CN111285936A

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

Info

Publication number: CN111285936A
Application number: CN202010165168.3A
Authority: CN
Inventors: 秦志海; 陈波; 陶宁; 李建茹; 李香群
Original assignee: Beijing Win Win Technology Co Ltd; Institute of Biophysics of CAS
Current assignee: Beijing Win Win Technology Co Ltd; Institute of Biophysics of CAS
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2020-06-16

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 nano peptide segment of targeted tumor and application thereof

Technical Field

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

Background

NK cells can exert their immune killing effect without primary immune stimulation and gene editing in a MHC-independent manner, including three ways, namely, ① NK cell-released killing mediators perforin and granzyme B to cause target cell apoptosis, the process requires direct contact between NK cell recognition receptor and target cell, ② NK cell can induce target cell apoptosis by binding membrane TNF family molecules (FasL, TRIAL, mTNF and the like) with target cell membrane ligand, the process does not require direct contact between NK cell receptor and target cell, ③ NK cell can also use anti-tumor antibodies IgG1 and IgG3 as a bridge, Fab segment of which binds to NK cell FcRIMA to generate antibody-dependent cell-mediated cytotoxicity (CD 16), and the function is mainly achieved by using CD16⁺NK cells were completed.

The tumor microenvironment is composed of tumor cells, a variety of stromal cells, and a range of cytokines and chemokines. Wherein the stromal cells comprise fibroblasts, tumor-infiltrating immune cells, endothelial cells, bone marrow-derived immature cells and the like; cytokines such as TNF, VEGF, IL-1, and the like; chemokines include CXCL12, CCL27, CCL21 and the like. Cytokines and chemokines can be secreted by tumor cells and can also be produced by stromal cells and the infiltrating immune cells described above. These cells and active mediators together form a stable tumor immune microenvironment, protecting tumor tissues from immune surveillance by the body and promoting tumor progression. The strong heterogeneity and proliferation ability of tumor cells, and the high mutation rate during their growth make it difficult to find specific tumor antigens that are stably expressed on multiple tumors or between different patients of the same tumor type during tumor therapy.

The tumor microenvironment has the trait of hypoxia and acidification, which are important causes for the progression from benign tumors to metastasized malignant tumors. Wherein acidification has three effects on tumor development: i.e., increased tolerance to chemotherapy, increased mutation rate, and increased invasiveness.

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

Low pH insertion peptides (PHLIPs) can undergo conformational transition from random coil to α helix in the acidic environment of the tumor microenvironment, and this conformational transition can allow insertion of themselves into the cell membrane of the tumor, with the C-terminus inside the cell and the N-terminus outside the cell.

For tumor and cancer treatment, the early treatment is selected by using a mode of radiotherapy or chemotherapy (paclitaxel and the like) by surgical removal; later on, targeted therapies were initiated to target tumor-specific targets, such as avastin, the first anti-tumor angiogenesis drug approved by FDA in 2004 in the united states, and oxitinib, the third-generation lung cancer drug in 2015; recent years have seen the development of treatments for tumors that regulate human immunity, and in 2018 nobel medical awarded james in the united states, illison and japan's own surmise, to highlight their pioneering work on cancer immunotherapy (the discovery of CTLA-4, PD-1). And CAR-T technology applied to the treatment of hematological neoplasms, but the final success was not as satisfactory for a variety of reasons, so we chose NK cells that are more potent 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 skilled in the art.

Disclosure of Invention

The invention aims to provide an acid-sensitive nano peptide segment targeting a tumor and application thereof, the acid-sensitive nano peptide segment targeting the tumor inserts a specific marker into tumor cells and other stromal cells in a microenvironment by utilizing the characteristic that the pH of the microenvironment of the tumor is less than 7 micro-acid environment, the specific marker can be attracted and combined onto CD16 molecules of NK cells, the ADCC function of the NK cells is recruited and activated by the specificity of the marker, and the killing function of the tumor cells is achieved.

In order to achieve the above object, the present invention provides an acidic sensitive nanopeptide segment for targeting tumor, comprising a low pH nanopeptide insert (pHLIP), a coupling agent, and an Fc fragment, which are sequentially connected in series; the low-pH nano insertion peptide comprises an amino acid sequence with a sequence of SEQ ID NO.1 or a variant thereof, and can be inserted into a tumor cell membrane by self-curling under a slightly acidic environment to play a targeting role.

In one embodiment of the invention, the Fc fragment is selected from the amino acid fragments of murine IgG2a or IgG2b, wherein IgG2a is selected from amino acid sequences 93-330 as shown in SEQ ID NO.2 and IgG2b is selected from amino acid sequences 97-335 as shown in SEQ ID NO. 3.

In one embodiment of the invention, the coupling agent is selected from the group consisting of sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) to link the low pH nano-insertion peptide to the ADCC-inducing Fc fragment.

In one embodiment of the present invention, the tumor-targeting acid-sensitive nanopeptide segment functions in an environment with a pH < 7.

The invention also provides a tumor marking system, which comprises the tumor-targeted acid-sensitive nano peptide segment.

The invention also provides a targeted tumor treatment system, which comprises the tumor marker system.

The invention also provides the application of the tumor-targeted acid-sensitive nano peptide segment in the preparation of the tumor labeling system.

The invention also provides the application of the acid sensitive nano peptide segment of the targeted tumor or the tumor marking system in the preparation of the targeted tumor treatment system.

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

(1) the acidic sensitive nano peptide segment of the target tumor mainly comprises three parts: the combination of the low-pH nano insertion peptide pHLIP, the coupling agent sulfo-SMCC and the Fc fragment can play a role respectively and can be coordinated with each other, so that effective and safe micro-environment insertion molecules are provided;

(2) the invention adopts the advanced nano peptide segment technology, reasonably utilizes the tumor hypoxia and acidification characteristics which originally affect the development and treatment effect of the tumor, enables the tumor hypoxia and acidification characteristics to become the target conditions for treatment, can insert the target molecule specificity into the cells of the leukemia bone marrow tumor microenvironment, provides a target for the tumor with specificity, and provides a target for target killing and treatment, thereby attracting immune cells (NK cells) to attack and kill in a targeted way;

(3) the acid sensitive nanometer peptide segment of the targeting tumor kills and treats the tumor by mobilizing the autoimmune system, and the method can be suitable for treating all tumors including solid tumors, different from the prior tumor targeting killing.

Drawings

FIG. 1A is a schematic illustration of a low pH nano-insertion peptide linked to an antibody via a coupling agent, according to one embodiment of the present invention;

FIG. 1B is a schematic diagram of a low pH nano-insertion peptide self-frizzling at low pH for insertion into a membrane surface according to an embodiment of the present invention;

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

FIG. 3 is a schematic illustration of tumor killing according to an embodiment of the present invention;

FIG. 4 is a time-of-flight mass spectrum of ionization using matrix assisted laser desorption in accordance with an embodiment of the present invention;

FIG. 5A is a graph of pHLIP-Fc mediated killing of melanoma cells (B16) by NK cells in a slightly acidic environment, according to one embodiment of the present invention;

FIG. 5B is a graph of pHLIP-Fc mediated killing of mouse breast cancer cells (4T1) by NK cells in a slightly acidic environment, according to one embodiment of the present invention;

FIG. 5C is a graph of pHLIP-Fc mediated killing of NK cells on human triple negative breast cancer in a slightly acidic environment, according to one embodiment of the present invention;

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

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

Detailed Description

The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the 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.

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The low pH nano-insert peptide pHLIP is synthesized by Shanghai Tanpu Biotech Co., Ltd, and the Fc fragment of mouse-derived IgG2a/b was purchased from Beijing Yi Qiao Shenzhou Tech Co., Ltd, and has the following sequence:

the sequence of the low-pH nano insertion peptide pHLIP is SEQ ID NO.1 acetylated at the N end;

the Fc fragment sequence of IgG2a from a murine source is shown as SEQ ID NO.2 from N-terminal to C-terminal;

the Fc fragment sequence of IgG2b from a murine source is shown as SEQ ID NO.3 from N-terminal to C-terminal;

the sequence of the human-derived CD16 protein is shown as SEQ ID NO. 4; the sequence of human Rit (anti-CD 20) protein is shown in SEQ ID NO. 5.

The tumor-targeting acid-sensitive nanopeptide segment of the present invention is described in detail in the following by way of preferred embodiments in terms of structural recombination, cell membrane insertion ability, binding ability to human CD16 protein, in vitro NK cell killing assay detection effect, and animal experiments.

Example 1

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

1.1 linking pHLIP to the coupling agent sulfo-SMCC

4800nmolsulfo-SMCC was dissolved in 300. mu.L PBS and 10nmol of Fc fragment was dissolved in 200. mu.L PBS, and the two solutions were mixed into a 1.5mL Eppendorf centrifuge tube and gently shaken at room temperature for 2 hours, with the pH being controlled at 7.4 throughout the procedure. The ligation product was then purified on a column of NAP-5(GE Healthcare, UK) pre-equilibrated with PBS buffer to give an Fc ligation solution.

1.2 ligation of the ligated Fc fragment to pHLIP

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

1.3 molecular masses of Fc/a, pHLIP-Fc/a, Fc/b, pHLIP-Fc/b were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (FIG. 4). As can be seen from FIG. 4, the m/z of the Fc/a fragment is 56780.7, and the m/z value is right-biased after pHLIP is ligated, which indicates that successful coupling of pHLIP to the Fc/a fragment is achieved; the Fc/b fragment showed a similar trend.

1.4 for labeling FITC or Cy5.5 to CD16 molecules, 100nmol FITC (Van Bo Biochemical Co., Ltd., China) was added to a sufficient solution of human-derived CD16(Sino Biological Inc., China) in CD16 by dissolving 10nmol CD16 molecules in 300. mu.L PBS buffer. The mixture was incubated in an Eppendorf centrifuge tube at room temperature with gentle shaking for 3 hours. The final product was purified by passing through a NAP-5 column pre-equilibrated with PBS buffer.

1.5 detection of the modification of the Fc molecule by sulfo-SMCC by tandem mass spectrometry (MS/MS). To follow the binding site of the sulfo-SMCC modification, SMCC-Fc/a and SMCC-Fc/b were digested with trypsin (Promage, USA). The digested peptides were analyzed by the nano-LC-LTQ-Orbitrap XL MS/MS system. MS data were analyzed by the Proteome discover software (version 1.4.0.288, Thermo Fischer Scientific). The second MS spectrum is searched in the Mus database using a sequence search engine. The sulfo-SMCC modification of lysine, glutathionylation or oxidation of cysteine and oxidation of methionine are set as variable modifications. 1.6 detection and calculation of pHLIP-Fc/a and pHLIP-Fc/b by Surface Plasmon Resonance Imaging (SPRi) detection applications. The binding affinity of Fc or pHLIP-Fc molecules to human recombinant CD16 (nano Biological Inc, china) and the binding affinity of Rit or pHLIP-Rit to human recombinant CD16a (nano Biological Inc, china) were measured using Biacore 8K SPR biosensor (GEHealthcare). All of the above human CD16 and human CD16a were attached to the CM5 sensor chip surface according to the manual instructions. All ligand proteins were spotted in PBST buffer (pH 6.8) in the range of 1000nM to 31.25nM at successive 2-fold dilutions. The flow rate was 30. mu.L/min and the buffer was PBST (0.01mM PBS plus 0.05% Tween 20). The contact and dissociation times were 60 seconds and 120 seconds, respectively. Regeneration of the protein chips was performed after 5mM NaOH for each sample. The above cycle was repeated for each concentration of Fc, pHLIP-Fc, Rit and pHLIP-Rit. Binding reactions were recorded continuously as Resonance Units (RU) and background binding was subtracted automatically. The BIAcore 8K evaluation software (GE Healthcare) was used with a 1: the 1Langmuir binding model calculates the affinity constant (KD).

Example 2

Cell transformation and detection of transformation conditions

2.1 recombinant pHLIP-Fc/a and pHLIP-Fc/b were transformed 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). Culturing in RPMI-1640 cell culture medium, adding 10% FBS (WISENT, Canada), and culturing at 37 deg.C and 5% CO₂A cell culture box.

2.1.2 cells were cultured on 35mm borosilicate chamber cover glass (Nunc, USA), then pHLIP-Fc/a was co-cultured with pHLIP-Fc/B and B16/F10 cells at pH 6.8 and 7.4 for 4 hours, respectively, and Cy5.5-labeled human recombinant CD16 molecules were added and co-cultured for 1 hour. During this process the pH of the solution is kept separate.

2.1.3 cells on coverslips were nuclear stained with HOECHST 33342(Invitrogen, USA) and observed using a Zeiss LSM710 confocal microscope (Carl Zeiss, Germany).

2.1.4K562 are human chronic myelogenous leukemia cells, purchased from the national scientific and technological cell experimental platform (China). Culturing in RPMI-1640 cell culture medium, adding 10% FBS (WISENT, Canada), and culturing at 37 deg.C and 5% CO₂A cell culture box.

2.1.5MDA-MB-231 is a human breast cancer cell line and purchased from the national science and technology cell experiment platform (China). Culturing in DMEM cell culture medium, adding 10% FBS (WISENT, Canada), and culturing at 37 deg.C and 5% CO₂A cell culture box.

Rit and pHLIP-Rit were added to MDA-MB-231 cell culture system for 4 hours under a cell culture environment at pH 6.8, respectively, and Cy5.5-labeled recombinant CD16 was added to the culture system and cultured for 1 hour.

The cells were then stained with HOECHST 33342 according to the same procedure and observed with a confocal microscope, while maintaining a pH of 6.8.

2.2 flow cytometry concentration-dependent kinetic Studies

2.2.1 different concentrations (0, 0.1, 0.5, 1, 2.5, 5, 10. mu.g/mL) of pHLIP-Fc/a or pHLIP-Fc/b were added to 24-well plates at 1X 10 per well⁵B16/F10 cells were cultured in 400 μ L of pH 6.8 medium for 5 hours.

2.2.2 cells were washed 3 times with PBS pH 6.8.

2.2.3 Induction by replacement with 20. mu.g/mL FITC-labeled recombinant human CD16 (400. mu.L) was performed for 1 hour.

2.2.4 cells were washed 3 times with PBS pH 6.8.

2.2.5 resuspension with PBS pH 6.8 was used for flow cytometry, 3 times for each sample.

2.3 flow cytometry time-dependent kinetic Studies

2.3.1 pHLIP-Fc/a or pHLIP-Fc/b was added to a 24-well cell culture dish at pH 6.8 at the same concentration (2.5. mu.g/mL) and the dish was plated at 1X 10 cells per well⁵A400. mu.L culture system of B16/F10 cells was used for different periods of time (5, 10, 30, 60, 120, 240 and 480 minutes).

2.3.2 cells were washed 3 times with PBS pH 6.8.

2.3.3 Induction by exchange with 20. mu.g/mL FITC-labeled recombinant human CD16 (400. mu.L) was performed for 1 hour.

2.3.4 cells were washed 3 times with PBS pH 6.8.

2.3.5 resuspension with PBS pH 6.8 was used for flow cytometry, 3 times for each sample.

Example 3

In vitro cytotoxicity assays

3.1 according to CytoTox-Glo^TMADCC function was tested using the instructions of the cytotoxicity assay kit (Promega, Madison, Wis., USA).

3.2 peripheral blood isolation kit (Miltenyi, Auburn, Canada) is used to isolate NK cells of mouse spleen, and the isolation purity reaches more than 90%. These NK cells can act as effector cells, B16/F10 tumor cells as target cells.

3.3 mix 2X 10⁴A B16/F10 tumor cell line was cultured in a 96-well plate and cultured with 100. mu.L pHLIP-Fc (2.5. mu.g/mL) (pH 6.8 or 7.4) for 2-4 hours

3.4 following effector cell: target cells ═ 1: 1 ratio of 2X 10⁴The individual NK cells were added to the culture system for induction culture for 4 hours.

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

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

3.7 mix 2X 10⁴MDA-MB-231 cells were plated in 96-well plates and incubated with 100. mu.L pHLIP-Rit (2.5. mu.g/mL) (pH 6.8 or 7.4) for 2-4 hours.

3.8 following effector cell: target cells ═ 1: 1 ratio of 2X 10⁴Individual NK cells were added to the culture system to induce culture for 4 hours.

The pHLIP-Fc molecule was incubated with tumor cells under slightly acidic and neutral conditions, respectively, followed by addition of NK cells. In a slightly acidic environment, pHLIP-Fc was able to effectively mediate NK cell killing of melanoma cells (B16) (FIG. 5A). In a slightly acidic environment, pHLIP-Fc could effectively mediate NK cell killing of mouse breast cancer cells (4T1) (FIG. 5B). In addition, research teams prove that pHLIP-Rit can induce human NK cells to kill human triple negative breast cancer (figure 5C), and the Fc or antibody modified by pHLIP has better clinical application prospect.

Example 4 in vivo animal experiments and assays

4.1 animal Experimental study and immunohistochemistry

4.1.1 in situ mouse model for B16/F10 melanoma, treatment was initiated 4 days after inoculation when tumor volume reached about 50mm³. The day of the first treatment was defined as day 0. By usingPBS, Fc/a + pHLIP, pHLIP-Fc/a, pHLIP-Fc/b or pHLIP-IgG3(n 6) were administered to (i.v.) mice. The therapeutic dose (similar molar dose) of pHLIP-Fc refers to the dose of rituximab (Rit) (500. mu.g/kg) used in the mouse model. The calculation method is as follows:

molecular weight (Mw) of Rit: -150 kDa; the Mw of pHLIP-Fc is about 60 kDa;

molar dose of Rit ═ (dose of Rit)/(Mw of Rit) ═ 500 μ g/kg/150kDa ═ 3.33nmol/kg

4.1.2A-2.5 nmol/kg molar dose (equal to 150. mu.g/kg) of pHLIP-Fc was chosen. pHLIP-Fc was administered every 2 days for a total of 4 injections.

Tumor size was measured by digital calipers and by the formula (L × W)²) The tumor volume was calculated as/2, where L is the longest and W is 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 before treatment began. For ethical reasons, when the implanted tumor volume reaches 1000mm³At that time, the animals were sacrificed.

For the 4T1 breast cancer model and the K562 leukemia model, treatment was started 5 days after implantation, when the tumor volume reached about 50mm³. The day of the first treatment was defined as day 0. Other conditions were identical to model B16/F10, 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 μ g/kg dose every 2 days for a total of 7 injections) with in vivo imaging by intraperitoneal injection of luciferase (n-3 per group); lungs from tumor bearing animals were dissected for tissue sections (n-3 per group).

4.1.3 in dose-dependent therapeutic efficacy assessment, 50 μ g/kg, 150 μ g/kg and 450 μ g/kg of pHLIP-Fc/a were tested in the K562 bone marrow tumor model (n ═ 6), and the other steps were the same as described 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) in the manner described in the instructions. A total of 100 nuclei on two separate slides were examined to obtain quantitative results.

4.1.5 for immunohistochemical staining, tumor sections (6 μm) were deparaffinized, rehydrated, and incubated with 0.3% H₂O₂Is incubated with the methanol solution of (3). After antigen retrieval at 95 ℃ for 15 min in 10mmol/L citrate buffer (pH6.0), sections were blocked with 5% goat serum/PBS for 1 h and incubated with primary antibodies (mouse monoclonal antibody vs. Proliferating Cell Nuclear Antigen (PCNA)) antibodies (1: 10,000, Abcam, UK) or rabbit polyclonal anti-CRTAM antibodies (1: 300, Abcam, UK) overnight at 4 ℃ followed by biotinylation of the secondary antibody for 1 h at room temperature and horseradish peroxidase conjugated streptavidin for 30 min at 37 ℃. DAB (R)&D system, USA) was used for visualization and sections were counterstained with hematoxylin. Data was analyzed by ImageJ.

As can be seen from FIGS. 6A and 6B, in vivo experiments, the pHLIP-Fc molecule achieved inhibition of various tumors (subcutaneous melanoma, in situ breast cancer).

4.2 blood detection, in vivo imaging and biodistribution detection

4.2.1 for Cy 7-labeled Fc/a and pHLIP-Fc/a couplings, we followed our previous work procedure. Cy7Mono NHS (Fanbobiochemicals, Beijing, China)0.1mg was added to Fc/a or pHLIP-Fc/PBS solution (100. mu.L, 0.5mg/mL, pH 7.4). The mixture was incubated in a 1.0ml Leppendorf tube at room temperature for 3 hours while gently shaking. The conjugated Fc/a and pHLIP-Fc/a were purified by passing through a NAP-5 column (GE Healthcare, UK) pre-equilibrated with PBS buffer.

4.2.2 to evaluate the 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 by an In Vivo Imaging System (IVIS) (PerkinElmer inc., USA) and fluorescence signals were detected. The wavelength of the excitation light is 740-760nm, and the emission wavelength is 780 nm.

4.2.3 to examine in vivo imaging and biodistribution of each formulation, nude mice bearing subcutaneous 4T1 tumor were injected intravenously with Fc-Cy7 and pHLIP-F/a-Cy7, respectively. 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. mu.g/kg) for 4 weeks (once a week). At week 5, whole blood was collected from these mice by retroorbital venipuncture and serum was collected after centrifugation. According to previous studies, anti-pHLIPOG titers were measured using an indirect ELISA assay. The absorbance was measured on a microplate reader at OD 450.

4.3.2 to assess the immune response of pHLIP-Fc, two groups of healthy BALB/c mice (female, n-6 per group) were pretreated with pHLIP-Fc/a for 4 weeks (150 μ g/kg, weekly injections)) before tumor implantation, and then treated in the same manner in a 4T1 breast tumor model (one group treated with PBS and the other with pHLIP-Fc/a). In the first 4 weeks, the control group (non-pretreated group) was dosed with PBS.

4.4 Biosafety assays

4.4.1 healthy BALB/c mice (female, 6 weeks old, 15-17g body weight) (iv)8 groups were given PBS, Fc/a + pHLIP, pHLIP-Fc/a, pHLIP-Fc/b or pHLIP-IgG3 (n). Every two days a dose of 50. mu.g/kg, for a total of 7 injections. Sera were collected for biochemical testing (n ═ 4) and organs (heart, liver, spleen, lung and kidney) were fixed, sectioned and H & E stained to investigate potential changes in organ morphology (n ═ 4) sections from the same organ from different groups.

4.4.2 to analyze acute immune responses after each formulation injection, healthy BALB/c mice (female, 6 weeks old, 15-17g body weight) (iv), pHLIP-Fc/b or pHLIP-IgG3(n ═ 4) (dose: 150. mu.g/kg), increased levels of interleukin-6 (IL-6) and tumor necrosis factor- β were tested with PBS, Fc/a + pHLIP, pHLIP-Fc/a (iv), pHLIP-Fc/b or pHLIP-IgG3(n ═ 4) (dose: 150. mu.g/kg), after 6 hours and 24 hours (TNF- α) in serum was measured with ELISA kits (EM1350 and 03EM 50, Solarbio, China).

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.

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Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys

195 200 205

Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu

210 215 220

Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Leu

225 230 235

<210>4

<211>534

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Met Thr Leu Asp Thr Gln Met Phe Gln Asn Ala His Ser Gly Ser Gln

1 5 10 15

Trp Leu Leu Pro Pro Leu Thr Ile Leu Leu Leu Phe Ala Phe Ala Asp

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

210 215 220

Lys Ser Pro Phe Arg 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

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.