor-infiltrating CD8 + T cells from human colorectal cancer samples to colorectal cancer cells from the same source was 5:1, *P ⁇ 0.05) (C);
  • Figure 23 shows imNA ⁇ PD1 & ⁇ PDL1 prolonging the retention of antibodies at tumor sites;
  • Figure 24 is a graph of bispecific Nanobodies inhibiting the growth of two types of tumors
  • Figure 25 is a graph of the body weight change of mice after bispecific Nanobody treatment
  • Figure 26 is a graph showing the survival curve of mice after bispecific Nanobody treatment
  • Figure 27 shows the B16-F10 tumor flow cytometry gating scheme
  • CD45 + is all immune cells, and the isotype control is used to distinguish non-specific fluorescent signals
  • Figure 28 shows that imNA ⁇ PD1 & ⁇ PDL1 reverses the immunosuppressive microenvironment of B16-F10 melanoma; the ratio of CD8 + T cells (A) and Treg cells (B) in CD3 + T cells in the tumor, and the ratio of CD8 + T cells to Treg cells (C), Proportion of subsets secreting Granzyme B (D), IFN- ⁇ (E) and IL-2 (F) in CD8 + T cells; **P ⁇ 0.01, ***P ⁇ 0.001, *** *P ⁇ 0.001;
  • Figure 30 shows H&E staining of lung sections in the 4T1-fLuc lung metastasis model treatment experiment; the scale bar is 5 mm.
  • the "plurality” mentioned in the present invention means two or more.
  • "And/or" which describes the association relationship of the associated objects means that there can be three kinds of relationships, for example, A and/or B, which can mean that A exists alone, A and B exist at the same time, and B exists alone.
  • the character "/" generally indicates that the associated objects are an "or" relationship.
  • This embodiment provides a nano-aptamer for multispecific antibody delivery, which is formed by linking a nanocarrier with an anti-Fc segment antibody or an anti-Fc segment antibody fragment through a chemical bond;
  • the Fab domain of the anti-Fc segment antibody or anti-Fc segment antibody fragment can non-covalently bind to the Fc domain of the delivered specific antibody; the delivered specific antibody is bound to the anti-Fc segment antibody or anti-Fc segment antibody.
  • Fc fragment The Fc fragment recognized by the antibody fragment has the same species origin.
  • Nanocarriers described herein refer to any system that can carry anti-Fc fragment antibodies or anti-Fc fragment antibody fragments and has nanoscale dimensions.
  • the surface of the nanocarrier has a functional group/linker that chemically reacts (couples) with the anti-Fc segment antibody or anti-Fc segment antibody fragment, so that the nanocarrier can react with the anti-Fc segment antibody or anti-Fc segment antibody fragment And form a chemical bond to obtain the nano-aptamer of the present invention.
  • the nanocarriers are preferably nanoparticles, which can be selected from but not limited to polymer nanoparticles, metal nanoparticles or protein nanoparticles.
  • the nano-carriers are nanoparticles with free amino groups on the surface, which can be selected from, but not limited to, nanoparticles with surface amination, surface chitosanization or surface albuminization, which are The free amino group on the surface reacts with the anti-Fc segment antibody or the anti-Fc segment antibody fragment to form a chemical bond connecting the two, thereby obtaining the nano-aptamer of the present invention.
  • the anti-Fc fragment antibody or anti-Fc fragment antibody fragment of the present invention is an anti-human IgG antibody Fc fragment antibody or an anti-human IgG antibody Fc fragment antibody fragment, an anti-rat IgG antibody Fc fragment antibody or an anti-rat IgG antibody Fc fragment antibody fragment , anti-mouse IgG antibody Fc fragment antibody or anti-mouse IgG antibody Fc fragment antibody fragment.
  • the specific antibody delivered by the present invention has the same species origin as the Fc segment recognized by the anti-Fc segment antibody or anti-Fc segment antibody fragment.
  • the Fc fragment antibody or anti-Fc fragment antibody fragment Select anti-human IgG antibody Fc fragment antibody or anti-human IgG antibody Fc fragment antibody fragment.
  • the nanocarriers in the present invention are further preferably spherical or quasi-spherical nanoparticles.
  • the particle size range of the nanocarriers in the present invention is preferably 25-500 nm, and more preferably, the particle size range is 80-200 nm.
  • the Fc region of the anti-Fc antibody or anti-Fc antibody fragment has glycosylation modification, and the terminal hydroxyl group of the glycosylation modification can be oxidized to form an aldehyde group, which can interact with nanoparticles Conjugate.
  • the nanoaptamers deliver at least 2 specific antibodies.
  • the chemical bond is selected from an alkylamino bond, an amide bond, or an imine bond.
  • the chemical bond is -CH 2 -NH-, and the amino terminus of the chemical bond is attached to the nanocarrier.
  • Another object of the present invention is to provide an application of the above-mentioned nano-aptamers in the preparation of immunotherapy drugs.
  • the immunotherapy drug is a tumor immunotherapy drug or an autoimmune disease treatment drug.
  • Another object of the present invention is to provide an application of the above-mentioned nano-aptamer in the preparation of a multispecific antibody delivery system.
  • Another object of the present invention is to provide a specific antibody delivery system, including the above-mentioned nano-aptamers, and specific antibodies.
  • the specific antibody delivery system includes at least 2 specific antibodies.
  • the Fc domain of the specific antibody is directed non-covalently to the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody fragment.
  • Another object of the present invention is to provide an application of the above-mentioned multispecific antibody delivery system in the preparation of immunotherapy drugs.
  • the immunotherapy drug is a tumor immunotherapy drug or an autoimmune disease treatment drug.
  • Another object of the present invention is to provide a method for constructing the above-mentioned nano-aptamer, wherein the nano-aptamer is formed by linking a nano-carrier with an anti-Fc segment antibody or an anti-Fc segment antibody fragment through a chemical bond; the An anti-Fc segment antibody or an anti-Fc segment antibody fragment reacts with the nanoparticle in one or more steps to form the chemical bond;
  • the Fab domain of the anti-Fc segment antibody or anti-Fc segment antibody fragment can non-covalently bind to the Fc domain of the specific antibody delivered by the nano-aptamer; the specific antibody delivered by the nano-aptamer is bound to the The Fc segment recognized by the anti-Fc segment antibody or the anti-Fc segment antibody fragment has the same species origin.
  • the nanocarrier is a nanoparticle with free amino groups
  • the method for constructing the nanoaptamer comprises the following steps:
  • Anti-Fc-segment antibodies are oxidized by oxidants to form aldehyde-containing anti-Fc-segment antibodies;
  • the nanoparticles with free amino groups on the surface of the step (1) are surface aminated, surface chitosanized or surface albuminated nanoparticles.
  • the nanoparticles with free amino groups on the surface select amino-functional polystyrene microspheres as an example, and the mass ratio of the amino-functional polystyrene microspheres to the anti-Fc segment antibody is 2-10 : 1, preferably 4 to 10:1. Further, the concentration of the nanoparticles with free amino groups on the surface in the condensation reaction system is 0.3-1.0 mg/mL.
  • the oxidant in step (1) is sodium periodate. Further, the concentration of the oxidant in the oxidation reaction system is 3-10 mM. Further, the oxidation conditions are as follows: the reaction is performed in a dark environment at 0-8° C. for 1-3 hours.
  • the concentration of the anti-Fc fragment antibody in step (1) in the oxidation reaction system is 0.3-1.0 mg/mL.
  • the concentration of the aldehyde group-containing anti-Fc segment antibody in the condensation reaction system in step (2) is 0.05-0.15 mg/mL.
  • the condensation conditions in step (2) are the reaction at 0-8° C. for 10-14 hours.
  • the reducing agent in step (3) is sodium borohydride or sodium cyanoborocyanide.
  • the concentration of the reducing agent in the reduction reaction system is 0.5-1.5 mg/mL.
  • the reduction conditions are as follows: the reaction is carried out at 0 to 8° C. for 0.5 to 1 h.
  • Goat anti-rat IgG Fc fragment antibody purchased from Rockland Company, USA;
  • Amino-functionalized polystyrene microspheres (25-300 nm) were purchased from Shanghai McLean Biochemical Technology Co., Ltd.;
  • Purpald solution 10 mg/mL 4-amino-3-hydrazino-5-mercapto-1,2,3-triazole (purchased from Bailingwei Technology Co., Ltd.), dissolved in 1N NaOH. Prepared before use. After reacting with an aldehyde group, it turned purple after the action of sodium periodate (Purpald: 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole, purchased from Beijing Bailingwei Technology Co., Ltd., China).
  • the goat anti-rat IgG-Fc antibody ( ⁇ Fc) was diluted with ultrapure water to 0.5 mg/mL, sodium periodate aqueous solution was added to the final concentration of 5 mM, and oxidized at 4°C for 2 h in the dark. After the oxidation reaction, the aldehyde group of oxidized ⁇ Fc antibody was rapidly detected by Purpald method. The detection results are shown in Figure 2, the reaction solution turned purple, and the absorbance at 550 nm increased, proving that the oxidized ⁇ Fc antibody has an aldehyde group.
  • the amount of goat anti-rat Fc antibody bound to the nanoparticles was tested by enzyme-linked immunosorbent assay (ELISA), and the free antibody remaining in the supernatant after centrifugation was subtracted from the total amount fed.
  • ELISA enzyme-linked immunosorbent assay
  • UPLC Ultra-performance liquid chromatography
  • the prepared ⁇ Fc-NP and anti-Fc antibody were tested by reducing SDS-APGE experiment.
  • the experimental method is as follows:
  • Sample processing Take 10 ⁇ g of ⁇ Fc antibody and dilute it with water to 20 ⁇ L, take 20 ⁇ L of ⁇ Fc-NP particle solution containing the same concentration of ⁇ Fc, add 5 ⁇ L of 5 ⁇ protein loading buffer (Biosharp, containing mercaptoethanol) and mix well, let stand for 10 min at room temperature , placed in a 99°C metal bath and heated for 10 min, and the samples were loaded after cooling; the sample bands were detected by SDS-PAGE, and the bands were observed by Coomassie brilliant blue staining.
  • 5 ⁇ protein loading buffer Biosharp, containing mercaptoethanol
  • the ⁇ Fc-NP group has significantly less heavy chains than free antibody, and the same intensity of the light chain bands, indicating that the ⁇ Fc-NP group is bonded to the nanoparticles through the sugar chain structure on the Fc segment of ⁇ Fc, and the light chain is cut by mercaptoethanol. After the disulfide bond between the heavy chain and the heavy chain, part of the heavy chain is bound to the nanoparticles and cannot enter the SDS-PAGE gel.
  • the experimental results can prove that ⁇ Fc is indeed a directional chemical binding.
  • the prepared ⁇ Fc-NPs were tested for particle size of drug-loaded nanoparticles by dynamic light scattering (DLS). As shown in Figure 4, the average hydrodynamic diameter of its ⁇ Fc-NP is about 130 nm.
  • the experimental method is as follows:
  • Anti-PD1 antibody and polystyrene nanoparticles were labeled with Alexa Flour 647 (green) and Alexa Flour 750 (red), respectively, as shown in Figure 7.
  • Alexa Flour 647 green
  • Alexa Flour 750 red
  • Figure 7 For the IgG-NP ⁇ PD1 group (left), the red surrounding particles were rarely observed The aggregation of green fluorescence (AF647), while the ⁇ Fc-NP ⁇ PD1 group (right) can observe greater co-localization of red (AF750) and green (AF647) fluorescence.
  • IgG is a control antibody, which cannot recognize the Fc segment of the antibody), indicating that ⁇ Fc-NP can specifically bind and carry monoclonal antibodies.
  • Nano-flow detector (NanoFCM) to verify the ability of ⁇ Fc-NP to bind two monoclonal antibodies at the same time
  • mice B16-F10 melanoma cell line and the mouse 4T1 orthotopic breast cancer cell line were obtained from the American Standard Biological Collection (ATCC).
  • ATCC American Standard Biological Collection
  • the mice were kept in the Laboratory Animal Center of South China University of Technology, and the animal experiment procedures followed the relevant regulations of the South China University of Technology Laboratory Animal Management Regulations.
  • the mean fluorescence intensity (MFI) of imNA ⁇ PD1 & ⁇ PDL1 in B16-F10 cells increased with the incubation time; and we confirmed that the extracellular fluorescence was quenched by trypan blue. The vast majority of particles were on the cell membrane surface rather than entering the cell ( Figure 14C).
  • CLSM images also showed that a large amount of imNA ⁇ PD1 & ⁇ PDL1 bound on the surface of B16-F10 cells (the cell membrane was labeled with PKH26 dye in red, and the NPs were labeled with FITC) ( FIG. 14B ).
  • imNA ⁇ PD1 & ⁇ PDL1 also bound in a time-dependent manner, and almost did not enter the granules into CD8 + T cells ( FIG. 15 ).
  • the control ⁇ Fc-NP IgG showed weak interaction with both cells ( Figure 14 and Figure 15), indicating that the binding of imNA ⁇ PD1 & ⁇ PDL1 to cells depends on the antigen specificity of the carried monoclonal antibodies Identify and combine.
  • the above results all prove that the co-inhibitory molecule PD1/PDL1 can serve as the binding site of imNA ⁇ PD1 & ⁇ PDL1 .
  • the binding of the ⁇ Fc-NP mAbs delivery platform to cells is based on the antigen-specific interaction of the carried monoclonal antibody, and this interaction is better limited to the cell surface, which is conducive to promoting Effector-target cell interactions.
  • the mouse melanoma cell line B16-F10 was selected to explore the interaction of multivalent Nanobodies conjugated with therapeutic antibodies with cells.
  • the CD8 + T cells isolated from the spleen were labeled with CFSE, and then co-cultured with B16-F10 cells (expressing mCherry fluorescent protein).
  • the sorted T cells were activated by CD3 and CD28 antibodies, fluorescently labeled with CellTrace Blue, and co-cultured with B16-F10 cells (expressing mCherry fluorescent protein).
  • ImNA ⁇ PD1 & ⁇ PDL1 have enhanced enrichment ability at tumor sites: In order to verify the efficacy in vivo, we need to determine whether imNA ⁇ PD1 & ⁇ PDL1 can simultaneously have the enhanced tumor penetration and enrichment (EPR) effect of nanomedicines Evaluate.
  • EPR tumor penetration and enrichment
  • the NP ⁇ PD1 &NP ⁇ PD1 experimental group had a certain inhibitory effect on tumor growth, which may be due to the weak enhancement of the therapeutic antibody effect due to the multivalent effect.
  • the imNA ⁇ PD1 & ⁇ PDL1 experimental group has a significant inhibitory effect on tumor growth, which is because the delivery vehicle can deliver antibody drugs to the tumor while enhancing the interaction of target cells.
  • the body weight of the mice in each group there was no significant change in the body weight of the mice in each group, which proved that the components in each group did not cause serious systemic toxicity to the mice.
  • the survival time of mice in the imNA ⁇ PD1 & ⁇ PDL1 experimental groups was significantly prolonged.
  • imNA ⁇ PD1 & ⁇ PDL1 significantly inhibited the formation of lung metastases in mouse 4T1-fLuc breast cancer.
  • imNA ⁇ PD1 & ⁇ PDL1 significantly inhibited the formation of lung metastases in mouse 4T1-fLuc breast cancer.
  • imNA ⁇ PD1 & ⁇ PDL1 can eliminate circulating tumor cells and inhibit tumor metastasis, but since the occurrence of orthotopic metastasis is difficult to monitor and control, we constructed by direct tail vein injection of firefly luciferase (fLuc)-expressing 4T1 cells Breast cancer lung metastasis model.
  • FIG. 30 demonstrated that the number and size of metastatic nodules were significantly reduced by imNA ⁇ PD1 & ⁇ PDL1 treatment.
  • the anti-metastatic ability of imNA ⁇ PD1 & ⁇ PDL1 may be partly due to its ability to promote the interaction of CD8 + T cells and tumor cells in the lungs and blood circulation.

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Abstract

La présente invention se rapporte à un nano-aptamère pour l'administration d'anticorps multi-spécifiques, à une application de celui-ci et à un procédé associé de construction génétique. Le nano-aptamère est formé par la liaison d'un nanovecteur à un anticorps à segment anti-Fc ou à une partie de fragment d'anticorps à segment anti-Fc au moyen d'une liaison chimique; un domaine Fab de l'anticorps à segment Anti-Fc ou du fragment d'anticorps à segment anti-Fc peut se lier de manière non covalente à un domaine Fc d'un anticorps spécifique administré; l'anticorps spécifique administré a la même origine d'espèce que le segment Fc reconnu par l'anticorps à segment anti-Fc ou le fragment d'anticorps à segment anti-Fc. Le nano-aptamère peut se lier rapidement, efficacement et de manière contrôlable à de multiples types d'anticorps, pour obtenir une "multivalence" et une "multi-spécificité" d'anticorps. Pour la première fois, ledit type de plate-forme d'administration de nanocorps construits génétiquement est appliqué de manière créative à la préparation d'un médicament immunothérapeutique ou d'un médicament destiné à traiter des tumeurs ou des maladies auto-immunes, et peut améliorer considérablement l'effet immunothérapeutique.
PCT/CN2020/122360 2020-08-04 2020-10-21 Nano-aptamère pour l'administration d'anticorps multi-spécifiques, application de celui-ci et procédé de construction associé WO2022027828A1 (fr)

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