CN114107435A - Activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy and application - Google Patents
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Abstract
The invention relates to the technical field of immune medical research, in particular to an activatable photoacoustic-fluorescent dual-mode probe for real-time monitoring of immunotherapy, and further discloses a preparation method and application thereof. The activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy consists of a PDL1 antibody and a granzyme B substrate peptide, wherein the surface of the granzyme B substrate peptide is modified with a fluorescent group and a quenching group, and the probe is an activatable photoacoustic/fluorescence dual-mode probe based on a PDL1 antibody and is used for monitoring the activity of the granzyme B.
Description
Technical Field
The invention relates to the technical field of immune medical research, in particular to an activatable photoacoustic-fluorescent dual-mode probe for real-time monitoring of immunotherapy, and further discloses a preparation method and application thereof.
Background
The immunotherapy of tumor is to stimulate and enhance the immune function of the organism and enhance the anti-tumor immunity of the tumor microenvironment so as to achieve the purpose of controlling and killing tumor cells. However, since immunotherapy can only eliminate a small amount of disseminated tumor cells and has limited efficacy on advanced solid tumors, it is often used as an adjuvant therapy in combination with conventional methods such as surgery, chemotherapy, and radiotherapy. In general, after a large amount of tumor cells are cleaned by a conventional method, the remaining tumor cells are cleaned by immunotherapy, so that the effect of comprehensive treatment of tumors can be improved.
The method for tumor immunological therapy is various, has been combined with modern biological high-tech technology, is developed into a fourth tumor therapy mode after surgery, chemotherapy and radiotherapy, namely a biological tumor therapy method, obtains remarkable treatment effect in clinical trials, and is the most potential tumor therapy strategy. However, due to the differences in heterogeneous immune responses, accurate monitoring of efficacy remains a major challenge. Traditional imaging techniques such as PET/CT and MRI greatly affect the evaluation of therapeutic efficacy and prognosis in early treatment due to lack of sensitivity and specificity of early response assessment, and are not favorable for optimization of clinical treatment protocols. Therefore, how to realize real-time monitoring of immunotherapy to guarantee clinical treatment effect has positive significance for development and application of tumor immunology.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide an activatable photoacoustic-fluorescent dual-mode probe for real-time monitoring of immunotherapy, which can monitor the immunotherapy reaction in real time and distinguish high-reactivity tumors from low-reactivity tumors;
the invention also aims to provide a preparation method and application of the activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy.
In order to achieve the aim, the invention provides an activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy, which consists of a PDL1 antibody and granzyme B substrate peptide, wherein the surface of the granzyme B substrate peptide is modified with a fluorescent group and a quenching group.
Specifically, the granzyme B substrate peptide has a core peptide segment structure shown as Ile-Glu-Pro-Asp, and the structures at other positions can be adaptively changed. Preferably, the granzyme B substrate peptide is selected to have a peptide fragment structure as shown by Gly-Lys (Dabcyl) -Ile-Glu-Pro-Asp-Ala-Pro-Cys (FITC). It is noted that dabcyl and FITC are not fixed as quenching and fluorescent groups and can be varied as desired. Normally dabcyl and FITC are used in vitro studies, while IRDye800CW and IRDye QC1 in the near infrared are generally needed in vivo studies because of the greater ease of detection of the nir in vivo.
The invention also discloses a method for preparing the activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy, which comprises the following steps:
(1) dissolving granzyme B substrate peptide with a selected peptide fragment structure in ultrapure water, and adding NHS and EDC for incubation treatment to activate carboxyl in the peptide fragment;
(2) and (3) uniformly mixing the activated peptide fragment with the PDL1 antibody, carrying out a light-shielding reaction at room temperature, removing impurities, and recovering the antibody to obtain the PDL1 antibody.
Specifically, the mass ratio of the PDL1 antibody, the granzyme B substrate peptide, NHS and EDC is 1: 1: 0.5: 0.5.
specifically, in the step (1), the incubation treatment step is carried out for 20-40 min.
Specifically, in the step (2), the time of the light-shielding reaction step is 10-15 h.
Specifically, in the step (2), the impurity removal step is to remove impurities such as free polypeptide by dialysis with a 1KD dialysis bag.
Specifically, the step (1) further comprises a step of synthesizing the granzyme B substrate peptide by a solid phase synthesis method.
The invention also discloses application of the activatable photoacoustic-fluorescent dual-mode probe for real-time monitoring of immunotherapy in the field of tumor immunotherapy.
Specifically, the probe is an injection and can be injected intravenously.
The activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy consists of a PDL1 antibody and a granzyme B substrate peptide, wherein the surface of the granzyme B substrate peptide is modified with a fluorescent group and a quenching group, and the probe is an activatable photoacoustic/fluorescence dual-mode probe based on a PDL1 antibody and is used for monitoring the activity of the granzyme B.
The activatable photoacoustic-fluorescence dual-mode probe disclosed by the invention blocks the inhibition effect of tumor cells on an immune system by sealing PDL1 on the surface of a tumor through an anti-programmed death ligand 1(PDL1) antibody, the activated immune system releases granzyme B to kill the tumor, and the activatable probe carried on the PDL1 antibody can release photoacoustic/fluorescence signals after being cut by the granzyme B, so that the activity of time-sensitive granzyme B is tracked and the activated photoacoustic-fluorescence dual-mode probe is used as a direct method for monitoring the starting of effective immune response. The double-mode probe disclosed by the invention has the advantages that the PDL1 antibody is coupled with the activatable probe to effectively inhibit the interaction of PD-1/PD-L1, the release of T cell mediated granzyme B is induced, and the activatable imaging probe can be used for imaging. The activatable dual-mode probe disclosed by the invention not only can feed back the activity of the granzyme B in real time, but also can monitor the immunotherapy reaction of a tumor-bearing mouse model in real time, and can be used as a direct method for monitoring the starting of effective immune response; in addition, the probe can perform high-sensitivity and high-specificity imaging on tumor tissues through a high-resolution fluorescence/photoacoustic imaging technology, so that high-reactivity and low-reactivity tumors can be distinguished, and the probe has important clinical significance.
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FIG. 1 is a synthetic HPLC report of a bimodal probe according to the present invention;
FIG. 2 is a schematic diagram of the construction and operation of the bimodal probe of the present invention;
FIG. 3 is the change of fluorescence intensity after incubation of the probe with different concentrations of granzyme according to the invention for the same time;
FIG. 4 is the change in fluorescence intensity after the response time of incubation of the dual mode probe with the enzyme according to the present invention;
FIG. 5 is a graph showing the change of activation of a probe observed in real time by fluorescence imaging of a small animal.
Detailed Description
The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
EXAMPLE 1 construction of activatable probes
Synthesizing the required granzyme B substrate peptide by using a conventional solid phase synthesis method, wherein the structure is as follows: Gly-Lys (Dabcyl) -Ile-Glu-Pro-Asp-Ala-Pro-Cys (FITC), and the successfully labeled peptide fragment was purified by high performance liquid chromatography for further use.
1mg of the above peptide fragment was dissolved in 1ml of ultrapure water, and incubated for 30 minutes with the addition of 0.5mg of NHS and 0.5mg of EDC to activate the carboxyl group in the peptide fragment. The activated peptide fragment and 1mg of PDL1 antibody are mixed uniformly, and then the mixture is magnetically stirred for 12 hours at 200 revolutions per minute at room temperature, and the whole process is reflected in a dark place. After the reaction is completed, dialyzing by using a 1KD dialysis bag to remove impurities such as free polypeptide and the like, and recovering the antibody to obtain the antibody.
The HPLC report of the probe synthesis is shown in the attached figure 1, and the structure of the probe is correct.
Working principle of the Probe of example 2
The activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy consists of a PDL1 antibody and a granzyme B substrate peptide, wherein the surface of the granzyme B substrate peptide is modified with a fluorescent group and a quenching group, and the probe is an activatable photoacoustic/fluorescence dual-mode probe based on a PDL1 antibody and is used for monitoring the activity of the granzyme B.
As shown in the schematic diagram of fig. 2, after intravenous injection of the composite probe, PDL1 antibody actively targets tumor surface PDL1 protein (as in a in fig. 2), by blocking the interaction of PDL1 with T cell surface PD1, the anti-tumor immunity of T cells is activated, and the activated T cells release granzyme B to kill tumor (as in B in fig. 2); meanwhile, after the granzyme B cuts the granzyme B substrate peptide on the probe, the fluorescent group on the probe is separated from the quenching group, and at the moment, a fluorescent signal (as c in figure 2) can be detected, so that the dynamic process and the curative effect of the immunotherapy can be reflected through fluorescence imaging.
Example 3 characterization of Probe functional Activity
The antibody marked by the probe and the recombinant granzyme B with different concentrations (0-1mg/ml) are incubated for 10 minutes, and the fluorescence intensity within the range of 460nm excitation and 480-600nm is detected by an enzyme-labeling instrument to verify the signal response characteristics of different probes to the granzyme B with different concentrations.
As shown in FIG. 3, the fluorescence intensity of the probe after incubation for the same time varies with the concentration of granzyme, and it can be seen that the probe can sensitively detect the activity of granzyme B and has a positive correlation with the concentration of granzyme B.
And (4) continuously incubating the probe-labeled antibody and 100ng/ml recombinant granzyme B, and detecting the signal response characteristics of the probe at different incubation times (0-130min) by using a microplate reader.
As shown in FIG. 4, the change of fluorescence intensity after the response time of the probe and the enzyme incubation is shown, and the probe can detect the time response process of the granzyme B.
Example 4 in vivo Effect verification
Subcutaneous tumor-bearing mice were constructed by inoculating 100 million 4T1 cells subcutaneously in the back of balbc mice. When the tumor grows to 5 mm in diameter, 10 mu g of probe is injected into the tumor-bearing mice by tail vein administration, and the activation of the probe is observed in real time by small animal fluorescence imaging.
As shown in FIG. 5, fluorescence imaging was performed before and one hour after the tail vein administration, and then, imaging was performed for 1 to 9 days. The mouse body had no fluorescent signal before probe injection and the fluorescent signal was not activated 1 hour after injection. As a result, it was found that significant activation of fluorescent signal began to appear in the tumor tissue 1 day after the injection of the probe and continued until the ninth day. This indicates that the laser probe has good tumor targeting and signal giving effects.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. An activatable photoacoustic-fluorescent dual-mode probe for real-time monitoring of immunotherapy, which is characterized in that the probe consists of a PDL1 antibody and a granzyme B substrate peptide, and the surface of the granzyme B substrate peptide is modified with a fluorescent group and a quenching group.
2. The activatable photoacoustic-fluorescent bimodal probe for real-time monitoring of immunotherapy according to claim 1, wherein the granzyme B substrate peptide has a core peptide stretch structure as shown in Ile-Glu-Pro-Asp.
3. A method for preparing the activatable photoacoustic-fluorescent bimodal probe for real-time monitoring of immunotherapy as set forth in claim 1 or 2, comprising the steps of:
(1) dissolving granzyme B substrate peptide with a selected peptide fragment structure in ultrapure water, and adding NHS and EDC for incubation treatment to activate carboxyl in the peptide fragment;
(2) and (3) uniformly mixing the activated peptide fragment with the PDL1 antibody, carrying out a light-shielding reaction at room temperature, removing impurities, and recovering the antibody to obtain the PDL1 antibody.
4. The preparation method of the activatable photoacoustic-fluorescent bimodal probe for real-time monitoring of immunotherapy according to claim 3, wherein the mass ratio of the PDL1 antibody, the granzyme B substrate peptide, NHS and EDC is 1: 1: 0.5: 0.5.
5. the method for preparing an activatable photoacoustic-fluorescent bi-modal probe for real-time monitoring of immunotherapy as claimed in claim 3 or 4, wherein the incubation treatment step in step (1) is performed for 20-40 min.
6. The method for preparing the activatable photoacoustic-fluorescent dual-mode probe for real-time monitoring of immunotherapy according to any one of claims 3 to 5, wherein in the step (2), the time for the light-shielding reaction step is 10 to 15 hours.
7. The method for preparing the activatable photoacoustic-fluorescent bi-modal probe for real-time monitoring of immunotherapy as recited in any one of claims 3-6, wherein in the step (2), the step of removing impurities is to remove impurities such as free polypeptides by dialysis with a 1KD dialysis bag.
8. The method for preparing an activatable photoacoustic-fluorescent bimodal probe for real-time monitoring of immunotherapy according to any one of claims 3 to 7, wherein the step (1) further comprises a step of synthesizing the granzyme B substrate peptide by solid-phase synthesis.
9. Use of the activatable photoacoustic-fluorescent dual mode probe for real-time monitoring of immunotherapy as defined in claim 1 or 2 in the field of tumor immunotherapy.
10. The use of claim 9, wherein the probe is an injection.
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