CD93 affinity peptide and application thereof

Abstract

The invention belongs to the technical field of protein polypeptides, and particularly discloses a CD93 affinity peptide and application thereof in preparation of antitumor drugs or detection reagents. The polypeptide with a CD93 protein affinity effect is obtained through repeated screening and optimization, and an affinity blocking activity experiment proves that the CD93 affinity peptide can affine an extracellular IgV-like structural domain of the CD93 protein and block the interaction of CD93 / IGFBP7 protein. An HUVECs cell in-vitro migration experiment and a tube formation experiment verify that the CD93 affinity peptide can inhibit migration of HUVECs cells and tube formation, promote normalization of tumor vascular functions and further inhibit growth of tumors, and has no obvious toxic or side effect. The CD93 affinity peptide provided by the invention can be used for preparing anti-tumor drugs (including anti-tumor immunotherapy drugs) or detection reagents taking CD93 as a target spot, has a better medical application prospect, and provides a new choice for tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of protein and polypeptide technology, specifically relating to a polypeptide with CD93 protein affinity and its application in the preparation of antitumor drugs or detection reagents. Background Technology

[0002] Tumor angiogenesis, as a core regulatory component of the tumor microenvironment, plays a crucial role in promoting tumor progression and metastasis, and is therefore considered a highly promising therapeutic target. Under continuous stimulation by vascular endothelial growth factor (VEGF), tumor tissue forms an abnormal vascular network with significant pathological characteristics. These vessels exhibit marked structural disorder, endothelial barrier dysfunction, and loss of pericyte coverage. This pathological vascular network not only leads to microcirculatory disturbances and hypoxia in tumor tissue, hindering the infiltration and effector function of immune cells, but also significantly enhances the metastatic potential of tumor cells. In response to these pathological changes, the "tumor angiogenesis normalization" treatment strategy has emerged. This strategy improves blood perfusion and endothelial barrier integrity in tumor tissue by reshaping vascular structure and function, thereby effectively enhancing the anti-tumor immune response and opening new avenues for tumor treatment.

[0003] CD93, a key regulatory molecule for tumor angiogenesis, possesses pleiotropic biological functions in the tumor microenvironment. Primarily located on the surface of vascular endothelial cells, it plays a crucial role in regulating angiogenesis, maintaining vascular structural stability, and modulating endothelial permeability. Clinicopathological analysis shows a significant positive correlation between CD93 overexpression and poor prognosis in various malignant tumors, resistance to immune checkpoint inhibitor therapy, and TNM stage progression. Molecular mechanism studies indicate that CD93, upon binding to its specific ligand, can activate downstream signal transduction pathways, inducing pathological tumor angiogenesis, leading to the formation of a hypoxic tumor microenvironment and immunosuppression. Blocking the CD93 pathway with monoclonal antibodies can effectively promote tumor angiogenesis, improve vascular permeability, and enhance the infiltration of effector T cells within the tumor, thereby improving the anti-tumor immune response. For example, Chinese patent CN113784983B (Stanford University) discloses an antibody or chimeric antigen receptor polypeptide containing a human CD93-specific antigen-binding domain (ABD). The ABD is composed of a heavy chain variable region (VH) domain and a light chain variable region (VL) domain defined by a specific sequence, and is mainly used for the treatment of cancer, especially malignant hematological diseases.

[0004] Previous studies have reported that IGFBP7, as an important functional ligand of CD93, exhibits significant high expression in the tumor vascular system. This molecule, through its specific binding to CD93, activates downstream signaling cascades, thereby promoting pathological changes in tumor angiogenesis, including abnormal proliferation and functional disorders. Molecular biological studies have shown that targeting and blocking the CD93-IGFBP7 protein interaction can significantly improve the pathological features of tumor angiogenesis, including: 1) promoting vascular remodeling; 2) enhancing vascular barrier function; and 3) improving blood perfusion. This vascular normalization effect can effectively promote tumor infiltration by various immune effector cells (such as cytotoxic T cells and natural killer cells), providing a theoretical basis for developing novel combined immunotherapy strategies.

[0005] Currently, peptide drugs exhibit unique therapeutic advantages in the field of cancer treatment. By specifically blocking key receptor-ligand interactions, they can effectively inhibit tumor growth and metastasis. As a promising class of therapeutic agents, peptide drugs have the following prominent characteristics: 1) excellent targeting binding ability and tumor tissue penetration; 2) highly specific mechanism of action, which can significantly reduce off-target effects; 3) low immunogenicity, improving the safety of clinical use; 4) mature synthesis processes and controllable production costs, which are conducive to large-scale production. Furthermore, peptide molecules are easy to modify structurally and optimize functionally, and their pharmacological properties can be further enhanced through amino acid sequence adjustments and chemical modifications. These advantages make them an important choice for modern targeted cancer therapy. However, current research lacks screening and structural optimization of peptides that block the CD93 pathway. Summary of the Invention

[0006] The main technical problem solved by this invention is to provide a CD93 affinity peptide that can bind to CD93 protein and block the interaction between CD93 and its ligand IGFBP-7, thereby promoting the normalization of tumor vascular function.

[0007] Meanwhile, this invention provides the application of CD93 affinity peptide in the preparation of antitumor drugs or detection reagents.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0009] A CD93 affinity peptide selected from any one or more of the following sequences:

[0010] (1) The amino acid sequence as shown in SEQ ID NO: 1;

[0011] (2) A truncated sequence of the amino acid sequence as shown in SEQ ID NO: 1;

[0012] (3) The reverse sequence of the truncated amino acid sequence as shown in SEQ ID NO: 1.

[0013] In a preferred embodiment of the present invention, the conformation of each amino acid in the CD93 affinity peptide is independently selected from either the L-configuration or the D-configuration. For example, the amino acid sequence shown in SEQ ID NO: 1 is the parent peptide CBP-1 (KWIFSPL), in which all amino acids are in the L-configuration. Uppercase single letters represent L-configuration amino acids, and lowercase single letters represent D-configuration amino acids.

[0014] In a preferred embodiment of the present invention, the truncated sequence of the amino acid sequence shown in SEQ ID NO: 1 is a polypeptide of the parent peptide CBP-1 truncated amino acid by amino acid from the C-terminus or N-terminus, including but not limited to the following truncated peptides:

[0015] CBP-C1 truncated peptide: KWIFSP;

[0016] CBP-C2 truncated peptide: KWIFS;

[0017] CBP-C3 truncated peptide: KWIF;

[0018] CBP-N1 truncated peptide: WIFSPL;

[0019] CBP-N2 truncated peptide: IFSPL;

[0020] All amino acids in the truncated peptide are in the L configuration.

[0021] In a preferred embodiment of the present invention, one or more L-configured amino acids in the truncated peptide CBP-C2 (KWIFS) are replaced with D-configured polypeptides, including but not limited to the following modified peptides:

[0022] Modified peptide CBP-D1:kWIFs;

[0023] Modified peptide CBP-D2: kWIfs;

[0024] Modified peptide CBP-D3:kWifs;

[0025] Modified peptide CBP-D4: kwIFs;

[0026] Modified peptide CBP-D5: kwIfs;

[0027] Modified peptide CBP-D6: kwifs;

[0028] Modified peptide CBP-D7: kwiFs;

[0029] Uppercase letters represent amino acids with the L configuration, and lowercase letters represent amino acids with the D configuration.

[0030] As a preferred embodiment of the present invention, the reverse sequence of the truncated sequence of the amino acid sequence shown in SEQ ID NO: 1 is a modified peptide CBP-D8 (sfIwk, also known as a reverse reverse polypeptide), and all amino acids in the sequence are in the D configuration.

[0031] Applications of a CD93 affinity peptide include, but are not limited to, one or more of the following:

[0032] (1) Application in the preparation of reagents that block the binding of CD93 protein to its ligand IGFBP-7;

[0033] (2) Application in the preparation of antitumor drugs.

[0034] As a preferred embodiment of the present invention, the application in the preparation of reagents that block the binding of CD93 protein to its ligand IGFBP-7 includes: labeling the CD93 affinity peptide (including but not limited to fluorescein, radionuclide, etc.) using labeling or conjugation methods known in the art to achieve imaging and tracing of the CD93 protein. The CD93 protein includes, but is not limited to, mammalian CD93 protein, such as human or mouse CD93 protein, and can be wild-type or a mutant protein retaining its activity.

[0035] As a preferred embodiment of the present invention, the anti-tumor effect includes, but is not limited to, promoting the normalization of tumor vascular function, improving the hypoxia of the tumor site, reducing tumor volume, and reducing tumor weight.

[0036] Specifically, the CD93 affinity peptide can be prepared by solid-phase synthesis, such as using the standard Fmoc synthesis method.

[0037] A reagent containing CD93 affinity peptide.

[0038] In a preferred embodiment of the present invention, the reagent is used to detect the affinity and / or blocking ability of the analyte for CD93 protein, or to qualitatively, quantitatively, and locally detect whether CD93 protein is expressed, its expression level, and its expression location in biological samples.

[0039] An antitumor drug containing CD93 affinity peptide.

[0040] In a preferred embodiment of the present invention, the CD93 affinity peptide in the antitumor drug is in a chemically modified form, including but not limited to cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin affinity peptide modification, NOA modification, radionuclide modification, tumor homing peptide coupling, membrane-penetrating peptide coupling, and nanocarrier coupling. Chemical modification can prolong the half-life of the CD93 affinity peptide and improve its stability.

[0041] In a preferred embodiment of the present invention, the CD93 affinity peptide in the antitumor drug exists in free form or in the form of its pharmaceutically acceptable salt.

[0042] In a preferred embodiment of the present invention, the content of CD93 affinity peptide in the antitumor drug is the pharmacodynamic amount, ranging from 0.01wt% to 99.99wt%.

[0043] In a preferred embodiment of the present invention, the antitumor drug further comprises other adjuvant antitumor pharmacological components, the content of which is a pharmacodynamic dose. Combined therapy with synergistic effects is achieved through combined drug use.

[0044] As a preferred embodiment of the present invention, the antitumor drug further includes pharmaceutically acceptable excipients, including but not limited to one or more of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, and buffer.

[0045] As a preferred embodiment of the present invention, the dosage form of the antitumor drug includes, but is not limited to, any one of tablets, powders, suspensions, granules, capsules, solutions, enemas, and emulsions.

[0046] The beneficial effects of this invention are:

[0047] This invention takes a unique approach, obtaining peptides with CD93 protein affinity through repeated screening and optimization. Affinity blocking activity experiments confirmed that the CD93 affinity peptide can bind to the extracellular IgV-like domain of the CD93 protein, blocking the interaction between CD93 and IGFBP7 proteins. In vitro migration and tube formation experiments of HUVECs cells verified that the CD93 affinity peptide can inhibit the migration and tube formation of HUVECs cells, promote the normalization of tumor vascular function, and thus inhibit tumor growth, without significant toxic side effects.

[0048] The CD93 affinity peptide provided by this invention has a strong affinity for CD93 protein and effectively blocks the binding of CD93 / IGFBP7. It can be used to prepare anti-tumor drugs (including anti-tumor immunotherapy drugs) or detection reagents targeting CD93, and has good prospects for medical applications, providing a new option for tumor immunotherapy. Attached Figure Description

[0049] Figure 1 The results show the affinity experiment of the parent peptide CBP-1 for human CD93 protein in the experimental example.

[0050] Figure 2The results show the experimental results of blocking the human CD93 / IGFBP7 protein interaction by C-terminal or N-terminal truncated peptides in the experimental examples.

[0051] Figure 3 The results show the experimental results of the modified peptide blocking the interaction between human or mouse CD93 / IGFBP7 proteins in the experimental examples.

[0052] Figure 4 The results show the affinity experiment of the modified peptide CBP-D8 for human CD93 protein in the experimental example.

[0053] Figure 5 The results of the enzymatic degradation experiment of the truncated peptide CBP-C2 and the modified peptide CBP-D8 in 10% human serum are presented in the experimental example.

[0054] Figure 6 The effect of the modified peptide CBP-D8 on the tube-forming ability of HUVECs in the experimental example.

[0055] Figure 7 The effect of the modified peptide CBP-D8 on tumor volume in the MC38 xenograft model C57BL / 6 mice was investigated in the experimental case.

[0056] Figure 8 The effect of the modified peptide CBP-D8 on the body weight of C57BL / 6 mice with MC38 xenograft tumor model was investigated in the experimental case.

[0057] Figure 9 The effect of the modified peptide CBP-D8 on blood perfusion at the tumor site in the MC38 xenograft model C57BL / 6 mice was investigated in the experimental case.

[0058] In the above figures, the significance indicators * indicate P < 0.05, ** indicate P < 0.01, and *** indicate P < 0.001.

[0059] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings obtained in the experimental examples have been briefly described above. It should be understood that the above drawings only show some experimental examples of the present invention and should not be considered as any limitation on the scope of protection of the claims. For those skilled in the art, other related drawings can be obtained based on these drawings without any creative effort. Detailed Implementation

[0060] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and experimental examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other implementation schemes obtained by those skilled in the art without creative effort, such as implementation schemes obtained by modification, variation or simple substitution, should fall within the scope of protection of the present invention.

[0061] Unless otherwise specified, the experimental methods used in the following examples and experimental cases are conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the field and commercially available to the public unless otherwise specified; the terms and abbreviations used have their conventional meanings in the field, such as PBS buffer being phosphate buffer.

[0062] 1. Main reagents and kits

[0063] Bovine serum albumin (BSA), Solarbio Biotechnology Co., Ltd.

[0064] Fetal bovine serum (FBS), BI, Israel.

[0065] MonolithNT TM His-Tag Labeling Kit RED-Tris-NTA protein labeling kit, Nortamp Technology (Beijing) Co., Ltd.

[0066] 2. Culture medium and solution

[0067] LB medium, top agar, LB / IPTG / X-gal plates, RMPI 1640 medium (containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin), Protein A / G Mix Magnetic Beads, TBS buffer (50 mM Tris-HCl (pH 7.5), containing 150 mM NaCl), Tris-HCl (pH 9.1) neutralization solution, PEG-8000 / NaCl precipitation solution, Tris-T buffer, elution buffer (0.2 M Glycine-HCl (pH 2.2), 1 mg / mL BSA), neutralization solution (1 M Tris-HCl (pH 9.1), PBS buffer (pH 7.2), PBST, BSA, Tween-20, TBST washing buffer, etc., were all prepared according to conventional techniques and will not be described in detail here.

[0068] 3. Main Instruments

[0069] MST Instruments, Nano Temper Technology GmbH, Germany.

[0070] Flow cytometer, BD Biosciences, USA.

[0071] 4. Biomaterials

[0072] MC38 cell line and HUVECs cell line were commercially purchased and preserved in our laboratory.

[0073] The CHO-K1-hCD93 cell line (cells overexpressing hCD93) was constructed using conventional techniques and was constructed and preserved in our laboratory.

[0074] C57BL / 6 mice (6-8 weeks old, female) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in an SPF-grade animal facility.

[0075] Experimental Example

[0076] I. Screening and Synthesis of CD93 Affinity Peptides

[0077] The steps for screening affinity peptides for human CD93 using a phage display heptapeptide library are briefly as follows:

[0078] (1) The phage display heptapeptide library was screened using a cell screening method;

[0079] (2) After several rounds of screening, phage monoclonals with affinity for the extracellular domain of the target protein human CD93 were enriched in each round.

[0080] (3) Select positive clones for sequencing to obtain multiple inserted heptapeptide sequences, namely human CD93 affinity peptide sequences, named CBP-1 to CBP-4 (amino acid sequences as shown in SEQ ID NO: 1-4), where the amino acid sequence of CBP-1 is: KWIFSPL, and the uppercase single letter represents the L-configuration amino acid.

[0081] Based on the affinity peptide CBP-1 obtained from the above screening, standard Fmoc solid-phase synthesis was performed. After being purified by high performance liquid chromatography and identified by mass spectrometry, the blocking ability of the peptide was tested by blocking experiments (see Table 1).

[0082] Determining the minimum active fragment of the CBP-1 peptide: The C-terminal or N-terminal amino acids of the CBP-1 peptide were truncated one by one to obtain truncated peptides CBP-C1 to CBP-C3, as well as CBP-N1 and CBP-N2 (amino acid sequences are shown in SEQ ID NO: 5-9). The blocking ability of the truncated peptides was then tested using blocking assays to determine the minimum active fragment of the CBP-1 peptide (see Table 2).

[0083] Peptide modification: The CBP-C2 peptide was modified using a D-configuration amino acid substitution strategy to obtain modified peptides CBP-D1 to CBP-D7, and simultaneously, the reverse-order peptide CBP-D8 was obtained. Its amino acid sequence is sfIwk, where lowercase letters represent D-configuration amino acids. The blocking ability of the modified peptides was then investigated using blocking and affinity assays, and the stability of the peptides was tested (see Table 3) to further confirm their antitumor effects.

[0084] II. Affinity Experiment

[0085] The affinity of the affinity peptides CBP-1 and CBP-D8 for hCD93 was detected using micro-thermophoresis (MST), as follows:

[0086] (1) Labeling the protein: Monolith NT was labeled with pure water. TM Dilute 5×PBST in the His-Tag Labeling Kit RED-Tris-NTA to 1×PBST. Dissolve the dye in 50 μL of 1×PBST to obtain a dye concentration of 5 μM. Then mix 2 μL of dye with 98 μL of 1×PBST to prepare a 100 nM dye solution. Adjust the protein concentration to 200 nM (volume 100 μL). Then mix the dye and protein at a volume ratio of 1:1 (i.e., 100 μL of 200 nM protein and 100 μL of 100 nM dye). Incubate at room temperature in the dark for 30 min. Centrifuge the labeled protein at 15000g for 10 min at 4℃. Retain the supernatant and discard the precipitate. Protein labeling is complete.

[0087] (2) Sample preparation: Dissolve the peptide to an appropriate concentration and perform 15 serial dilutions to obtain 16 concentration gradients of samples, each with a volume of 5 μL. Then add 5 μL of labeled protein sample to each tube, mix well, centrifuge to remove air bubbles, incubate on ice for 5 min, and then use an MST-specific capillary tube to aspirate the incubated liquid and place it on the instrument holder.

[0088] (3) On-machine testing: Open the computer and start the MO.Control software, select the Red channel BindingAffinity mode for testing.

[0089] (4) Analysis Results: The binding dissociation constant (K0) was calculated using the Nano Temper analysis software MO.AffinityAnalysis v2.2.4. D (value), the affinity experiment results of CBP-1 are as follows: Figure 1 As shown, the affinity experiment results for CBP-D8 are as follows: Figure 4 As shown.

[0090] III. Blocking CD93 / IGFBP7 interaction

[0091] (1) Culturing CHO-K1-hCD93 or CHO-K1-mCD93 cells in RMPI 1640 medium (containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin), collecting cells in the logarithmic growth phase, counting them, and then aliquoting them into 3×10⁻⁶ cells / mL containers. 5 Add pre-cooled PBS 7.2 buffer to each tube, centrifuge at 3000 rpm for 5 min at 4°C, wash, and place on ice.

[0092] (2) Dissolve the affinity peptides in PBS 7.2 buffer and serially dilute to obtain 6 concentration gradients. Take 50 μL of each gradient concentration affinity peptide solution and incubate with CHO-K1-hCD93 or CHO-K1-mCD93 cells for 30 min. Add only 50 μL of PBS 7.2 solution to both negative and positive controls.

[0093] (3) Add an appropriate amount of recombinant hIGFBP7-Fc or mIGFBP7-Fc protein to the incubated system. Add an equal amount of protein solution to the positive control tube and an equal amount of PBS 7.2 solution to the negative control tube. Mix well and incubate in an ice-water bath at 4°C for 30 min.

[0094] (4) Add 5 μL of flow cytometry antibody (anti-human Fc PE) to the incubated solution, mix well and incubate at 4°C for 30 min.

[0095] (5) Add 1 mL of pre-cooled PBS 7.2 buffer to the incubated solution, wash and centrifuge, then resuspend the cells in 200 μL of PBS 7.2 solution, and then detect the average fluorescence intensity of the cells using flow cytometry. Calculate the blocking rate based on the average fluorescence intensity of each group. The formula for calculating the blocking rate is as follows:

[0096] Blocking rate = (average fluorescence value of positive control tube - fluorescence value of sample tube) / average fluorescence value of positive control tube × 100%.

[0097] The blocking effects of C-terminal amino acid truncated peptides CBP-C1 to CBP-C3 and N-terminal amino acid truncated peptides CBP-N1 and CBP-N2 on human CD93 / IGFBP7 interaction are as follows: Figure 2 As shown. The blocking results of modified peptides CBP-D1 to CBP-D8 on the human CD93 / IGFBP7 interaction, and the blocking results of gradient concentrations of modified peptides CBP-D6 to CBP-D8 on the mouse CD93 / IGFBP7 interaction are shown in the figure. Figure 3 As shown.

[0098] Table 1. CD93 affinity peptide blocks CD93 / IGFBP7 protein interaction at a concentration of 400 μM.

[0099]

[0100] Note: " / " indicates that there is no blocking effect.

[0101] Table 2 shows how C-terminal or N-terminal truncated peptides block CD93 / IGFBP7 protein interaction at a concentration of 200 μM.

[0102]

[0103] Table 3 shows that peptides with D conformation or reverse modification block CD93 / IGFBP7 protein interaction at a concentration of 200 μM.

[0104]

[0105] Note: Lowercase letters represent D-configuration amino acids.

[0106] IV. Enzyme Degradation Stability Experiment

[0107] (1) Weigh the truncated peptide CBP-C2 and modified peptide CBP-D8 dry powder, dissolve them in physiological saline to 200 μM, add human serum to prepare a mixed solution containing 10% (V / V) serum, mix quickly and place in a metal bath, incubate at 37℃ for 48 h, and take out a portion of the sample at 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h and 48 h for subsequent detection.

[0108] (2) Add 1 / 2 volume of 10% perchloric acid solution to the samples taken at different time points, shake and mix quickly to terminate the proteolytic digestion, centrifuge at 4℃ and 12000rpm for 15min, and collect the supernatant.

[0109] (3) RP-HPLC analysis of peptide-serum mixture samples was performed, and the experimental results were statistically analyzed. The enzymatic degradation results of CBP-C2 and CBP-D8 in 10% serum are as follows: Figure 5 As shown.

[0110] Enzymatic degradation stability experiments showed that the modified peptide CBP-D8 had significantly improved serum stability compared to CBP-C2, with only a slight decrease in solubility at 48 h.

[0111] V. In vitro migration experiment of HUVECs cells

[0112] (1) Plate laying: Transfer the matrix gel from -80℃ to 4℃ for slow thawing. Dilute the matrix gel 1:1 with culture medium and add 50μL to each well. Place it in an incubator to solidify.

[0113] (2) Peptide dissolution: Weigh the CBP-D8 peptide and dissolve it in DMEM medium as a solvent. Prepare three working concentrations of peptide solution: 100μM, 200μM and 400μM. Filter the solution with a 0.22μm filter membrane to remove bacteria.

[0114] (3) Treatment: The cultured cells were digested with 0.5% trypsin and the density was adjusted to 2×10⁻⁶. 5 Cells / mL, take out a 96-well plate, add 100 μL to each well, then add 100 μL of peptide solution of different concentrations to each well, and after 30 min, add 60 ng of IGFBP7 protein to each well.

[0115] (4) Detection: After 4 hours of culture, images were acquired from three randomly selected non-overlapping fields of view using an inverted microscope. ImageJ software was used to analyze the branch length and number of branch points formed in the tubes. The effect of CBP-D8 peptide on HUVECs cell migration was as follows: Figure 6 As shown.

[0116] In vitro HUVECs cell migration experiments showed that CBP-D8 peptide could significantly inhibit the migration of HUVECs cells.

[0117] VI. Anti-tumor animal experiments

[0118] To detect the antitumor activity of the modified peptide CBP-D8, the experimental procedure is as follows:

[0119] (1) Inoculate the right back of each C57BL / 6 mouse with 1×10 6 MC38 tumor cells were used to treat mice until the tumor volume reached 40-80 mm. 3 At that time, the tumors were grouped in an S-shape according to their size. For the MC38 tumor model, 3 mg / kg CBP-D8 peptide (low-dose group), 9 mg / kg CBP-D8 peptide (high-dose group), or PBS buffer were injected daily as negative controls for 14 consecutive days.

[0120] (2) Measure the length (a), width (b), and height (c) of the tumor every other day, and calculate the tumor volume according to the formula (calculation formula: V=1 / 2×a×b×c). Plot the tumor growth curve, and the results are as follows. Figure 7 As shown. Mouse body weight was measured and recorded every other day, and a curve was plotted to examine the toxic side effects of the modified peptide CBP-D8. The results are as follows. Figure 8 As shown in the figure. To investigate the effect of CBP-D8 peptide on blood vessels in tumor sites, laser blood flow imaging analysis was performed on the tumor sites of mice after drug administration, and changes in blood perfusion function were recorded in real time. The results are as follows. Figure 9 As shown.

[0121] from Figure 7It can be seen that in the MC38 tumor model, compared with the PBS group, the tumor volume of mice in the high- and low-dose groups of modified peptide CBP-D8 was significantly reduced, and the tumor volume of mice in the high-dose group was the smallest.

[0122] from Figure 8 It can be seen that in the MC38 tumor model, the weight change trends of mice in the high- and low-dose CBP-D8 modified peptide groups were almost the same as those in the PBS group, indicating that the injection of modified peptide CBP-D8 had no toxic side effects on the growth of mice.

[0123] from Figure 9 It can be seen that in the MC38 tumor model, the modified peptide CBP-D8 can increase the blood perfusion of the tumor site, which helps to improve the hypoxia of the tumor site and transform the immunosuppressive tumor microenvironment into an immune-supportive microenvironment.

[0124] The above experimental results show that the CD93 affinity peptide provided by this invention can block the binding of CD93 / IGFBP7 and can be used to prepare anti-tumor drugs targeting CD93, providing a new option for tumor immunotherapy.

[0125] Example 1

[0126] This embodiment provides a CD93 affinity peptide, selected from any one or more of the following sequences:

[0127] (1) The amino acid sequence as shown in SEQ ID NO: 1;

[0128] (2) A truncated sequence of the amino acid sequence as shown in SEQ ID NO: 1;

[0129] (3) The reverse sequence of the truncated amino acid sequence as shown in SEQ ID NO: 1.

[0130] This embodiment also provides an application of the CD93 affinity peptide, including but not limited to one or more of the following aspects:

[0131] (1) Application in the preparation of reagents that block the binding of CD93 protein to its ligand IGFBP-7;

[0132] (2) Application in the preparation of antitumor drugs.

[0133] This embodiment also provides a detection reagent containing CD93 affinity peptide.

[0134] This embodiment also provides an antitumor drug containing CD93 affinity peptide.

[0135] Example 2

[0136] This embodiment provides a CD93 affinity peptide, selected from any one or more of the following sequences:

[0137] Mother peptide CBP-1: KWIFSPL;

[0138] CBP-C1 truncated peptide: KWIFSP;

[0139] CBP-C2 truncated peptide: KWIFS;

[0140] CBP-C3 truncated peptide: KWIF;

[0141] CBP-N1 truncated peptide: WIFSPL;

[0142] CBP-N2 truncated peptide: IFSPL;

[0143] Modified peptide CBP-D1:kWIFs;

[0144] Modified peptide CBP-D2: kWIfs;

[0145] Modified peptide CBP-D3:kWifs;

[0146] Modified peptide CBP-D4: kwIFs;

[0147] Modified peptide CBP-D5: kwIfs;

[0148] Modified peptide CBP-D6: kwifs;

[0149] Modified peptide CBP-D7: kwiFs;

[0150] Modified peptide CBP-D8: sfIwk.

[0151] In the CD93 affinity peptides above, uppercase letters represent amino acids with the L configuration and lowercase letters represent amino acids with the D configuration. All peptides were prepared using the standard Fmoc synthesis method.

[0152] This embodiment also provides the application of CD93 affinity peptide in the preparation of reagents that block the binding of CD93 protein to its ligand IGFBP-7.

[0153] This embodiment also provides a detection reagent containing CD93 affinity peptide, which is used to detect the affinity and / or blocking ability of the analyte to CD93 protein, or to qualitatively, quantitatively, and locally detect whether CD93 protein is expressed, the amount of expression, and the location of expression in biological samples.

[0154] Example 3

[0155] This embodiment provides a CD93 affinity peptide, specifically a modified peptide CBP-D8:sfIwk.

[0156] This embodiment also provides the application of CD93 affinity peptide in the preparation of antitumor drugs, the antitumor effects including but not limited to promoting the normalization of tumor vascular function, improving the hypoxia of the tumor site, reducing tumor volume, and reducing tumor weight.

[0157] This embodiment also provides an antitumor drug containing CD93 affinity peptide, wherein the content of CD93 affinity peptide is at a clinically effective level; the drug also contains pharmaceutically acceptable excipients for preparing the drug into an injectable dosage form.

[0158] In other embodiments of the present invention, the CD93 affinity peptide in the antitumor drug is in a chemically modified form, including but not limited to cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, etc., to prolong the half-life of the CD93 affinity peptide and improve its stability.

[0159] In other embodiments of the present invention, the antitumor drug, in addition to containing CD93 affinity peptide, also contains other pharmacologically active ingredients that assist in antitumor activity, at a pharmacodynamic concentration. Combined therapy with synergistic effects is achieved through combined drug use.

[0160] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A CD93 affinity peptide, characterized in that: Selected from any one or more of the following sequences: (1) The amino acid sequence as shown in SEQ ID NO: 1; (2) A truncated sequence of the amino acid sequence as shown in SEQ ID NO: 1; (3) The reverse sequence of the truncated amino acid sequence as shown in SEQ ID NO:

1.

2. The CD93 affinity peptide according to claim 1, characterized in that: The configuration of each amino acid in the CD93 affinity peptide is independently selected from either the L or D configuration; And / or, all amino acids in the amino acid sequence shown in SEQ ID NO: 1 are L-configured.

3. The CD93 affinity peptide according to claim 1, characterized in that: The truncated sequence of the amino acid sequence shown in SEQ ID NO: 1 is a polypeptide that is truncated one amino acid at the C-terminus or N-terminus of the parent peptide CBP-1, including but not limited to the following truncated peptides: CBP-C1 truncated peptide: KWIFSP; CBP-C2 truncated peptide: KWIFS; CBP-C3 truncated peptide: KWIF; CBP-N1 truncated peptide: WIFSPL; CBP-N2 truncated peptide: IFSPL; All amino acids in the truncated peptide are in the L configuration.

4. The CD93 affinity peptide according to claim 3, characterized in that: A polypeptide that replaces one or more L-configured amino acids in the truncated peptide CBP-C2 with D-configured amino acids, including but not limited to the following modified peptides: Modified peptide CBP-D1:kWIFs; Modified peptide CBP-D2: kWIfs; Modified peptide CBP-D3:kWifs; Modified peptide CBP-D4: kwIFs; Modified peptide CBP-D5: kwIfs; Modified peptide CBP-D6: kwifs; Modified peptide CBP-D7: kwiFs; Uppercase letters represent amino acids with the L configuration, and lowercase letters represent amino acids with the D configuration.

5. The CD93 affinity peptide according to claim 1, characterized in that: The reverse sequence of the truncated amino acid sequence shown in SEQ ID NO: 1 is the modified peptide CBP-D8:sfIwk, in which all amino acids are in the D configuration.

6. The application of a CD93 affinity peptide as described in any one of claims 1-5, characterized in that: Including but not limited to one or more of the following aspects: (1) Application in the preparation of reagents that block the binding of CD93 protein to its ligand IGFBP-7; (2) Application in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that: The application of the reagent in the preparation of the reagent that blocks the binding of CD93 protein to its ligand IGFBP-7 includes: labeling CD93 affinity peptides with fluorescein or radionuclide to achieve imaging and tracing of CD93 protein. And / or, the anti-tumor effects include, but are not limited to, promoting the normalization of tumor vascular function, improving the hypoxia at the tumor site, reducing tumor volume, and reducing tumor weight.

8. A reagent comprising the CD93 affinity peptide as described in any one of claims 1-5, characterized in that: The reagent is used to detect the affinity and / or blocking ability of the analyte for CD93 protein, or to qualitatively, quantitatively, and locally detect whether, how much, or where CD93 protein is expressed in biological samples.

9. An antitumor drug comprising the CD93 affinity peptide as described in any one of claims 1-5.

10. The antitumor drug according to claim 9, characterized in that: The CD93 affinity peptide in the antitumor drug is in a chemically modified form, including but not limited to cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin affinity peptide modification, Nota modification, radionuclide modification, tumor homing peptide coupling, membrane-penetrating peptide coupling, and nanocarrier coupling. And / or, the CD93 affinity peptide in the said antitumor drug exists in free form or in the form of its pharmaceutically acceptable salt; And / or, the content of CD93 affinity peptide in the antitumor drug is the pharmacodynamic amount, ranging from 0.01wt% to 99.99wt%; And / or, the antitumor drug may also contain other adjuvant antitumor pharmacological components, the content of which is a pharmacologically effective amount; And / or, the antitumor drug further includes pharmaceutically acceptable excipients, including but not limited to one or more of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, solvent, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, and buffer. And / or, as a preferred embodiment of the present invention, the dosage form of the antitumor drug includes, but is not limited to, any one of tablets, powders, suspensions, granules, capsules, solutions, enemas, and emulsions.

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