CN117624293A - DDR1 affinity peptide and application thereof - Google Patents

Abstract

The invention relates to the field of biopharmaceuticals, in particular to DDR1 affinity peptide and application thereof. The DDR1 affinity peptide is selected from the following peptides a1, a2 or a combination thereof: peptide a1: (D-Phe) -Trp- (D-Cha); peptide a2: (D-Phe) - (D-Phe) -Trp- (D-Phe) or a truncated peptide thereof, and an alanine scanning peptide. The DDR1 affinity peptide provided by the invention has better DDR1 inhibition effect, and in vitro experiments prove that the DDR1 affinity peptide can promote CD8 ⁺ Chemotaxis and infiltration of T cells and inhibition of activation of DDR1 downstream pathways are expected to be developed as drugs for the treatment of solid tumors.

Description

DDR1 affinity peptide and application thereof

Technical Field

The invention relates to the field of biopharmaceuticals, in particular to DDR1 affinity peptide and application thereof.

Background

Breast cancer is the most common cancer in women, and in 2020 breast cancer has replaced lung cancer as the first cancer worldwide. 10% -20% of milkAdenocarcinomas are triple negative breast cancers that are insensitive to common breast cancer therapeutic agents due to the lack of Estrogen Receptor (ER), progestogen Receptor (PR) and human epidermal factor growth receptor-2 (HER-2). In addition, the extracellular matrix limits tissue infiltration of immune cells, further rendering triple negative breast cancers difficult to treat. How to break through the physical barrier of triple negative breast cancer and promote infiltration of immune cells is a major difficulty in the field at present. Disc domain receptor 1 (DDR 1) is a tyrosine kinase receptor that is highly expressed in a variety of malignant tumors and regulates various signaling pathways between cells, cell migration, and adhesion functions. In 2021, researchers at university of washington, university of texas, and the institute of houston health science, university of american, have conducted studies on breast cancer, including triple negative breast cancer, where DDR1 was found to prevent immune cells from entering the tumor and kill cancer cells therein. This study demonstrates that the extracellular domain of DDR1 binds collagen and aligns it to form a physical barrier inhibiting CD8 ⁺ Infiltration of T cells. The physical barrier of the solid tumor is destroyed, so that the method has an important effect on the treatment of triple negative breast cancer, and the DDR1 blocking-based immunotherapy has a wide application prospect.

DDR was discovered in 1993 by Johnson et al in studying tyrosine phosphorylation in breast cancer cells, and two subtypes, DDR1 and DDR2, have been identified. DDR1 can be combined with collagen types I, II, III and IV, and tyrosine kinase can recognize a specific amino acid sequence GVGGFO on the collagen, so that the tyrosine kinase is activated slowly and continuously. DDR1 is expressed in various tumors, such as lung cancer, brain tumor, esophageal cancer, liver cancer and the like, and the high expression of DDR1 is often related to poor prognosis of the tumors. According to literature reports, DDR1 function mainly depends on two different mechanisms: (1) The classical pathway, which depends on the activity of the collagen-DDR1 kinase, activates DDR1, for example by activating a down stream Src kinase, which phosphorylates DDR1 kinase specific tyrosine; (2) Independent of DDR1 kinase activity, for example, activated DDR1 recruits PSD4 to bind to ARF6, thereby activating ARF6 and its downstream MAPK (mitogen activated protein kinase signaling pathway), and thus promoting lung metastasis of hepatoma cells. DDR1 can promote proliferation and metastasis of tumors from various aspects. DDR1 activates the Ras/Raf/ERK and PI3K/Akt pathways in human breast and colon cancer cells, thereby upregulating the anti-apoptotic protein Bcl-xL and allowing the cells to survive under genotoxic stress conditions. DDR1 can promote migration of pancreatic cancer cells by up-regulating the expression of MMP2 and MMP9 proteins. In summary, blocking the DDR1 pathway is an effective strategy to inhibit tumor growth.

Drugs targeting DDR1 as a target are currently available. In terms of antibodies, caracoli et al developed in 2012 a monoclonal antibody 3E3 that binds to the discoid protein domain of DDR 1. The Fab fragment of the antibody binds to the DS-like domain of DDR1 and inhibits activation of DDR1 without interfering with collagen binding. However, the antibody has no later description. In 2019, YIRAn Tao et al developed an ADC drug targeting DDR1 (T4H 11-DM 4), and found an anti-tumor effect in colon cancer mice, whose monoclonal antibodies acted on DS domain of DDR1 extracellular region, but did not overlap with collagen site. In 2021, the neutralizing antibody PRTH-101 targeting DDR1 developed by researchers at university of washington and university of houston health science, texas, has entered the phase of clinical phase i trials, which mainly overlapped with collagen sites, demonstrated that in vivo experiments were able to inhibit tumor growth in immunocompetent hosts, but no significant inhibition in immunodeficient hosts. In terms of small molecules, in 2017, liu et al developed and evaluated some novel dasatinib analogs as potent DDR1 and DDR2 dual inhibitors, found that 2-morpholinoethyl-4- (6- (5- (2-chloro-6-methylphenylcarbamoyl)) thiazol-2-ylamino) -2-me-thlypyrimidin-4-yl) piprazine-1-carboxylate was a more potent DDR1 and DDR2 inhibitor, kinase inhibition IC ₅₀ The values were 2.26.+ -. 0.46nM and 7.04.+ -. 2.90nM, respectively. In addition, ICP-033, a multi-target receptor tyrosine kinase independently developed by Nochengjianhua, was also tested in clinical phase I, and the antibody was mainly directed against DDR1 and VEGFR to inhibit tumor angiogenesis. Therefore, drugs inhibiting DDR1 are expected to provide a safe and effective new scheme for tumor immunotherapy.

There are a number of drugs currently under study for DDR1, mostly focusing on the field of tumor and anti-fibrotic therapy. In the aspect of DDR1 antibody research, DDR1 monoclonal antibody PRTH-101 has been proved to be capable of inhibiting tumor proliferation in a mouse breast cancer model, and has entered clinical stage I; while other antibody drugs remain in the preclinical study stage.

Compared with antibodies, the small molecule drug has better permeability, can be orally taken, is easy to synthesize and has smaller side effect, the current DDR1 small molecule inhibitor is mainly a tyrosine kinase inhibitor targeting DDR1 intracellular structure, and inhibits the activation of DDR1 downstream channels by inhibiting kinase domains so as to inhibit the proliferation and migration of tumors, on one hand, the small molecule has serious side effect caused by off-target effect in clinical application, and on the other hand, the small molecule can not target the extracellular region of DDR1, and blocks the combination of DDR1 and collagen so as to influence the physical barrier formed by cell matrixes.

Therefore, a small molecular medicine aiming at the DDR1 extracellular domain is developed, and the combination of collagen and DDR1 is inhibited, so that the infiltration of immune cells is improved, and the method has important clinical significance for tumor immunotherapy.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the DDR1 affinity peptide which can well inhibit the combination of collagen and DDR1, thereby improving infiltration of immune cells and having important clinical significance for tumor immunotherapy.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the present invention provides a DDR1 affinity peptide selected from the following peptides a1, a2 or a combination thereof:

peptide a1: (D-Phe) -Trp- (D-Cha);

peptide a2: (D-Phe) - (D-Phe) -Trp- (D-Phe) or a truncated peptide thereof, and an alanine scanning peptide.

DDR1 degrades the extracellular segment, so that the extracellular segment is free in the tumor environment, and the extracellular segment can combine with extracellular collagen when the extracellular segment is free, so that the collagen surrounds the outside of the tumor to form a barrier, thereby influencing infiltration of T cells. The DDR1 affinity peptide provided by the invention: the polypeptide (D-Phe) - (D-Phe) -Trp- (D-Cha), (D-Phe) - (D-Phe) -Trp- (D-Phe) and its derivative peptide (including truncated peptide and scan peptide) are low molecular weight compounds, can be affinitized with DDR1 protein, the main binding region is DDR1 extracellular segment, MST experiment proves that it can bind to the free extracellular domain part, thus blocking the barrier formed by collagen; the WB experiment also demonstrates that low molecular weight compounds can inhibit activation of the DDR1 downstream pathway, thereby inhibiting proliferation and migration effects caused by DDR1 activation in tumors.

Preferably, the truncated peptide of the peptide a2 comprises at least one of (D-Phe) - (D-Phe), (D-Phe) -Trp, trp- (D-Phe), (D-Phe) -Trp, (D-Phe) -Trp- (D-Phe).

Truncated peptides, in the art, refer to a series of shorter peptides obtained by truncating 1 to more amino acids from the N-or C-terminus of the protein precursor.

Preferably, the alanine scanning peptide of the peptide a2 includes at least one of (D-Ala) - (D-Phe) -Trp- (D-Phe), (D-Phe) - (D-Ala) -Trp- (D-Phe), (D-Phe) -Ala- (D-Phe), (D-Phe) - (D-Phe).

Alanine scanning peptides refer in the art to a series of single mutant peptides obtained by substituting alanine for any one of the amino acids of the protein precursor.

Preferably, the DDR1 may be wild type of human and murine origin or mutant proteins that still retain their activity.

In a second aspect, the invention provides the use of a DDR1 affinity peptide in the manufacture of a medicament for the treatment of a tumor or a probe for the diagnosis of a tumor; the DDR1 affinity peptide comprises at least one of the following polypeptides: (D-Phe) - (D-Phe) -Trp- (D-Cha), (D-Phe) - (D-Phe) -Trp- (D-Phe), (D-Phe) -Trp, trp- (D-Phe), (D-Phe) - (D-Phe) -Trp, (D-Phe) -Trp- (D-Phe), (D-Ala) - (D-Phe) -Trp- (D-Phe), (D-Phe) - (D-Ala) -Trp- (D-Phe), (D-Phe) - (D-Phe) -Ala- (D-Phe), (D-Phe) - (D-Phe) -Trp- (D-Ala).

The DDR1 affinity peptide provided by the invention is a small molecular medicine aiming at DDR1 extracellular domain, inhibits the combination of collagen and DDR1, and inhibits the activation of DDR1, thereby improving the infiltration of immune cells, and has important clinical significance for tumor immunotherapy.

Preferably, the DDR1 affinity peptide inhibits tumor growth by blocking DDR1 binding to collagen or inhibiting activation of the DDR1 downstream pathway.

Preferably, the tumor is any one of colon cancer and breast cancer.

In a third aspect, the invention provides a pharmaceutical composition comprising a DDR1 affinity peptide as described above, or a pharmaceutically acceptable salt form thereof, or a free form thereof, or a chemically modified derivative thereof.

Preferably, the chemical modification is any one of cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin affinity peptide coupling, tumor homing peptide coupling, transmembrane peptide coupling, nanocarrier coupling, radionuclide coupling, small molecule compound coupling, nucleotide coupling, and protein coupling.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

The invention has the advantages that:

according to the invention, through MOE virtual screening, a low molecular weight compound capable of specifically binding to DDR1 extracellular domain to block collagen interaction sites is obtained.

The DDR1 affinity peptide prepared by the invention has the following effects:

(1) Can inhibit DDR1 in tumor, thereby promoting infiltration of immune cells;

(2) Can inhibit DDR1 downstream channel activation so as to inhibit tumor cell migration;

(3) Avoiding off-target side effects of tyrosine kinase inhibition in the technical defects;

(4) Avoid the broad-spectrum side effects of antibody-induced immune reaction, platelet thrombotic diseases and the like and promote the infiltration of the medicine into tumor tissues.

Drawings

FIG. 1 is a graph showing the results of MST validation of low molecular weight compound I affinity for hDDR1 protein.

FIG. 2 is a mass spectrum (theoretical molecular weight 651.1) of the polypeptide of SEQ ID NO. 1.

FIG. 3 is a mass spectrum (theoretical molecular weight 645.7) of the polypeptide of SEQ ID NO. 2.

FIG. 4 is a mass spectrum (theoretical molecular weight 312.4) of the polypeptide of SEQ ID NO. 3.

FIG. 5 is a mass spectrum (theoretical molecular weight 351.4) of the polypeptide of SEQ ID NO. 4.

FIG. 6 is a mass spectrum (theoretical molecular weight 351.4) of the polypeptide of SEQ ID NO. 5.

FIG. 7 is a mass spectrum (theoretical molecular weight 498.6) of the polypeptide of SEQ ID NO. 6.

FIG. 8 is a mass spectrum (theoretical molecular weight 498.6) of the polypeptide of SEQ ID NO. 7.

FIG. 9 is a mass spectrum of the polypeptide of SEQ ID NO.8 (theoretical molecular weight 569.5).

FIG. 10 is a mass spectrum of the polypeptide of SEQ ID NO.9 (theoretical molecular weight 569.5).

FIG. 11 is a mass spectrum (theoretical molecular weight 530.5) of the polypeptide of SEQ ID NO. 10.

FIG. 12 is a mass spectrum of the polypeptide of SEQ ID NO.11 (theoretical molecular weight 569.5).

FIG. 13 is a graph showing the results of enhancing immune cell infiltration in tumor spheres by low molecular weight compounds I, SEQ ID NO.1 and SEQ ID NO. 2.

FIG. 14 is a graph showing the results of low molecular weight compound I inhibiting CT26 and 4T1 cell migration.

FIG. 15 is a graph showing the results of SEQ ID NO.2 inhibiting CT26 and 4T1 cell migration.

FIG. 16 is a graph showing the effect of SEQ ID NO.2-7 on CT26 migration.

FIG. 17 is a graph showing the effect of SEQ ID NO.2 and SEQ ID NO.8-11 on CT26 migration.

FIG. 18 is a graph showing the effect of SEQ ID NO.2 on the volume of transplanted tumors in BABL/c mice vaccinated with CT 26.

FIG. 19 is a graph showing the effect of SEQ ID NO.2 on body weight of BABL/c mice at a dose of 0.48 mg/kg.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.

The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.

The invention refers to polypeptides by sequence numbers, and the corresponding relationship is shown in table 1:

TABLE 1

Example 1: screening for compounds that affinity hDDR1 proteins and affinity assays

2862 drugs on the market in the drug library Approved Drug Library of the compound library are initially screened by adopting molecular docking software MOE to obtain drugs with molecular weight of 200-750, and 15 compounds are selected from compounds with the front scoring function to carry out a micro thermophoresis experiment (MST). From these, compound I, which was found to be more preferable, was selected, and its name and structure are shown in Table 2.

TABLE 2 names and structures of Compound I

(1) Labeling of Human DDR1 protein: labeling Monolith NT His-tag in kit

The solid fluorescent dye is taken out, 50 mu L of PBST is added, the dye is fully dissolved by light-proof vortex, and split charging is carried out. The concentration of dye was adjusted to 100nM using PBST and 100. Mu.L of diluted dye was taken for use. The hDDR1 protein concentration was diluted to 200nM with PBST and 100. Mu.L of diluted protein was taken for use. The volume ratio is 1:1, mixing the dye and the protein in proportion, and incubating for 30min at room temperature in dark. After incubation, centrifuging at 4 ℃ for 10min at 15000g, transferring the supernatant to a new EP tube, and finishing protein labeling;

(2) Dilution of low molecular weight compound i: preparing 16 200 mu L EP pipes, marking as 1-16, adding 10 mu L of 200 mu M small molecule solution into the 1 st EP pipe, taking the solution in the pipe 1 as the highest concentration, diluting with PBST in the 2-16 pipes to obtain 16 concentration small molecules diluted with 5 mu L of the ratio in each pipe, mixing uniformly, and centrifuging to remove bubbles;

(3) Co-incubation of small molecules with fluorescent marker proteins: adding 5 mu L of marked hDDR1 protein into a 1-16 tube, and incubating for 10min at room temperature in a dark place after vortex mixing;

(4) And (3) detecting: after the incubation, the mixed solution in 16 tubes was sequentially aspirated with a capillary tube, subjected to MST instrument detection, and the binding dissociation constant (K) was calculated using NanoTemper Analysis software MO.affinity Analysis v2.2.4 _D Values).

The results of MST validation of the affinity of low molecular weight compound I for hDDR1 protein are shown in FIG. 1.

The pocket combined with collagen in the DDR1 extracellular region is selected for screening, 15 low molecular weight compounds are obtained through virtual screening in an FDA approved drug library on the market by a computer, and the low molecular weight compound I is calculated according to the affinity provided by the computer to be possibly combined with the DDR1 extracellular region. The parent molecule of the compound I is of a peptide amide structure, the parent molecule contains Phe-Phe, and the parent molecule is modified to obtain a derivative peptide, namely the affinity peptide of DDR 1: SEQ ID No.1 and SEQ ID No.2 and variants thereof were subjected to subsequent in vitro screening.

Example 2: synthesis and purification of DDR1 affinity peptide SEQ ID NO.1-11

(1) Rink resin is used as a carrier, and the synthesis direction is from the C end to the N end of the sequence.

Using 2mL of DMF-dissolved amino acid (SEQ ID NO: 1-11) with Fmoc (fluorenylmethoxycarbonyl) or Boc (t-butyloxycarbonyl) protection as starting material, 1mL of condensing agent HCTU (6-chlorobenzotriazole-1, 3-tetramethylurea hexafluorophosphate) and 200. Mu.L of DIEA (N, N-diisopropylethylamine) were added for coupling, and after 1h of reaction, the indene assay resin was not changed to blue after alternate washing with DMF (N, N-dimethylformamide) and DCM (dichloromethane), and then the reaction was performed with DMF: piperidine = 1:1 removing Fmoc protecting groups, and then: washing, coupling, indene detection and deprotection until the last amino acid coupling is finished;

(2) Cutting polypeptide for 4h by using a peptide cutting reagent (TFA: phenyl sulfide: phenol: ethanedithiol: double distilled water=79:6:6:3), adding diethyl ether precooled at 4 ℃ to precipitate the polypeptide, centrifuging at 4 ℃ at 2000rpm to collect precipitate, washing 3 times by diethyl ether, and airing by a fume hood to obtain a crude polypeptide;

(3) Purifying the crude polypeptide by preparative reverse phase liquid chromatography (RP-HPLC), and analyzing and identifying by HPLC and MS.

Mobile phase a:0.1% TFA/water, mobile phase B: acetonitrile; linear elution gradient: 30% A-70% A; the flow rate is 10mL/min, and the detection wavelength is 220nm; and freeze-drying to obtain the refined peptide.

And (3) carrying out mass spectrometry on the obtained refined peptide, wherein the mass spectrograms of the polypeptides of SEQ ID NO.1-11 are shown in the figures 2-12.

Example 3: tumor ball immune cell infiltration experiment

(1) Preparation of tumor spheres: preparing 1% agarose with PBS, sterilizing at 121deg.C for 30min, adding 100 μl/well into 96U-shaped pore plate while it is hot, adding 8000 CT26 cells into each well in refrigerator at 4deg.C, centrifuging at 1000g for 10min, standing for 10min, and culturing for 5 days for experiment;

(2) And (3) drug treatment: adding 50 mu M low molecular weight drugs including compound I, SEQ ID NO.1 and SEQ ID NO.2 polypeptide for 24 hours on the fifth day of culturing the cell pellet, removing the drugs, and washing with PBS three times;

(3) Acquisition of immune cells: BALB/c mice were dissected after cervical removal and surface disinfection, and inguinal lymph nodes, axillary lymph nodes, facial lymph nodes, mesenteric lymph nodes and spleens were taken, ground with glass slides, filtered with a filter screen, centrifuged at 4℃for 5min, collected cells at 1500g, lysed erythrocytes at 4℃for 7min, and washed 2 times with PBS buffer;

(4) CFSE labeling immune cells: DMSO was added to CFSE fluorescent dye to allow the dye to be fully dissolved and mixed well to prepare a 5mM stock solution. Collecting immune cells to be marked by centrifugal washing with PBS buffer solution, and adjusting cell density to 1×10 ⁷ Adding CFSE dye into the solution at a volume of per mL to make the final concentration of the solution be 0.5 mu M, slowly covering a release tube, rapidly inverting a centrifuge tube, vortex-shaking to quickly mix the dye uniformly, and incubating the solution at room temperature in a dark place for 10min; to the direction ofAdding precooled complete culture medium with volume of more than 6 times of cell sap into the centrifuge tube, fully and uniformly mixing, incubating in ice-water bath for 5min, and stopping marking; washing the cells with complete medium at 4deg.C, 15000g for 5min for 2 times;

(5) Co-incubation and tumor ball photography: the CFSE-labeled immune cells were used at 1X 10 ⁶ Adding tumor balls into each hole for co-incubation for 48 hours, then gently sucking the tumor balls, washing the tumor balls with PBS for 3 times, and washing out non-infiltrated immune cells; the sucked-out pellets are placed in a confocal dish, 5% paraformaldehyde is added for fixation for 30min, PBS is used for washing three times after fixation is finished, and a proper amount of anti-quenching agent is added for Z-axis scanning by an Olinbus confocal microscope. The results are shown in FIG. 13.

The results show that the low molecular weight compounds I, SEQ ID NO.1 and SEQ ID NO.2 enhance immune cell infiltration in tumor spheres.

Example 4: tumor cell migration experiments

(1) Tumor cell migration: culturing and collecting logarithmic growth phase CT26 cells, centrifuging and washing with precooled PBS buffer solution, re-suspending with serum-free DMEM medium, counting and adjusting cell density to 2×10 ⁵ individual/mL; 4T1 cells were resuspended in serum-free DMEM medium, counted and cell densities were adjusted to 5X 10 ⁵ individual/mL; 200. Mu.L of the cell suspension was added to the upper layer of the 8 μm cell, and 600. Mu.L of complete medium containing 50. Mu.M of the low molecular weight compound (I) or its derivative was added to the lower layer, and the mixture was allowed to migrate in an incubator at 37℃for 24 hours;

(2) Cell fixation and staining: taking out the cell after 24 hours, washing 3 times by PBS, adding 5% paraformaldehyde for fixation for 30 minutes, gently wiping the non-migrated cells on the upper layer after fixation, and washing 3 times by PBS; 1% crystal violet was added for 20min and washed 3 times with PBS. Shooting with Color Camera Nikon DS-Fi3 20X magnification lens. The results are shown in FIGS. 14-17.

The results in FIGS. 14 and 15 show that low molecular weight compound I with SEQ ID NO.2 inhibits CT26 and 4T1 cell migration. FIG. 16 shows that the truncated peptide of SEQ ID No.2 also has an inhibitory effect on CT26 migration, but that the inhibitory effect is not as pronounced as the intact tetrapeptide, indicating that the tetrapeptide structure of SEQ ID No.2 is crucial for its function in inhibiting tumor cell metastasis. FIG. 17 shows that the alanine scanning peptide of SEQ ID NO.2 has significantly reduced inhibition of CT26 migration, indicating that the four amino acids of the SEQ ID NO.2 polypeptide are critical for inhibiting tumor cells.

Example 5: the anti-tumor effect of SEQ ID NO.2 is explored in CT26 colon cancer transplantation tumor model

(1) Tumor-bearing: CT26 colorectal cancer cells with good growth status were collected, and tumor-bearing BABL/c mice (5X 10) ⁵ cells/cells only);

(2) After 5 days of tumor bearing, the tumor volume of the mice is about 40-80mm ³ Starting S-shaped group and daily intraperitoneal administration, continuously administering for 2 weeks, weighing the weight of the mice with an electronic balance at intervals of day, measuring the tumor of the mice with a digital vernier caliper, calculating according to the formula v=1/2×a (length) ×b (width) ×c (height) and recording the tumor volume change of the mice. The results are shown in FIGS. 18-19.

The tumor volume curve of FIG. 18 shows that SEQ ID NO.2 can well inhibit the growth of CT26 colorectal cancer transplants at the administration dosage of 0.48 mg/kg. FIG. 19 shows that the body weight of BABL/c mice is not significantly reduced at the dose of 0.48mg/kg of SEQ ID NO.2, and the mental state of the mice is good during the administration period.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (9)

1. A DDR1 affinity peptide, wherein said DDR1 affinity peptide is selected from the group consisting of the following peptides a1, a2, or a combination thereof:

peptide a1: (D-Phe) -Trp- (D-Cha);

peptide a2: (D-Phe) - (D-Phe) -Trp- (D-Phe) or a truncated peptide thereof, and an alanine scanning peptide.

2. The DDR1 affinity peptide of claim 1, wherein the truncated peptide of peptide a2 comprises at least one of (D-Phe) - (D-Phe), (D-Phe) -Trp, trp- (D-Phe), (D-Phe) -Trp, (D-Phe) -Trp- (D-Phe).

3. The DDR1 affinity peptide of claim 1, wherein the alanine scanning peptide of peptide a2 comprises at least one of (D-Ala) - (D-Phe) -Trp- (D-Phe), (D-Phe) - (D-Ala) -Trp- (D-Phe), (D-Phe) -Ala- (D-Phe).

Use of a DDR1 affinity peptide for the preparation of a medicament for the treatment of a tumor or a probe for the diagnosis of a tumor, characterized in that said DDR1 affinity peptide comprises at least one of the following polypeptides: (D-Phe) - (D-Phe) -Trp- (D-Cha), (D-Phe) - (D-Phe) -Trp- (D-Phe), (D-Phe) -Trp, trp- (D-Phe), (D-Phe) - (D-Phe) -Trp, (D-Phe) -Trp- (D-Phe), (D-Ala) - (D-Phe) -Trp- (D-Phe), (D-Phe) - (D-Ala) -Trp- (D-Phe), (D-Phe) - (D-Phe) -Ala- (D-Phe), (D-Phe) - (D-Phe) -Trp- (D-Ala).

5. The use of claim 4, wherein the DDR1 affinity peptide inhibits tumor growth by blocking DDR1 binding to collagen or inhibiting activation of the DDR1 downstream pathway.

6. The use of claim 4, wherein the tumor is any one of colon cancer, breast cancer.

7. A pharmaceutical composition comprising a DDR1 affinity peptide of any one of claims 1-3, or a pharmaceutically acceptable salt-forming form thereof, or a free form thereof, or a chemically modified derivative thereof.

8. The pharmaceutical composition of claim 7, wherein the chemical modification is any one of cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin affinity peptide coupling, tumor homing peptide coupling, transmembrane peptide coupling, nanocarrier coupling, radionuclide coupling, small molecule compound coupling, nucleotide coupling, protein coupling.

9. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable excipient.