WO2022027828A1

WO2022027828A1 - Nano-aptamer for multi-specific antibody delivery, application thereof and construction method therefor

Info

Publication number: WO2022027828A1
Application number: PCT/CN2020/122360
Authority: WO
Inventors: 沈松; 王均; 江澄涛; 许从飞; 杨显珠
Original assignee: 华南理工大学
Priority date: 2020-08-04
Filing date: 2020-10-21
Publication date: 2022-02-10
Also published as: CN112336872B; CN112336872A

Abstract

The present invention relates to a nano-aptamer for multi-specific antibody delivery, an application thereof and a construction method therefor. The nano-aptamer is formed by connecting a nanocarrier to an anti-Fc segment antibody or an anti-Fc segment antibody fragment portion by means of a chemical bond; a Fab domain of the anti-Fc segment antibody or the anti-Fc segment antibody fragment can non-covalently bind to an Fc domain of a delivered specific antibody; the delivered specific antibody has the same species origin as the Fc segment recognized by the anti-Fc segment antibody or the anti-Fc segment antibody fragment. The nano-aptamer can quickly, efficiently, and controllably bind to multiple types of antibodies, achieving the "multivalence" and "multi-specificity" of antibodies. For the first time, said type of constructed nanobody delivery platform is creatively applied to the preparation of an immunotherapeutic drug or therapeutic drug for tumors or autoimmune diseases, and can significantly improve the effect of immunotherapy.

Description

Nano aptamer for multispecific antibody delivery and its application and construction method

technical field

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.

Background technique

Malignant tumors are still a major public health problem that seriously threatens the health of residents. In recent years, tumor immunotherapy, especially immune checkpoint blockade therapy, has developed rapidly, and is profoundly changing the treatment landscape of malignant tumors. 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. Responses to immunotherapies such as blockade vary widely, and clinical response rates are generally low. The low clinical response rate and treatment effect severely limit the scope of patients benefiting from immune checkpoint blockade therapy, and it is urgent to develop new strategies to improve the antitumor effect of immune checkpoint antibodies.

In recent years, 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. Although 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. It has been greatly increased, and most of them are prepared by chemical coupling and DNA recombination technology. It is necessary to chemically modify the monoclonal antibody that produces the effect, which will inevitably affect the antigen-binding ability of the antibody itself. At the same time, there are short half-lives and complicated administration methods. , poor stability, poor solubility, and high cost. Currently, no bispecific/multispecific antibody has been approved for the treatment of solid tumors. Therefore, if the design concept of 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.

SUMMARY OF THE INVENTION

Based on this, 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.

The specific technical solutions are as follows:

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;

Wherein, 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;

Wherein, 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.

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

The inventors of the present invention have constructed a universal nanobody delivery platform (named as nanoaptamers, αFc-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.

In addition, 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 _{αPD1 & αPDL1} 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.

Description of drawings

Figure 1 is a schematic diagram of the preparation of nano-aptamer αFc-NP;

Fig. 2 is an aldehyde group detection diagram after oxidation of anti-IgG Fc antibody (αFc);

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 αFc-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 αFc 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 αFc-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 αPD1 and αPDL1 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 αFc-NP _αPD1 ;

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 _{αPD1 & αPDL1} 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 _{αPD1 & αPDL1} , the scale bar is 5 μm; 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 _{αPD1 & αPDL1} to CD8 ⁺ T cells;

Figure 16 shows the binding of NP _αPD1 or NP _αPD1 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 _αPD1 or NP _αPD1 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 _{αPD1 & αPDL1} in vitro to promote the killing of B16-F10 cells by CD8 ⁺ T cells (ELISA kit detects IFN-γ (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 αFc(H)-NP and imNA Keytruda _{& Tecentiq} (A and B): αFc(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 αPD1 & αPDL1} prolonging the retention of antibodies at tumor sites; A) Small animal imager image of in vitro 4T1 breast cancer antibody Cy5 fluorescence intensity distribution, **P<0.01, ***P<0.001; B) Small animals Analysis of the antibody Cy5 fluorescence signal intensity in the imager image; C) Immunofluorescence imaging: the distribution of the antibody Cy5 fluorescence in the tumor, the scale bar is 50 μm;

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; A) T cell subsets: CD3 ⁺ is further circled in the CD45 ⁺ population CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ CTL, and then circle CD3 ⁺ CD4 ⁺ CD25 ⁺ Treg cells; B) CD8 ⁺ CTL subset: further circle CD3 ⁺ CD8 ⁺ CTL in the CD45 ⁺ population, and finally analyze CD45 ⁺ CD3 ⁺ CD8 ⁺ CTL subsets of CTL secreting cytokines IL2 ⁺ , IFN-γ ⁺ and Gran B ⁺ ;

Figure 28 shows that imNA _{αPD1 & αPDL1} 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-γ (E) and IL-2 (F) in CD8 ⁺ T cells; **P<0.01, ***P<0.001, *** *P<0.001;

Figure 29 shows that imNA _{αPD1 & αPDL1} inhibits the formation of lung metastases; A) small animal in vivo imaging to observe the fLuc fluorescence of mouse breast cancer lung metastases; B) in vitro observation of the fLuc fluorescence distribution of lung metastatic nodules; C) Statistics of the number of lung metastases, *P<0.05, **P<0.01;n=5;

Figure 30 shows H&E staining of lung sections in the 4T1-fLuc lung metastasis model treatment experiment; the scale bar is 5 mm.

detailed description

The experimental methods of unreceipted specific conditions in the following examples of the present invention are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York:Cold Spring Harbor Laboratory Press, 1989), or Follow the conditions recommended by the manufacturer. Various common chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, 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 terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention.

The terms "comprising" and "having" and any variations thereof of the present invention are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but optionally also includes unlisted steps, or optionally also includes steps for these processes, other steps inherent in the method, product or device.

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;

Wherein, 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. Preferably, 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. As an example of the nano-aptamers of the present invention, 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. For example, when a humanized anti-PD1 antibody is selected for the delivered specific antibody, 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.

In some of these embodiments, 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.

In some of these embodiments, the nanoaptamers deliver at least 2 specific antibodies.

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

In some of these embodiments, the chemical bond is -CH ₂ -NH-, and the amino terminus of the chemical bond is attached to the nanocarrier.

In some of these embodiments, the immunotherapy drug is a tumor immunotherapy drug or an autoimmune disease treatment drug.

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

In some of these embodiments, 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 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;

Wherein, 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.

In some embodiments, the nanocarrier is a nanoparticle with free amino groups, and the method for constructing the nanoaptamer comprises the following steps:

(1) Anti-Fc-segment antibodies are oxidized by oxidants to form aldehyde-containing anti-Fc-segment antibodies;

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

(3) The Schiff base is reduced by a reducing agent to form nano-aptamers.

In some embodiments, the nanoparticles with free amino groups on the surface of the step (1) are surface aminated, surface chitosanized or surface albuminated nanoparticles.

In the following examples, 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.

In some embodiments, 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.

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

In some of the embodiments, 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.

In some of the embodiments, the condensation conditions in step (2) are the reaction at 0-8° C. for 10-14 hours.

In some embodiments, the reducing agent in step (3) is sodium borohydride or sodium cyanoborocyanide. 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: the reaction is carried out at 0 to 8° C. for 0.5 to 1 h.

The present invention will be further described in detail below in conjunction with specific embodiments.

Example 1 Construction and characterization of nano-aptamer αFc-NP

Source of raw materials used in the embodiment and processing method:

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.;

Sodium periodate (NaIO ₄ ), used and prepared now, avoid light (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China);

Sodium borohydride, currently used and prepared (purchased from Saen Chemical Technology (Shanghai) Co., Ltd., China);

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).

1. Anti-Fc antibody oxidation treatment

As shown in Figure 1, the goat anti-rat IgG-Fc antibody (αFc) 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 αFc 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 αFc antibody has an aldehyde group.

Removal of sodium periodate: In the condition of 0.01M acetic acid-sodium acetate buffer (pH 4.2), use a 100kDa ultrafiltration tube for 2 to 3 times to remove sodium periodate. After recovery of oxidized αFc antibody, Nanodrop A280 was used to rapidly detect the concentration of oxidized αFc antibody.

2. Preparation of nano-aptamer αFc-NP carrier

Dilute the 100nm amino-functionalized polystyrene microspheres with ultrapure water to 0.5mg/mL, add the above oxidized anti-Fc antibody to 0.1mg/mL, mix thoroughly and react at 4°C for 12h; after the reaction, add hydroboration Sodium to 1mg/mL, react at 4°C for 0.5-1.0h; after the reaction, centrifuge at 15000rpm × 1.5h at 4°C, discard the supernatant, resuspend with an equal volume of ultrapure water, and centrifuge again for 2 times, and finally as needed Resuspend in a volume of 5% glucose solution.

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. Ultra-performance liquid chromatography (UPLC) was used to assist in the testing.

3. Characterization of Nanoaptamers

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

Sample processing: Take 10 μg of αFc antibody and dilute it with water to 20 μL, take 20 μL of αFc-NP particle solution containing the same concentration of αFc, add 5 μL 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.

As shown in Figure 3, the αFc-NP group has significantly less heavy chains than free antibody, and the same intensity of the light chain bands, indicating that the αFc-NP group is bonded to the nanoparticles through the sugar chain structure on the Fc segment of αFc, 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 αFc is indeed a directional chemical binding.

2. The prepared αFc-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 αFc-NP is about 130 nm.

As shown in Figure 5, observed under a scanning electron microscope (SEM), αFc-NP has a rougher surface compared with the polystyrene microspheres without antibody binding, and the particle size increases from about 100 nm to about 110 nm, The electron microscope results were basically consistent with the DLS results, indicating that a layer of antibody was indeed attached to the surface of the NP.

3. Use ELISA detection kit (Alpha Diagnostic International) to measure the content of oxidized αFc in the supernatant after centrifugation, and calculate the difference between the input amount A and the residual amount B of the supernatant by formula (2-1) to obtain the binding on the αFc-NP particles. αFc binding efficiency.

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

The experimental method is as follows:

(1) According to the above method 2.3.1, different αFc-NPs were prepared according to the mass ratio NP:αFc of 3:1, 4:1, 5:1...9:1; wherein, the concentration of αFc was fixed and the total reaction volume was The same, 3 copies in each group; at the same time, a blank control group was set up, that is, the same concentration of oxidized αFc was added to the same volume of MiliQ ultrapure water (Millipore);

(2) Centrifuge the prepared αFc-NP at high speed, 15000rpm×1.5h, 4°C; collect the supernatant, use an ELISA kit to detect the concentration of αFc antibody in the supernatant, and calculate the amount of antibody bound to the particles according to formula (2-1).

The results in Figure 6 show that when the mass ratio of NP to αFc is greater than or equal to 5:1, more than 80% of αFc is bound to the nanoparticles. Considering the binding efficiency of αFc and the loading capacity of NP, the ratio of NP:αFc=5:1 was chosen to construct αFc-NP, at which 1 mg of NP can bind about 160 μg of αFc.

4. The ability of αFc-NP to bind therapeutic monoclonal antibodies was tested by stochastic optical reconstruction microscopy (STORM).

Anti-PD1 antibody and polystyrene nanoparticles were labeled with Alexa Flour 647 (green) and Alexa Flour 750 (red), respectively, as shown in Figure 7. For the IgG-NP _αPD1 group (left), the red surrounding particles were rarely observed The aggregation of green fluorescence (AF647), while the αFc-NP _αPD1 group (right) can observe greater co-localization of red (AF750) and green (AF647) fluorescence. On the NP, it cannot bind to the surface of IgG-NP (IgG is a control antibody, which cannot recognize the Fc segment of the antibody), indicating that αFc-NP can specifically bind and carry monoclonal antibodies.

5. Nano-flow detector (NanoFCM) to verify the ability of αFc-NP to bind two monoclonal antibodies at the same time

Two antibodies, PerCP-Cy5.5-αPDL1 and FITC-αPD1, were incubated with αFc-NP alone or together, and incubated at 4°C in the dark for more than 4 h; transferred to a 0.5 mL EP tube, and NanoFCM (Xiamen Fuliu) was used to detect the NPs respectively. _{PP-Cy5.5-αPDL1} , NP _FITC-αPD1 , NP _{PP-Cy5.5-αPDL1&FITC-αPD1} were detected, and the results were analyzed and processed by FlowJo v10 (Tree Star) software.

The results of the double-positive particle population of FITC ⁺ PerCP-Cy5.5 ⁺ appearing in the mixed group (Figure 8), the double-positive particle population of FITC ⁺ PerCP-Cy5.5 ⁺ (upper right quadrant) represent particles that bind both antibodies at the same time, It is verified that αFc-NP can simultaneously bind to at least two different monoclonal antibodies of the same species, and can be used as a universal platform for monoclonal antibody-based combination therapy.

6. Test the binding amount of αPD1 and αPDL1 bound to αFc-NP by ELISA. According to the ratio of one monoclonal antibody carried by one αFc, that is, the ratio of αFc:αPD1:αPDL1=1:0.5:0.5 to αFc-NP. Into αPD1 and αPDL1, as shown in Figure 9, the binding amount of therapeutic monoclonal mAb increases with the increase of incubation time, approaching 80% at about 4h; as shown in Figure 10, according to different ratios αPD1:αPDL1(αFc:( When αPD1&αPDL1)=1:1), it is interesting to find that the purpose of predicting the binding ratio can be achieved by changing the feeding ratio of αPD1 and αPDL1, that is, the feeding ratio is basically equal to the binding ratio. At the same time, this finding proves that our platform has good selectivity and controllability.

7. The dissociation constants of αPD1 and αPDL1 bound to αFc-NP were tested by a non-competitive ELISA method. As shown in Figure 11, the dissociation constants of the two are basically similar, indicating that binding to αFc-NP will not affect αPD1 antigen-binding capacity.

8. Validation of αFc-NP as a universal delivery platform for binding mAbs

After verifying the successful construction of αFc-NP, we further investigated whether αFc-NP can be used as a general delivery platform for binding mAb. DLS measurement showed that after incubation with αPD1 at 4°C for 4 h, the particle size of αFc-NPs increased by about 30 nm (Fig. 12B), and its zeta potential also changed significantly (Fig. 12C), indicating that αPD1 binds to αFc-NPs. On the NP, αFc-NP _αPD1 is formed. We monitored the change in particle size of αFc-NP _αPD1 in storage solution (isotonic 5% glucose solution), which remained stable for 3 days ( FIG. 12D ) with some stability.

Example 2. Application of dual multispecific nanobodies

The mouse B16-F10 melanoma cell line and the mouse 4T1 orthotopic breast cancer cell line were obtained from the American Standard Biological Collection (ATCC). SPF grade female C57BL/6 mice and female BALB/C mice, 5-6 weeks old, were purchased from Hunan Slike Jingda Laboratory Animal Co., Ltd. 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.

Rat anti-mouse PD1 (CD279) antibody (αPD1), rat anti-mouse PDL1 (B7-H1) antibody (αPDL1) and rat isotype control (anti-trinitrophenol) antibody (IgG2a): all purchased from the United States Bio X Cell Inc.

1. Binding of bispecific nanobodies to tumor cells and T cells

1. To mimic the tumor microenvironment in vitro, we induced high expression of PDL1 in B16-F10 cells (1.0×10 ⁵ cells/well) by stimulation with IFN-γ, and CD8 ⁺ T cells sorted from the spleen using αCD3ε and αCD28 Activated (5.0×10 ⁵ cells/well), the concentration of αPD-1 or αPD-L1 was 25 μg/mL, and a significant up-regulation of PDL1 expression in B16-F10 cells and a decrease in PD1 expression in CD8 ⁺ T cells were observed by flow cytometry. upregulated (Figure 13). These two stimulated cells can be used as effector-target cells to mimic the tumor microenvironment in in vitro experiments.

2. To evaluate the superiority of this delivery platform, we first explored the ability of bispecific nanobodies to interact with cells. PDL1 ^high B16-F10 cells (1.0×10 ⁵ cells/well) and PD1 ^high CD8 ⁺ T cells (2.0×10 ⁵ cells/well) were co-incubated with αFc NP _IgG2a and FITC-labeled imNA _{αPD1 & αPDL1} (IgG2a or αPDL1), respectively. The concentration of αPD-1 & αPD-L1 was 20 μg/mL), and the targeting ability of imNA _{αPD1 & αPDL1} was evaluated by flow cytometry and laser scanning confocal microscopy (CLSM). As shown in Fig. 14A, the mean fluorescence intensity (MFI) of imNA _{αPD1 & αPDL1} 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 _{αPD1 & αPDL1} 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 ). For CD8 ⁺ T cells, imNA _{αPD1 & αPDL1} also bound in a time-dependent manner, and almost did not enter the granules into CD8 ⁺ T cells ( FIG. 15 ). In contrast, the control αFc-NP _IgG showed weak interaction with both cells (Figure 14 and Figure 15), indicating that the binding of imNA _{αPD1 & αPDL1} 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 _αPD1 _{& αPDL1} .

3. In addition, we also found that FITC-labeled αFc-NP _αPDL1 can only effectively bind to B16-F10 cells, while αFc-NP _αPD1 can only effectively bind to CD8 ⁺ T cells, and the two nanoparticles Neither can effectively bind to another cell at the same time (Figure 16 and Figure 17, the concentration of αPD-1 or αPD-L1 is 25 μg/mL); indicating that the binding of αFc-NP _αPD1 or αFc-NP _αPDL1 to cells is antigen-specific of. In general, the binding of the αFc-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.

We selected the mouse melanoma cell line B16-F10 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 IgG control group was set, the free αPD1 and αPDL1 mixed group, αFc-NP carrying αPD1 (NP _αPD1 ) and αFc -NP carrying αPDL1 (NP _αPDL1 ) mixed group (NP _αPD1 & NP _αPD1 ), bispecific nanobody group that simultaneously carries αPD1 and αPDL1 group (imNA _{αPD1 & αPDL1} ) ([IgG]=20μg/mL, [αPD1] , [αPDL1] 10 μg/mL each) four experimental groups. The corresponding antibody components were added respectively, and after 4 hours of culture, the unbound nanoparticles and CD8 ⁺ T cells that did not interact with tumor cells were washed away. As shown in Figure 18, compared with other groups, imNA _{αPD1 & αPDL1 had} more CD8 ⁺ T cells Cells (green) co-localize with tumor cells (red), suggesting that the particle facilitates the interaction of the two cells.

2. In vitro cell killing experiments of bispecific nanobodies

1. After demonstrating the ability of imNA _{αPD1 & αPDL1} to enhance the physical interaction of effector-target cells, we hope to understand whether it can further activate CD8 ⁺ T cells in vitro and promote their mediated cytotoxic effects. We spiked activated CD8 ⁺ T cells and PDL1 ^high B16-F10 cells with different groups of antibodies or particles and examined cytokine production in the medium after 24 h of co-culture. Mixed cell culture media treated with imNA _{αPD1 & αPDL1} showed higher levels of IFN-γ secretion compared to Free _{αPD1 & αPDL1} and NP _αPD1 & NP _αPDL1 with equal amounts of antibodies (Figure 19), which are recognized as representative functional activation of CTLs. As shown in B and C of Figure 19, we also detected increases in granzyme B and perforin, which represent the direct killing ability of CTL cells, in the imNA _{αPD1 & αPDL1} group media simultaneously.

2. 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). Set up IgG control group, mixed free αPD1 and αPDL1 group, αFc-NP carrying αPD1 (NP _αPD1 ) and αFc-NP carrying αPDL1 (NP _αPDL1 ) mixed group (NP _αPD1 & NP _αPDL1 ), and bispecific nanobody group were synchronized. There were four experimental groups carrying αPD1 and αPDL1 groups (imNA _{αPD1 & αPDL1} ) ([IgG]=20 μg/mL, [αPD1], [αPDL1] 10 μg/mL each. The antibody components were added, and Annexin V FITC apoptotic cell dye was added, and the cells were continuously observed with a high-content imaging analysis system at 37°C under 5% CO ₂ . As shown in Figure 20, Free _{αPD1 & αPDL1} group showed only few apoptotic tumor cells (green FITC-positive large cells), NP _αPD1 & NP _αPD1 observed partial inhibition of tumor cell growth (red), while imNA _αPD1 A significant killing effect was observed with _&αPDL , which sharply induced apoptosis of tumor cells over time, and there were few surviving tumor cells after 48 h ( FIG. 20 ). This may be due to the fact that imNAs particles promote the interaction between T cells and tumor cells, and simultaneously inhibit the PD1/PDL1 pathway.

3. Considering the specific recognition of CD8 ⁺ T cells in the in vivo environment, we used ovalbumin-specific OT-1CD8 ⁺ T cells and B10-F10-OVA cells for cell killing experiments, as shown in Figure 21. The experimental group in which imNA _{αPD1 & αPDL1} participated detected more fluorescence release in tumor cells, showing more effective killing effect; and as the ratio of T cells to tumor cells increased, the killing effect was enhanced.

4. In addition, we immobilized anti-human IgG Fc [αFc(H)] on nanoparticles to prepare αFc(H)-NP (αFc is goat anti-human IgG Fc antibody) that can bind to humanized antibodies. Characterization is shown in Figure 22, A and B. We sorted out the infiltrating CD8 ⁺ T cells and cancer cells from the patients' colorectal cancer samples obtained during the operation, and performed the H33342 release experiment (Keytruda and Tecentiq concentrations were 10 μg/mL each), which also showed that imNA Keytruda _{& Tecentiq} increased the The excellent effect (Fig. 22C) indicates that the monoclonal antibody to the immune checkpoint used in clinic is also applicable and has certain clinical application prospects. In conclusion, imNA _{αPD1 & αPDL1} achieved higher antitumor activity than NP _αPD1 & NP _αPDL1 combination in vitro by enhancing the interaction between CD8 ⁺ T cells and tumor cells.

5. ImNA _{αPD1 & αPDL1} have enhanced enrichment ability at tumor sites: In order to verify the efficacy in vivo, we need to determine whether imNA _{αPD1 & αPDL1} can simultaneously have the enhanced tumor penetration and enrichment (EPR) effect of nanomedicines Evaluate. We used BALB/C mice to construct an orthotopic 4T1 breast cancer model, labeled αPD1 and αPDL1 with Cy5, injected two free antibodies or imNA _{αPD1 & αPDL1} through the tail vein, and collected tumor tissue at predetermined time points. The analysis of the fluorescence signal was performed by an animal in vivo imaging system (IVIS). As shown in Figure 23A, Free _{αPD1 & αPDL1} and imNA _{αPD1 & αPDL1} exhibited similar tumor enrichment at 12h and 24h after drug injection; but unlike the rapid clearance of free antibody drug, imNA _{αPD1 & αPDL1} continued after 24h Accumulated at the tumor site, and the retention time was more than 72h. After analysis, the fluorescence intensity of imNA αPD1 & αPDL1 was about 100.3% and 936.9% higher than that of Free _{αPD1 & αPDL1} _at the 48h and 72h time points, respectively ( FIG. 23B ). Afterwards, we performed immunofluorescence staining on tumor tissue to confirm the intratumoral distribution of monoclonal antibodies, and observed experimental results consistent with IVIS (Fig. 23C). It shows that imNA _{αPD1 & αPDL1} does retain a certain EPR effect of nano-drugs, and due to the nano-characteristics, the antibody drug stays in the tumor site for a longer time, which is beneficial for the drug to exert its effect for a longer time.

3. Anti-tumor treatment experiments at animal level

1. 40 C57BL/6 mice implanted with B16-F10 subcutaneous melanoma model were randomly divided into 4 groups, 10 mice in each group, and 400 μL of IgG control, free αPD1 and αPDL1 mixed groups were injected into the tail vein respectively ( Free _{αPD1 & αPDL1} ), αFc-NP carrying αPD1 and αFc-NP carrying αPDL1 mixed group (NP _αPD1 & NP _αPD1 ), bispecific nanobody group synchronously carrying αPD1 and αPDL1 group (imNA _{αPD1 & αPDL1} ) ([ IgG] = 5 mg/kg, [αPD1], [αPDL1] each 2.5 mg/kg), administered once every three days for a total of 3 administrations. During the whole treatment process, the tumor size was measured with a vernier caliper every 2-3 days, and the body weight of the mice in each group was monitored. The calculation formula of tumor volume is as follows: volume (mm ³ )=0.5×length×width ² . Another 40 BALB/C mice implanted with the 4T1 orthotopic breast cancer model were selected and divided into the same groups as above, and administered twice, and the tumor size and body weight were recorded. Finally, the survival of mice was observed.

As shown in Figure 24, the tumors in the IgG control control group and the free antibody group grew rapidly. The NP _αPD1 &NP _αPD1 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 _{αPD1 & αPDL1} 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. As shown in Figure 25, during the whole treatment process, 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. As shown in Figure 26, under the two tumor models, the survival time of mice in the imNA _{αPD1 & αPDL1} experimental groups was significantly prolonged.

2. In order to further clarify the mechanism of imNA _{αPD1 & αPDL1} improving the anti-tumor activity of antibody drugs, we analyzed the infiltrating T cells and their subpopulations in tumor tissue by flow cytometry. The flow gate scheme is shown in Figure 27. As shown in Figure 28A, the frequencies of CTLs (CD45 ⁺ CD3 ⁺ CD8 ⁺ T cells) in _{imNA αPD1} _& _αPDL1 _- treated tumors were 4.7, 2.3, and 1.8 times. At the same time, it was found that in the tumors treated with imNA _αPD1 _{& αPDL1} , the proportion of Treg cells (CD45 ⁺ CD3 ⁺ CD4 ⁺ CD25 ⁺ T cells) that exerted immunosuppressive function was also significantly decreased, and the increased CTL/Treg ratio indicated that immunosuppression was less effective. Reversal of the environment (Figure 28B and C). Another important result showed that treatment with imNA _{αPD1 & αPDL1} was more able to induce an increase in the subset of CD8 ⁺ T cells secreting granule B, IFN-γ and IL-2 than the other groups (Fig. 28D-F), suggesting that CTL The anti-tumor ability and the enhancement of proliferation ability. Overall, the enhanced antitumor effect is the result of imNA _{αPD1 & αPDL1} improving and reversing the tumor suppressive immune microenvironment.

3. imNA _{αPD1 & αPDL1} significantly inhibited the formation of lung metastases in mouse 4T1-fLuc breast cancer. In a pilot experiment of orthotopic 4T1 breast cancer treatment, we found differences in lung metastases by dissecting mice, and the orthotopic carcinomas treated with imNA _{αPD1 & αPDL1} had significantly fewer lung metastases. We guessed that imNA _{αPD1 & αPDL1} 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. ivq2d×3 treatment was started on day 1 after tail vein injection of 4T1-fLuc. From the results of IVIS in vivo and in vitro, in mice treated with IgG2a cotrol, Free _αPD1 _{& αPDL1} , and NP _αPD1 & NP _αPDL1 , bioluminescence signal was evident in the lungs of mice on day 15 after tumor implantation, compared with , the bioluminescence signal was the weakest in the lungs of mice treated with imNA _{αPD1 & αPDL1} (Figure 29A and B). Direct enumeration of whole lung tumor nodules (FIG. 29C) and hematoxylin-eosin (H&E) staining of sections (FIG. 30) demonstrated that the number and size of metastatic nodules were significantly reduced by imNA _αPD1 _{& αPDL1} treatment. The anti-metastatic ability of imNA _{αPD1 & αPDL1} may be partly due to its ability to promote the interaction of CD8 ⁺ T cells and tumor cells in the lungs and blood circulation.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

A nanometer aptamer for multispecific antibody delivery, characterized in that it is formed by linking a nanocarrier with an anti-Fc segment antibody or an anti-Fc segment antibody fragment through a chemical bond;

Wherein, 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 specific antibody delivered is of the same species origin as the Fc segment recognized by the anti-Fc segment antibody or anti-Fc segment antibody fragment.
The nano-aptamer of claim 1, wherein the nano-aptamer delivers at least two specific antibodies.
The nano-aptamer according to claim 1, wherein the anti-Fc segment antibody or the Fc segment region of the anti-Fc segment antibody fragment has glycosylation modification.
The nano-aptamer according to claim 1, wherein the chemical bond is selected from an alkylamino bond, an amide bond or an imine bond.
The nano-aptamer according to any one of claims 1-4, characterized in that, the nano-carriers are nanoparticles with a particle size range of 25-500 nm, preferably a particle size range of 80-200 nm.
Application of the nano-aptamer according to any one of claims 1 to 5 in the preparation of a multispecific antibody delivery system.
A multispecific antibody delivery system, characterized by comprising the nano-aptamer according to any one of claims 1 to 5, and a specific antibody.
The multispecific antibody delivery system of claim 7, comprising at least two specific antibodies.
Application of the nano-aptamer according to any one of claims 1 to 5 or the multispecific antibody delivery system according to any one of claims 7 to 8 in the preparation of immunotherapy drugs.
The application according to claim 9, wherein the immunotherapy drug is a tumor immunotherapy drug or an autoimmune disease therapeutic drug.
A method for constructing a nano-aptamer for multispecific antibody delivery, characterized in that, 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 anti-Fc segment antibody or the anti-Fc segment antibody fragment reacts with the nanoparticle in one or more steps to form the chemical bond;

Wherein, 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 fragments recognized by the antibody fragments are of the same species origin.
The construction method according to claim 11, wherein the nanoparticle is a nanoparticle with free amino groups on the surface, and the construction method comprises the following steps:

(1) Anti-Fc-segment antibodies are oxidized by oxidants to form aldehyde-containing anti-Fc-segment antibodies;

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

(3) The Schiff base is reduced by a reducing agent to form nano-aptamers.
The construction method according to claim 12, wherein the nanoparticles with free amino groups on the surface of the step (1) are nanoparticles with surface amination, surface chitosanization or surface albuminization;

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

And/or, the conditions for the oxidation in step (1) are: reaction in a dark environment at 0-8° C. for 1-3 hours.
The construction method according to claim 12 or 13, wherein the condensation condition in step (2) is a reaction at 0-8° C. for 10-14 h;

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

And/or, the reduction conditions in step (3) are: reaction at 0-8° C. for 0.5-1 h.