CN116549660A - Azobenzene-mediated hypoxia-responsive antibody cross-linked material and composition containing same - Google Patents
Azobenzene-mediated hypoxia-responsive antibody cross-linked material and composition containing same Download PDFInfo
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- CN116549660A CN116549660A CN202310406476.4A CN202310406476A CN116549660A CN 116549660 A CN116549660 A CN 116549660A CN 202310406476 A CN202310406476 A CN 202310406476A CN 116549660 A CN116549660 A CN 116549660A
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Classifications
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- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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Abstract
The invention discloses an azobenzene-mediated hypoxia response type antibody crosslinking product and a composition containing the same, and the azobenzene-mediated hypoxia response type antibody crosslinking product is called H103-azo-PEG for short 5000 Wherein H103 is shorthand for H103 monoclonal antibody; the amino acid sequence of the heavy chain variable region of the H103 monoclonal antibody is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the H103 monoclonal antibody is shown as SEQ ID NO. 2. The antibody cross-linked product of the invention can effectively prolong the half-life of the antibody, improve the bioavailability, and in addition, can effectively reduce the nonspecific cross-binding of the antibody with the antigen of non-tumor cells, thereby eliminating the cross-off-target effect of the antibody, reducing the toxic and side effects to the greatest extent, andand the clinical treatment effect is synergistically improved. The composition containing the azobenzene-mediated hypoxia response type antibody cross-linked compound has the advantage that the curative effect is fully verified in a liver cancer-bearing mouse model. Is beneficial to developing a series of safer and more effective similar medicaments and promotes the application of the medicaments to clinical practice of accurate tumor treatment.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to an azobenzene-mediated hypoxia response type antibody cross-linked compound, a composition containing the cross-linked compound and application thereof.
Background
Worldwide, the incidence of tumors has risen year by year, and due to the difficulty of early diagnosis, some patients have progressed to late stages at the time of diagnosis, with very limited treatment options. Therapeutic antibody drugs are one of the more effective methods for treating tumors at present, and the antibody drugs regulate signal transduction by combining antigens on tumor cells, so that the tumor cells die or mobilize the immune system of a patient, and the immune cells are utilized to kill the tumor cells with high efficiency, thereby preventing disease progression and playing a therapeutic role.
The antibody drug targeted therapy is used clinically, and toxic and side effects brought by the antibody drug targeted therapy are alert. For antibody drugs that exert therapeutic effects by binding to solid tumor cell membrane antigens, their target antigens are expressed not only on tumor cells, but also on normal cells of various healthy tissues. This results in the inability to accurately recognize the target antigen in the tumor area upon systemic administration of the antibody drug, which is cross-linked to the normal tissue target antigen. Thus, various toxic side effects occur, which limit the maximum tolerated dose and therapeutic window for clinical application of antibody drugs. Therefore, there is an urgent need to design new tumor targeted therapeutic strategies and accurate antibody drugs for cancer treatment. The core of solving the problem is how to enhance the accurate targeting of the therapeutic antibody drug and simultaneously minimize the toxic and side effects.
Research at home and abroad shows that the tumor cells have metabolic changes different from normal cells, so that the microenvironment where the tumor cells are located is obviously changed. Meanwhile, the interaction of tumor cells and tumor microenvironment depends on each other, and some characteristics of the tumor microenvironment can promote the malignant transformation and progress of tumors. According to the characteristics of tumor tissue microenvironment, the design of more accurate targeted and safe antibody drugs is the leading trend in the field of current biomacromolecule drugs, and also meets the urgent requirements of actual clinical treatment. The tumor tissue microenvironment is obviously different from the main characteristics of normal tissues, including the characteristics of hypoxia, subacidity, high expression of certain proteases, immunosuppression and the like. Such unique tumor microenvironment characteristics may be exploited to trigger specific stimulus-responsive chemical responses. Therefore, by utilizing the specific microenvironment characteristics of tumor tissues, the antibody drug is highly selectively targeted to tumor cells without affecting surrounding normal cells, and is a safe and efficient treatment strategy.
Due to rapid proliferation of tumor cells, incomplete vascular development and disturbed distribution, oxygen is insufficient inside tumor tissues, and a hypoxic region exists widely in tumors. Hypoxia is a common feature of solid tumors and is a key physiological feature that distinguishes tumor tissue from normal tissue. Hypoxia not only can cause the tumor to resist radiotherapy, but also can increase the drug resistance of tumor chemotherapy and malignant invasiveness. The azobenzene aromatic derivative mainly comprises an azo bond (-N=N-) connected with a benzene ring structure. Because of its own special molecular structure, it has received extensive attention in the fields of chemical and biological basic research and multifunctional material design and application. Under hypoxic conditions, azo bonds are more prone to reduction to aniline structures, i.e., azo bonds are more easily broken in hypoxic environments. From a biological perspective, the oxygen-dependent behavior of azobenzene reduction provides a new idea for developing an accurate diagnosis probe and a targeted therapeutic drug aiming at the solid hypoxic tumor.
The current clinical methods for treating solid tumors include radiotherapy, surgical treatment, chemotherapy, various biological drug treatments including immunotherapy, and the like. Different treatment strategies are suitable for different links of tumorigenesis progress. Antitumor drugs with different mechanisms of action are combined or applied in combination, so that the actual curative effect of the drugs can be enhanced and toxic and side effects can be reduced. The combined use of therapeutic antibody drugs and small molecule chemotherapeutic drugs is a mainstream trend in the field of tumor targeted therapy in recent years. However, the combined application of antibody drugs and small molecule drugs for the hypoxic tumor, especially the combined application of the hypoxic response type antibody drugs and the hypoxia activated small molecule prodrugs has not been reported in the literature before.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide an azobenzene-mediated hypoxia response type antibody cross-linked product.
The second object of the present invention is to provide a method for producing the azobenzene-mediated hypoxia responsive antibody cross-linked material.
The third object of the invention is to provide the application of the azobenzene-mediated hypoxia response type antibody cross-linked compound in preparing medicaments for accurately diagnosing and targeting tumor treatment.
It is a fourth object of the present invention to provide a composition comprising the azobenzene-mediated hypoxia responsive antibody conjugate described above.
It is a fifth object of the present invention to provide the use of the above composition for the preparation of a medicament for targeted treatment of tumors.
The technical scheme of the invention is summarized as follows:
azobenzene-mediated hypoxia response type antibody cross-linked product, H103-azo-PEG for short 5000 Wherein H103 is shorthand for H103 monoclonal antibody; the amino acid sequence of the heavy chain variable region of the H103 monoclonal antibody is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the H103 monoclonal antibody is shown as SEQ ID NO. 2.
The preparation method of the azobenzene-mediated hypoxia response type antibody cross-linked material comprises the following steps: h103 mab was combined with PEG in carbonate buffer at ph=9.0 5000 Mixing azo-NHS uniformly, fixing in a shaking table for reaction, and centrifugally ultrafiltering the reaction product to obtain a purified azobenzene-mediated hypoxia response type antibody cross-linked product; the PEG 5000 The structure of the-azo-NHS is shown in formula 7:
the azobenzene-mediated hypoxia response type antibody cross-linked compound is applied to preparation of drugs for accurate diagnosis and targeted treatment of tumors.
Compositions comprising azobenzene-mediated hypoxia responsive antibody cross-links, including H103-azo-PEG 5000 And a hypoxia activated small molecule prodrug TH302.
Preferably, the H103-azo-PEG 5000 And the mass ratio of the hypoxia activated small molecule prodrug TH302 is 1:10.
the application of the composition in preparing a drug for targeted treatment of tumors.
H103 mab heavy chain variable region amino acid sequence (SEQ ID No. 1):
QVQLQESGPGLVRPSGTLSLICAVSGDSISSSIWWSWVRQSPGKGLEWIGYIYHNGNTYYNPSLESRVTISVDTSE NQFSLKLSSVTAADTAVYYCARGYDSSGYYWTDDRYYFDYWGQGTLVTVSS
h103 mab light chain variable region amino acid sequence (SEQ ID No. 2):
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPPGVPDRFSGSIDSSSNSASL TISALETEDEADYYCQSYDSRNIDVVFGGGTKVTVLGQ
the invention has the advantages that:
the azobenzene-mediated hypoxia response type antibody cross-linked compound can effectively prolong the half life of an antibody and improve the bioavailability, and can effectively reduce the nonspecific cross-binding of the antibody with antigens of non-tumor cells, so that the cross-off-target effect of the antibody is eliminated, the toxic and side effects are reduced to the greatest extent, and the clinical treatment effect is synergistically improved. In addition, the antibody modification scheme provided by the invention can be also used for other medicinal proteins and active small molecule drugs, such as other cytokines, antibody derivatives, polysaccharides and nucleic acid macromolecular drugs, and can be subjected to similar modification and combined treatment.
The composition containing the azobenzene-mediated hypoxia response type antibody cross-linked compound has the advantage that the curative effect is fully verified in a liver cancer-bearing mouse model. Is beneficial to developing a series of safer and more effective similar medicaments and promotes the application of the medicaments to clinical practice of accurate tumor treatment.
Drawings
FIG. 1 is a design of azobenzene mediated hypoxia responsive antibody cross-links;
FIG. 2 is a graph showing the measurement of the crosslinking rate of azobenzene mediated hypoxia responsive antibody crosslinks;
FIG. 3 is an SDS-PAGE electrophoretic detection of azobenzene mediated hypoxia responsive antibody cross-links;
FIG. 4 is an ELISA assay for azobenzene mediated hypoxia responsive antibody cross-link binding antigen PKM 2;
FIG. 5 is a Biacore analysis of azobenzene mediated hypoxia responsive antibody cross-link affinity assay;
FIG. 6 is a confocal laser microscopy analysis of azobenzene mediated hypoxia responsive antibody cross-link binding to liver cancer cells;
FIG. 7 is a flow cytometry assay for azobenzene mediated hypoxia responsive antibody cross-link binding assay to liver cancer cells;
FIG. 8 is a CCK-8 experimental analysis of azobenzene mediated hypoxia responsive antibody cross-link killing of liver cancer cells;
FIG. 9 is an azobenzene mediated hypoxia responsive antibody cross-link mediated Antibody Dependent Cellular Cytotoxicity (ADCC) assay;
FIG. 10 is an azobenzene mediated hypoxia responsive antibody cross-link mediated Complement Dependent Cytotoxicity (CDC) analysis;
FIG. 11 is an azobenzene mediated hypoxia responsive antibody cross-link binding assay for human liver cancer tissue and paracancel tissue;
FIG. 12 is an in vivo imaging and single treatment effect analysis of azobenzene mediated hypoxia responsive antibody conjugate and TH302 small molecule prodrug in nude mice model of liver cancer;
FIG. 13 is a graph showing the continuous therapeutic effect of azobenzene-mediated hypoxia responsive antibody conjugate and TH302 small molecule prodrug in liver cancer bearing nude mice;
FIG. 14 is an analysis of toxicity of azobenzene mediated hypoxia responsive antibody cross-links and/or TH302 to tumor or normal tissue organ after treatment;
FIG. 15 Compound 2 1 H NMR spectrum (delta, ppm, cdcl3,400 mhz);
FIG. 16 Compound 5 1 H NMR spectrum (delta, p)pm,DMSO,400MHz)；
FIG. 17 Compound 6 1 H NMR spectrum (delta, ppm, cdcl3,400 mhz);
mass spectrometry identification of compound 6 of fig. 18;
FIG. 19 end product Compound 7 (PEG 5000 -azo-NHS);
FIG. 20 pair of products of Compound 7 (PEG 5000 -azo-NHS) for continuous HPLC preparative analysis.
Detailed Description
Preferred embodiments of the present invention will be described below with reference to examples. The description is only intended to further illustrate the features and advantages of the invention and is not intended to limit the claims of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Hypoxia activated small molecule prodrug TH302 (Ai Fulin amide, evofosfamide, commercially available)
Example 1
PEG 5000 Azobenzene-N-hydroxysuccinimide ester (PEG) 5000 -azo-NHS), comprising the steps of:
1)PEG 5000 adding TsCl:
under nitrogen protection, 300 ml of dichloromethane and 30 g of mPEG are added into a 1000 ml four-port bottle under magnetic stirring 5000 1, cooling to 5 ℃ by using an external ice salt bath, adding an aqueous solution of sodium hydroxide (25.5 g of sodium hydroxide solid is added into 60 ml of water), controlling the temperature to 5 ℃, dropwise adding a dichloromethane solution of p-toluenesulfonyl chloride (5.45 g of p-toluenesulfonyl chloride is dissolved in 180 ml of dichloromethane) for about 2 hours, carrying out heat preservation reaction for 5 hours at 5 ℃ after the completion of the addition, monitoring the disappearance of raw materials by TLC (MeOH/DCM=1:10), adding 240 ml of dichloromethane and 60 ml of saturated sodium chloride aqueous solution into the reaction solution, layering, washing an organic phase with 150X 2 ml of saturated sodium chloride, drying by using anhydrous sodium sulfate, carrying out suction filtration and spin-drying to 42 g of oily matter, adding 180 ml of ethyl acetate, carrying out suction filtration after the mixture is placed in a refrigerator for 30 minutes, and obtaining 32 g of white solidCompound 2 (i.e. PEG 5000 P-toluenesulfonate, for characterization see figure 15);
2) Diazotizing to synthesize azo compound:
under the protection of nitrogen, 30 ml of water and 1.85 g of p-aminobenzyl alcohol 3 are added into a 250 ml four-port bottle under the magnetic stirring, 3.2 ml of concentrated hydrochloric acid is added into the four-port bottle at the temperature of 5 ℃ of an ice salt bath, the temperature is controlled to be 5 ℃, an aqueous solution of sodium nitrite (1.1 g of sodium nitrite is dissolved in 7.5 ml of water) is dropwise added, the temperature is kept for 1 hour after the addition, an aqueous solution of sodium carbonate of phenol 4 (1.5 g of phenol is dissolved in 24 ml of aqueous solution of 10% sodium carbonate) is dropwise added, the temperature is naturally raised to room temperature after the addition is completed, the room temperature is stirred for 3 hours, after TLC monitors the disappearance of raw materials, the pH=7 is regulated by 5% hydrochloric acid, and the phenol is pumped and filtered, and washed by water to obtain 2.3 g of brown solid compound 5, namely 4- ((4- (hydroxymethyl) phenyl) azobenzene) phenol, and the characteristics of the phenol are shown in fig. 16;
3)PEG 5000 and (3) receiving an azo compound:
under the protection of nitrogen, 300 ml of DMF (dimethylformamide) and 30 g of compound 2,1.5 g of compound 5,5.2 g of potassium carbonate are added into a 1000 ml four-port bottle under the magnetic stirring condition, the reaction is carried out for 5 hours at 80 ℃, TLC monitors the disappearance of raw materials, the reaction liquid is poured into 300 ml of saturated ammonium chloride, 300 ml of dichloromethane is added, the layers are separated, the aqueous phase is extracted by 150 ml of x 2 dichloromethane, dichloromethane phases are combined, 150 ml of x 2 saturated ammonium chloride is used for washing, anhydrous sodium sulfate is used for drying, 35 g of brown oily matter is obtained by suction filtration and spin-drying, 100 ml of ethyl acetate is added, and after the mixture is put into a refrigerator for 30 minutes, 24 g of yellow solid compound 6, namely PEG is obtained by suction filtration 5000 Azobenzene-benzyl alcohol, for characterization see fig. 17, 18);
4) Compound 6 (10 g, 2 mmol) was dissolved in 200 ml dry toluene and the solvent was removed under reduced pressure. This process was repeated three times to remove moisture traces in compound 6; redissolved in dry DCM (dichloromethane) (300 ml); n, N' -disuccinylcarbonate (DSC, 1.53 g, 0.6 mmol) was added followed by triethylamine (TEA, 405 mg, 6 mmol). The mixture was stirred at room temperature overnight. The crude product was purified using a pre-equilibrated PD10 column to give compound 7 as a yellow solid. I.e. PEG 5000 Azobenzene-N-hydroxysuccinimide ester, abbreviated as PEG 5000 -azo-NHS; see fig. 19 and 20 for their characterization).
The synthetic route is as follows:
example 2
A preparation method of an azobenzene-mediated hypoxia response type antibody cross-linked material comprises the following steps:
h103 mab was diluted to 1mg/mL with carbonate buffer ph= 9.0,0.1M, 200ul was taken and 200ul15.6mg PEG was added 5000 azo-NHS (i.e. H103 mab with PEG 5000 The molar ratio of azo to NHS is 1:50, at this time PEG 5000 -azo-NHS excess), fixing in a shaker, reacting at 100rpm for 12h, placing the obtained product into an ultrafiltration centrifuge tube, centrifuging and ultrafiltering at 4000 Xg centrifugal force using a swing-barrel rotor for 20min, and filtering to remove unreacted PEG 5000 azo-NHS, and re-suspending with 200ul cold PBS buffer to obtain purified azobenzene-mediated hypoxia responsive antibody cross-linked Product (PEG) 5000 The ester in-azo-NHS reacts with the free amino group in H103 monoclonal antibody to form an amide bond, namely H103-azo-PEG 5000 Or H103-A-P; stored at 4℃for further use. See fig. 1.
H103 mab consists of a heavy chain amino acid sequence and a light chain amino acid sequence; the amino acid sequence of the heavy chain variable region of the H103 monoclonal antibody is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the H103 monoclonal antibody is shown as SEQ ID NO. 2.
H103 mab heavy chain variable region amino acid sequence (SEQ ID No. 1):
QVQLQESGPGLVRPSGTLSLICAVSGDSISSSIWWSWVRQSPGKGLEWIGYIYHNGNTYYNPSLESRVTISVDTSE NQFSLKLSSVTAADTAVYYCARGYDSSGYYWTDDRYYFDYWGQGTLVTVSS
h103 mab light chain variable region amino acid sequence (SEQ ID No. 2):
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPPGVPDRFSGSIDSSSNSASL TISALETEDEADYYCQSYDSRNIDVVFGGGTKVTVLGQ
h103 mab is composed of heavy and light chains, which are screened from a repertoire of fully human phage antibodies and cloned. The light and heavy chain variable region genes are reconstructed into heavy chain variable region amino acid sequences shown in SEQ ID NO.1 and light chain variable region amino acid sequences shown in SEQ ID NO.2, and can be directly used for accurate diagnosis and targeted treatment of liver tumors. In addition, the H103 monoclonal antibody is used as a carrier, and is crosslinked with cytotoxic drugs, toxins, radionuclides, enzymes, biological response modifiers and the like to be used as guiding drugs, and can also be used for diagnosing and treating hypoxia-related diseases including but not limited to liver tumors.
By Na 2 S 2 O 4 In vitro simulated hypoxia activation is carried out on H103-A-P in the following specific modes:
dissolving and diluting Na with carbonate buffer having pH= 9.0,0.1M 2 S 2 O 4 To a concentration of 200mM, freshly prepared 100. Mu. LNa was taken 2 S 2 O 4 The solution was mixed with 100. Mu.L of a 4℃stock H103-A-P solution and suspended with a vertical suspension at 4℃for 6 hours. After the incubation, the reaction system was placed in an ultrafiltration centrifuge tube, and centrifuged and ultrafiltered at 4000 Xg for 20 minutes using a swing-barrel rotor to remove unreacted Na by filtration 2 S 2 O 4 Then washing the membrane with cold PBS buffer solution, re-suspending and recovering to obtain the product Na 2 S 2 O 4 Simulating hypoxia reduction activated H103-A-P. And (5) storing at 4 ℃.
Example 3
The crosslinking rate was measured by the 2,4, 6-trinitrobenzenesulfonic acid (TNBSA) method:
free amino group in H103 monoclonal antibody combined with PEG 5000 Will cause a decrease in the free amino content and thus the determination of PEG by measuring the free amino groups remaining in the H103-A-P antibody 5000 Cross-linking conditions of (a). The crosslinking rate is defined as the ratio of PEG in the antibody molecule 5000 The percentage of cross-linked amino groups over all amino groups is: (crosslinking NH) 2 Number of groups/NH in antibody 2 Total number of groups) x 100%. The specific operation of this embodiment is as follows:
H103-A-P and Na 2 S 2 O 4 H103-A-P respectively simulating hypoxia reduction activationDiluted to 200 μg/mL with sodium bicarbonate buffer ph=8.5. The 5-amino-1-pentanol standard was diluted in a gradient starting from 20 μg/mL with sodium bicarbonate buffer at ph=8.5 for preparing the standard curve. 5% (w/v) TNBSA was diluted to 0.1% with sodium bicarbonate buffer. 100. Mu.L of diluted H103-A-P and Na were taken 2 S 2 O 4 The simulated hypoxia reduction activated H103-A-P was mixed with 100. Mu.L of 5-amino-1-pentanol dilutions at different dilution gradients, added to 96-well plates, followed by 50. Mu.L of 0.1% TNBSA solution, respectively, and mixed well, three wells per group. The 96-well plates were then placed in a 37 ℃ oven for 2 hours of incubation. The 96-well plate was removed, and 50. Mu.L of 10% (w/v) SDS solution and 25. Mu.L of 1mol/L aqueous HCl solution were added to each well to terminate the reaction. UV absorbance at wavelength 335nm was measured using a microplate reader, and finally the antibodies (H103-A-P and Na) were calculated using a calibration curve for the 5-amino-1-pentanol standard 2 S 2 O 4 The concentration of primary amine in the solution of H103-A-P) activated by hypoxia reduction is simulated, and the crosslinking rate can be calculated.
See FIG. 2A, which shows a standard curve obtained by measuring 5-amino-1-pentanol standards at different concentrations. From H103-A-P and Na 2 S 2 O 4 The results of simulating the oxygen deficiency reduction activated H103-A-P absorbance and standard curve calculation to obtain the crosslinking rate of the two are shown in figure 2B. The crosslinking rate of H103-A-P is 75.259% which indicates that the H103 monoclonal antibody and azobenzene PEG 5000 Successfully cross-link with Na 2 S 2 O 4 The decrease in crosslinking rate of H103-A-P activated by simulated hypoxia reduction to 9.830% suggests that H103-A-P can revert to H103 mab under simulated hypoxia reduction conditions.
Example 4
SDS-PAGE electrophoresis identifies H103-A-P:
to further verify the prepared H103-A-P, SDS-PAGE electrophoresis was used to verify the H103 monoclonal antibody cross-linked PEG 5000 -azo-NHS before and after and Na-passing 2 S 2 O 4 The molecular weight change of H103-A-P after hypoxia activation was simulated.
H103, H103-A-P and Na 2 S 2 O 4 Simulated hypoxia activated H103-A-P and 5 Xreduction protein loading buffer and 5 Xnon-reduction eggThe white loading buffer was mixed and denatured by placing in a metal bath at 100deg.C for 10 min. Preparing 12% separating gel and 5% concentrating gel, performing electrophoresis at 80V until bromophenol blue just runs out. After electrophoresis, the gel plate is taken down, the glass plate is carefully pried off, the gel is peeled off into a coomassie brilliant blue staining solution dye dish, the staining solution can be ensured to fully cover the gel, the gel is placed on a horizontal shaking table to shake slowly for 45min, the staining solution is poured out, after rinsing with water for several times, the gel is decolorized in a horizontal shaking table in a decolorizing solution for 20min, the decolorizing solution is poured out, and a new decolorizing solution is added until protein strips are clear, and the background is transparent and colorless.
The results of SDS-PAGE are shown in FIG. 3, and the increase in molecular weight of H103-A-P compared to H103 mab demonstrates successful production of H103-A-P.
In addition, na 2 S 2 O 4 H103-A-P, which mimics hypoxia reduction activation, was restored to parental antibody levels.
Example 5
Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of H103-A-P antibody crosslinks:
PKM2 antigen (commercial) was diluted to 10 μg/mL with carbonate buffer at ph= 9.6,0.1M, coated onto 96-well elisa plate at 50 μl per well, 3 duplicate wells per treatment, tapped with hand to homogenize, fixed with disposable glove bags around the rubber band, and incubated overnight at 4 ℃. The wells were discarded and the wells were gently rinsed 3 times with PBST buffer for 3min each. 50 μLPBS/5% BSA was added to each well, blocked at 37℃for 1h, and the wells were gently rinsed 3 times with PBST buffer for 3min each. H103, H103-A-P and Na were buffered with PBS/0.1% BSA 2 S 2 O 4 Three antibodies H103-A-P after simulated hypoxia activation were added to wells at the set dilution, 50. Mu.L per well, incubated for 1H at 37℃and wells were rinsed 3 times with PBST for 3min each. HRP-labeled goat anti-mouse IgG (h+l) secondary antibody (1:2000 dilution) was added at 50 μl per well and after incubation for 1H at 37 ℃ the wells were washed 3 times with PBST for 3min each time. TMB substrate chromogenic solution 50. Mu.L was added to each well in sequence and incubated at 37℃for 30min until a visible color gradient was obtained. The reaction was terminated by adding 50. Mu.L of 2M sulfuric acid to each reaction well. On a microplate reader at a wavelength of 450nmThe absorbance value of each well was measured after zeroing the blank wells. The obtained data were processed and analyzed by GraphPad Prism 8.
The ELISA results are shown in FIG. 4, wherein the affinity of H103-A-P is significantly reduced compared to that of H103 mab, and the affinity is restored to the level of H103 mab after in vitro activation of H103-A-P.
Example 6
Biacore analysis of affinity of H103-A-P
The experiment adopts a method of Protein A chip capture method to fix the antibody in an indirect way, fixes the antibody Protein on the chip surface, takes PKM2 antigen as a mobile phase, uses the Protein A chip to detect the kinetics ka, KD and affinity data KD of the combination of the antibody and the antigen, and analyzes H103, H103-A-P and Na 2 S 2 O 4 The difference of affinities of H103-A-P after hypoxia activation to a target PKM2 antigen is simulated, and the interaction with the PKM2 antigen is further known, and the specific method is as follows:
h103, H103-A-P and Na were buffered with PBS-P (pH 7.4) 2 S 2 O 4 The three H103-A-P antibodies after simulated hypoxia activation are diluted to 19.8 mu M, and Protein A chip pre-enrichment crosslinking is carried out on Biacore T200. PKM2 antigen proteins were diluted in PBS-P buffer at concentrations of 1000nM, 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.6nM, 7.8nM, and blank wells were set up without PKM2 antigen protein, i.e., at a concentration of 0 nM. The contact time was set at 120s, the flow rate was 20. Mu.L/min, and the dissociation time was 300s. After each cycle, the chip was regenerated with PBS-P buffer as EXTRA WASH buffer at a flow rate of 20. Mu.L/min for 30 seconds to clear the tubing of residual small molecules. The five concentrations of 125nM, 62.5nM, 31.25nM, 15.6nM and 7.8nM were finally selected and the obtained data were processed and analyzed by GraphPad Prism 8.
See FIG. 5, A, B, C for H103, H103-A-P and Na, respectively 2 S 2 O 4 The affinity assay curves for H103-A-P after hypoxia activation were simulated. Consistent with the ELISA results, this example also demonstrates that H103-A-P has significantly reduced affinity for the target antigen PKM2, and can recover its affinity after hypoxia reduction.
Example 7
Verification of binding of H103-A-P to cells by normoxic and hypoxic conditions: confocal laser microscopy observed binding of antibodies (H103, H103-A-P, with IgG as negative control) to HepG2 cells (purchased from Wohpnuosai Life technologies Co., ltd., website, https:// www.procell.com.cn) under normoxic or hypoxic conditions.
A 24-well plate was used for the climbing plate. Placing 1 round cover glass in each well, seeding pancreatin digested HepG2 cells in each well, adding DMEM complete culture solution, culturing overnight, waiting for cell adhesion until complete expansion, and placing in normoxic (37deg.C, 5% CO) respectively 2 ) And hypoxia (37 c,<0.1％O 2 ) Culturing for 12h in an incubator environment. The medium was aspirated and washed 3 times with pre-chilled PBS, 200. Mu.L of 4% paraformaldehyde was added to each well, and after 20min fixation at room temperature, the slides were aspirated and washed 3 times with PBS for 3min each. 0.05% saponin was allowed to pass through for 10min, and the slides were washed 3 times with PBS for 3min each. Next, 200. Mu.L of blocking agent [ PBS (pH 7.4)/10% (V/V) FBS was added to each well]Blocking for 1h, then 200. Mu.L of antibody diluted with PBS (pH 7.4)/10% (V/V) FBS was added dropwise to each well cover slide, covered, and the incubation in incubator with normoxic or hypoxic environment was continued for one hour. Carefully aspirate the liquid and add PBST to wash 3 times for 3min each. rhodamine-X anti-human secondary antibody was incubated for 1 hour. Hochest 33342 (1:2000 dilution) was incubated at room temperature in the dark for 5min for staining nuclei, carefully blotted with liquid, and rinsed 3 times with PBST for 3min each. The slide was marked and a drop of anti-fluorescence quencher was placed on top of the slide for sealing, the coverslip was removed from the 24-well plate, excess liquid was aspirated off the edge of the wipe, and the cells were then facing down and placed into the sealing drop. Removing the sealing tablet on the cover glass, coating nail polish on the edge of the cover glass for sealing the edge of the cover glass, preserving in a dark place at 4 ℃, drying, placing a sample under a confocal microscope for observation, photographing and collecting an image, and processing and analyzing the obtained image by using GIMP software.
The binding of different antibodies to HepG2 cells is shown in FIG. 6, and H103-A-P does not bind to HepG2 cells under normoxic conditions, but rather H103-A-P binds to HepG2 cells under hypoxic conditions, as compared to H103.
Example 8
Flow cytometry determines the binding of H103-A-P to cells under normoxic and hypoxic conditions:
advanced for 24 hours, 5×10 5 Huh-7 (9 months in 2020, available from the Living technologies Co., ltd., withannomunosai CL-0120, website, https:// www.procell.com.cn) and HepG2 cells were each seeded in 24 well plates with 1mL of complete culture medium per well. Cells were gently washed twice with PBS, 100. Mu.L of PBS/EDTA was added to each well and placed in an incubator for digestion for 10 minutes until gaps appeared between cells were detected under the microscope, 200. Mu.L of DMEM complete medium was immediately added to each well after removal to terminate digestion, cells were collected into a 1.5mL centrifuge tube, centrifuged at 800rpm for 5 minutes, resuspended by careful pipetting with PBS, centrifuged at 800rpm for 5 minutes again, the supernatant was carefully discarded, 1mL of PBS/0.5% BSA was added to each tube, and centrifuged after shaking for 2 minutes, the supernatant was discarded. 500 mu L H103 and H103-A-P, na were added to the corresponding tubes of the experimental group respectively 2 S 2 O 4 Incubation of H103-A-P after hypoxia activation with human IgG (control) was simulated, and the control was supplemented with the same volume of PBS. The resuspension was performed with sufficient shaking and gentle shaking at 4℃for 1.5h (< 100 rpm) to allow sufficient binding. Centrifuge at 800rpm for 5min and discard supernatant. The centrifugation was repeated 1 time with 500. Mu.L PBS/0.5% BSA per tube. Antibody treatment group plus rhodamine-X anti-human secondary antibody (diluted in PBS/0.5% BSA), control group plus the same volume of PBS,4 ℃ light shaking 1h to fully bind (light shielding), 800rpm centrifugation for 5min, discarding supernatant, repeating centrifugation and washing 2 times as above, finally re-suspending cells with 500 muLPBS-0.5% BSA. Analysis was performed on a flow cytometer (FACS), sheath fluid was checked, pressurized on start-up, software was turned on, software was set, and the instrument tubing was thoroughly washed. FSC, SSC channel voltage was adjusted using the control group. The FL2-H channel is switched to a data collection mode by taking rhodamine as an abscissa, samples of the experimental group are detected, and data analysis is performed by using FlowJo software.
The binding of different antibodies to Huh-7 and HepG2 cells is shown in FIG. 7A, B, H103-A-P cannot bind to the surface of liver cancer cells under normoxic conditions, and the binding activity to cells can be recovered under hypoxic conditions.
Example 9
CCK-8 assay to determine cell viability:
to examine the effect of antibodies on cancer cell cytotoxicity, cell number assays were performed by CCK-8 experiments using both Huh-7 and HepG2 cell lines.
For cell culture, the cells were cultured in an incubator (normoxic (37 ℃,5% co) 2 ) And hypoxia (37 c,<0.1％O 2 ) Cells are cultured. When the cell growth density reaches 80% -90%, carefully sucking the culture medium in the culture dish by using a pipette, slowly adding PBS along the side wall of the culture dish and gently shaking the culture dish, and then discarding the PBS to clean the residual serum in the culture medium. mu.L of pancreatin was pipetted into the petri dish and gently shaken to cover the cell surface, followed by digestion for 3min in an incubator. The dish was removed, the outer wall of the dish was gently tapped, and the digestion of the cells was observed under a microscope. The digestion was stopped by adding 4mL of complete medium, the pellet was gently blown into individual cells by a pipette, cell counts were performed, and the cell suspension was diluted to 5X 10 4 100 μl of cell suspension (about 5000 cells/well) was seeded per well in 96-well plates, three wells per group. Plates were pre-incubated in an incubator for 24 hours. H103, H103-A-P and Na were added to the wells at final concentrations of 0.001. Mu.g/mL, 0.01. Mu.g/mL, 0.1. Mu.g/mL, 1. Mu.g/mL, respectively 2 S 2 O 4 Antibody dilutions of H103-A-P after hypoxia activation were simulated while IgG was used as antibody control and blank wells (wells containing medium and CCK-8) and control wells (wells containing cells, medium and CCK-8) were set. The plates were incubated in an incubator for 48 hours. To each well of the plate, 10. Mu. LCCK-8 solution was added (no air bubbles were introduced into the wells) and the plate was incubated in an incubator for 4 hours. Before reading the plates, mix gently on a shaker and then measure absorbance at 450nm using a microplate reader. The obtained data were processed and analyzed by GraphPad Prism 8. Cell viability (%) = [ (As-Ab)/(Ac-Ab)]X 100, where as=experimental well absorbance (absorbance of wells containing cells, medium, CCK-8 and test compound), ab=blank well absorbance (absorbance of wells containing medium and CCK-8), ac=control well absorbance (absorbance of wells containing cells, medium and CCK-8)
Huh-The results of CCK-8 for both 7 and HepG2 cells are shown in FIG. 8A, B, C, D, H103 and Na under normoxic conditions compared to H103-A-P 2 S 2 O 4 The H103-A-P after simulated hypoxia activation can generate stronger cell killing effect, so that the cell activity is obviously reduced.
Example 10
Experiments to verify antibody-dependent cell-mediated cytotoxicity (ADCC) effect of H103-a-P production:
cytotoxicity assays were performed using Lactate Dehydrogenase (LDH) activity assay kits, with LDH release as an indicator. Collecting venous blood of a liver cancer patient by adopting a sterile sampling method, collecting the venous blood in a centrifuge tube containing heparin, transferring 10mL into a 50mL centrifuge tube, adding 10mL of PBS (phosphate buffered saline) with pH of 7.4 for dilution, gently mixing, taking two 15mL centrifuge tubes, and adding 5mL of human peripheral blood lymphocyte separation liquid (Ficoll). Then the diluted blood was gently added to the Ficoll upper layer of two separate tubes, 10mL each tube was gently handled to avoid mixing the two solutions together, centrifuged at 2000rpm for 20min (no break in the deceleration setting), and after centrifugation, the layers were formed, the upper layer was serum (for example 11), and the cell layer where peripheral blood lymphocytes were white. At this time, the cells were pipetted into another clean 15mL centrifuge tube, PBS was added to 15mL, thoroughly mixed, centrifuged at 1500rpm for 10min, the supernatant was removed, RPMI 1640 complete medium was added for the same washing, 10mL complete medium was added to resuspend the cells, and inoculated into a T25 flask at 37℃in 5% CO 2 The cell incubator is ready for use. After the cell state is stable, a part is taken for the experiment. ADCC effect was then detected by LDH method. Taking HepG2 and Huh-7 cells in logarithmic growth phase as target cells, digesting with pancreatin, collecting in a 1.5mL centrifuge tube, centrifuging, discarding supernatant, washing with PBS for 2 times, removing phenol red in pancreatin, re-suspending in RPMI 1640 complete culture solution without phenol red, counting cells, and adjusting cell concentration to 1 x 10 5 mu.L/mL, added to 96-well plates, and 100. Mu.L/well. Cell-free medium wells (background blank control), control cell wells without drug treatment (sample control), cell wells without drug treatment for subsequent lysis (sample maximum enzyme activity control), and drugsThe treated cells were plated (experimental group) with 3 duplicate wells in each case. H103, H103-A-P, na with Medium 2 S 2 O 4 H103-A-P and human IgG after simulated hypoxia activation were diluted in respective fold ratios to give 5 concentrations (10. Mu.g/mL, 1. Mu.g/mL, 0.1. Mu.g/mL, 0.01. Mu.g/mL and 0.001. Mu.g/mL) of 100. Mu.L each added to the well plate. The cultured peripheral blood lymphocytes were digested with pancreatin, and the complete medium was adjusted to a concentration of 5X 10 6 mu.L of each well was added per mL. 37 ℃,5% CO 2 Culturing for 5h. Centrifuging the 96-well plate at 800rpm for 5min, sucking out supernatant, diluting LDH release agent in kit (lactate dehydrogenase (LDH) activity detection kit) with PBS by 10 times, adding 150 μl of diluted LDH release agent per well, and shaking culture plate appropriately. Incubation was then continued for 1 hour in the cell incubator, after which the cell culture plates were centrifuged for 5min at 800rpm with a centrifuge. The supernatants from each well were taken 120 μl into a new 96-well plate, respectively, and prepared for assay. Adding 60 mu L of LDH detection working solution into each well, uniformly mixing, incubating for 30min at room temperature in a dark place, slowly shaking the well plate on a horizontal shaking table, and measuring the absorbance value, cytotoxicity or death rate (%) = (absorbance of treated sample-absorbance of sample control well)/(absorbance of maximum enzyme activity of cell-absorbance of sample control well) ×100 at 490nm
The results of ADCC effect experiments are shown in FIG. 9A, B, and H103-A-P cannot exert its ADCC effect in normoxic conditions, so that the cell killing effect on liver cancer cells is significantly reduced. Through Na 2 S 2 O 4 After simulated hypoxia reduction activation, H103-A-P can restore the ADCC effect on liver cancer cells, and the killing effect on the cells is obviously enhanced.
Example 11
Complement Dependent Cytotoxicity (CDC) experiments to verify H103-a-P production:
collecting HepG2 and Huh-7 target cells in logarithmic growth phase, washing with PBS, digesting with pancreatin for 3min, adding DMEM complete culture solution, stopping digestion, blowing, resuspension, centrifuging at 800rpm for 5min, discarding supernatant, resuspension cells in serum-free RPMI 1640 complete culture medium, counting, and adjusting cell density to 5×10 4 /mL, target cells are addedInto 96-well plates, 100. Mu.L (5000) per well, cell incubator (normoxic 37 ℃,5% CO 2 ) Culturing for 24H, diluting fresh human serum (serum obtained in example 10) with serum-free RPMI 1640 complete medium, and concentrating H103, H103-A-P, na with this serum 2 S 2 O 4 The simulated hypoxia activated H103-A-P and human IgG were diluted in respective ratios to give 5 concentrations (10. Mu.g/mL, 1. Mu.g/mL, 0.1. Mu.g/mL, 0.01. Mu.g/mL and 0.001. Mu.g/mL), and added to the corresponding wells after thoroughly mixing, 100. Mu.L per well, gently mixed, and the well plates were placed in an incubator for incubation for 4H. Cytotoxicity assays were performed using LDH activity assay kits according to the instructions.
The results of the CDC effect experiment are shown in FIG. 10A, B, in which H103-A-P almost loses CDC effect on liver cancer cells under normoxic conditions, na 2 S 2 O 4 The simulated hypoxia reduction activation can restore the CDC effect.
Example 12
Binding of H103-A-P to liver cancer tissue and paracancerous tissue in clinical patients:
immunohistochemical assay for H103, H103-A-P, and Na 2 S 2 O 4 The combination of H103-A-P after hypoxia activation to human liver cancer tissues is simulated.
In a fume hood, the prepared paraffin sections of human liver cancer tumor tissue were sequentially placed in xylene I for 10min, xylene II for 10min, absolute ethanol I for 5min, absolute ethanol II for 5min,95% ethanol for 10min,75% ethanol for 10min, and dd water for 1min, and then soaked in PBS at a speed of 100rpm on a shaker to dewax. The prepared 1 x antigen retrieval liquid (pH=6.0) is heated by a microwave oven with high fire until the antigen retrieval liquid is boiled, then the antigen retrieval liquid is put into a tissue slice, the tissue slice is thawed by the microwave oven, taken out and cooled to room temperature after 20min, washed by PBS for 5min,3 times and 100rpm, and the antigen retrieval is completed. The tissue was circled out with an immunohistochemical pen, placed on a wet box, and incubated at room temperature for 10min to remove endogenous peroxidase by dropping endogenous peroxidase blocked until the surface of the tissue was completely covered, washed 5min with PBS for 3 times at 100rpm. Taking out the tissue slice, spin-drying, wiping the liquid around the tissue with paper towel, placing in a wet box, and dripping normal goat serum working solution for sealing until the surface of the tissue is completely coveredThe tissue was closed for 1h. H103, H103-A-P, na 2 S 2 O 4 The simulated hypoxia-activated H103-A-P and human IgG were diluted with 5% goat serum. The tissue sections were spun dry, the liquid surrounding the tissue was wiped dry with a paper towel, and placed in a wet box. And (3) dripping diluted antibody on the surface of the tissue until the surface of the tissue is completely covered. The wet box was placed in a refrigerator at 4 ℃ for overnight incubation. The next day the tissue sections were removed and left to stand at room temperature, washed with PBS, 10min,3 times, 100rpm. HRP-labeled anti-human secondary antibodies were diluted with 5% goat serum (1:1000), tissue sections were removed, spun dry, surrounding liquid was wiped dry with paper towels and placed in a wet box. The prepared secondary antibody is dripped until the secondary antibody covers all tissues, and the secondary antibody is incubated for 1h at room temperature. Tissues were washed with PBS for 5min,3 times, 100rpm. Spin-drying, and wiping the tissue with liquid. Dripping horseradish enzyme-labeled streptavidin until the surface of the tissue is completely covered, and incubating for 15min. The tissue sections were washed with PBS for 5min,3 times, 100rpm. Simultaneously preparing DAB chromogenic liquid, spin-drying tissue slices, wiping redundant liquid with paper towel, placing on white paper, and dripping the prepared DAB chromogenic liquid onto the tissue until the surface is completely covered. The specific staining was observed under a microscope and no staining was observed on the background (about 1 min), and the tissue was placed in tap water to terminate the staining. PBS was washed 2min at 100rpm 3 times. Excess liquid around the tissue was wiped off, the sections were counterstained with nuclei in hematoxylin for 2min, the tissue sections were placed in PBS and blued for 5min, and rinsed with tap water for 5min. The tissue was removed from tap water and in a fume hood, 80% ethanol for 10min,95% ethanol for 5min, absolute ethanol for 5min, xylene for 5min, and xylene for 5min. Finally, taking out the tissue slice from the dimethylbenzene, airing in a fume hood, dripping a drop of neutral gum on the tissue, and covering a cover glass, thereby avoiding air bubbles in the process. The next day was microscopy overnight in a fume hood, image acquisition analysis.
The immunohistochemical results are shown in FIG. 11A, B, and the results show that H103-A-P almost loses the cooperation and Na of human liver cancer tissues under the normoxic condition 2 S 2 O 4 The combination effect of the liver cancer cells on liver cancer tissues or tissues beside the cancer can be recovered after the simulated hypoxia reduction activation.
Example 13
A composition comprising an azobenzene-mediated hypoxia responsive antibody cross-link prepared in example 2, in a mass ratio of 1:10, including H103-azo-PEG 5000 And a hypoxia activated small molecule prodrug TH302.
For a liver cancer-bearing nude mouse model animal living body imaging experiment, the specific steps are as follows:
huh-7 cells in the logarithmic growth phase are taken, and the density of the cells reaches about 80-90%. The fresh serum-free medium was changed the evening before the cells were collected. The following day, cells were digested with pancreatin, washed twice with pre-chilled PBS at ph=7.4, and cell pellet was blown with PBS, typically the amount of cells inoculated for subcutaneous tumors was 1-5×10 6 The inoculation volume is 0.1mL per cell, so that the concentration of the cell suspension is 1-5×10 7 Individual cells/mL. The cell suspension is placed on ice to reduce the metabolism of cells, and the cell suspension is fully blown away before inoculation, so that the cell is prevented from agglomerating, and the cell survival rate is reduced. Balb/c-nu mice with rich blood supply were selected at the planting sites, and tumors were seen to begin to appear subcutaneously about 10 days after inoculation. Tumor bearing nude mice were then randomly divided into 6 groups: cy-7hIgG (5 mg/kg), TH-302 (50 mg/kg), cy7-H103 (5 mg/kg), cy7-H103-A-P (5 mg/kg), TH-302 (50 mg/kg) +Cy7-H103 (5 mg/kg), TH-302 (50 mg/kg) +Cy7-H103-A-P (5 mg/kg). The nude mice were marked by shearing.
Tumor size measurements are typically twice weekly, with vernier calipers measuring the longest and shortest tumor sites. V=1/2×a×b 2 (a is the major axis and b is the minor axis).
The mice were weighed according to the group and each dose was given once by tail vein injection. Imaging was performed at 12, 36, 84, 132, 180 hours after dosing, respectively, the mice were weighed before each imaging, anesthetized with 4% chloral hydrate, left to stand for observation of loss of mobility and stable signs, and then placed on a black background cardboard and imaged in an imager. And photographing to collect data. After imaging, tumor tissues are dissected and washed by PBS, surface moisture is absorbed by water absorbing paper, and a scale is placed for photographing and comparing the tumor sizes.
The results are shown in FIG. 12 and A, B, and FIG. 12A illustrates that H103-A-P can precisely target tumors and reduce the binding to non-tumor organs, thereby reducing the toxic and side effects of antibodies on the body and prolonging the half-life of H103-A-P. FIG. 12B illustrates that the TH-302 (50 mg/kg) +Cy7-H103-A-P (5 mg/kg) combination was more tumor-inhibiting than the control.
Example 14
The H103-A-P and TH302 small molecule prodrug has the therapeutic effect on nude mice bearing liver cancer, and is specifically as follows:
the steps of establishing and grouping the nude mice model of liver cancer are described in the twelve embodiments.
The dosing was performed weekly in the corresponding group, 2 times weekly, for 21 days. During this period, nude mice were weighed every 3 days of interval and tumor volume data was collected by vernier caliper measurement calculation. 21 days after treatment, mice were sacrificed and tumors were weighed (tissues and organs were taken for example 15).
The results are shown in FIG. 13A, B, C, D, and FIG. 13A, C, D together demonstrate that the TH-302 (50 mg/kg) +Cy7-H103-A-P (5 mg/kg) combination treatment group has the best effect of inhibiting tumors of nude mice bearing liver cancer and can remarkably inhibit the development and growth of the tumors after 21 days of treatment. Fig. 13B illustrates during treatment. The nude mouse model did not show weight loss or death.
Example 15
HE staining verifies differences in toxicity to tumors or normal tissues and organs following H103-a-P and/or TH302 treatment. The method comprises the following steps:
tissue organs taken out in example 14 were fixed with 4% PFA for 24 hours, and tissue pieces with a cut diameter of about 5mm were placed in an embedding cassette, marked, and immersed in PBS for 2 hours. Tissue dehydration, placing the embedding cassette into a beaker for the following dehydration operations:
heart, kidney and spleen: sequentially using 70% ethanol water solution with volume concentration for 1h, 80% ethanol water solution with volume concentration for 1h, 90% ethanol water solution with volume concentration for 1h, 95% ethanol water solution with volume concentration for 30min, absolute ethanol for 20min again, xylene for 30min and xylene for 30min again.
Lung liver tumor: sequentially using 70% ethanol water solution with volume concentration for 1h, 80% ethanol water solution with volume concentration for 1h, 90% ethanol water solution with volume concentration for 1h, 95% ethanol water solution with volume concentration for 40min, absolute ethanol for 30min again, xylene for 30min, and xylene for 30min again.
The embedding machine is started 3 hours in advance, clean wax is put into the embedding machine for melting 30 minutes before embedding, and then embedding operation is carried out. Slicing is soaked in ice water at 4 ℃ for 12 hours in advance, slicing is carried out on the next day, the thickness is 5um, and the slices are baked for 6 hours. Dewaxing and hydration: xylene 10min×3 times, absolute ethanol 3min×3 times, 95% ethanol 3min×2 times, 75% ethanol 3min×2 times, distilled water 1min, and placing in PBS buffer; hematoxylin-stained nuclei: the sections were stained with hematoxylin for 3min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for 2-3 seconds, and rinsed with tap water for 6 min. Eosin-stained cytoplasm: the sections were stained in eosin dye solution for 2min. The water is washed once (for 5-20 s). Transparent sealing piece: sequentially placing the slices into 95% alcohol I for 5min,95% alcohol II for 5min, absolute alcohol I for 5min, absolute alcohol II for 5min, xylene I for 5min, and xylene II for 5min for dehydration and transparency, taking out the slices from xylene, slightly airing, and sealing with neutral resin. Microscopic examination, image acquisition and analysis.
The results are shown in fig. 14, and fig. 14 shows that H103-a-P can reduce the damage to other organs and tissues, can realize highly selective and accurate targeting of the position of the hypoxic tumor (T), and can also reduce the toxic and side effects on normal organs such as heart (H), liver (Li), spleen (Sp), lung (Lu), kidney (K), stomach (St), and the like, thereby realizing the effect of accurate treatment of antibodies.
We show by the series of experiments described above that: successfully prepares H103-azo-PEG 5000 . This engineering strategy enables the antibody conjugate to bind the target antigen PKM2 with high selectivity in the solid tumor hypoxic microenvironment or under in vitro reducing conditions. This not only does not affect the drug effect function of the antibody, but also prolongs the half-life period of the antibody. More importantly, the chemical modification strategy for improving the targeting selectivity of the antibody drug based on the tumor hypoxia microenvironment can increase the maximum tolerance dose of the in vivo treatment of the antibody drug, eliminate various toxic and side effects of the parent antibody caused by the cross off-target effect, and realize the accurate targeting of the hypoxic solid tumorCurative effect. The conditional activation type antibody medicine and the preparation method thereof can be applied to the imaging and treatment of various solid tumors with hypoxia characteristics, and can also be applied to the imaging and treatment of other diseases with hypoxia characteristics.
In the combination with the hypoxia activated small molecule prodrug TH302, we find that the combined administration can inhibit the progress of tumor to the greatest extent, and the treatment effect is obviously stronger than that of single-drug treatment, and simultaneously, the injury or toxic and side effects of the drug on other non-tumor organs can be completely eliminated.
The foregoing description is only a partial embodiment of the present invention to enable those skilled in the art to understand or practice the present invention. Based on the dual drug synergistic condition activated hypoxia targeted combination treatment scheme and embodiments presented in this invention, related industry personnel can reproduce the claims of this invention in other embodiments without conflict with the protection of the rights of this invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. Azobenzene-mediated hypoxia response type antibody cross-linked product, H103-azo-PEG for short 5000 Wherein H103 is shorthand for H103 monoclonal antibody; the amino acid sequence of the heavy chain variable region of the H103 monoclonal antibody is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the H103 monoclonal antibody is shown as SEQ ID NO. 2.
2. The method for preparing the azobenzene-mediated hypoxia response type antibody cross-linked matter according to claim 1, which is characterized by comprising the following steps: h103 mab was combined with PEG in carbonate buffer at ph=9.0 5000 Mixing azo-NHS uniformly, fixing in a shaking table for reaction, and centrifugally ultrafiltering the reaction product to obtain a purified azobenzene-mediated hypoxia response type antibody cross-linked product; the PEG 5000 The structure of the-azo-NHS is shown as a formula (7):
3. use of the azobenzene mediated hypoxia responsive antibody cross-link of claim 1 for preparing a medicament for accurate diagnosis and targeted treatment of tumor.
4. A composition comprising the azobenzene-mediated hypoxia responsive antibody cross-linked material of claim 1, characterized by comprising H103-azo-PEG 5000 And a hypoxia activated small molecule prodrug TH302.
5. The composition according to claim 4, wherein said H103-azo-PEG 5000 And the mass ratio of the hypoxia activated small molecule prodrug TH302 is 1:10.
6. use of a composition according to claim 4 or 5 for the preparation of a medicament for targeted treatment of a tumor.
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