CN112336872B

CN112336872B - Nano-aptamer for multi-specific antibody delivery and application and construction method thereof

Info

Publication number: CN112336872B
Application number: CN202010773467.5A
Authority: CN
Inventors: 沈松; 王均; 江澄涛; 许从飞; 杨显珠
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-08-04
Filing date: 2020-08-04
Publication date: 2022-05-06
Anticipated expiration: 2040-08-04
Also published as: WO2022027828A1; CN112336872A

Abstract

The invention relates to a nano aptamer for multi-specific antibody delivery and an application and a construction method thereof. The nano aptamer is formed by connecting a nano carrier with an anti-Fc segment antibody or an anti-Fc segment antibody fragment part through a chemical bond; wherein the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody is capable of non-covalent binding to the Fc domain of the specific antibody delivered; the specific antibody delivered is of the same generic origin as the Fc fragment recognized by the anti-Fc fragment antibody or anti-Fc fragment antibody fragment. The nano-aptamer can be rapidly, efficiently and controllably combined with various types of antibodies, so that the multivalence and the multispecific performance of the antibodies are realized. The invention creatively applies the constructed nano antibody delivery platform to the preparation of immunotherapy drugs or treatment drugs for tumors or autoimmune diseases for the first time, and can obviously improve the immunotherapy effect.

Description

Nano-aptamer for multi-specific antibody delivery and application and construction method thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a nano aptamer for multi-specific antibody delivery and an application and construction method thereof.

Background

Malignant tumors remain a significant public health problem that seriously threatens the health of residents. In recent years, the development of tumor immunotherapy, especially immune checkpoint blockade therapy, has progressed rapidly and has profoundly changed the therapeutic pattern of malignant tumors. Immune checkpoint blocking antibodies have become a new hotspot in the world 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 years. Although multiple immune checkpoint blocking antibodies are sequentially approved for the treatment of multiple types of tumors and multiple indications, and show great commercial value and clinical application prospect, different types of tumors and patients with the same type of tumors have great response to immune therapies such as immune checkpoint blocking, and the clinical response rate is generally low. The low clinical response rate and therapeutic effect severely limit the range of patients who benefit from immune checkpoint blockade therapy, and new strategies for improving the anti-tumor effect of immune checkpoint antibodies are urgently needed.

In recent years, bispecific antibodies, even multispecific antibodies, have been gaining attention as an effective strategy, and have been developed to overcome the problem of insufficient drug potency of monoclonal antibodies. Whereas traditional monoclonal antibodies consist of two identical heavy and light chains, bispecific antibodies comprise two different H and L chains, capable of specifically targeting two different antigens or two different epitopes of an antigen simultaneously. Researchers have developed over 100 bispecific antibody construction models and over 85 bispecific antibodies are in clinical development. Although the bispecific/multispecific antibody can greatly improve the titer and disease treatment effect of the antibody through dual or multiple recognition, the structural design complexity is high, the complexity of the processes of design, preparation, purification and the like is greatly increased compared with that of a monoclonal antibody, the bispecific/multispecific antibody is mostly prepared by chemical coupling and DNA recombination technology, the monoclonal antibody which generates the effect needs to be chemically modified, the antigen binding capacity of the antibody is inevitably influenced, and the bispecific/multispecific antibody has the defects of short half life, complex administration mode, poor stability, poor solubility, high cost and the like. Therefore, if a novel and simple strategy can be developed to achieve "multivalence", "multispecific", and "multifunctional" of monoclonal antibodies by utilizing the design concept of bispecific/multispecific antibodies, the clinical efficacy of monoclonal antibodies can be greatly improved, and more monoclonal antibodies under development or clinically approved can be applied to the treatment of solid tumors.

Disclosure of Invention

Based on the above, the invention aims to provide a universal nano antibody delivery platform (named as a nano aptamer) capable of greatly improving the antibody curative effect, which can be used for quickly, efficiently and controllably directionally and non-covalently combining multiple and multiple types of specific antibodies, so that the disease treatment effect of an antibody drug is remarkably enhanced.

The specific technical scheme is as follows:

a nano-aptamer for multi-specific antibody delivery is formed by connecting a nano-carrier with an anti-Fc fragment antibody or an anti-Fc fragment antibody part through a chemical bond;

wherein the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody is capable of non-covalent binding to the Fc domain of a specific antibody delivered by the nano-aptamer; the specific antibody delivered by the nano-aptamer has the same species source as the Fc fragment which can be recognized by the anti-Fc fragment antibody or the anti-Fc fragment antibody fragment.

The invention also aims to provide application of the nano-aptamer in preparation of immunotherapy drugs.

Another object of the present invention is to provide a use 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, comprising the above-mentioned nano-aptamer, and a specific antibody.

Another object of the present invention is to provide a use of the above-mentioned multispecific antibody delivery system in the preparation of an immunotherapeutic drug.

The invention also aims to provide a construction method of the nano aptamer, wherein the nano aptamer is formed by connecting a nano carrier with an anti-Fc segment antibody or an anti-Fc segment antibody fragment part through a chemical bond; the anti-Fc fragment antibody or anti-Fc fragment antibody portion reacts with the nanoparticle in one or more steps to form the chemical bond;

wherein the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody is capable of non-covalent binding to the Fc domain of a specific antibody to be delivered; the specific antibody delivered is of the same species as the Fc fragment recognized by the anti-Fc fragment antibody or anti-Fc fragment antibody fragment.

Compared with the prior art, the invention has the following beneficial effects:

based on abundant experience accumulation and a large number of creative experiments, the inventor constructs a universal nano antibody delivery platform (named as a nano aptamer, alpha Fc-NP or imNAs) capable of greatly improving the antibody curative effect, and the anti-Fc fragment antibody or anti-Fc fragment antibody of the nano aptamer and the delivered specific antibody can be quickly, efficiently and controllably combined with 1 or more types of therapeutic monoclonal antibodies only through simple physical mixing, so that the 'multivalence' and 'multispecific' of the antibody are simply and conveniently realized. The invention creatively applies the constructed nano antibody delivery platform to the preparation of immunotherapy drugs or treatment drugs for tumors or autoimmune diseases for the first time.

The anti-Fc fragment antibody or anti-Fc fragment antibody of the nano-aptamer is combined with the antibody in an antibody-antigen specific recognition mode, the structure of the antibody cannot be damaged, the defects that the traditional chemical bonding and fixing mode can damage the structure of an antibody drug, seal the antibody recognition area of the antibody drug, obviously influence the function of the antibody drug, and is high in complexity, high in difficulty and the like are overcome, and a brand-new simple structural design is provided for development of combined antibody therapy.

In addition, the nano-aptamer can expose the Fab segment of the antibody outwards, so that the function of the antibody can be retained to the maximum extent.

A large number of in vivo and in vitro pharmacological tests prove that the multispecific antibody delivery system imNA obtained by combining the nano aptamer and the specific antibody_αPD1&αPDL1Compared with free monoclonal antibody combined therapy, the monoclonal antibody has obvious superiority, can obviously promote the interaction of effect-target cells, enhances the anti-tumor capability mediated by T cells, and has good clinical transformation prospect and great practical significance.

Drawings

FIG. 1 is a schematic diagram of preparation of nano-aptamer alpha Fc-NP;

FIG. 2 is a photograph of aldehyde detection after oxidation of anti-IgG Fc antibody (. alpha.Fc);

FIG. 3 is an SDS-PAGE picture of the manner in which anti-Fc antibodies bind to nanoparticles;

FIG. 4 shows the particle size of the nano-aptamer alpha Fc-NP;

FIG. 5 is a scanning electron microscope image of the nano-aptamer and the conjugated antibody;

FIG. 6 is a graph of the binding efficiency of α Fc measured by ELISA;

FIG. 7 is an ultra-high resolution photomicrograph of the nano-aptamer binding therapeutic monoclonal antibody;

FIG. 8 is a graph showing the ability of NanoFCM to detect alpha Fc-NP binding to two monoclonal antibodies simultaneously;

FIG. 9 is the efficiency of time-varying nano-aptamer binding to therapeutic monoclonal antibodies;

fig. 10 is the ratio of antibodies bound to the nano-aptamers at different ratios of the α PD1, α PDL1 dosing;

FIG. 11 is a graph showing the ability of a therapeutic monoclonal antibody binding to a nano-aptamer to bind to the corresponding antigen;

FIG. 12 is α Fc-NP_αPD1The basic characterization of (1);

FIG. 13 is an in vitro stimulation of B16-F10 melanoma cells and CD8⁺Expression of PDL1 in T cells, PD 1;

FIG. 14 shows imNA_αPD1&αPDL1Binding to B16-F10 melanoma cells (A) extracellular fluorescence intensity versus time curve; B) B16-F10 cells and imNA_αPD1&αPDL1Combined CLSM images, scale bar 5 μm; C) flow histogram of fluorescence intensity versus time before and after trypan blue quenching: trypan blue can quench extracellular fluorescence, so that fluorescence detectable by flow cytometry after quenching is considered as intracellular fluorescence; FITC fluorescent label on NP);

FIG. 15 shows imNA_αPD1&αPDL1And CD8⁺(ii) T cell binding status;

FIG. 16 shows NP_αPD1Or NP_αPD1Binding to B16-F10 melanoma cells (histogram A, statistics of B mean fluorescence intensity, P < 0.0001; FITC fluorescence labeling on NPs);

FIG. 17 shows NP_αPD1Or NP_αPD1And CD8⁺T cell binding (histogram A, statistics of B mean fluorescence intensity. x. P < 0.001; FITC fluorescence labeling on NP);

FIG. 18 shows tumor cells and CD8 mediated by confocal laser observation bispecific nanobody⁺T cell interactions;

FIG. 19 shows imNA_αPD1&αPDL1Promotion of CD8 in vitro⁺Cytokine release by T cells for killing B16-F10 cells (ELISA kit for detecting IFN-. gamma. (A), granzyme B (B) and perforin (C) concentrations in cell culture supernatant;. P)<0.01，***P<0.001，****P<0.0001)；

FIG. 20 is a high content imaging analysis system continuously observing images of T cell killing on tumor cells;

FIG. 21 is a graph of the activity of B16-F10-OVA melanoma cells as determined by the H33342 Release assay (OVA-specific CD 8)⁺The ratio of T cells to B16-F10-OVA tumor cells was A) 5: 1 and B) 10: 1; p<0.01，***P<0.001，****P<0.0001)；

FIG. 22 is a drawing showing. alpha. Fc (H) -NP and imNA_{Keytruda&Tecentiq}Particle size distribution (a and B): alpha Fc (H) -NP is 141.7 + -4.3 nm, imNA_{Keytruda&Tecentiq}158.7 +/-7.0 nm; h33342 Release assay for CD133⁺Human colorectal cancer cell viability: tumor infiltration CD8 from human colorectal cancer sample⁺The ratio of T cells to colorectal cancer cells from the same sample is 5: 1, P<0.05)(C)；

FIG. 23 shows imNA_αPD1&αPDL1Prolonged retention of the antibody at the tumor site; A) ex vivo 4T1 breast cancer antibody Cy5 fluorescence intensity profile small animal imager images<0.01，***P<0.001; B) analyzing the intensity of a fluorescence signal of an antibody Cy5 in an image of a small animal imager; C) immunofluorescence imaging: cy5 fluorescence of the antibody was distributed within the tumor at a scale of 50 μm;

figure 24 is a graph of bispecific nanobody inhibition of growth of two tumors;

figure 25 is a graph of the change in body weight of mice after bispecific nanobody treatment;

figure 26 is a graph of survival of mice after bispecific nanobody treatment;

FIG. 27 is a B16-F10 tumor flow cytometry gate protocol; CD45⁺For all immune cells, isotype controlTo distinguish non-specific fluorescent signals; A) t cell subsets: further on CD45⁺Circle CD3 in the group⁺CD4⁺T cells and CD3⁺CD8⁺CTL, Re-encirclement of CD3⁺CD4⁺CD25⁺The Treg cell of (4); B) CD8⁺CTL subgroup: further on CD45⁺Circle CD3 in the group⁺CD8⁺CTL of (5), analysis of CD45⁺CD3⁺CD8⁺Cytokine-secreting IL2 in CTL cell populations⁺、IFN-γ⁺And Gran B⁺A subset of CTLs of (a);

FIG. 28 is imNA_αPD1&αPDL1Reversing the B16-F10 melanoma immunosuppressive microenvironment; CD3 in tumors⁺CD8 in T cells⁺Ratio of T cells (A) and Treg cells (B), CD8⁺Ratio of T cells to Treg cells (C), CD8⁺The ratio of subpopulations secreting Granzyme B (D), IFN- γ (E) and IL-2(F) in T cells; p<0.01，***P<0.001，****P<0.001；

FIG. 29 shows imNA_αPD1&αPDL1Inhibiting the formation of pulmonary metastases; A) fLuc fluorescence of a mouse breast cancer lung metastasis range is observed by in vivo imaging of a small animal; B) observing the fluorescence distribution condition of the pulmonary metastasis nodule fLuc in vitro; C) statistics of number of lung metastasis nodules<0.05，**P<0.01；n＝5；

FIG. 30 is H & E staining of 4T1-fLuc lung metastasis model treatment experimental lung sections; the scale bar is 5 mm.

Detailed Description

The experimental procedures of the present invention, without specifying the specific conditions in the following examples, are generally carried out according to conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or apparatus that comprises a list of steps is not limited to only those steps or modules recited, but may alternatively include other steps not recited, or may alternatively include other steps inherent to such process, method, article, or apparatus.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The embodiment provides a nano aptamer for multi-specific antibody delivery, which is formed by connecting a nano carrier with an anti-Fc fragment antibody or an anti-Fc fragment antibody part through a chemical bond;

wherein the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody is capable of non-covalent binding to the Fc domain of the specific antibody delivered; the specific antibody delivered is of the same species origin as the Fc fragment recognized by the anti-Fc fragment antibody or anti-Fc fragment antibody fragment.

The nanocarrier as described herein refers to any system that can support an anti-Fc fragment antibody or an anti-Fc fragment antibody fragment and has a nanoscale size. Preferably, the surface of the nano-carrier has a functional group/linker chemically reacting (coupling) with the anti-Fc fragment antibody or the anti-Fc fragment antibody fragment, so that the nano-carrier can react with the anti-Fc fragment antibody or the anti-Fc fragment antibody fragment and form a chemical linkage, thereby obtaining the nano-aptamer of the present invention. The nano-carrier is preferably a nano-particle, and may be selected from, but not limited to, a polymer nano-particle, a metal nano-particle, a protein nano-particle, or the like. As an example of the nano-aptamer of the present invention, the nano-carrier is a nanoparticle having a free amino group on the surface, which may be selected from, but not limited to, a surface aminated, surface chitosan, or surface albuminated nanoparticle, and the free amino group on the surface reacts with the anti-Fc fragment antibody or the anti-Fc fragment antibody 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 of the invention is an anti-human IgG antibody Fc fragment antibody or anti-human IgG antibody Fc fragment antibody fragment, an anti-rat IgG antibody Fc fragment antibody or anti-rat IgG antibody Fc fragment antibody fragment, an anti-mouse IgG antibody Fc fragment antibody or anti-mouse IgG antibody Fc fragment antibody fragment.

The specific antibody delivered by the invention and the Fc fragment which can be recognized by the anti-Fc fragment antibody or the anti-Fc fragment antibody fragment have the same species source, and for example, when the delivered specific antibody is humanized anti-PD 1 antibody, the anti-Fc fragment antibody or the anti-Fc fragment antibody is anti-human IgG antibody Fc fragment antibody or anti-human IgG antibody Fc fragment antibody fragment.

The nanocarrier of the present invention is further preferably spherical or spheroidal nanoparticles. The preferable particle size range of the nano-carrier in the invention is 25-500 nm, and the more preferable particle size range is 80-200 nm.

In some of these embodiments, the Fc region of the anti-Fc fragment antibody or anti-Fc fragment antibody fragment has a glycosylation modification, and the terminal hydroxyl group of the glycosylation modification is oxidized to form an aldehyde group, so that the conjugation to the nanoparticle can be performed.

In some of these embodiments, the nano-aptamer delivers at least 2 specific antibodies.

In some of these embodiments, the chemical bond is selected from an alkyl amino bond, an amide bond, or an imine bond.

In some of these embodiments, the bond is-CH₂-NH-, said chemical bond having its amino terminus attached to said nanocarrier.

In some of these embodiments, the immunotherapeutic agent is a tumor immunotherapeutic agent or an autoimmune disease therapeutic agent.

In some of these embodiments, the specific antibody delivery system comprises at least 2 specific antibodies.

In some of these embodiments, the Fc domain of the specific antibody is non-covalently directed to bind to the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody.

It is a further object of the present invention to provide a use of the above-described multispecific antibody delivery system in the preparation of an immunotherapeutic drug.

The invention further aims to provide a construction method of the nano-aptamer, wherein the nano-aptamer is formed by connecting a nano-carrier with an anti-Fc fragment antibody or an anti-Fc fragment antibody part through a chemical bond; the anti-Fc fragment antibody or anti-Fc fragment antibody reacts with the nanoparticle in one or more steps to form the chemical bond;

wherein the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody is capable of non-covalent binding to the Fc domain of an antibody specific for delivery by the nano-aptamer; the specific antibody delivered by the nano-aptamer has the same species source as the Fc fragment which can be recognized by the anti-Fc fragment antibody or the anti-Fc fragment antibody fragment.

In some embodiments, the nano-carrier is a nanoparticle having a free amino group, and the method for constructing the nano-aptamer comprises the following steps:

(1) oxidizing the anti-Fc fragment antibody by an oxidant to form an aldehyde group-containing anti-Fc fragment antibody;

(2) condensing the aldehyde group-containing anti-Fc segment antibody with nanoparticles with free amino groups on the surface to form Schiff base;

(3) and reducing the Schiff base by a reducing agent to form the nano aptamer.

In some of these embodiments, the nanoparticles having free amino groups on the surface in step (1) are surface aminated, surface chitosan, or surface albuminated nanoparticles.

The nanoparticles with free amino groups on the surface are exemplified by amino-functionalized polystyrene microspheres in the following examples, and the mass ratio of the amino-functionalized polystyrene microspheres to the anti-Fc fragment antibody is 2-10: 1, preferably 4-10: 1. Further, the concentration of the nanoparticles with free amino groups on the surface in a condensation reaction system is 0.3-1.0 mg/mL.

In some of these embodiments, the oxidizing agent of 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: and reacting for 1-3 h in a dark environment at 0-8 ℃.

In some embodiments, the concentration of the anti-Fc fragment antibody in the oxidation reaction system in the step (1) is 0.3-1.0 mg/mL.

In some embodiments, the concentration of the aldehyde group-containing anti-Fc region antibody in the condensation reaction system in the step (2) is 0.05-0.15 mg/mL.

In some embodiments, the condensation in step (2) is carried out at 0-8 ℃ for 10-14 h.

In some of these embodiments, the reducing agent of step (3) is sodium borohydride or sodium cyanoborohydride. Further, the concentration of the reducing agent in the reduction reaction system is 0.5-1.5 mg/mL. Further, the reduction conditions are as follows: reacting for 0.5-1 h at 0-8 ℃.

The present invention will be described in further detail with reference to specific examples.

Example 1 construction and characterization of Nanopadapter alpha Fc-NP

The raw material sources and treatment methods used in the examples were:

goat anti-rat IgG Fc fragment antibody, purchased from Rockland, USA;

amino-functionalized polystyrene microspheres (25-300 nm) purchased from Shanghai Michelin Biotechnology, Inc.;

sodium periodate (NaIO)₄) Prepared at present, and should be protected from light (purchased from Shanghai Aladdin Biotechnology GmbH, China);

sodium borohydride, now available (purchased from sahn chemical technology (shanghai) ltd., china);

purplad solution: 10mg/mL 4-amino-3 hydrazino-5 mercapto-1, 2, 3-triazole (available from carbofuran technologies, Inc.) was dissolved in 1N NaOH. It is prepared before use. The reaction with aldehyde group is purple after the action of sodium periodate (purple: 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole, purchased from Beijing carbofuran technologies Ltd., China).

Oxidation treatment of anti-Fc antibodies

As shown in FIG. 1, goat anti-rat IgG-Fc antibody (. alpha.Fc) was diluted to 0.5mg/mL with ultrapure water, and then an aqueous solution of sodium periodate was added thereto to a final concentration of 5mM, followed by oxidation at 4 ℃ for 2 hours in the absence of light. And after the oxidation reaction is finished, quickly detecting aldehyde groups of the oxidized alpha Fc antibody by using a Purpald method. As shown in FIG. 2, the reaction solution became purple and the absorbance increased at 550nm, indicating that the oxidized α Fc antibody had an aldehyde group.

Removing sodium periodate: and then carrying out ultrafiltration for 2-3 times by using a 100kDa ultrafiltration tube under the condition of 0.01M acetic acid-sodium acetate buffer solution (pH 4.2) to remove the sodium periodate. Oxidized alpha Fc antibody concentration was rapidly determined with Nanodrop A280 after recovery of oxidized alpha Fc antibody.

Preparation of di-nano aptamer alpha Fc-NP carrier

Diluting 100nm amino-functionalized polystyrene microspheres to 0.5mg/mL by using ultrapure water, adding the oxidized anti-Fc antibody to 0.1mg/mL, fully and uniformly mixing, and reacting at 4 ℃ for 12 h; after the reaction is finished, adding sodium borohydride to 1mg/mL, and reacting for 0.5-1.0 h at 4 ℃; after the reaction, the reaction mixture was centrifuged at 15000rpm × 1.5h at 4 ℃, the supernatant was discarded, and the reaction mixture was resuspended in an equal volume of ultrapure water, centrifuged again for 2 times, and finally resuspended in a volume of 5% glucose solution as needed.

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 dosed. Ultra-performance liquid chromatography (UPLC) assisted testing.

Characterization of the Tri, Nano-aptamer

1. The prepared alpha Fc-NP and anti-Fc antibody are tested by a reducing SDS-APGE experiment. The experimental method is as follows:

sample treatment: diluting 10 μ g of alpha Fc antibody with water to 20 μ L, adding 5 μ L of 5 × protein loading buffer (Biosharp, containing mercaptoethanol) into 20 μ L of alpha Fc-NP particle solution containing alpha Fc with the same concentration, mixing, standing at room temperature for 10min, heating in 99 deg.C metal bath for 10min, cooling, and loading; SDS-PAGE detects the sample bands, and Coomassie brilliant blue staining observes the bands.

As shown in fig. 3, the heavy chains of the α Fc-NP group are significantly reduced compared to the free antibody, and the light chain bands have the same intensity, which indicates that the α Fc is bonded to the nanoparticle through the sugar chain structure on the Fc segment of the α Fc, and after the disulfide bond between the light chain and the heavy chain is cut by mercaptoethanol, part of the heavy chain is bonded to the nanoparticle and cannot enter the SDS-PAGE gel.

2. The particle size of the prepared alpha Fc-NP is detected by a Dynamic Light Scattering (DLS) instrument. As shown in FIG. 4, the average hydrodynamic diameter of α Fc-NP was around 130 nm.

As shown in FIG. 5, when observed under a Scanning Electron Microscope (SEM), α Fc-NP showed a rough surface, increasing the particle size from about 100nm to about 110nm, compared to polystyrene microspheres with no bound antibody, and the results of the SEM were substantially identical to those of the DLS, indicating that a layer of antibody was indeed attached to the surface of NP.

3. The content of oxidized α Fc in the supernatant after centrifugation was measured by an ELISA detection kit (Alpha Diagnostic International), and the difference between the input amount a and the residual amount B of the supernatant was calculated by the formula (2-1) to obtain the binding efficiency of α Fc bound to the α Fc-NP particles.

Binding efficiency (%) ═ A-B/A formula (2-1)

The experimental method is as follows:

(1) according to the method 2.3.1, respectively according to the mass ratio NP: α Fc is 3: 1,4: 1,5: 1 … … 9: 1 preparing different alpha Fc-NPs; wherein the concentration of the alpha Fc is fixed, the total reaction volume is the same, and each group contains 3 parts; meanwhile, a blank control group is arranged, namely oxidized alpha Fc with the same concentration is added into MilliQ ultrapure water (Millipore) with the same volume;

(2) centrifuging the prepared alpha Fc-NP at high speed, 15000rpm multiplied by 1.5h, 4 ℃; the supernatant was collected, the alpha Fc antibody concentration of the supernatant was measured using an ELISA kit, and the amount of the antibody bound to the particles was calculated according to the formula (2-1).

As shown in fig. 6, when the mass ratio of NP to α Fc is 5: 1, more than 80% of the α Fc is bound to the nanoparticle. Considering the binding efficiency of α Fc in combination with the loading capacity of NP, the selection was made as NP: α Fc ═ 5: 1 ratio at which 1mg of NP can bind approximately 160 μ g α Fc.

4. Ultrahigh resolution microscopy (STORM)) tested the ability of α Fc-NPs to bind therapeutic monoclonal antibodies.

anti-PD 1 antibody and polystyrene nanoparticles were labeled with Alexa Flour 647 (green) and Alexa Flour 750 (red), respectively, as shown in FIG. 7 for IgG-NP_αPD1Group (left), aggregation of green fluorescence (AF647) was rarely observed around red particles, whereas α Fc-NP_αPD1The group (right) was observed to have larger co-localization of both red (AF750) and green (AF647) fluorescence, and it was revealed from the figure that the anti-PD 1 antibody bound to α Fc-NP but not to the surface of IgG-NP (IgG is a control antibody and does not recognize the Fc region of the antibody), indicating that α Fc-NP was able to specifically bind and carry monoclonal antibody.

5. Nanoflow detector (NanoFCM) for verifying capacity of alpha Fc-NP for simultaneously binding two monoclonal antibodies

The PerCP-Cy5.5-alpha PDL1 and FITC-alpha PD1 antibodies are singly or jointly incubated with alpha Fc-NP, and are incubated for more than 4 hours at 4 ℃ in a dark place; transferred to a 0.5mL EP tube, and each NP was treated with NanoFCM (Xiamenfu flow)_{PP-Cy5.5-αPDL1}、NP_FITC-αPD1、NP_{PP-Cy5.5-αPDL1&FITC-αPD1}The results of the detection were analyzed and processed by using FlowJo v10(Tree Star) software.

Appearance of FITC in Mixed groups⁺PerCP-Cy5.5⁺Results of double positive particle population of (1) (FIG. 8), FITC⁺PerCP-Cy5.5⁺The double positive population of particles (upper right quadrant) of (a) represents particles that bind both antibodies simultaneously, demonstrating that α Fc-NP can simultaneously bind at least two different monoclonal antibodies to the same species, and can serve as a platform for the versatility of monoclonal antibody-based combination therapies.

6. The amount of binding of α PD1 and α PDL1 to α Fc-NP was tested by ELISA, as a ratio of one α Fc carrying one monoclonal antibody, i.e., as α Fc: α PD 1: α PDL1 ═ 1: 0.5: a ratio of 0.5 incorporating α PD1 and α PDL1 into α Fc-NP as shown in fig. 9, the amount of binding of therapeutic monoclonal mab increased with increasing incubation time, approaching 80% around about 4 h; as shown in fig. 10, the α PD 1: when feeding α PDL1(α Fc: (α PD1& α PDL1) ═ 1: 1), we found interestingly that the predicted binding ratio could be achieved by changing the feeding ratios of α PD1 and α PDL1, i.e. the ratio was substantially equal to the binding ratio. At the same time, this finding demonstrates that our platform has good selectivity and controllability.

7. The dissociation constants of α PD1 and α PDL1 bound to α Fc-NP were tested by a non-competitive ELISA method, as shown in fig. 11, and were substantially similar, indicating that binding to α Fc-NP does not affect the antigen binding ability of α PD 1.

8. Validation of alpha Fc-NPs as a Universal delivery platform for binding mAbs

After verifying the success of the α Fc-NP construction, we further investigated whether α Fc-NP could serve as a universal delivery platform for binding mabs. Measurement of DLS showed that after 4h incubation with α PD1 at 4 deg.C, the particle size of α Fc-NP increased by about 30nm (FIG. 12B) and the zeta potential changed significantly (FIG. 12C), indicating that α PD1 binds to α Fc-NP, i.e., α Fc-NP is formed_αPD1. We monitored α Fc-NP_αPD1The particle size of (2) was stable for 3 days (fig. 12D) with a certain stability, due to the change in the storage solution (isotonic 5% glucose solution).

Example 2 application of Bimultispecific Nanobodies

Both the mouse B16-F10 melanoma cell line and the mouse 4T1 in situ breast cancer cell line were derived from the American Standard Biotech Collection (ATCC). SPF-grade female C57BL/6 mice and female BALB/C mice, 5-6 weeks old, were purchased from Schlekschada laboratory animals Co., Ltd, Hunan. The mice are bred in the center of experimental animals of the university of southern China, and the animal experimental flow conforms to the relevant regulations of the experimental animal management of the university of southern China.

Rat anti-mouse PD1(CD279) antibody (α PD1), rat anti-mouse PDL1(B7-H1) antibody (α PDL1) and rat isotype control (anti-trinitrophenol) antibody (IgG2 a): all purchased from Bio X Cell, USA.

Combination of bispecific nano antibody and tumor cell and T cell

1. To mimic the tumor microenvironment in vitro, we induced high expression of PDL1 in B16-F10 cells by IFN- γ stimulation (1.0X 10)⁵Cells/well), and induction of CD8 sorted from spleen using α CD3 epsilon and α CD28⁺T cell activation (5.0X 10)⁵Cells/well), alpha PD-1 or alpha PD-L1 concentration was 25 μ g/mL, significant PDL1 expression up-regulation by B16-F10 cells and CD8 were observed by flow cytometry detection⁺Upregulation of T cell PD1 expression (fig. 13). These two stimulated cells can act as target cells that mimic the tumor microenvironment in vitro.

2. To assess the superiority of this delivery platform, we first explored the ability of bispecific nanobodies to interact with cells. PDL1^highB16-F10 cells (1.0X 10)⁵Cells/well) and PD1^high CD8⁺T cells (2.0X 10)⁵Cell/well) with α Fc NPs, respectively_IgG2aAnd FITC-labeled imNA_αPD1&αPDL1Co-incubation (IgG2a or. alpha. PD-1)&α PD-L1 concentration of 20 μ g/mL) and evaluation of imNA by flow cytometry and laser scanning confocal microscopy (CLSM)_αPD1&αPDL1The targeting ability of (a). As shown in FIG. 14A, imNA of B16-F10 cells_αPD1&αPDL1Mean Fluorescence Intensity (MFI) with incubation timeIs increased by elongation; and we confirmed that most of the particles were on the cell membrane surface rather than entering the cell as a method of trypan blue quenching extracellular fluorescence (fig. 14C). CLSM images also show a large number of imNA_αPD1&αPDL1Bound to the surface of B16-F10 cells (cell membranes labeled with PKH26 dye, red, NPs labeled with FITC) (fig. 14B). For CD8⁺T cell, imNA_αPD1&αPDL1Also time-dependent, and little to no particle entry to CD8⁺In T cells (fig. 15). In contrast, control group α Fc-NP_IgGShows a weaker interaction with both cells (FIGS. 14 and 15), indicating that the imNA_αPD1&αPDL1Binding to cells depends on antigen-specific recognition and binding of the carried monoclonal antibody. The above results all prove that the co-inhibiting molecule PD1/PDL1 can be used as imNA_αPD1&αPDL1The binding site of (3).

3. In addition, we have found experimentally that FITC-labeled α Fc-NPs_αPDL1Can only effectively bind to B16-F10 cells, while the alpha Fc-NP_αPD1Can only be connected with CD8⁺T cells bound efficiently, and neither nanoparticle could bind efficiently to the other cell at the same time (fig. 16 and 17, with a PD-1 or a PD-L1 concentration of 25 μ g/mL); description of alpha Fc-NP_αPD1Or alpha Fc-NP_αPDL1Binding to cells is antigen specific. Overall, α Fc-NPs_mAbsThe binding of the delivery platform to the cells is based on antigen-specific interactions of the carried monoclonal antibodies, and such interactions are better confined to the cell surface, facilitating efficient-to-target cell interactions.

We selected the mouse melanoma cell line B16-F10 for exploring the interaction of multivalent nanobodies bound with therapeutic antibodies with cells. CD8 isolated from spleen⁺After labeling CFSE with T cells, they were incubated with B16-F10 cells (expressing mCherry fluorescent protein), and an IgG control group was set, a free α PD1 and α PDL1 mixed group, and α Fc-NP carried α PD1 (NP)_αPD1) Alpha PDL1 with alpha Fc-NP (NP)_αPDL1) Mixed group (NP)_αPD1&NP_αPD1) The bispecific nanobody group synchronously carries alpha PD1 and alpha PDL1 group (imNA)_αPD1&αPDL1)([IgG]＝20μg/mL，[αPD1]、[αPDL1]10 μ g/mL each) of the four experimental groups. Adding corresponding antibody components, culturing for 4 hr, and washing away unbound nanoparticles and CD8 without effect on tumor cells⁺T cells, shown in FIG. 18, imNA_αPD1&αPDL1More CD8 than other groups⁺The co-localization of T cells (green) with tumor cells (red) suggests that the particles can promote the interaction of both cells.

In vitro cell killing experiment of bispecific nano antibody

1. In the presence of imNA_αPD1&αPDL1Having the ability to enhance the potent-target cell physical interaction, we wanted to see if it could further activate CD8 in vitro⁺T cells and promote their mediated cytotoxic effects. We directed to activated CD8⁺T cells and PDL1^highB16-F10 cells were spiked with different groups of antibodies or particles and the medium was examined for cytokine production after 24h of co-culture. With Free with equal amounts of antibody_αPD1&αPDL1And NP_αPD1&NP_αPDL1In contrast, with imNA_αPD1&αPDL1The treated mixed cell culture medium showed higher levels of secretion of IFN- γ (fig. 19), which are recognized to represent functional activation of CTLs. As shown in B and C of FIG. 19, we are also at imNA at the same time_αPD1&αPDL1An increase in granzyme B and perforin, which represent the direct killing ability of CTL cells, was detected in the group media.

2. The sorted T cells were activated with antibodies to CD3 and CD28, fluorescently labeled with CellTrace Blue, and co-cultured with B16-F10 cells (expressing mCherry fluorescent protein). IgG control group was set, free alpha PD1 was mixed with alpha PDL1, and alpha Fc-NP carried alpha PD1 (NP)_αPD1) Alpha PDL1 with alpha Fc-NP (NP)_αPDL1) Mixed group (NP)_αPD1&NP_αPDL1) The bispecific nanobody group synchronously carries alpha PD1 and alpha PDL1 (imNA)_αPD1&αPDL1)([IgG]＝20μg/mL，[αPD1]、[αPDL1]10 μ g/mL each) of the four experimental groups. Adding antibody components, Annexin V FITC apoptotic cell dye, and 5% CO at 37 deg.C₂Under the environment of the environment, the air conditioner is provided with a fan,and continuously observing by using a high content imaging analysis system. Free, as shown in FIG. 20_αPD1&αPDL1Groups showed only few apoptotic tumor cells (green FITC-positive large cells), NPs_αPD1&NP_αPD1A partial inhibition of the growth of tumor cells (red) was observed, whereas imNA_αPD1&αPDLA significant killing effect was observed, apoptosis of tumor cells was strongly induced over time, and there were few surviving tumor cells after 48h (fig. 20). This is probably due to the fact that imNAs particles promote T cell and tumor cell interactions and simultaneously inhibit the PD1/PDL1 pathway.

3. Considering CD8 in an in vivo environment⁺Specific recognition of T cells, we identified ovalbumin-specific OT-1CD8⁺Cell killing experiments with T cells and B10-F10-OVA cells, as shown in FIG. 21, via imNA_αPD1&αPDL1The participating experimental groups detect the release of more fluorescence in the tumor cells, and show more effective killing effect; and the killing effect is enhanced as the ratio of the T cells to the tumor cells is increased.

4. In addition, we will anti-human IgG Fc [ α Fc (H)]Alpha Fc (H) -NP (alpha Fc is goat anti-human IgG Fc antibody) capable of binding to the humanized antibody was prepared immobilized on nanoparticles, and the particle size characterization thereof was as shown in A and B of FIG. 22. We treated CD8 infiltrated in colorectal cancer samples from patients obtained intraoperatively⁺T cells and cancer cells were separately sorted and subjected to H33342 release assay (Keytruda, Tementiq concentrations 10. mu.g/mL each) which also showed ImNA_{Keytruda&Tecentiq}The better effect (figure 22C) shows that the monoclonal antibody is also applicable to clinically used immune checkpoint monoclonal antibodies, and has certain clinical application prospect. In summary, imNA_αPD1&αPDL1By enhancing CD8⁺The interaction between T cell and tumor cell reaches specific NP level in vitro_αPD1&NP_αPDL1Combined with higher antitumor activity.

5、imNA_αPD1&αPDL1Has enhanced ability to concentrate at the tumor site: to verify the therapeutic effect in vivo, we need to test the imNA_αPD1&αPDL1Whether or not to have the same toolThe enhanced tumor penetration and Enrichment (EPR) effect of the nano-drugs is evaluated. We constructed an in situ 4T1 breast cancer model using BALB/C mice, Cy 5-labeled with α PD1 and α PDL1, and injected free two antibodies or imNA via tail vein_αPD1&αPDL1And tumor tissue was collected at predetermined time points and analyzed for fluorescence signal by a small animal In Vivo Imaging System (IVIS). As shown in FIG. 23A, Free was administered 12h and 24h after drug injection_αPD1&αPDL1And imNA_αPD1&αPDL1Exhibit similar tumor enrichment; however, unlike the rapid clearance of free antibody drugs, imNA_αPD1&αPDL1Accumulation at the tumor site continued after 24h and the retention time exceeded 72 h. Upon analysis, imNA at 48h and 72h time points_αPD1&αPDL1Specific Free_αPD1&αPDL1The fluorescence intensity of (A) was higher by about 100.3% and 936.9%, respectively (FIG. 23B). Thereafter, we immunofluorescent-stained the tumor tissue to confirm the intratumoral distribution of the monoclonal antibody, and observed the experimental results consistent with IVIS (fig. 23C). Description of imNA_αPD1&αPDL1The method really keeps certain EPR effect of the nano-drug, and the nano-property ensures that the antibody drug has longer residence time at the tumor part, thereby being beneficial to the longer time for the drug to act.

Animal level antitumor therapy experiment

1. 40C 57BL/6 mice implanted with the B16-F10 subcutaneous melanoma model were randomly divided into 4 groups of 10 mice each, and 400. mu.L of IgG control, Free alpha PD1 and alpha PDL1 mixed groups (Free) were injected into the tail vein of each of the 10 mice_αPD1&αPDL1) Alpha Fc-NP carrying alpha PD1 and alpha Fc-NP carrying alpha PDL1 mixed group (NP)_αPD1&NP_αPD1) The bispecific nanobody group synchronously carries alpha PD1 and alpha PDL1 (imNA)_αPD1&αPDL1)([IgG]＝5mg/kg，[αPD1]、[αPDL1]2.5mg/kg each), once every three days for a total of 3 administrations. During the whole treatment process, the tumor size is measured by using a vernier caliper every 2 to 3 days, and the weight of each group of mice is detected for monitoring. The formula for tumor volume is as follows: volume (mm)³) 0.5 x length x width². Another 40 BALB/C mice planted with 4T1 in-situ breast cancer model were selectedMice, grouped with the above treatments, were dosed 2 times and tumor size and body weight were recorded. Finally, the survival of the mice is observed.

As shown in fig. 24, the IgG control group and the free antibody group showed rapid tumor growth. NP_αPD1&NP_αPD1The experimental group had some inhibitory effect on tumor growth, probably due to the slightly enhanced effect of the therapeutic antibody due to the multivalent effect. imNA_αPD1&αPDL1The experimental group has obvious effect of inhibiting the growth of the tumor, because the delivery vector can deliver the antibody drug to the tumor and simultaneously enhance the interaction of target cells. As shown in fig. 25, there was no significant change in body weight of each group of mice throughout the treatment, demonstrating that each component did not cause severe systemic toxicity to the mice. As shown in FIG. 26, imNA was observed in two tumor models_αPD1&αPDL1The survival time of the experimental group mice is obviously prolonged.

2. To further elucidate the imNA_αPD1&αPDL1The mechanism for improving the anti-tumor activity of antibody drugs, we analyzed infiltrated T cells and their subsets in tumor tissues by flow cytometry, and the flow gating scheme is shown in fig. 27. As shown in FIG. 28A, in imNA_αPD1&αPDL1Among the tumors treated, CTL (CD 45)⁺CD3⁺CD8⁺T cells) were IgG2a control, Free, respectively_αPD1&αPDL1And NP_αPD1&NP_αPDL14.7, 2.3 and 1.8 times of tumors. At the same time, it was found that_αPD1&αPDL1Treg cells exerting immunosuppressive function in the treated tumor (CD 45)⁺CD3⁺CD4⁺CD25⁺T cells) also decreased significantly, while an increased CTL/Treg ratio indicates a reversal of the immunosuppressive microenvironment (fig. 28B and C). Another important result shows that imNA_αPD1&αPDL1Compared with other groups, the treatment of (A) is more capable of inducing CD8 secreting granules B, IFN-gamma and IL-2⁺The increase of T cell subsets (FIGS. 28D-F) suggests an increase in the antitumor ability and proliferative ability of CTLs. Overall, the enhancement of the antitumor effect was imNA_αPD1&αPDL1The result of an immune microenvironment that ameliorates and reverses tumor suppressive properties.

3、imNA_αPD1&αPDL1Remarkably inhibits the formation of the pulmonary metastasis of the mouse 4T1-fLuc breast cancer. In a preliminary experiment with in situ 4T1 breast cancer treatment, we found differences in pulmonary metastasis, imNA, by dissecting mice_αPD1&αPDL1The treated carcinoma in situ occurred with significantly fewer lung metastases. We guess to be imNA_αPD1&αPDL1Circulating tumor cells can be eliminated to inhibit tumor metastasis, but since the occurrence of in situ metastasis is difficult to monitor and control, a lung metastasis model of breast cancer was constructed by direct tail vein injection of firefly luciferase (ffluc) -expressing 4T1 cells. I.v. q2d x 3 treatment was started on day 1 after tail vein injection of 4T 1-ffluc. From the results of IVIS in vivo and in vitro, IgG2a cotrol, Free_αPD1&αPDL1And NP_αPD1&NP_αPDL1In treated mice, bioluminescent signals were evident in the lungs of mice at day 15 post-tumor implantation, in contrast to imNA-treated mice_αPD1&αPDL1The bioluminescent signal was the weakest in the lungs of the treated mice (fig. 29A and B). Direct enumeration of whole lung tumor nodules (FIG. 29C) and hematoxylin-eosin (H) in sections&E) Observation of the staining (FIG. 30) demonstrates that, imNA_αPD1&αPDL1The number and size of metastatic nodules treated was significantly reduced. imNA_αPD1&αPDL1The anti-metastatic ability of the compound is probably partly attributed to the fact that the compound can promote CD8 in lung and blood circulation⁺T cell and tumor cell interaction.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Use of a nano-aptamer for the preparation of a multi-specific antibody delivery system in the manufacture of an immunotherapeutic drug, said multi-specific antibody delivery system comprising a nano-aptamer for the delivery of specific antibodies, and at least 2 specific antibodies;

the nano aptamer is formed by connecting nanoparticles with free amino on the surface and an anti-Fc fragment antibody or an anti-Fc fragment antibody part through a Schiff base reaction;

wherein the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody fragment is capable of non-covalent binding to the Fc domain of the specific antibody delivered;

the specific antibody delivered is of the same species origin as the Fc fragment recognized by the anti-Fc fragment antibody or anti-Fc fragment antibody fragment;

the Fc fragment region of the anti-Fc fragment antibody or anti-Fc fragment antibody has a glycosylation modification.

2. The use according to claim 1, wherein the nanoparticles have a particle size in the range of 25 to 500 nm.

3. The use according to claim 2, wherein the nanoparticles have a particle size in the range of 80 to 200 nm.

4. The use according to claim 1, wherein the nanoparticle is linked to the anti-Fc fragment antibody or anti-Fc fragment antibody moiety by a chemical bond, said nanoparticle being linked to the amino terminus of the chemical bond.

5. The use of claim 1, wherein the anti-Fc fragment antibody or anti-Fc fragment is an anti-human IgG antibody Fc antibody or anti-human IgG antibody Fc fragment, an anti-rat IgG antibody Fc antibody or anti-rat IgG antibody Fc fragment, an anti-mouse IgG antibody Fc antibody or anti-mouse IgG antibody Fc fragment.

6. A multi-specific antibody delivery system in an immunotherapy drug, comprising the nano-aptamer according to any one of claims 1 to 5, and at least 2 specific antibodies.

7. Use of the multispecific antibody delivery system of claim 6 in the manufacture of a medicament for immunotherapy.

8. The use of claim 7, wherein the immunotherapeutic agent is a tumor immunotherapeutic agent or an autoimmune disease therapeutic agent.

9. A method for constructing a multi-specific antibody delivery system in an immunotherapeutic drug according to claim 6, wherein a nanoparticle is linked to an anti-Fc fragment antibody or an anti-Fc fragment antibody moiety by Schiff's base reaction to form a nano aptamer;

wherein the Fab domain of the anti-Fc fragment antibody or anti-Fc fragment antibody is capable of non-covalent binding to the Fc domain of a specific antibody to be delivered; the specific antibody delivered is of the same species origin as the Fc fragment recognized by the anti-Fc fragment antibody or anti-Fc fragment antibody fragment;

and (3) incubating the delivered specific antibody and the nano-aptamer to obtain the multi-specific antibody delivery system.

10. The method for constructing, according to claim 9, a nanoparticle having a surface with free amino groups, comprising the steps of:

(3) and reducing the Schiff base by a reducing agent to form the nano aptamer.

11. The method of claim 10, wherein the nanoparticles having free amino groups on the surface are surface aminated, surface chitosan, or surface albuminated nanoparticles;

and/or, the oxidant in the step (1) is sodium periodate, and the concentration of the oxidant in the oxidation reaction system is 3-10 mM;

and/or, the oxidation conditions in the step (1) are as follows: and reacting for 1-3 h in a dark environment at 0-8 ℃.

12. The construction method according to claim 10 or 11, wherein the condensation in the step (2) is carried out at 0-8 ℃ for 10-14 h;

and/or the reducing agent in the step (3) is sodium borohydride or sodium cyanoborohydride, and the concentration of the reducing agent in a reduction reaction system is 0.5-1.5 mg/mL;

and/or, the reduction conditions in the step (3) are as follows: reacting for 0.5-1 h at 0-8 ℃.