CN111285936A - Acid sensitive nano peptide segment of targeted tumor and application thereof - Google Patents

Abstract

The invention discloses an acid-sensitive nano peptide segment targeting a tumor and application thereof. The acidic sensitive nano peptide segment targeted to the tumor comprises a low-pH nano insertion peptide, a coupling agent and an Fc segment which are sequentially connected in series; wherein the low pH nano-insertion peptide comprises an amino acid sequence having the sequence of SEQ ID No.1 or a variant thereof. The acid-sensitive nano peptide segment of the targeted tumor inserts specific markers into tumor cells and other stromal cells in a microenvironment by utilizing the characteristic that the pH of the tumor microenvironment is less than 7 micro-acid environment, can attract and combine the markers to CD16 molecules of NK cells, and specifically recruits and activates ADCC function of the NK cells by the markers, thereby achieving the killing function of the tumor cells.

Description

Acid-sensitive nanopeptides targeting tumors and their applications

technical field

The present invention relates to the field of immune cells, in particular to a tumor-targeting acid-sensitive nano-peptide segment and its application.

Background technique

Natural killer cells (NK cells) are a subset of lymphocytes. It plays an important role in the immune response of the human body, whether it is innate immunity or specific immunity. NK cells can exert their immune killing effect independently of MHC without primary immune stimulation and gene editing. There are three ways for NK cells to kill cells: (1) the killing mediators perforin and granzyme B released by NK cells cause apoptosis of target cells, which requires direct contact between NK cell recognition receptors and target cells; (2) NK cells can pass Membrane TNF family molecules (FasL, TRIAL, mTNF, etc.) bind to target cell membrane ligands to induce target cell apoptosis, which does not require direct contact between NK cell receptors and target cells; ③ NK cells can also pass anti-tumor antibodies IgG1 and IgG3 As a bridge, its Fab segment specifically recognizes tumors, and its Fc segment binds to NK cell FcRγIIIa to produce antibody-dependent cell-mediated cytotoxicity (ADCC), which is mainly performed by CD16 ⁺ NK cells.

The tumor microenvironment is composed of tumor cells, a variety of stromal cells, and a series of cytokines and chemokines. The stromal cells include fibroblasts, tumor-infiltrating immune cells, endothelial cells, bone marrow-derived immature cells, etc.; cytokines such as TNF, VEGF, IL-1, etc.; chemokines such as CXCL12, CCL27, CCL21, etc. Cytokines and chemokines can be secreted by tumor cells and can also be produced by stromal cells and the aforementioned infiltrating immune cells. Together, these cells and active mediators form a stable tumor immune microenvironment that protects tumor tissues from immune surveillance and promotes tumor progression. Tumor cells have strong heterogeneity and proliferation ability, and their high mutation rate in the process of growth makes it difficult for us to find a variety of tumors or different patients of the same tumor type in the process of tumor treatment. stably expressed specific tumor antigens.

The tumor microenvironment is characterized by hypoxia and acidification, which are important reasons for the development of benign tumors to metastatic malignant tumors. Among them, acidification has three effects on tumor development: increasing resistance to chemotherapy, increasing mutation rate, and enhancing invasiveness.

The bone marrow (hematopoietic) microenvironment refers to the network system that can regulate the proliferation, differentiation and function of hematopoietic cells, including cellular components and cellular products. The cellular components include stromal cells and accessory cells. The stromal cells include fibroblasts, osteoblasts, macrophages, endothelial cells, etc. The accessory cells are mainly monocytes and lymphocytes. Bone marrow stromal cells can generate and precipitate complex extracellular matrix.

The pH low insertion peptide (PHLIP) can undergo a conformational transition from random coil to α-helix in the acidic environment of the tumor microenvironment, and this conformational transition allows it to insert itself into the tumor cell membrane Above, the C-terminus is intracellular and the N-terminus is extracellular. This self-assembly at low pH has been shown to occur efficiently in a variety of solid tumors.

For tumor and cancer treatment, surgical removal followed by radiotherapy or chemotherapy (paclitaxel, etc.) will be used in the early stage of treatment; later, targeted therapy will be completed for tumor-specific targets, such as the first anti-tumor therapy approved by the US FDA in 2004. The angiogenesis drug, Avastin, and the third-generation lung cancer drug osimertinib were launched in 2015. Recently, people have begun to regulate the human immune system for the treatment of tumors. In 2018, the Nobel Prize in Medicine was awarded to James Ai of the United States. Leeson and Japan's Shusuke Honjo, for their pioneering work on cancer immunotherapy (discovery of CTLA-4, PD-1). And CAR-T technology was applied to the treatment of hematological tumors, but the final results were not so satisfactory due to various reasons, so we chose NK cells that have more potential for tumor cell killing.

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 of ordinary skill in the art.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a tumor-targeting acid-sensitive nano-peptide segment and its application. The tumor-targeting acid-sensitive nano-peptide segment utilizes the characteristics of a tumor microenvironment pH<7 and a slightly acidic environment to provide the tumor in the microenvironment. Cells and other stromal cells insert specific markers, which can attract and bind to the CD16 molecule of NK cells, and the markers can specifically recruit and activate the ADCC function of NK cells to achieve the killing function of tumor cells.

In order to achieve the above object, the present invention provides an acid-sensitive nanopeptide targeting tumor, comprising serially connected low pH nanoinsert peptide (pHLIP), a coupling agent, and an Fc fragment; wherein, the low pH nanoinsert The peptide includes the amino acid sequence of SEQ ID NO. 1 or a variant thereof, and the low pH nano-inserted peptide can be inserted into the cell membrane of tumor cells by self-coiling in a slightly acidic environment to play a targeting role.

In one embodiment of the present invention, the Fc fragment is selected from the amino acid fragment of murine IgG2a or IgG2b, wherein IgG2a is selected from the amino acid sequence of positions 93-330, as shown in SEQ ID NO. 2, and IgG2b is selected from the 97th amino acid sequence - The amino acid sequence at position 335 is shown in SEQ ID NO.3.

In one embodiment of the present invention, the coupling agent is selected from sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) , to ligate the low pH nanoinsert peptide and the ADCC-inducible Fc fragment.

In one embodiment of the present invention, the acid-sensitive nanopeptide segment targeting tumors can function in an environment with a pH value of <7.

The present invention also provides a tumor marker system, which comprises the above acid-sensitive nanopeptide targeting tumors.

The present invention also provides a targeted tumor therapy system, which includes the above tumor marker system.

The present invention also provides the application of the above acid-sensitive nano-peptide targeting tumors in the preparation of the above tumor marker system.

The present invention also provides the application of the above-mentioned acid-sensitive nano-peptide targeting tumors or the above-mentioned tumor labeling system in preparing the above-mentioned tumor-targeting treatment system.

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

(1) The acid-sensitive nanopeptide targeting tumor used in the present invention is mainly composed of three parts: pHLIP, a low pH nano-insertion peptide, a coupling agent sulfo-SMCC and an Fc fragment, which can not only function separately, but also interact with each other. Coordinate to provide an effective and safe microenvironment to insert molecules;

(2) The present invention adopts the cutting-edge nano-peptide technology, and rationally utilizes the characteristics of tumor hypoxia and acidification that originally affect the development and therapeutic effect of tumors, making it a target condition for treatment, and can make the target molecule specific Inserted into the cells of the leukemia bone marrow tumor microenvironment, giving specific markers to the tumor, providing targets for targeted killing and treatment, thereby attracting immune cells (NK cells) to target attack and killing;

(3) The acid-sensitive nanopeptides targeting tumors involved in the present invention can kill and treat tumors by mobilizing the autoimmune system. Different from the existing targeted killing of tumors, this method can be applied to all tumors, including solid tumor treatment.

Description of drawings

Figure 1A is a schematic diagram of a low pH nanoinsert peptide linked to an antibody through a coupling agent according to an embodiment of the present invention;

1B is a schematic diagram of the low pH nano-inserted peptide according to an embodiment of the present invention self-coiling at low pH to insert into the membrane surface;

2 is a schematic diagram of the effect of NK cells exerting ADCC effects according to an embodiment of the present invention;

3 is a schematic diagram of tumor killing according to an embodiment of the present invention;

4 is a time-of-flight mass spectrogram of matrix-assisted laser desorption ionization according to an embodiment of the present invention;

5A is a detection diagram of the killing of melanoma cells (B16) by pHLIP-Fc mediated by NK cells in a slightly acidic environment according to an embodiment of the present invention;

5B is a detection diagram of the killing of mouse breast cancer cells (4T1) by pHLIP-Fc-mediated NK cells in a slightly acidic environment according to an embodiment of the present invention;

5C is a detection diagram of pHLIP-Fc-mediated killing of human triple-negative breast cancer by pHLIP-Fc in a slightly acidic environment according to an embodiment of the present invention;

6A is a graph showing the inhibition of melanoma by pHLIP-Fc molecules according to an embodiment of the present invention;

Figure 6B is a graph of the inhibition of breast cancer by pHLIP-Fc molecules according to one embodiment of the present invention.

Detailed ways

The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

Unless expressly stated otherwise, throughout the specification and claims, the term "comprising" or its conjugations such as "comprising" or "comprising" and the like will be understood to include the stated elements or components, and Other elements or other components are not excluded.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the following examples can be purchased from commercial sources unless otherwise specified. The low pH nano-insertion peptide pHLIP was synthesized by Shanghai Taopu Biotechnology Co., Ltd., and the Fc segment of mouse IgG2a/b was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. The sequence is as follows:

The low pH nano-insert peptide pHLIP sequence is SEQ ID NO.1 acetylated at the N-terminus;

Fc fragment sequence from murine IgG2a, the sequence from N-terminal to C-terminal is shown in SEQ ID NO.2;

Fc fragment sequence from murine IgG2b, the sequence from N-terminal to C-terminal is shown in SEQ ID NO.3;

The human-derived CD16 protein sequence is shown in SEQ ID NO.4; the human-derived Rit (anti-CD20) protein sequence is shown in SEQ ID NO.5.

In the following, the acid-sensitive nanopeptides targeting tumors claimed in the present invention will be analyzed in terms of structural recombination, cell membrane insertion ability, binding ability to human CD16 protein, detection effect of in vitro NK cell killing experiments, and animal experiments by means of preferred embodiments. Discuss in detail.

Example 1

pHLIP-Fc, pHLIP-Rit construction and labeling of CD16 with Cy5.5 or FITC

1.1 Connect pHLIP to the coupling agent sulfo-SMCC

Dissolve 4800 nmol sulfo-SMCC in 300 μL PBS, and dissolve 10 nmol Fc fragment in 200 μL PBS, respectively. Mix the two solutions into a 1.5 mL Eppendorf centrifuge tube and shake gently at room temperature for 2 hours. During the whole process, control pH=7.4. The ligation product was then purified on a column of NAP-5 (GE Healthcare, UK) pre-equilibrated with PBS buffer to obtain an Fc ligation solution.

1.2 Connect the ligated Fc fragment to pHLIP

A total of 4800 nmol of pHLIP dissolved in PBS buffer was added to the Fc ligation solution obtained in the previous step, and the ligation reaction was induced by gentle shaking at room temperature for 5 hours. The final pHLIP-Fc product was purified and filtered using a column of NAP-10 (GE Healthcare, UK) pre-equilibrated with PBS buffer. The same method was applied to the ligation of IgG3 and Rit.

1.3 The molecular mass of Fc/a, pHLIP-Fc/a, Fc/b, pHLIP-Fc/b was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Fig. 4). It can be seen from Figure 4 that the m/z of the Fc/a fragment is 56780.7, and the m/z value is shifted to the right after pHLIP is connected, indicating that the successful coupling of pHLIP to the Fc/a fragment is achieved; the Fc/b fragment shows a similar trend.

1.4 In order to label FITC or Cy5.5 on the CD16 molecule, 100 nmol FITC (Fanbo Biochemical Co., Ltd., China) was added to a human-sufficient CD16 (Sino Biological Inc., China) solution, which was prepared by 10 nmol CD16 molecules were dissolved in 300 μL of PBS buffer. The mixture was shaken gently for 3 hours at room temperature in an Eppendorf centrifuge tube to induce the reaction. The final product was purified on a NAP-5 column pre-equilibrated with PBS buffer.

1.5 Detection of the modification of Fc molecules by sulfo-SMCC by tandem mass spectrometry (MS/MS). To track the sulfo-SMCC modified binding site, SMCC-Fc/a and SMCC-Fc/b were digested with trypsin (Promage, USA). Digested peptides were analyzed by nano-LC-LTQ-Orbitrap XL MS/MS system. MS data were analyzed by Proteome Discoverer software (version 1.4.0.288, Thermo Fischer Scientific). The second MS spectrum was searched in the Mus database using the SEQUEST search engine. Sulfo-SMCC modification of lysine, glutathionylation or oxidation of cysteine, and oxidation of methionine were set as variable modifications. 1.6 Detection and calculation of pHLIP-Fc/a and pHLIP-Fc/b by surface plasmon resonance imaging (SPRi) detection application. The binding affinity of Fc or pHLIP-Fc molecules to human recombinant CD16 (Sino Biological Inc, China) and the binding affinity of Rit or pHLIP-Rit to human recombinant CD16a (Sino Biological Inc, China) were measured using a Biacore 8K SPR biosensor (GE Healthcare). binding affinity. All of the above human CD16 and human CD16a were connected to the surface of the CM5 sensor chip according to the instructions in the user manual. All ligand proteins were spotted at serial 2-fold dilutions in PBST buffer (pH 6.8) ranging from 1000 nM to 31.25 nM. The flow rate was 30 μL/min and the buffer was PBST (0.01 mM PBS plus 0.05% Tween 20). Contact and dissociation times were 60 s and 120 s, respectively. Regeneration of the protein chip was performed after each sample with 5 mM NaOH. The above cycle was repeated for each concentration of Fc, pHLIP-Fc, Rit and pHLIP-Rit. Binding responses were recorded continuously as resonance units (RU) and background binding was automatically subtracted. Affinity constants (KD) were calculated using a 1:1 Langmuir binding model using BIAcore 8K evaluation software (GE Healthcare).

Example 2

Cell transformation and detection of transformation conditions

2.1 Transformation of recombinant pHLIP-Fc/a and pHLIP-Fc/b to the cell surface.

2.1.1B16/F10 is a mouse melanoma cell line, purchased from the National Science and Technology Cell Experiment Platform (China). The cells were cultured in RPMI-1640 cell culture medium with 10% FBS (WISENT, Canada) added, and cultured at 37°C in a 5% CO ₂ cell incubator.

2.1.2 Cells were cultured on 35 mm borosilicate chamber coverslips (Nunc, USA), then pHLIP-Fc/a and pHLIP-Fc/b and B16/F10 cells were cultured at pH=6.8 and 7.4, respectively The cells were co-cultured and induced in the environment for 4 hours, and then Cy5.5-labeled human recombinant CD16 molecules were added, and the cells were co-cultured and induced for 1 hour. The pH of the solution has been kept separately during this process.

2.1.3 The cells on the coverslip were stained with HOECHST 33342 (Invitrogen, USA) for nuclear staining, and the cells were observed with a Zeiss LSM710 confocal microscope (Carl Zeiss, Germany).

2.1.4K562 is a human chronic myeloid leukemia cell, purchased from the National Science and Technology Cell Experiment Platform (China). The cells were cultured in RPMI-1640 cell culture medium with 10% FBS (WISENT, Canada) added, and cultured at 37°C in a 5% CO ₂ cell incubator.

2.1.5 MDA-MB-231 is a human breast cancer cell line, purchased from the National Science and Technology Cell Experiment Platform (China). The cells were cultured in DMEM cell culture medium with 10% FBS (WISENT, Canada) added, and cultured at 37°C in a 5% CO ₂ cell incubator.

In the cell culture environment of pH=6.8, Rit and pHLIP-Rit were added to the MDA-MB-231 cell culture system for 4 hours, and then Cy5.5-labeled human recombinant CD16 was added to the culture system for 1 culture. Hours.

The same procedure was followed by staining with HOECHST 33342 and observation with a confocal microscope, maintaining pH=6.8 during this process.

2.2 Flow cytometry for concentration-dependent kinetic studies

2.2.1 Add pHLIP-Fc/a or pHLIP-Fc/b with different concentrations (0, 0.1, 0.5, 1, 2.5, 5, 10 μg/mL) into a 24-well plate, and spread 1×10 ⁵ in each well B16/F10 cells were cultured in 400 μL of pH=6.8 medium for 5 hours.

2.2.2 Cells were washed 3 times with PBS pH=6.8.

2.2.3 Replace with 20 μg/mL FITC-labeled human recombinant CD16 (400 μL) to induce 1 hour.

2.2.4 Cells were washed 3 times with PBS pH=6.8.

2.2.5 Resuspend with pH=6.8 PBS for flow cytometry, and each sample is tested 3 times.

2.3 Time-dependent kinetic studies by flow cytometry

2.3.1 Add pHLIP-Fc/a or pHLIP-Fc/b at the same concentration (2.5 μg/mL) to a 24-well cell culture dish with pH=6.8, and plate 1×10 ⁵ B16/F10 cells per well, 400 μL of culture system was used for different time (5, 10, 30, 60, 120, 240 and 480 minutes).

2.3.2 The cells were washed 3 times with PBS pH=6.8.

2.3.3 Replace with 20 μg/mL FITC-labeled human recombinant CD16 (400 μL) for induction for 1 hour.

2.3.4 Cells were washed 3 times with PBS pH=6.8.

2.3.5 Resuspend with pH=6.8 PBS for flow cytometry, and each sample is tested 3 times.

Example 3

In vitro cytotoxicity assay

3.1 ADCC function was detected according to the instruction manual of CytoTox-Glo ^™ Cytotoxicity Assay Kit (Promega, Madison, WI, USA).

3.2 The NK cells from the mouse spleen were isolated with a peripheral blood isolation kit (Miltenyi, Auburn, Canada), and the isolation purity reached more than 90%. These NK cells can act as effector cells and B16/F10 tumor cells as target cells.

3.3 2×10 ⁴ B16/F10 tumor cells were cultured in a 96-well plate and incubated with 100 μL pHLIP-Fc (2.5 μg/mL) (pH=6.8 or 7.4) for 2-4 hours

3.4 According to the ratio of effector cells:target cells=1:1, add 2×10 ⁴ NK cells into the culture system to induce culture for 4 hours.

3.5 Fluorescence values were detected with a multi-template reader (PerkinElmer, USA). Lactate dehydrogenase (LDH) release was determined and percent cytotoxicity was calculated after correcting for background absorbance values according to the following formula:

3.6 Using MDA-MB-231 as target cells and NK cells as effector cells. NK cells were isolated from human peripheral blood (PBMC) using lymphocyte isolation medium (Corning, USA) according to the instructions, and then the isolated NK cells were purified by MACS NK cell isolation kit to a purity greater than 90%.

3.7 2×10 ⁴ MDA-MB-231 cells were plated in a 96-well plate and incubated with 100 μL of pHLIP-Rit (2.5 μg/mL) (pH 6.8 or 7.4) for 2-4 hours.

3.8 According to the ratio of effector cells:target cells=1:1, add 2×10 ⁴ NK cells into the culture system to induce culture for 4 hours.

This pHLIP-Fc molecule was incubated with tumor cells under slightly acidic and neutral conditions, respectively, followed by the addition of NK cells. In a slightly acidic environment, pHLIP-Fc could effectively mediate the killing of melanoma cells (B16) by NK cells (Fig. 5A). In a slightly acidic environment, pHLIP-Fc could effectively mediate the killing of mouse breast cancer cells (4T1) by NK cells (Fig. 5B). In addition, the research team demonstrated that pHLIP-Rit can mediate the killing of human triple-negative breast cancer by human NK cells (Figure 5C), indicating that pHLIP-modified Fc or antibody has good clinical application prospects.

Example 4. Animal in vivo experiment and detection

4.1 Animal research and immunohistochemistry

4.1.1 For the orthotopic mouse model of B16/F10 melanoma, treatment was started 4 days after inoculation, when the tumor volume reached approximately 50 mm ³ . The day on which the first treatment was administered was defined as day 0. (iv) Mice were dosed with PBS, Fc/a+pHLIP, pHLIP-Fc/a, pHLIP-Fc/b or pHLIP-IgG3 (n=6). The therapeutic dose (similar molar dose) of pHLIP-Fc refers to the dose of rituximab (Rit) used in the mouse model (500 μg/kg). It is calculated as follows:

Molecular weight (Mw) of Rit: ~150kDa; Mw of pHLIP-Fc: ~60kDa;

Molar dose of Rit=(Dose of Rit)/(Mw of Rit)=500μg/kg/150kDa=3.33nmol/kg

4.1.2 Select pHLIP-Fc at a molar dose of ~2.5 nmol/kg (equivalent to 150 μg/kg). pHLIP-Fc was administered every 2 days for a total of 4 injections.

Tumor size was measured by digital calipers, and tumor volume was calculated by the formula (L×W ² )/2, where L was the longest and W was the shortest of the tumor diameters (mm). Relative tumor volume (RTV) is equal to the tumor volume at a given time point divided by the tumor volume prior to the start of treatment. For ethical reasons, animals were sacrificed when the implanted tumor volume reached 1000 ^mm3 .

For the 4T1 breast cancer model and the K562 leukemia model, treatment was started 5 days after implantation, at which point the tumor volume reached approximately 50 ^mm3 . The day on which the first treatment was administered was defined as day 0. Other conditions were the same as for the B16/F10 model, but 7 injections were given. For the metastatic tumor model, treatment was started 4 days after tumor cell injection (n=6). Two weeks later (150 μg/kg dose every 2 days for a total of 7 injections), luciferase was administered by intraperitoneal injection for in vivo imaging (n=3 per group); lungs of tumor-bearing animals were dissected for tissue sections ( n=3 per group).

4.1.3 50 μg/kg, 150 μg/kg and 450 μg/kg of pHLIP-Fc/a were tested in K562 myeloid tumor model (n=6) in dose-dependent therapeutic effect evaluation, and other steps were the same as above.

4.1.4 To assess apoptosis in tumors, tumor tissue sections were stained by terminal deoxynucleotidyl transferase dUTP nick end labeling kit (TUNEL, KeyGENBioTECH, Nanjing, China) according to the instructions. A total of 100 nuclei on two separate slides were examined for quantitative results.

4.1.5 For immunohistochemical staining, tumor sections ( ₆ μm) _were deparaffinized, rehydrated, and incubated with 0.3% H2O2 in methanol. After antigen retrieval at 95°C for 15 minutes in 10 mmol/L citrate buffer (pH 6.0), sections were blocked with 5% goat serum/PBS for 1 hour and incubated with primary antibodies (mouse monoclonal antibody and proliferating cell nuclei) Antigen (PCNA) antibody (1:10,000, Abcam, UK) or rabbit polyclonal anti-CRTAM antibody (1:300, Abcam, UK) overnight at 4°C followed by biotinylated secondary antibody for 1 hr at room temperature, horseradish Peroxidase-conjugated streptavidin for 30 min at 37 °C. DAB (R&D system, USA) was used for color development, and sections were counterstained with hematoxylin. Data were analyzed by ImageJ.

It can be found from Figure 6A and Figure 6B that pHLIP-Fc molecule can inhibit various tumors (subcutaneous melanoma, breast cancer in situ) in vivo experiments.

4.2 Blood testing, in vivo imaging and biodistribution testing

4.2.1 For Cy7-labeled Fc/a and pHLIP-Fc/a conjugation, our previous working procedure was followed. Cy7Mono NHS (FanboBiochemicals, Beijing, China) 0.1 mg was added to Fc/a or pHLIP-Fc/PBS solution (100 μL, 0.5 mg/mL, pH 7.4). The mixture was incubated in a 1.0 mL eppendorf tube at room temperature for 3 hours with gentle shaking. Conjugated Fc/a and pHLIP-Fc/a were purified by NAP-5 columns (GE Healthcare, UK) pre-equilibrated with PBS buffer.

4.2.2 To evaluate in vivo blood clearance, Fc-Cy7 and pHLIP-Fc/a-Cy7 were injected intravenously into BALB/c mice bearing 4T1 tumors. At different time points, blood was collected and fluorescent signals were detected by an in vivo imaging system (IVIS) (PerkinElmer Inc., USA). The wavelength of excitation light was 740-760 nm, and the emission wavelength was 780 nm.

4.2.3 To examine in vivo imaging and biodistribution of each formulation, Fc-Cy7 and pHLIP-F/a-Cy7 were injected intravenously into nude mice bearing subcutaneous 4T1 tumors, respectively. Mice were scanned with IVIS. At 12 hours, organs and tumors were excised for ex vivo imaging.

4.3 Immune Response Studies

4.3.1 Healthy BALB/c mice (female, n=6) were immunized with pHLIP (20 μg/kg) for 4 weeks (once a week). At week 5, whole blood was collected from these mice by retro-orbital venipuncture, and serum was collected after centrifugation. Anti-pHLIPIgG titers were measured using an indirect ELISA assay according to a previous study. Absorbance was measured at OD450 on a microplate reader.

4.3.2 To assess the immune response to pHLIP-Fc, two groups of healthy BALB/c mice (female, n=6 per group) were pretreated with pHLIP-Fc/a for 4 weeks (150 μg/kg, weekly injection)) The treatment program was then performed in the same manner in the 4T1 breast tumor model before tumor implantation (one group was treated with PBS, the other group was treated with pHLIP-Fc/a). During the first 4 weeks, the control group (unpretreated group) was administered with PBS.

4.4 Biosafety testing

4.4.1 Administer healthy BALB/c mice (female, 6 weeks old, 15-17 g body weight) with PBS, Fc/a+pHLIP, pHLIP-Fc/a, pHLIP-Fc/b or pHLIP-IgG3(n) (iv) 8 per group). A dose of 50 μg/kg every two days for a total of 7 injections. Serum was collected for biochemical testing (n=4) and organs (heart, liver, spleen, lung and kidney) were fixed, sectioned and stained with H&E to study potential changes in organ morphology (n=4) Sections were from different groups of the same organ.

4.4.2 To analyze the acute immune response after each formulation injection, healthy BALB/c mice (female, 6 weeks old, 15-17 g body weight) were treated with PBS, Fc/a+pHLIP, pHLIP-Fc/a (iv ), pHLIP-Fc/b or pHLIP-IgG3 (n=4) (dose: 150 μg/kg). Levels of interleukin-6 (IL-6) and tumor necrosis factor-beta were elevated after 6 and 24 hours. (TNF-α) in serum was determined with ELISA kits (EM1350 and EM0350, Solarbio, China).

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many changes and modifications are possible in light of the above teachings. The exemplary embodiments were chosen and described for the purpose of explaining certain principles of the invention and their practical applications, to thereby enable others skilled in the art to make and utilize various exemplary embodiments and various different aspects of the invention. Choose and change. The scope of the invention is intended to be defined by the claims and their equivalents.

sequence listing

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Institute of Biophysics, Chinese Academy of Sciences

<120> Acid-sensitive nanopeptides targeting tumors and their applications

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20 25 30

Arg Gln Ser Ala Ala Leu Pro Lys Ala Val Val Lys Leu Asp Pro Pro

35 40 45

Trp Ile Gln Val Leu Lys Glu Asp Met Val Thr Leu Met Cys Glu Gly

50 55 60

Thr His Asn Pro Gly Asn Ser Ser Thr Gln Trp Phe His Asn Trp Ser

65 70 75 80

Ser Ile Arg Ser Gln Val Gln Ser Ser Tyr Thr Phe Lys Ala Thr Val

85 90 95

Asn Asp Ser Gly Glu Tyr Arg Cys Gln Met Glu Gln Thr Arg Leu Ser

100 105 110

Asp Pro Val Asp Leu Gly Val Ile Ser Asp Trp Leu Leu Leu Gln Thr

115 120 125

Pro Gln Arg Val Phe Leu Glu Gly Glu Thr Ile Thr Leu Arg Cys His

130 135 140

Ser Trp Arg Asn Lys Leu Leu Asn Arg Ile Ser Phe Phe His Asn Glu

145 150 155 160

Lys Ser Val Arg Tyr His His Tyr Lys Ser Asn Phe Ser Ile Pro Lys

165 170 175

Ala Asn His Ser His Ser Gly Asp Tyr Tyr Cys Lys Gly Ser Leu Gly

180 185 190

Ser Thr Gln His Gln Ser Lys Pro Val Thr Ile Thr Val Gln Asp Pro

195 200 205

Ala Thr Thr Ser Ser Ile Ser Leu Val Trp Tyr His Thr Ala Phe Ser

210 215 220

Leu Val Met Cys Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Tyr

225 230 235 240

Val Arg Arg Asn Leu Gln Thr Pro Arg Asp Tyr Trp Arg Lys Ser Leu

245 250 255

Ser Ile Arg Lys His Gln Ala Pro Gln Asp Lys Met Thr Leu Asp Thr

260 265 270

Gln Met Phe Gln Asn Ala His Ser Gly Ser Gln Trp Leu Leu Pro Pro

275 280 285

Leu Thr Ile Leu Leu Leu Phe Ala Phe Ala Asp Arg Gln Ser Ala Ala

290 295 300

Leu Pro Lys Ala Val Val Lys Leu Asp Pro Pro Trp Ile Gln Val Leu

305 310 315 320

Lys Glu Asp Met Val Thr Leu Met Cys Glu Gly Thr His Asn Pro Gly

325 330 335

Asn Ser Ser Thr Gln Trp Phe His Asn Trp Ser Ser Ile Arg Ser Gln

340 345 350

Val Gln Ser Ser Tyr Thr Phe Lys Ala Thr Val Asn Asp Ser Gly Glu

355 360 365

Tyr Arg Cys Gln Met Glu Gln Thr Arg Leu Ser Asp Pro Val Asp Leu

370 375 380

Gly Val Ile Ser Asp Trp Leu Leu Leu Gln Thr Pro Gln Arg Val Phe

385 390 395 400

Leu Glu Gly Glu Thr Ile Thr Leu Arg Cys His Ser Trp Arg Asn Lys

405 410 415

Leu Leu Asn Arg Ile Ser Phe Phe His Asn Glu Lys Ser Val Arg Tyr

420 425 430

His His Tyr Lys Ser Asn Phe Ser Ile Pro Lys Ala Asn His Ser His

435 440 445

Ser Gly Asp Tyr Tyr Cys Lys Gly Ser Leu Gly Ser Thr Gln His Gln

450 455 460

Ser Lys Pro Val Thr Ile Thr Val Gln Asp Pro Ala Thr Thr Ser Ser

465 470 475 480

Ile Ser Leu Val Trp Tyr His Thr Ala Phe Ser Leu Val Met Cys Leu

485 490 495

Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Tyr Val Arg Arg Asn Leu

500 505 510

Gln Thr Pro Arg Asp Tyr Trp Arg Lys Ser Leu Ser Ile Arg Lys His

515 520 525

Gln Ala Pro Gln Asp Lys

530

<210> 5

<211> 638

<212> PRT

<213> Artificial Sequence

<400> 5

Met Glu Arg Trp Leu Phe Leu Gly Ala Thr Glu Glu Gly Pro Lys Arg

1 5 10 15

Thr Met Asp Ser Gly Thr Arg Pro Val Gly Ser Cys Cys Ser Ser Pro

20 25 30

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

35 40 45

Val Gly Lys Ser Ala Met Thr Met Gln Phe Ile Ser His Arg Phe Pro

50 55 60

Glu Asp His Asp Pro Thr Ile Glu Asp Ala Tyr Lys Ile Arg Ile Arg

65 70 75 80

Ile Asp Asp Glu Pro Ala Asn Leu Asp Ile Leu Asp Thr Ala Gly Gln

85 90 95

Ala Glu Phe Thr Ala Met Arg Asp Gln Tyr Met Arg Ala Gly Glu Gly

100 105 110

Phe Ile Ile Cys Tyr Ser Ile Thr Asp Arg Arg Ser Phe His Glu Val

115 120 125

Arg Glu Phe Lys Gln Leu Ile Tyr Arg Val Arg Arg Thr Asp Asp Thr

130 135 140

Pro Val Val Leu Val Gly Asn Lys Ser Asp Leu Lys Gln Leu Arg Gln

145 150 155 160

Val Thr Lys Glu Glu Gly Leu Ala Leu Ala Arg Glu Phe Ser Cys Pro

165 170 175

Phe Phe Glu Thr Ser Ala Ala Tyr Arg Tyr Tyr Ile Asp Asp Val Phe

180 185 190

His Ala Leu Val Arg Glu Ile Arg Arg Lys Glu Lys Glu Ala Val Leu

195 200 205

Ala Met Glu Lys Lys Lys Ser Lys Pro Lys Asn Ser Val Trp Lys Arg Leu

210 215 220

Lys Ser Pro Phe Arg Lys Lys Lys Lys Asp Ser Val Thr Met Asp Ser Gly

225 230 235 240

Thr Arg Pro Val Gly Ser Cys Cys Ser Ser Pro Ala Gly Leu Ser Arg

245 250 255

Glu Tyr Lys Leu Val Met Leu Gly Ala Gly Gly Val Gly Lys Ser Ala

260 265 270

Met Thr Met Gln Phe Ile Ser His Arg Phe Pro Glu Asp His Asp Pro

275 280 285

Thr Ile Glu Asp Ala Tyr Lys Ile Arg Ile Arg Ile Asp Asp Glu Pro

290 295 300

Ala Asn Leu Asp Ile Leu Asp Thr Ala Gly Gln Ala Glu Phe Thr Ala

305 310 315 320

Met Arg Asp Gln Tyr Met Arg Ala Gly Glu Gly Phe Ile Ile Cys Tyr

325 330 335

Ser Ile Thr Asp Arg Arg Ser Phe His Glu Val Arg Glu Phe Lys Gln

340 345 350

Leu Ile Tyr Arg Val Arg Arg Thr Asp Asp Thr Pro Val Val Leu Val

355 360 365

Gly Asn Lys Ser Asp Leu Lys Gln Leu Arg Gln Val Thr Lys Glu Glu

370 375 380

Gly Leu Ala Leu Ala Arg Glu Phe Ser Cys Pro Phe Phe Glu Thr Ser

385 390 395 400

Ala Ala Tyr Arg Tyr Tyr Ile Asp Asp Val Phe His Ala Leu Val Arg

405 410 415

Glu Ile Arg Arg Lys Glu Lys Glu Ala Val Leu Ala Met Glu Lys Lys

420 425 430

Ser Lys Pro Lys Asn Ser Val Trp Lys Arg Leu Lys Ser Pro Phe Arg

435 440 445

Lys Lys Lys Asp Ser Val Thr Met Thr Met Gln Phe Ile Ser His Arg

450 455 460

Phe Pro Glu Asp His Asp Pro Thr Ile Glu Asp Ala Tyr Lys Ile Arg

465 470 475 480

Ile Arg Ile Asp Asp Glu Pro Ala Asn Leu Asp Ile Leu Asp Thr Ala

485 490 495

Gly Gln Ala Glu Phe Thr Ala Met Arg Asp Gln Tyr Met Arg Ala Gly

500 505 510

Glu Gly Phe Ile Ile Cys Tyr Ser Ile Thr Asp Arg Arg Ser Phe His

515 520 525

Glu Val Arg Glu Phe Lys Gln Leu Ile Tyr Arg Val Arg Arg Thr Asp

530 535 540

Asp Thr Pro Val Val Leu Val Gly Asn Lys Ser Asp Leu Lys Gln Leu

545 550 555 560

Arg Gln Val Thr Lys Glu Glu Gly Leu Ala Leu Ala Arg Glu Phe Ser

565 570 575

Cys Pro Phe Phe Glu Thr Ser Ala Ala Tyr Arg Tyr Tyr Ile Asp Asp

580 585 590

Val Phe His Ala Leu Val Arg Glu Ile Arg Arg Lys Glu Lys Glu Ala

595 600 605

Val Leu Ala Met Glu Lys Lys Ser Lys Pro Lys Asn Ser Val Trp Lys

610 615 620

Arg Leu Lys Ser Pro Phe Arg Lys Lys Lys Asp Ser Val Thr

625 630 635

Claims (8)

1. An acid-sensitive nano peptide segment targeting a tumor is characterized by comprising a low-pH nano insertion peptide, a coupling agent and an Fc segment which are sequentially connected in series;

wherein the low pH nano-insertion peptide comprises an amino acid sequence having the sequence of SEQ ID No.1 or a variant thereof.

2. The tumor-targeting acid-sensitive nanopeptide segment of claim 1, wherein the Fc fragment is selected from the group consisting of murine IgG2a and IgG2b, wherein the amino acid fragment selected from IgG2a is represented by SEQ ID No.2 and the amino acid fragment selected from IgG2b is represented by SEQ ID No. 3.

3. The tumor-targeting acid-sensitive nanopeptide segment of claim 1, wherein the coupling agent is selected from the group consisting of sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate.

4. The tumor-targeting acid-sensitive nanopeptide segment of claim 1, wherein the tumor-targeting acid-sensitive nanopeptide segment functions in an environment with a pH < 7.

5. A tumor marker system comprising the tumor-targeting acid-sensitive nanopeptide segment of any one of claims 1-4.

6. A targeted tumor therapy system comprising the tumor marker system of claim 5.

7. Use of the tumor-targeting acid-sensitive nanopeptide segment of claim 1 in the preparation of the tumor labeling system of claim 5.

8. Use of the tumor-targeting acid-sensitive nanopeptide segment of claim 1 or the tumor labeling system of claim 5 in the preparation of the tumor-targeting therapeutic system of claim 6.

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