CN116769047A

CN116769047A - Immune activating molecule and preparation method and application thereof

Info

Publication number: CN116769047A
Application number: CN202210248964.2A
Authority: CN
Inventors: 倪健; 梁辉
Original assignee: Yourui Biomedical Technology Shenzhen Co ltd
Current assignee: Yourui Biomedical Technology Shenzhen Co ltd
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2023-09-19

Abstract

The application belongs to the technical field of immunoregulation, and discloses an immune activating molecule, a preparation method and application thereof. The immune activating molecule is formed by connecting CpG molecules with an anti-CD 32 single-chain antibody, and the amino acid sequences of a light chain variable region and a heavy chain variable region in the anti-CD 32 single-chain antibody are respectively shown in SEQ ID NO: 1-2. The immune activating molecule can activate humoral immunity and cellular immunity signals simultaneously, and has excellent specificity and targeting property. The immune activating molecules can also be combined with antigens of different specificities, resulting in therapeutic products suitable for different indications.

Description

Immune activating molecule and preparation method and application thereof

Technical Field

The application belongs to the technical field of immunoregulation, and particularly relates to an immune activating molecule, a preparation method and application thereof.

Background

In recent years, tumor immunotherapy techniques represented by monoclonal antibodies, small molecular drugs, tumor vaccines and cellular immunity are rapidly developed, and the method is the fifth largest therapy after surgery, radiotherapy, chemotherapy and targeted therapy, and tumor immunotherapy is mainly used for killing tumors by activating the immune system of organisms, and is recognized as the most active and most development-promoting method in the twenty-first century tumor comprehensive treatment mode, and the only hope of completely killing tumor cells at present. Since the mechanism of tumor immunotherapy consists in activating the autoimmune system to recognize and eliminate tumor cells, once it is effective, it is possible for the patient to obtain a dynamic, sustained anti-tumor immune response, with a low tumor recurrence rate, and even for advanced cancers, clinical cure can be achieved, thus having unique advantages over traditional therapies.

At present, related products are marketed for tumor immunotherapy aiming at different mechanisms, but the related products of the tumor immunotherapy which are marketed at present have respective problems: (1) Monoclonal antibodies against tumor-associated protein targets (first generation immunotherapy), which are currently limited in targets, and only against epitopes of 1 target, have limited antitumor effects, and are prone to drug resistance and side effects; (2) Monoclonal antibodies against immune checkpoints (second generation immunotherapy), activating non-specific T cells against tumors (not specific for tumor antigens), may risk autoimmune disease side effects, and have uncertainty in efficacy; (3) Bispecific (functional) antibodies, which are directed against non-tumor specific T cells, have only 1 or 2 epitopes, and there is a risk of overactivating non-specific T cells, resulting in a large side effect; (4) Cell therapy, CAR-T cells express artificial tumor specific receptors, which are currently mainly aimed at blood tumors, have limited therapeutic effects in solid tumors, and are too personalized in production cost and expensive in production cost; (5) Tumor vaccine can excite specific immune reaction of human body to kill tumor by utilizing natural immune process of human body, but general tumor vaccine can exert therapeutic effect in 3-4 months, and several times of immunity may be needed, and the period required for acting is too long, and the specific antibody titer produced by stimulation is too low.

Therefore, aiming at the limitations of the existing tumor immunotherapy technology, the application hopes to provide a novel immunotherapy product with obvious curative effect, thereby providing assistance for enhancing the treatment effect of tumors.

Disclosure of Invention

The present application aims to solve at least one of the technical problems in the prior art described above. Therefore, the application provides an immune activating molecule, and a preparation method and application thereof. The immune activating molecule can activate humoral immunity and cellular immunity signals simultaneously, and has excellent specificity and targeting property. The immune activating molecules can also be combined with antigens of different specificities, resulting in therapeutic products suitable for different indications.

The application provides an immune activating molecule, which is formed by connecting CpG molecules with an anti-CD 32 single-chain antibody, wherein the amino acid sequences of a light chain variable region and a heavy chain variable region in the anti-CD 32 single-chain antibody are respectively shown as SEQ ID NO: 1-2.

Amino acid sequence of the light chain variable region: EVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGESIYPDDFKGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTSVTVSS (SEQ ID NO: 1);

amino acid sequence of heavy chain variable region: DIVMTQAAPSVPVTPGESVSISCRSSKSLLHTNGNTYLHWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTAFTLSISRVEAEDVGVFYCMQHLEYPLTFGAGTKLELKGGGSKGP (SEQ ID NO: 2).

Preferably, the amino acid sequence of the anti-CD 32 single chain antibody is shown in SEQ ID NO: 3.

The amino acid sequence of the anti-CD 32 single chain antibody is: MEFGLSWLFLVAILKGVQCEVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGESIYPDDFKGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKSLLHTNGNTYLHWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTAFTLSISRVEAEDVGVFYCMQHLEYPLTFGAGTKLELKGGGSKGPHHHHHHEVSALEKEVSALEKEVSALEKEVSALEKEVSALEKA(SEQ ID NO：3)。

Compared with the existing CD32 antibody, the anti-CD 32 single-chain antibody designed and screened by the application has better affinity, specificity, targeting property and other performances, and can target plasma cell-like dendritic cells (pDC cells) to realize specific immune activation. The light chain variable region and the heavy chain variable region in the anti-CD 32 single-chain antibody are connected by a connecting peptide (with the sequence of GGGGSGGGGSGGGGS), the tail end of the anti-CD 32 single-chain antibody contains a pepK/E helical peptide (underlined part), and the automatic assembly of the immune activating molecule and various immunogens can be realized through the pepK/E helical peptide when the anti-CD 32 single-chain antibody is applied, so that the effect of enhancing or expanding the indication is achieved. In addition, the application connects the anti-CD 32 single-chain antibody with the oligodeoxynucleotide (CpG) with the function of activating the immune system of the organism, thereby realizing the effect of activating the immune system rapidly and efficiently.

The immune activating molecule can be used in combination with different technical methods for treating tumors, allergies, infectious diseases, inflammations, autoimmune diseases and other indications.

Preferably, the nucleotide sequence encoding the anti-CD 32 single chain antibody is as set forth in SEQ ID NO: 4.

The nucleotide sequence encoding the anti-CD 32 single chain antibody is:

5’-GAATTCGCCGCCACCATGGAGTTCGGACTCAGTTGGCTGTTCCTGGTGGCCATCCTGAAGGGTGTGCAGTGTGAGGTGCAGCTGCAGCAGTCCGGCCCCGAGCTGAAGAAGCCCGGCGAGACCGTGAAGATCTCCTGCAAGGCCTCCGGCTACACCTTCACCAACTACGGCATGAACTGGGTGAAGCAGGCCCCCGGCAAGGGCCTGAAGTGGATGGGCTGGCTGAACACCTACACCGGCGAGTCCATCTACCCCGACGACTTCAAGGGCCGCTTCGCCTTCTCCTCCGAGACCTCCGCCTCCACCGCCTACCTGCAGATCAACAACCTGAAGAACGAGGACATGGCCACCTACTTCTGCGCCCGCGGCGACTACGGCTACGACGACCCCCTGGACTACTGGGGCCAGGGCACCTCCGTGACCGTGTCCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGACATCGTGATGACCCAGGCCGCCCCCTCCGTGCCCGTGACCCCCGGCGAGTCCGTGTCCATCTCCTGCCGCTCCTCCAAGTCCCTGCTGCACACCAACGGCAACACCTACCTGCACTGGTTCCTGCAGCGCCCCGGCCAGTCCCCCCAGCTGCTGATCTACCGCATGTCCGTGCTGGCCTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGCCTTCACCCTGTCCATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTTCTACTGCATGCAGCACCTGGAGTACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGCGGCGGCTCCAAGGGCCCCCACCACCACCACCACCACGAGGTGTCCGCCCTGGAGAAGGAGGTGTCCGCCCTGGAGAAGGAGGTGTCCGCCCTGGAGAAGGAGGTGTCCGCCCTGGAGAAGGAGGTGTCCGCCCTGGAGAAGGCCTGAGCGGCCGC-3’(SEQ ID NO：4)。

preferably, the CpG molecule is coupled to the anti-CD 32 single chain antibody in a manner.

Preferably, the CpG molecules consist of Class A CpG ODNs, class B CpG ODNs, and Class C CpG ODNs. The application combines CpG molecules of different subtypes, can transmit various signals of immune activation, and can activate signals of humoral immunity and cellular immunity at the same time, thereby playing a role of immune activation better.

Preferably, the mass ratio of CpG molecules to anti-CD 32 single chain antibodies in the immune activating molecule is (3-10): 1

The application also provides a preparation method of the immune activating molecule, which comprises the following steps:

the CpG molecule reacts with amine-sulfhydryl crosslinking agent and then reacts with anti-CD 32 single chain antibody to produce the immune activating molecule.

Preferably, the amine-mercapto cross-linker is sulfo-SMCC.

Preferably, the CpG molecules are subjected to a pretreatment operation of reduction and desalting prior to their reaction with the amine-sulfhydryl crosslinking agent.

Preferably, the preparation method further comprises the step of purifying the immune activating molecule by chromatography, wherein the mobile phase of the chromatography is phosphate buffer solution, and the filler of the chromatography is Superdex 200.

The application also provides application of the immune activating molecule in preparing immune activating agent.

The application also provides an immune activator, which comprises an immunogen and the immune activating molecule.

Preferably, the immunogen is selected from at least one of proteins, carbohydrates or nucleic acids.

Wherein the immunogens may be either mono-epitope or multi-epitope or a combination of different immunogens with multiple epitopes. Depending on the immunogen, a combination of an immune activating molecule with different immunogens may form an immune activator formulation for different indications.

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

(1) The application connects the anti-CD 32 single-chain antibody with the oligodeoxynucleotide (CpG) with the function of activating the immune system of the organism, thereby realizing the effect of activating the immune system rapidly and efficiently;

(2) Compared with the existing CD32 antibody, the anti-CD 32 single-chain antibody adopted by the application has higher affinity, better specificity, better targeting performance and the like, and can target plasma cell-like dendritic cells (pDC cells) to realize specific immune activation;

(3) The tail end of the anti-CD 32 single-chain antibody contains the pepK/E helical peptide, and the assembly of the immune activating molecule and various immunogens can be realized through the pepK/E helical peptide when the anti-CD 32 single-chain antibody is applied, so that the effect of enhancing or expanding indication is achieved;

(4) The application combines CpG molecules of different subtypes, can transmit various signals of immune activation, and can activate signals of humoral immunity and cellular immunity at the same time, thereby playing a role of immune activation better.

Drawings

FIG. 1 is a molecular sieve chromatographic profile of PTIA;

FIG. 2 shows the result of SDS-PAGE electrophoresis of PTIA;

FIG. 3 shows the fit of CpG concentration to absorbance;

FIG. 4 shows the results of the detection of PTIA activity by the U937 fluorescein reporter gene method;

FIG. 5 shows the change in cytokines after co-culture of cells using PTIA and pDC;

FIG. 6 is a graph showing the results of an experiment using PTIA to immunize mice;

fig. 7 shows the results of an assay using PTIA in combination with gastrin antigen.

Detailed Description

In order to make the technical solutions of the present application more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following embodiments are only preferred embodiments of the present application, and the scope of the present application is not limited to the following embodiments, and any modifications, substitutions, and combinations made without departing from the spirit and principles of the present application are included in the scope of the present application.

The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.

Example 1

The present example provides an immune activating molecule PTIA, which is formed by coupling CpG molecules with an anti-CD 32 single chain antibody (named CD32 scFv);

wherein, the amino acid sequence of the anti-CD 32 single-chain antibody is as follows:

MEFGLSWLFLVAILKGVQCEVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGESIYPDDFKGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKSLLHTNGNTYLHWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTAFTLSISRVEAEDVGVFYCMQHLEYPLTFGAGTKLELKGGGSKGPHHHHHHEVSALEKEVSALEKEVSALEKEVSALEKEVSALEKA(SEQ ID NO：3)。

the nucleotide sequence encoding the anti-CD 32 single chain antibody is:

the CpG molecules consist of Class A CpG ODNs, class B CpG ODNs and Class C CpG ODNs, and phosphorothioate modification is carried out among the nucleotide molecules; the specific nucleotide sequences are shown in Table 1;

TABLE 1 CpG nucleotide sequence

The specific preparation method of the immune activating molecule PTIA in the embodiment comprises the following steps:

synthesis of cpg molecules and anti-CD 32 single chain antibodies: the CpG molecules are obtained by adopting a chemical synthesis method, the amounts of the Class A CpG ODNs, the Class B CpG ODNs and the Class C CpG ODNs in the CpG molecules are equal molar ratios, the amounts of the ODNs 1585, the ODNs 2216 and the ODNs 2336 in the C lass A CpG ODNs are equal molar ratios, and the amounts of the ODNs in the Class B CpG ODNs and the Class C CpG ODNs are equal molar ratios;

the nucleotide sequence of the CD32scFv is obtained by a total gene synthesis method (shown as SEQ ID NO: 4), the nucleotide sequence is constructed into a PTT5 carrier, 293T cells are transiently transfected, after cell culture for 2-3 days, cell fermentation supernatant is collected, and the CD32scFv antibody protein is obtained by purification through an affinity column.

CpG dissolution: 27.7mg of CpG was weighed into a 15mL centrifuge tube, 4.82mL of purified water was added, and the mixture was dissolved with stirring to obtain a CpG solution.

3. Trichloroethyl phosphate (TCEP) reduction: 48.2 mu L of TCEP (trichloroethyl phosphate) with the concentration of 0.5M is added into the CpG solution, and the mixture is uniformly mixed and reacted for 30min in a water bath at 25 ℃.

4. Desalting: TCEP was removed using 2 10mL Zeba desalting columns.

Buffer:25mM Phosphate Buffer (PB), pH6.5;

pretreatment: centrifuging at 1000 x g for 2min to remove the storage solution in the desalting column;

balance: adding 5mL of buffer solution into the desalting column, centrifuging for 2min at 1000 x g, repeating for 4 times, discarding the collected liquid, and replacing the centrifuge tube;

desalting: slowly adding the reaction solution into two desalting columns, centrifuging for 2min at 1000 g, mixing the collected liquid, and completing the desalting of the C pG.

Sulfo-SMCC crosslinking: 7.68mg of Sulfo-SMCC (4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt) was weighed into a 15mL centrifuge tube, desalted CpG was added, and dissolved and mixed well. Placed in a microplate thermostatted shaker at 25℃and incubated at 600rpm for 20min.

6. Alcohol precipitation and centrifugation: (1) Aliquoting 9 parts of the crosslinked product from step 5 into centrifuge tubes (about 0.64 mL/tube); then 0.08mL of sodium acetate (NaAc-HAc) (pH 5.6) at a concentration of 3M was added, respectively;

(2) Adding 1.8mL of absolute ethyl alcohol (precooled by ice), uniformly mixing, and carrying out ice bath reaction for 5min;

(3) 16100 Xg, centrifuging at 4deg.C for 30min, and pouring out supernatant;

(4) After washing with 700. Mu.L of 70% ethanol, 16100 Xg was centrifuged at 4℃for 10min, the supernatant was decanted. Repeating the washing operation once;

(5) And (5) airing the sample, and preserving at the temperature of minus 20 ℃ to finally obtain the SMCC-CpG.

7. Crosslinking reaction: (1) Taking 3 SMCC-CpG pipes, absorbing 0.904mL CD32scFv protein (3 mg,3.32 mg/mL), adding into the 1 st SMCC-CpG, adding 0.1mL PB (pH 7.5) buffer solution with the concentration of 0.5M, uniformly mixing, transferring into a 12-pore plate, and reacting at 38 ℃ and 700rpm for 1h to obtain a first-step reaction solution;

(2) Adding the first step reaction solution into a2 nd SMCC-CpG pipe, uniformly mixing, and transferring to a 12-pore plate to react for 1h at 38 ℃ C.and 700rpm to obtain a second step reaction solution;

(3) And adding the reaction solution obtained in the second step into a3 rd SMCC-CpG pipe, uniformly mixing, transferring into a 12-pore plate, and reacting at 38 ℃ and 800rpm for 3 hours to obtain a solution containing the immune activating molecule PTIA.

The solution can be subjected to molecular sieve by SEC chromatographic column to obtain PTIA1-1/PTIA1-2/PTIA2-1/PTIA2-2/PTIA3-1/PTIA3-2, and molecular sieve chromatography map is shown in figure 1. The purity of the solution can be identified by electrophoresis on SDS-PAGE, and the results of the test are shown in FIG. 2, which shows that PTIA samples collected at different time points are more uniform, indicating that PTIA samples have been successfully prepared.

To assess the progress of the reaction, a3 μl aliquot of the reaction solution was also taken after the end of each incubation period, and 1 μl of Tris (hydroxymethyl) aminomethane (Tris) buffer (ph=8) at a concentration of 1M was added and mixed.

PTIA purification:

instrument: an AKTA pure protein purification system;

chromatography column: 16/600;

and (3) filling: superdex 200 (120 mL);

mobile phase: d-phosphate buffer;

flow rate: 1mL/min;

A. pre-treating the column with 0.2M sodium hydroxide (NaOH), washing with purified water to neutrality, and equilibrating at least one column volume with buffer;

B. the solution containing the immune activating molecule PTIA was aspirated by syringe, filtered through 0.22 μm filter into 2mL loop, loaded onto equilibrated Superdex-200SEC column and eluted with buffer.

C. The first two main peaks (designated PTIA pooled sample 1 and PTIA pooled sample 2) were collected by the split tube according to the a280 absorbing component.

PTIA concentration: the collected fractions were concentrated to about 0.5ml using a 30kDa ultrafiltration tube and the relevant fractions were pooled to give PTIA and stored at 4 ℃.

Example 2

Cross-linking ratio (CpG/CD 32 scFv) assay

(1) 38.4mg CpG was weighed into a 15mL centrifuge tube, 6.68mL water for injection was added, and dissolved with stirring. After the impurity is subtracted (about 13.1%) to a concentration of 5mg/mL, the mixture is diluted to 0.5mg/mL with water for injection, and diluted 16, 32 and 64 times respectively, the test results under the condition are shown in Table 2, the fitting results are shown in FIG. 3, and the extinction coefficient at 260nm is 26.336 mL.mg ^-1 ·cm-1。

Table 2 absorbance test results

CpG content mg/mL	Absorbance (Abs 260-1)	Absorbance (Abs 260-2)	Absorbance (Abs 260) mean
				0.031	0.813	0.812	0.8125
0.016	0.413	0.411	0.412
				0.008	0.192	0.193	0.1925

(2) Establishing a standard curve by taking CD32scFv as a standard substance, and measuring the protein content in PTIA by using a BCA method; and then the absorption value of the diluted PTIA at 260nm is measured by an extinction coefficient method, the CpG content in the diluted sample is calculated, and the CpG/CD32 scFv ratio is further calculated, and the result is shown in Table 3.

TABLE 3 CpG/ScFv ratio results

Example 3

Identification of PTIA in vitro Activity

Detection of PTIA Activity by U937 fluorescein reporter Gene method

PTIA activity was first identified using U937 cells containing a reporter gene (luciferase) and overexpressing CD32 molecules and TLR9 (CpG molecular receptor), giving a cell density of 5X 10 ⁵ U937 cells were cultured in 6-well plates at V bottom for 12 hours, and PTIA was added at 1. Mu.g/mL to 100. Mu.g/mL, respectively, at 37℃and 5% CO ₂ The incubation was continued for 24 hours under conditions, followed by addition of substrate fluorescein and shaking on a shaker (200 rpm) for 3 minutes, followed by incubation for 10 minutes, and finally fluorescence values were detected using a multifunctional microplate reader. The results of the experiment are shown in FIG. 4, which shows the trend of increasing fluorescence intensity produced by U937 cells with increasing PTIA concentration.

Cytokine changes after co-culture of PTIA and pDC cells

pDC cells (plasmacytoid dendritic cells) were isolated from human PBMC using a MicroBead kit (available from Miltenyi) following the protocol described, and cultured in a medium containing X-VIVO15 (available from thermo), 2% human serum albumin and 10ng/mL IL2 for 24 hours, followed by 1X 10 ⁵ pDC cells of (1) were plated into 6-well plates, and PTIA molecules were added at 5. Mu.g/mL to 1. Mu.g/mL, respectively, and after 24 hours of stimulation, culture supernatants were collected and were tested for changes in IL-12 cytokines using cytokine ELISA test kits (purchased from Daidae). The results of the experiment are shown in FIG. 5, which shows that the addition of PTIA can inhibit the production of IL-12 cytokine in pDC cells, and that the inhibition effect exhibits a dose-dependency.

Example 4

Immunoassay of PTIA

PTIA immunized mouse test

PTIA samples with aluminum adjuvant (alum) were immunized with 6-8 week old Balb/c mice at the following doses: 30 μg and 150 μg PTIA, the immunization results are shown in FIG. 6, experimental data show: after 2 weeks of immunization, 30 mug and 150 mug of PTIA can stimulate the organism to generate good immune response, and high-titer specific antibodies are generated, so that the PTIA has good immunogenicity, and the immune response initiated along with the increase of the PTIA dosage is higher.

Test for antibody production in PTIA-conjugated gastrin (G17) antigen-immunized mice

Classical antigen G17 aiming at gastrin is selected to be mixed with PTIA to prepare an immune activator (named PTIAG), and the PTIAG added with aluminum adjuvant (alum) is used for immunizing Balb/c mice, wherein the immune doses are respectively as follows: 30 μg and 150 μg PTIAG, immunization results are shown in FIG. 7, experimental data showing: after 2 weeks immunization, both 30 μg and 150 μg PTIAG stimulated the body to generate a significant immune response against the G17 antibody and showed dose-dependent effects of the immune stimulation.

The embodiments of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.

SEQUENCE LISTING

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<213> artificial sequence

<400> 14

tcgtcgtttt cggcgcgcgc cg 22

<210> 15

<211> 25

<212> DNA

<213> artificial sequence

<400> 15

tcgtcgtcgt tcgaacgacg ttgat 25

<210> 16

<211> 29

<212> DNA

<213> artificial sequence

<400> 16

tcgcgaacgt tcgccgcgtt cgaacgcgg 29

Claims

1. An immune activating molecule is characterized in that the immune activating molecule is formed by connecting CpG molecules with an anti-CD 32 single-chain antibody, and the amino acid sequences of a light chain variable region and a heavy chain variable region in the anti-CD 32 single-chain antibody are respectively shown in SEQ ID NO: 1-2.

2. The immune activating molecule of claim 1, wherein the anti-CD 32 single chain antibody has an amino acid sequence as set forth in SEQ ID NO: 3.

3. The immune activating molecule of claim 1, wherein the CpG molecule is coupled to the anti-CD 32 single chain antibody in a manner.

4. The immune activating molecule of claim 1, wherein the CpG molecules consist of Class a CpG ODNs, class B CpG ODNs, and Class C CpG ODNs.

5. The immune activating molecule according to claim 1, wherein the mass ratio of CpG molecules to anti-CD 32 single chain antibodies in the immune activating molecule is (3-10): 1.

6. a method for the preparation of an immune activating molecule according to any one of claims 1 to 5, comprising the steps of:

7. The method of claim 6, wherein the CpG molecules are subjected to a pretreatment operation of reduction and desalting prior to reacting the CpG molecules with the amine-sulfhydryl crosslinking agent.

8. The method of claim 6, further comprising the step of purifying the immunoactive molecule using chromatography, wherein the mobile phase of the chromatography is phosphate buffer, and wherein the filler of the chromatography is Superdex 200.

9. Use of an immune activating molecule according to any one of claims 1 to 5 for the preparation of an immune activator.

10. An immune activator comprising an immunogen and an immune activating molecule of any one of claims 1-5.