CN111166727A - Tumor immunotherapy compound and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of tumor medicaments, in particular to a tumor immunotherapy compound and a preparation method and application thereof. The tumor immunotherapy compound comprises a nano-carrier and CpG encapsulated in the nano-carrier, wherein the nano-carrier mainly consists of HBc-144, and the CpG is C-class CpG. The tumor immunotherapy compound can be used for inhibiting tumor growth and reducing tumor volume, and can be used for preparing antitumor drugs.

Description

Tumor immunotherapy compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of tumor medicaments, in particular to a tumor immunotherapy compound and a preparation method and application thereof.

Background

the CpG ODN is classified into 3 types, A-type CpGODN, containing CG dinuclear in Palindromic sequence, capped at the 5 ' and/or 3 ' end with 3-6 PoGuanoides, capable of activating plasmacytoid dendritic cells to produce large amounts of interferon- α (IFN- α), indirectly activating NK cells, promoting induction of cytotoxic T lymphocytes, B-type CpG ODNPS residues and requiring at least one CpG dinuclear and preferably two or more, which can stimulate proliferation and activation of B cells and directly trigger differentiation of ants, according to their structural and functional characteristics, the CpG ODN residues have the properties of A-type and B-type CpGODN, the CpG dinuclear residues in 5 ' -end costimulatory sequences and the CpG dinuclear sequences "HES, the immunostimulatory properties of the CpG dinuclear DNA sequences which require the development of a single-CpG dinuclear DNA sequences for the treatment of various cancers, the discovery of a single-nucleotide sequence CpG dinuclear DNA sequences, and the discovery of inducing a significant decrease in the stem length of CpG dinuclear DNA sequences, the inhibitory sequence of CpG dinuclear DNA sequences, the induction of the activity of CpG dinuclear DNA sequences, the induction of the high- α.

CPG-ODN are Toll-like receptor 9(TLR9) agonists, and induce inflammatory cytokines and type I Interferon (IFN) via TLR9 to activate humoral or cellular immunity. TLR9 is expressed only in plasmacytoid dendritic cells (pdcs) and B cells, which are able to respond directly to the stimulatory effects of CPG, in humans. CPG developed to date is a single-stranded synthetic ODN with 20 bases. And they are roughly classified into four groups according to their differences in structure and immunoreactivity.

Since CpG ODN can not only promote the production of Thl and proinflammatory cytokines and the maturation and activation of APC, but also enhance the immunogenicity of polypeptide tumor vaccines. It induces a cytokine microenvironment that promotes helper T cell responses, properties that make it useful as an anti-tumor immunopotentiator in clinical studies. However, when CpG ODN is used independently for the treatment of cancer, the intensity of immune response induced by CpG ODN is not sufficient enough to eliminate tumors (Advances in the study of novel immunopotentiators CpG ODN, Xunning, et al, agricultural science of Anhui, 2019,47 (6): 8-10). There are currently some studies aimed at increasing the antitumor effect of CpG ODN by screening for sequences of CpG oddn. For example: chinese patent document CN 109022431A discloses a CPG oligonucleotide, the sequence of the CPG-B type oligonucleotide is TCGTCGTTTGTCGTTTGACGTT, TCGTCGTTTGACGTTTCGTTTG or TGCTGCTTTGAGCTTTGCTTTG, and the research shows that the 3 CPG-B type oligonucleotides can be used for inhibiting the volume of lung cancer; chinese patent document CN 109593765a discloses a CpG oligodeoxynucleotide, which can be used to induce a TLR 9-activated immune response, a TLR 21-activated immune response or a combination thereof in a host, comprising: one or more repeated GTCGTT sequences; one or more repeated GTT sequences; and one or more repeated TTTT sequences, wherein at least one repeated GTCGTT sequence is encoded between a GTT sequence and a TTTT sequence. The CpG oligonucleotide can be used for treating various tumors. Although the above CPG oligonucleotides can be used for treating tumors, the individual ODNs have poor water solubility and stability, are difficult to permeate cell membranes, and are easily cleared by serum and cytoplasmic neutralizing nucleases, which greatly hinders the immunological applications of the ODNs.

Chinese patent document CN 108578694a discloses a CpG oligodeoxynucleotide modified hollow gold nanosphere, which is obtained by shake culturing a mixed solution prepared from but not limited to a hollow gold nanosphere solution, CpG oligodeoxynucleotide, a surfactant, and a buffer solution. The CpG oligodeoxynucleotide modified hollow gold nanospheres can ablate primary tumors by means of near infrared light thermal effect, generate tumor-associated antigens in situ, and activate Dendritic Cells (DC) under the promotion of the CpG oligodeoxynucleotide to increase the stimulation of tumor-specific T cells to promote the regression of metastatic tumors. The preparation process of the CpG oligodeoxynucleotide modified hollow gold nanosphere is complex, and when the CpG oligodeoxynucleotide modified hollow gold nanosphere is used for treating tumors, the near infrared light thermal effect is needed, so that the treatment is inconvenient.

The hepatitis B core protein virus-like particle is a hollow shell structure without virus nucleic acid, and a plurality of virus structural proteins have the capacity of automatically assembling VLPs, are similar to natural virus particles in morphological structure, and have strong immunogenicity and biological activity. Since VLPs do not contain viral genetic material and are therefore not infectious, some of them have been successfully used clinically as vaccines. VLPs structurally allow insertion of foreign genes or gene fragments to form chimeric VLPs and display of foreign antigens on their surfaces.

Hepatitis B virus core antigen (HBcAg) was first used as a foreign antigen virus-like particle vector in 1987. Expression systems for HBcAg expression are diverse, including prokaryotic expression systems, mammalian cell expression systems, transgenic plant expression systems, yeast expression systems, insect cell expression systems, and the like, and can achieve higher yields and form natural granular structures. Hbcags produced by expression in e.coli expression systems are predominantly of two particle sizes, one comprising 180 subunits (T ═ 3, T ═ 4) and the other 240 subunits (T ═ 4), with part of the particles encapsulating e.coli nucleic acids. Each particle-forming subunit is a dimer formed from HBcAg monomers, constituting spikes on the outside of the particle. Two HBcAg monomers form a subunit through disulfide bonds, each HBcAg monomer having 4 Cys residues including Cys48, Cys61, Cys107, Cys 183. Wherein Cys107 is encapsulated within the granule; the Cys48 part is involved in the formation of inter-monomer disulfide bonds, and the remaining residues are free on the particle surface; cys61 and Cys183 are all involved in the formation of disulfide bonds between monomers or dimers, and have the same binding pattern as wild-type HBcAg. For a full length of 183 amino acids of HBcAg, the first 144 amino acids belong to the particle assembly region, with major functions associated with viral particle formation. And the 145-183 amino acids belong to nucleic acid binding regions rich in arginine, have three repeated SPRRR structures and mainly have the functions of binding virus nucleic acid and wrapping the virus nucleic acid into virus particles to protect the virus nucleic acid and provide replication sites. Between 78-82 amino acids in the HBcAg amino acid sequence is a Main Immune Region (MIR), and the Region is presented on the top of a spike on the surface of a particle; amino acids 127-133 form a small spike next to the main spike, which are the major B cell recognition sites on the HBcAg surface. The MIR region has the advantage of being easily bound to the receptor and not affecting the natural conformation of the exogenous fragment, due to its distribution on the top of the spikes on the surface of the particle. However, for the N-terminal and C-terminal of the HBcAg monomer, the insertion fragment needs to be considered in terms of whether the fragment is present on the surface of the particle, whether the assembly of the particle structure is affected, and whether the insertion fragment is capable of maintaining the native conformation. The insertion of a small fragment sequence after 144 amino acids at the C-terminus does not affect particle self-assembly, but the size of the insert has a large impact on whether the insert is correctly folded and can be presented on the particle surface. The N-terminal insertion of foreign sequences is difficult to maintain the correct conformation of the sequences and to present on the surface of the particles, so that there is a strict limit to the size of the inserted fragment. The possibility of presenting the N-terminal insert on the surface of the particle is enhanced by using a particle with a more loosely constructed truncated form, i.e., the first 144 amino acids.

Currently, when hepatitis b virus-like particles are used for treating tumors, hepatitis b virus-like particles are generally used as carriers of antitumor drugs or by inserting antigens, antibodies or corresponding nucleotide sequences of tumors between hepatitis b virus-like particles, and virus-like particle vaccines are prepared to achieve the purpose of treating tumors. For example, chinese patent document CN 105497886 a discloses a technical scheme of using hepatitis b core antigen virus-like particles as a carrier of a tumor therapeutic vaccine, and the document discloses a method for preparing a vaccine by using HPV16E7_49-57CTLs epitope peptide segment is inserted between 78-79 amino acids of hepatitis B virus core antigen through gene recombination to obtain recombinant plasmid pHBcAg-E7_49-57transformed into Escherichia coli DH5 α, induced expression and purification to obtain presentation E7_49-57H of (A) to (B)The bcAg virus-like particle vaccine can induce an organism to generate stronger HPV16E7 specific cellular immune response after a tumor-bearing mouse is immunized by the virus-like particle vaccine, and obviously inhibit the growth of tumors. However, in the case of the N-terminal and C-terminal of the HBcAg monomer, the three problems of whether the insert is present on the surface of the particle, whether the assembly of the particle structure is affected, and whether the insert is capable of maintaining the native conformation are required to be considered, so that in practical application, specific consideration needs to be taken in combination with the insert to be inserted, and the application difficulty is high.

Disclosure of Invention

The inventor creatively prepares the tumor immunotherapy compound by using the truncated hepatitis B core antigen HBc-144 as a carrier of C-class CpG, and the immunotherapy compound can achieve the purpose of treating tumors under the condition of not adopting antitumor drugs, tumor antigens or antibodies.

The tumor targeted immunotherapy compound adopts the following technical scheme: a tumor-targeted immunotherapeutic complex comprising a nanocarrier and a CpG encapsulated in the nanocarrier, the nanocarrier consisting essentially of HBc-144, the CpG being a C-class CpG.

Preferably, amino acids 78-82 of HBc-144 are substituted with the targeting peptide.

Preferably, both ends of the targeting peptide are respectively connected with the 77 th amino acid and the 83 th amino acid of the HBc-144 through connecting peptides, the amino acid sequence of the targeting peptide is RGD, and the amino acid sequence of the connecting peptides is GTSGSSGSGSGGSGSGGGG.

Preferably, the nanocarrier further comprises a pH sensitive polypeptide linked to HBc-144, wherein the pH sensitive polypeptide is a polyhistidine polypeptide consisting of 5-15 histidines.

Preferably, the C class CpG includes, but is not limited to, any one or a combination of several of the following:

D-SL03, the sequence is: tcgcgaacgttcgccgcgttcgaacgcgg, respectively;

D-SL02, the sequence is: tcgcgtcgttcgcccgtcgttcggta, respectively;

D-SL01, the sequence is: tcgcgacgttcgcccgacgttcggta, respectively;

and the sequence of the GC-ODN is tgcccaagcttgccccccttgcaagcgcgg.

Preferably, the CpG is a thio or non-thio CpG.

The second objective of the present invention is to provide a method for preparing the tumor immunotherapy complex, which comprises the following steps: the method comprises the following steps: (1) expressing and purifying the nano-carrier by a genetic engineering method; (2) loading the CpG to the nano-carrier obtained by the step (1) through a urea depolymerization method or a heat shock method.

Preferably, the urea depolymerization process comprises the steps of: (1) depolymerization: incubating the purified nanocarrier with a dissociation solution at 4 ℃; (2) and (3) recombination: transferring the mixed solution obtained by the treatment in the step (1) into a dialysis bag with the molecular weight of 8000-14000 Da, and sequentially placing the dialysis bag into a recombinant buffer solution 1 and a recombinant buffer solution 2 for dialysis; (3) adding dissociation liquid into the solution obtained by the treatment of the step (2), incubating for 2.5h at 4 ℃, adding CpG, continuing to culture for 30min under the shaking condition, and then transferring into a dialysis bag with the molecular weight of 8000-14000 Da; (4) putting the dialysis bag into the recombinant buffer solution 1 and the recombinant buffer solution 2 in sequence for dialysis, and obtaining the tumor immunotherapy compound after the dialysis is finished; the dissociation solution is urea with pH8.0 and containing 50mM Tris-HCl, 150mM NaCl and 8.0M; the reconstitution buffer 1 was pH8.0 containing 50mM Tris-HCl, 150mM NaCl, 10% glycerol, and 1% glycine; the reconstitution buffer 2 was a pH8.0 solution containing 50mM Tris-HCl, 150mM NaCl, and 1% glycine.

Preferably, the heat shock method comprises the following specific steps: (1) mixing the nano-carrier and CpG in a ratio of 2: 1; (2) heating the mixture obtained in the step (1) at 60-80 ℃ for 70-100 min; (3) naturally cooling the mixture obtained in the step (2) to room temperature; (4) filling the mixture obtained in the step (3) into a dialysis bag, and dialyzing the bag in a dialysate to remove the unencapsulated free CpG.

Preferably, the nanocarrier and the CpG in step (1) are mixed in a ratio of 2: 1; in the step (2), the heating temperature is 70 ℃, the heating time is 90min, and the heating mode is water bath, oil bath or sand bath; and (3) the dialysis bag in the step (3) is a dialysis bag with the interception amount of 3.5kDa, and the dialysate is PBS or normal saline.

The invention also provides an application of the tumor immunotherapy compound, and the specific technical scheme is as follows: the tumor immunotherapy compound is applied to the preparation of a medicine for treating breast cancer.

The invention has the beneficial effects that: the tumor immunotherapy compound can achieve the purposes of inhibiting the growth of tumor cells and reducing the tumor volume by activating in-vivo immune response.

The invention selects HBc-144 as the carrier of CpG ODN, and the HBc-144 is protein, is easy to biodegrade in vivo and has good safety. The particle size of the HBc-144 nano-carrier is 30-40nm, and the nano-carrier particles are stable in the range and have a tumor enrichment effect.

According to the invention, the targeting peptide is inserted into the main immune region of the nano-carrier HBc-144, so that HBc-144 has targeting property and the treatment effect is improved.

The RGD is selected as a targeting peptide, and the two ends of the RGD are respectively connected with connecting peptides, so that the connecting peptides at the two ends are respectively connected with 77 th amino acid and 83 th amino acid of HBc-144, and the tumor immunotherapy compound can target tumor cells expressed by high integrin receptors, and further target CpGODN to the surfaces of the tumor cells. The HBc-144 nano-carrier can target a plurality of tumor cells with high integrin receptor expression, such as melanoma, colon cancer, breast cancer, lung cancer, prostate cancer, brain cancer or head and neck cancer.

The invention can encapsulate CpGODN in nano carrier HBc-144 by adopting heat shock method, and the preparation method is simple.

Compared with the pure C-class CpG, the tumor immunotherapy compound has better performance of inhibiting the growth of the tumor.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a SDS-PAGE gel electrophoresis of the nanocarriers of the invention;

FIG. 2 is a morphology of the purified nanocarrier of example one observed by an electron microscope;

FIG. 3 is a graph showing IFN-. alpha.curves obtained by treating human PBMCs with HBc-VLPs/D-SL03 and D-SL03 CpGODNs according to example.

FIG. 4 is a graph showing the results of the immunostimulatory activity of D-SL03 CpGODNs on lymphocytes from different animals as measured in example three;

FIG. 5 is a photograph of targeted fluorescence imaging of the tumor immunotherapy complexes of the invention measured in example four;

FIG. 6 is a graph showing the results of the anti-breast cancer effect of the tumor immunotherapy complex of the present invention measured in example five.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows: preparation of nanocarriers

1. Constructing the amino acid sequence of the nano-carrier: HBc-144 (amino acid sequence isMDIDHYKEFG ASVELLSFLPSDFFPSIRDL LDTASALYRE ALESPEHCSP HHTALRQAIL CWGELMNLAT WVGSNLEDPASRELVVGYVN VNMGLKIRQI LWFHISCLTF GRETVLEYLV SFGVWIRTPP AYRPPNAPILSTLP) The amino acids from 78 th to 82 th (namely DPASR) of the nano-carrier are replaced by a targeting peptide RGD and a connecting peptide (GTSGSSGSGSGGSGSGGGG) connected to both sides of the targeting peptide RGD, namely the amino acid sequence of the nano-carrier isMDIDHYKEFGASVELLSFLP SDFFPSIRDL LDTASALYRE ALESPEHCSP HHTALRQAIL CWGELMNLATW VGSNLEGTSGSSGSGSGGSGSGGGGRGDGGGGSGSGGSGSGSSGSTGELVVGYVNVNMGLKIRQI LWFHISCLTF GRETVLEYLV SFGVWIRTPP AYRPPNAPIL STLP

2. On the basis of the nano-carrier, HBc-144 can be connected with a pH sensitive polypeptide (polyhistidine polypeptide), and the amino acid sequence of the HBc-144 isMDIDHYKEFG ASVELLSFLP SDFFPSIRDL LDTASALYRE ALESPEHCSPHHTALRQA ILCWGELMNLATWVGSNLEGTSGSSGSGSGGSGSGGGGRGDGGGGSGSGGSGSGSSGSTGELVVGYVN VNMGLKIRQI LWFHISCLTF GRETVLEYLV SFGVWIRTPP AYRPPNAPIL STLPHHHHHHHH; wherein HHHHHHHHH can be replaced by HHHHH, HHHHHHHHHHHHH, HHHHHHHHHHHHHHH (selected from any polyhistidine polypeptide with 5-15H connected) etc.

3. The amino acid sequences designed in (1) and (2) above were supplied to jireri biotechnology limited, shanghai, where nucleotides encoding the amino acid sequences were synthesized based on the supplied amino acid sequences, and the synthesized nucleotide sequences were constructed into expression vector plasmid pet43.1(a) via XhoI/Nde I cleavage sites for future use.

4. Transforming the expression vector plasmid pEt43.1(a) into escherichia coli BL21(DE3) to express a target protein (nano-vector), and separating and purifying to obtain the target protein (nano-vector), wherein the method comprises the following specific steps:

4.1 transformation of plasmids:

4.1.1. 100 μ l of competent cells were thawed on an ice bath.

4.1.2. Add 2. mu.l of pure plasmid or ligation product (which can be done by dipping the tip of the pipette) and blow gently and mix them well, ice-wash for 30 min.

4.1.3. The tube was placed in a 42 deg.C (thermometer temperature) water bath and heat-shocked for 90 seconds

4.1.4. Immediately placed in an ice bath for 2min (all above).

4.1.5. Add 800ul LB liquid medium (without antibiotics) gently mixed, at 37 degrees C constant temperature shaking bed 200rpm temperature in 45 min. Table, wiping and coating rod

4.1.6. Centrifuging the bacterial solution at 4000rpm/min for 5min, collecting 300 μ l of supernatant, gently scattering thallus, spreading 50 μ l, 100 μ l and 150 μ l of bacterial solution on the surface of agar plate containing antibiotic (1:1000, AMP),

4.1.7. the plates were cultured overnight (12-16 h) in an inverted format at 37 ℃.

4.2 inducible expression of the protein of interest (nanocarrier): (1) BL21(DE3) single colonies containing the expression vector were picked and cultured in LB medium (containing 100. mu.g/mL ampicillin) at 37 ℃ and 255rpm to log phase;

(2) diluting the bacterial liquid and the culture medium according to the proportion of 1:1000, culturing overnight at 37 ℃ and 255rpm to enable the OD600 value to reach 0.5-0.6; diluting the strain to fresh culture medium at a ratio of 1:1, and culturing at 37 deg.C and 255rpm for 2 h;

(3) inducing at 37 ℃ with a final concentration of 1mM IPTG;

(4) after 4 hours of induction, 1mL of the bacterial suspension was taken out, centrifuged at 12,000rpm for 1min, the supernatant was removed (the supernatant was collected and subjected to SDS-PAGE gel electrophoresis as a sample, 2 samples were taken, see the band labeled "supernatant" in FIG. 1), 100. mu.L of PBS was added to resuspend the cells, and the samples (the resuspension was collected and subjected to SDS-PAGE gel electrophoresis as a sample, 2 samples were taken, see the band labeled "precipitate" in FIG. 1) were analyzed by 12% SDS-PAGE gel electrophoresis.

The specific method of SDS-PAGE gel electrophoresis analysis is as follows (the detailed electrophoresis chart is shown in FIG. 1):

(1) mu.L of the collected solution was added with 5. mu.L of 5 Xsample buffer solution and boiled in a water bath at 80 ℃ for 5 min. SDS-PAGE gels were prepared according to the recipe, the electrophoresis apparatus was mounted and the electrophoresis buffer was added to the cell.

(2) Adding samples into the sample adding holes according to a certain sequence, and selecting the adding amount of the samples according to the difference of the gap thickness of the SDS-PAGE electrophoresis glass plate, wherein the sample adding amount of 15 holes with the gap of 0.75mm is generally less than 10 mu L/hole, and the sample adding amount of 10 holes with the gap of 1mm is less than 20 mu L/hole. The loading for this study was 15 μ L per well.

(3) The power supply is turned on to adjust the voltage to 80v (generally about 15 min), and after the sample runs through the compressed gel, the voltage is adjusted to 120v until the sample is electrophoresed to the bottom of the gel.

(4) Carefully taking down the gel, placing into a container, adding staining solution, and shaking with a shaker for 2-3 h

(5) Pouring out the staining solution after the band is clear, adding the destaining solution overnight

(6) The destaining solution was decanted and the imaging system photographed, analyzed, and protein concentration estimated.

Remarking: (1) collecting liquid which is a sample for gel electrophoresis;

(2) the 2 bands identified with the supernatant in FIG. 1 are the same samples; the 2 bands identified in FIG. 1 with the precipitate are also identical samples. As can be seen from FIG. 1, the target protein (nanocarrier) obtained in example one is mainly present in the supernatant, and the protein concentration is between 17 kD and 25 kD.

4.3 ammonium sulfate precipitation of primary pure protein of interest (nanocarrier): (1) centrifuging the induced bacterial liquid at 4000rpm for 10min, collecting thalli, and blowing the thalli by using 10mM Tris and 0.5% Triton pH8.0 buffer solution;

(2) crushing thallus by ultrasonication method, performing ultrasonication at power of 300W for 30min to obtain thick and semitransparent thallus, centrifuging at 12000rpm for 10min, and collecting supernatant (soluble protein in the supernatant);

(3) the supernatant was dispensed into 1.5ml centrifuge tubes, 500. mu.L each, and protein salting-out was performed with 10%, 20%, 30%, 40%, 50% saturated ammonium sulfate solutions, respectively, and the precipitates were collected by salting-out at 4 ℃ for 30min, centrifuged at 12,000rpm for 10min, and the precipitate samples at each concentration were subjected to SDS-PAGE to detect the distribution of fusion proteins.

4.4 DEAE ion exchange chromatography purification

(1) Column equilibration with base solution: the column was washed with 10 column volumes of a base solution containing 10mM Tris-Cl (pH8.0) until the effluent pH was consistent with the base solution pH.

(2) Loading: the collected sample, i.e., the crude protein extract after dialysis by ammonium sulfate precipitation, was loaded at a flow rate of 3 mL/min.

(3) Loading and eluting: the column was washed with 5 column volumes of base solution until the UV detector reading returned to baseline.

(4) And (3) eluting by eluent I: the column was washed with 5 column volumes of eluent I (containing 10mM Tris-Cl (pH8.0), 50mM NaCl) and the eluted peak was collected.

(5) And (3) eluting with an eluent II: the column was washed with 5 column volumes of eluent II (containing 10mM Tris-Cl (pH8.0), 100mM NaCl) and the eluted peak was collected.

(6) And (3) eluting with eluent III: the column was washed with 5 column volumes of eluent III (containing 10mM Tris-Cl (pH8.0), 200mM NaCl) and the eluted peak was collected.

(7) And (3) eluting with eluent III: the column was washed with 5 column volumes of eluent IV (containing 10mM Tris-Cl (pH8.0), 300mM NaCl) to collect the eluted peak.

(8) And (3) eluting with eluent III: the column was washed with 5 column volumes of eluent IV (containing 10mM Tris-Cl (pH8.0), 400mM NaCl) to collect the peak.

4.5 protein dialysis and concentration

(1) Putting the dialysis bag containing the protein solution into the precooled dialysate, and dialyzing in a refrigerator at 4 ℃;

(2) the dialysis solution is changed for 4 times in the whole dialysis process until the dialysis is complete;

(3) putting the completely dialyzed protein solution into a new big beaker, adding polyethylene glycol powder to cover the dialysis bag, quickly sucking out the solvent in the bag by polyethylene glycol when the solvent seeps out, and putting the beaker into a refrigerator at 4 ℃ for concentration;

(4) during the concentration process, adding a small amount of dried polyethylene glycol powder for multiple times to increase the concentration speed, and replacing the saturated polyethylene glycol with new polyethylene glycol until the required concentration volume is reached;

(5) and after the concentration is finished, washing the dialysis bag by using distilled water, and taking out the concentrated protein solution to obtain the purified target protein (nano carrier). The protein concentration can be quantitatively determined by BCA method to be 0.5mg/ml as required, and the obtained morphological structure of the target protein can be observed by electron microscope (see figure 2 in the attached figure of the specification)

EXAMPLE two drug Loading (including two methods)

1. Urea depolymerization process

(1) Depolymerization: taking the target protein prepared in the example 1, and incubating the target protein and the dissociation solution for 3h at 4 ℃;

(2) and (3) recombination: transferring the mixed solution obtained by the treatment in the step (1) into a dialysis bag with the molecular weight of 8000-14000 Da, placing the dialysis bag into 100mL of recombinant buffer solution 1, dialyzing the dialysis bag overnight at 4 ℃, and then placing the dialysis bag into recombinant buffer solution 2 for dialysis. The dialysis time was 48h in total (total time of dialysis in recombinant buffer 1 and recombinant buffer 2). The molecular weight of the prepared protein monomer is verified by SDS-PAGE electrophoresis.

(3) CpG loading: 1mL of the target protein (0.5mg/mL) obtained after the recombination in the step (2) and a prepared dissociation solution are incubated for 2.5h at 4 ℃, and the total volume of the solution is 10 mL. At this time, 500mg of CPG (C group) was added to the above dissociation solution, and the resulting mixed solution was further co-cultured for 30min with gentle shaking. Then, the membrane was transferred to a dialysis bag with a molecular weight of 8000-14000 Da, and the membrane was first placed in a recombinant buffer solution 1 and dialyzed overnight at 4 ℃ before the dialysis bag was placed in a recombinant buffer solution 2 for dialysis. The dialysis time is totally 48h (the total dialysis time in the recombinant buffer solution 1 and the recombinant buffer solution 2), the recombinant buffer solution 2 is replaced once after dialysis is carried out for 12h in the recombinant buffer solution 2, the tumor targeted immunotherapy compound is obtained, and the obtained tumor targeted immunotherapy compound is stored at the temperature of minus 20 ℃ for later use.

The dissociation solution, the recombinant buffer solution 1 and the recombinant buffer solution 2 comprise the following specific components:

preparing dissociation liquid: the pH of the dissociation solution was 8.0, and the dissociation solution contained 50mM Tris-HCl, 150mM NaCl and 8.0M urea (the concentrations of the above components were all final concentrations);

recombinant buffer 1: recombinant buffer 1, pH8.0, containing 50mM Tris-HCl pH8.0, 150mM NaCl, 10% glycerol and 1% glycine (the concentrations of the above components are final concentrations);

recombinant buffer 2: recombinant buffer 1, pH8.0, contains 50mM Tris-HCl pH8.0, 150mM NaCl and 1% glycine (the concentrations of the above components are final concentrations).

2. Thermal shock method

heating ② mixing heating ②, heating ② mixing heating ② the heating ② prepared heating ② nano heating ② -heating ② carrier heating ② (heating ② 0.5 heating ② mg heating ②/heating ② ml heating ②) heating ② and heating ② CpG heating ② (heating ② 250 heating ② mu heating ② g heating ②/heating ② ml heating ②, heating ② C heating ② class heating ②) heating ② according heating ② to heating ② the heating ② mass heating ② ratio heating ② of heating ② 2 heating ②: heating ② 1 heating ②, heating ② heating heating ② the heating ② mixture heating ② at heating ② 70 heating ② ℃ heating ② for heating ② 90 heating ② min heating ②, heating ② cooling heating ②, heating ② standing heating ② at heating ② room heating ② temperature heating ② for heating ② 10 heating ② -heating ② 30 heating ② min heating ②, heating ② cooling heating ② to heating ② room heating ② temperature heating ②, heating ② dialyzing heating ②, heating ② filling heating ② the heating ② mixture heating ② into heating ② a heating ② dialysis heating ② bag heating ② with heating ② the heating ② cut heating ② -heating ② off heating ② of heating ② 3.5 heating ② kD heating ②, heating ② putting heating ② the heating ② dialysis heating ② bag heating ② into heating ② PBS heating ②, heating ② removing heating ② unencapsulated heating ② free heating ② drugs heating ② to heating ② obtain heating ② the heating ② tumor heating ② targeted heating ② immunotherapy heating ② compound heating ②, heating ② and heating ② storing heating ② at heating ② -heating ② 20 heating ② ℃ heating ② for heating ② later heating ② use heating ②. heating ②

EXAMPLE two Interferon- α detection

Human PBMC (peripheral blood mononuclear cells) (6X 10 per well) were treated with D-SL03 CpG ODNs (3. mu.g/ml) and HBc-VLPs/D-SL03 (3. mu.g/ml, prepared by heat shock method), respectively⁵/ml)36h, taking the supernatant to measure IFN- α, performing immunohistochemical detection by using an ELISA kit, and detecting interferon- α in the supernatant, wherein the detected IFN- α is shown in figure 3.

Example three: immunostimulatory Activity of D-SL03 on lymphocytes from different animals

To verify breed specificity, spleen cell proliferation capacity of mouse (mouse), rat (rat), rabbit, and pig PBMC was evaluated using D-SL03 and HBc-VLPs/D-SL03, respectively. D-SL03 (3. mu.g/ml) and HBc-VLPs/D-SL03 (3. mu.g/ml) were injected at the same dose. As shown in FIG. 4, it is understood from FIG. 4 that both D-SL03 and HBc-VLPs/D-SL03 induce the vigorous proliferation of lymphocytes of mice, rats, rabbits, pigs, etc. Interestingly, D-SL03 and HBc-VLPs/D-SL03 consistently induced a response of strong rabbit splenocytes and porcine PBMCs. D-SL03 and HBc-VLPs/D-SL03 had effects on lymphocytes from various animals (FIG. 4).

Example four: targeting of the tumor targeting immunotherapeutic complexes of the invention

(1) Preparation of Cy 5-labeled nanocarriers

Cy5 dissolved in a small amount of DMSO was mixed with the nanocarrier to be labeled at a molar ratio of 1.5:1, and after reacting at room temperature for 30min, the nanocarrier was dialyzed in PBS buffer solution at pH 7.4 for 24h to remove the excess Cy5, thereby obtaining Cy 5-labeled nanocarrier.

(2) And (3) CPG encapsulation: mixing the nano-carrier (0.5mg/ml) prepared in the first example with CpG (250 mu g/ml, C class CPG) according to the mass ratio of 2: 1; heating the mixture at 70 deg.C for 90min, and naturally cooling to room temperature; the mixture was filled into dialysis bags with a cut-off of 3.5kD, dialyzed against PBS solution to remove unencapsulated CpG, yielding a tumor-targeted immunotherapeutic complex labeled with Cy 5.

(3) The tumor targeted immunotherapy compound labeled with Cy5 prepared in the step (2) above was injected into tumor-bearing mice (mouse breast cancer tumor EMT6) at 200 μ L, these mice were anesthetized with 7% chloral hydrate, and the fluorescence intensity of the tumor and the whole body was monitored by a small animal living body imager at 12h and 24h after administration. After 24h, the cervical vertebrae were removed and the mice were sacrificed, and the main organs, tissues and tumors were collected, and fluorescence imaging pictures were taken to measure the fluorescence intensity (fig. 5).

Example five: anti-breast cancer effect of tumor immunotherapy compound

In order to confirm the immunostimulation effect of the tumor targeting immunotherapy compound in vivo, an antitumor activity test was performed on the mouse breast cancer cell line EMT6 cells. HBc-VLPs/CpG (experimental group) and intratumoral injection are injected from 8 days after tumor challenge, and are injected for 1 time every other day and 6 times in total. Animals were monitored for survival 100 days after tumor inoculation. Wherein, the HBc-VLPs/CpG (experimental group) is divided into two groups, one group is injected with HBc-VLPs/CpG (D-SL02), and the other group is injected with HBc-VLPs/CpG (D-SL03), and the experimental result is detailed in figure 6 of the attached figure of the specification.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> research center of bioengineering technology in Henan province

<120> tumor immunotherapy compound and preparation method and application thereof

<130>AJ192658

<140>2019111209579

<141>2019-11-15

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<213> Artificial Sequence (Artificial Sequence)

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tcgcgtcgtt cgcccgtcgt tcggta 26

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tgcccaagct tgcccccctt gcaagcgcgg 30

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Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

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Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala

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Ser Arg Glu Leu Val Val Gly Tyr Val Asn Val Asn Met Gly Leu Lys

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100 105 110

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

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Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

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Gly Thr Ser Gly Ser Ser Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly

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Gly Gly Gly

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Met Asp Ile Asp His Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu

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Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp

20 25 30

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35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

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Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Gly Thr Ser

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Gly Ser Ser Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Gly Gly

85 90 95

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180

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Gly Ser Ser Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Gly Gly

85 90 95

Arg Gly Asp Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly

100 105 110

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115 120 125

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Ser Thr Leu Pro His His His His His His

180 185

Claims (10)

1. A tumor-targeted immunotherapy complex, which comprises a nano-carrier and CpG encapsulated in the nano-carrier, wherein the nano-carrier mainly comprises HBc-144, and the CpG is C-class CpG.

2. The tumor immunotherapeutic complex of claim 1, wherein amino acids 78 to 82 of HBc-144 are substituted with the targeting peptide.

3. The tumor immunotherapy complex according to claim 2, wherein both ends of the targeting peptide are connected to amino acid 77 and amino acid 83 of HBc-144 via a linking peptide, respectively, the amino acid sequence of the targeting peptide is RGD, and the amino acid sequence of the linking peptide is GTSGSSGSGSGGSGSGGGG.

4. The tumor immunotherapeutic complex of claim 1, wherein the nanocarrier further comprises a pH sensitive polypeptide linked to HBc-144, the pH sensitive polypeptide being a polyhistidine polypeptide consisting of 5-15 histidines.

5. The tumor immunotherapeutic complex of any one of claims 1 to 4, wherein the C class CpG includes, but is not limited to, any one or a combination of:

D-SL03, the sequence is: tcgcgaacgttcgccgcgttcgaacgcgg, respectively;

D-SL02, the sequence is: tcgcgtcgttcgcccgtcgttcggta, respectively;

D-SL01, the sequence is: tcgcgacgttcgcccgacgttcggta, respectively;

and the sequence of the GC-ODN is tgcccaagcttgccccccttgcaagcgcgg.

6. The tumor immunotherapeutic complex of claim 1, wherein the CpG is a thio or non-thio CpG.

7. The method of preparing a tumor immunotherapeutic complex according to any one of claims 1 to 6, comprising the steps of: (1) expressing and purifying the nano-carrier by a genetic engineering method; (2) loading the CpG to the nano-carrier obtained by the step (1) through a urea depolymerization method or a heat shock method.

8. The method for preparing a tumor immunotherapeutic complex according to claim 7, wherein the heat shock method comprises the specific steps of: (1) mixing the nano-carrier and CpG in a ratio of 2: 1; (2) heating the mixture obtained in the step (1) at 60-80 ℃ for 70-100 min; (3) naturally cooling the mixture obtained in the step (2) to room temperature; (4) filling the mixture obtained in the step (3) into a dialysis bag, and dialyzing the bag in a dialysate to remove the unencapsulated free CpG.

9. The method for preparing tumor immunotherapeutic complexes according to claim 8, wherein the nanocarriers and the CpG are mixed in a ratio of 2:1 in the step (1); in the step (2), the heating temperature is 70 ℃, the heating time is 90min, and the heating mode is water bath, oil bath or sand bath; and (3) the dialysis bag in the step (3) is a dialysis bag with the interception amount of 3.5kDa, and the dialysate is PBS or normal saline.

10. Use of a tumor immunotherapeutic complex according to any one of claims 1 to 6 in the preparation of a medicament for the treatment of breast cancer.

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