CN110551177A

CN110551177A - Polypeptide, polypeptide derivative, polypeptide-antibody compound, preparation and application

Info

Publication number: CN110551177A
Application number: CN201910808761.2A
Authority: CN
Inventors: 吴疆; 刘丹; 邓蒙蒙; 王保如
Original assignee: Anhui Ruida Health Industry Co Ltd
Current assignee: Anhui Ruida Health Industry Co Ltd
Priority date: 2019-08-29
Filing date: 2019-08-29
Publication date: 2019-12-10

Abstract

The invention discloses a polypeptide, and belongs to the field of biological medicines. Aiming at the problems of complicated production process, high cost and low targeting property of the existing method for enhancing the targeting property of the T cell, the invention provides the polypeptide, the polypeptide can remarkably enhance the targeting property of the immune cell, and meanwhile, the preparation process of the polypeptide is simple, and the preparation cost is lower than the cost for preparing the CAR-T cell and the cost for preparing the bispecific antibody cell.

Description

polypeptide, polypeptide derivative, polypeptide-antibody compound, preparation and application

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a polypeptide, a polypeptide derivative, a polypeptide-antibody compound, and preparation and application thereof.

background

at present, tumors become one of serious diseases which harm human health, according to the annual report of Chinese tumor registration in 2017, about 1 million people are diagnosed as tumors every day, about 7 people suffer from cancer every minute, compared with 2012, the number of new cancer cases continues to rise, about 368 thousand, in the same year, 1409 thousand new worldwide cancer cases, and 1/4 about the world is occupied by new Chinese cancer cases. At present, the treatment means of tumors mainly comprises surgical excision, radiotherapy, chemotherapy, immunotherapy and the like, and the immunotherapy is increasingly paid more attention. At present, there are two methods for enhancing the targeting of T cells, specifically as follows:

A chimeric antigen receptor modified T cell (CAR-T) is characterized in that a chimeric antigen receptor consisting of an ScFv segment, a transmembrane region, a costimulatory signal and an intracellular signaling region of an antibody is transferred into the T cell by means of viral transfection, so that the receptor is expressed on the surface of the T cell, on one hand, the capacity of the T cell to specifically recognize tumors is enhanced, on the other hand, the T cell is activated, and the CAR-T plays a role without being limited by MHC molecules. Grupp SA showed that complete remission was achieved after CAR-T cell therapy with CTL019 in two relapsed and therapy-resistant pre-B cell acute lymphoblastic leukemia child patients (N Engl JMed.2013; 368(16): 1509-. Porter DL developed CART19 cells targeting CD19, in response to relapsed chronic lymphocytic leukemia, at the time of publication, complete remission rates had persisted for 10 months (N Engl J Med.2011; 365(8): 725-. The study of Maude SL showed that complete remission rates were 90% (27) in 30 total children and adults treated with CD 19-targeted CAR-T cell CTL019 against relapsed and refractory acute lymphoblastic leukemia. Disease-free survival rate at 6 months was 67% (N Engl J Med.2014; 371(16): 1507-17.).

Bispecific antibodies are another type of method to enhance immune cell targeting, e.g., bispecific antibodies that enhance T cell targeting, typically consisting of an antibody that recognizes the T cell surface antigen CD3 and recognizes a tumor cell surface antigen, to target T cells to tumor cells. The study by Reusch U et al showed that the CD33/CD3 bispecific antibody significantly enhanced T cell killing of the CD33+ acute myelogenous leukemia cell line, and the T cell + CD33/CD3 bispecific antibody significantly inhibited the growth of HL-60 transplantable tumors in an immunodeficient mouse tumor model (Clin Cancer Res.2016; 22(23): 5829-). 5838.). Another CD3/EGFR bispecific antibody developed against EGFR positive tumors by Reusch U et al showed good results in both in vivo and in vitro experiments (Clin Cancer Res.2006; 12(1): 183-90.). The research shows that the target property of the immune cells is enhanced, and the target killing activity of the immune cells to the tumor is obviously enhanced.

At present, CAR-T cell treatment is only effective on blood tumors but not on solid tumors, and the CAR-T cell preparation needs to construct a lentiviral vector and purify viruses, so that the production process is complicated and the cost is high.

At present, no clinical report of the bispecific antibody exists, and the production and preparation process of the bispecific antibody is relatively complicated and high in cost.

In summary, the existing methods for enhancing T cell targeting are as follows:

1. The chimeric antigen receptor modifies T cells to enhance the targeting property of the T cells, and has the problems of complicated production process, high cost and low targeting property of CAR-T cells;

2. the bispecific antibody enhances the targeting of T cells, and has the problems of complicated production process, high cost and low targeting of the bispecific antibody cells.

Disclosure of Invention

1. Problems to be solved

Aiming at the problems of complicated production process, high cost and low targeting property of the existing method for enhancing the targeting property of the T cell, the invention provides the polypeptide, the polypeptide can remarkably enhance the targeting property of the immune cell, and meanwhile, the preparation process of the polypeptide is simple, and the preparation cost is lower than the cost for preparing the CAR-T cell and the cost for preparing the bispecific antibody cell.

2. Technical scheme

In order to solve the above problems, the present invention adopts the following technical solutions.

The polypeptide is any one of amino acid sequences shown in (I) and (II):

(I) has the sequence of SEQ ID NO: 1;

And (II) has a sequence obtained by modifying, substituting or adding one or more amino acids to the amino acid sequence shown in (I).

Preferably, the substitution is that K in the amino acid sequence is substituted by R, and/or S in the amino acid sequence is substituted by T, the proportion of K in the polypeptide after substitution is 40-47%, and the amino acid close to the carboxyl terminal of the polypeptide is lysine.

Preferably, the modification is acetylation.

"the amino acid near the carboxy terminus of a polypeptide is lysine" means that the polypeptide is counted from the left (the amino terminus is the left) to the right, and the rightmost amino acid is lysine. Preferably, the addition is of 1, 2, 3, 4 or 5 amino acids.

Preferably, the amino acid sequence shown in (II) is any one of the amino acid sequences shown in (III) or (IV):

(iii) has SEQ ID NO: 2;

(iv) has the sequence of SEQ ID NO: 3.

the polypeptide derivative is any one of amino acid sequences shown in (V), (VI), (VII) and (VIII):

(VII) having a sequence obtained by modifying, substituting or adding one or more amino acids to the amino acid sequence shown in (V);

(VIII) has a sequence obtained by modifying, substituting or adding one or more amino acids to the amino acid sequence shown in (VI);

Wherein,

the R is₀Is alkynyl, any one of formula (1) or formula (2);

The R is₁Is alkyl or alkenyl.

Preferably, the modification is acetylation.

Preferably, the substitution is that K in the amino acid sequence is substituted by R, and/or S in the amino acid sequence is substituted by T, the proportion of K in the substituted polypeptide derivative is 40-47%, and the amino acid close to the carboxyl terminal of the polypeptide derivative is lysine.

"the amino acid near the carboxy terminal of the polypeptide is lysine" means that the polypeptide derivative is counted from the left (the amino terminal is the left) to the right, and the rightmost amino acid is lysine.

Preferably, the addition is of 1, 2, 3, 4 or 5 amino acids.

Preferably, the amino acid sequence shown in (VII) is any one of formula (3) and formula (4), the amino acid sequence shown in (VIII) is any one of formula (5) and formula (6),

Wherein,

the R is₀Is alkynyl, any one of formula (1) or formula (2);

the R is₁Is alkyl or alkenyl.

preferably, the first and second liquid crystal materials are,

When said R is₀When it is alkynyl, said R₀Is any one or more of formula (10), formula (11), formula (12), formula (13) and formula (14);

The R is₁is any one or more of formula (15), formula (16), formula (17) and formula (18);

A polypeptide derivative-antibody complex having the general formula:

Polypeptide derivative-R₂；

The polypeptide derivative is the polypeptide derivative of any one of the above;

the R is₂Is an antibody.

R₂Is an antibody capable of recognizing a tumor cell surface antigen.

Preferably, said R is₂is any one of Her2 antibody, EGFR antibody, 5T4 antibody, VEGFR antibody, CD19 antibody and CD20 antibody.

A cellular complex having the general formula:

Immunocyte-polypeptide derivative-R₂；

The R is₂is an antibody.

Preferably, the immune cell is any one of NK cell, NKT cell, alpha beta T cell and gamma delta T cell, and R is₂Is any one of Her2 antibody, EGFR antibody, 5T4 antibody, VEGFR antibody, CD19 antibody and CD20 antibody.

The preparation method of the polypeptide derivative-antibody compound comprises the steps of dissolving the polypeptide derivative, adding a buffer solution, mixing, adding an NHS active ester modified antibody for reaction, and obtaining the polypeptide derivative-antibody compound.

Preferably, the mass ratio of the polypeptide derivative to the antibody modified by the NHS active ester is 0.5-10:1, the reaction time is 0.5-24h, and the reaction temperature is 0-50 ℃.

Preferably, the mass ratio of the polypeptide derivative to the antibody modified by the NHS active ester is 2.5:1, the reaction time is 2 hours, the reaction temperature is 37 ℃, the buffer solution is D-PBS, and the solvent for dissolving the polypeptide derivative is dimethyl sulfoxide.

Preferably, the compound is prepared by any one of the preparation methods.

The polypeptide of any one of the above items, the polypeptide derivative-antibody complex of any one of the above items, and the use of the cell complex of any one of the above items in the preparation of an anti-tumor drug.

The polypeptide, the polypeptide derivative-antibody complex and the cell complex are preferably used for preparing an anti-tumor medicament, and the tumor comprises any one or more of breast cancer, lung cancer, colorectal cancer, liver cancer, glioma, pancreatic cancer, ovarian cancer, prostate cancer, kidney cancer, melanoma, sarcoma, myeloma, leukemia and gastric cancer.

3. Advantageous effects

compared with the prior art, the invention has the beneficial effects that:

(1) The polypeptide derivative can obviously enhance the targeting property of immune cells, and meanwhile, the preparation process of the polypeptide derivative is simple, and the preparation cost is lower than the cost for preparing the CAR-T cells and the cost for preparing bispecific antibody cells in the prior art;

(2) The polypeptide derivative can obviously enhance the targeting property of immune cells, and meanwhile, the preparation process of the polypeptide derivative is simple, and the preparation cost is lower than the cost for preparing the CAR-T cells and the cost for preparing bispecific antibody cells in the prior art;

(3) The polypeptide derivative-antibody compound can be almost 100 percent combined on the surface of immune cells, so that the targeting property and the killing activity of the immune cells are enhanced, and the aim of treating tumors is fulfilled;

(4) the amino group of the carboxyl-terminal side chain of the polypeptide can be conveniently modified with NHS active ester through modification of NHS active ester or modification of acetylene NHS active ester₂the antibody is connected to form a polypeptide derivative-antibody compound, and the connection mode can promote the connection performance of the polypeptide derivative and the antibody and is more favorable for the activity of the antibody;

(5) The amino terminal of the polypeptide of the invention is connected with saturated fatty acid or unsaturated fatty acid, which can anchor the polypeptide on the surface of immune cell membrane, thereby enhancing the targeting effect and lethality of immune cells.

Drawings

FIG. 1 is a confocal inverted microscope showing the distribution of 17-sequence polypeptide derivatives on the cell surface;

FIG. 2 shows the distribution of the 17-sequence polypeptide derivative-5T 4 antibody complex on the NK cell surface detected by Confocal;

FIG. 3 is a flow cytometer detecting the proportion of 17-sequence polypeptide derivative-5T 4 antibody complex-FITC positive cells;

FIG. 4 is a CFSE + PI double staining method for detecting the killing activity of NK cell-17 sequence polypeptide derivative-5T 4 antibody complex on non-small cell lung cancer cell H-1975 cells;

FIG. 5a is the inhibition of the growth of H-1975 cell transplantable tumors by NK cell-17 sequence polypeptide derivative-5T 4 antibody complex;

FIG. 5b is the change of NK cell-17 sequence polypeptide derivative-5T 4 antibody complex to H-1975 cell transplanted tumor volume;

FIG. 5c is the change in weight of NK cell-17 sequence polypeptide derivative-5T 4 antibody complex against H-1975 cell transplant tumor;

FIG. 6 shows the killing activity of NK cell-20 sequence polypeptide derivative-5T 4 antibody complex against breast cancer cell HCC 1954;

FIG. 7 shows the killing activity of gamma delta T cell-20 sequence polypeptide derivative-Her 2 antibody complex against breast cancer cell HCC 1954;

FIG. 8 shows the killing activity of gamma delta T cell-22 sequence polypeptide derivative-Her 2 antibody complex against cancer cell NCI-N87.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

EXAMPLE 1 Synthesis of polypeptide derivatives

The polypeptide derivative of the invention completes a peptide chain by utilizing Fmoc solid-phase polypeptide synthesis technology, and the purity is higher than 95% through HPLC detection.

raw materials and related reagents for synthesis:

Fmoc-L-Asp (Otbu) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Lys (Boc) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Gly-OH, Gill Biochemical (Shanghai) Ltd

Fmoc-Pro-OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-Ser (Tbu) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Lys (Dde) -OH, Gill Biochemical (Shanghai) Co., Ltd

A source of myristic acid,

Azoic acetic acid NHS active ester

HBTU (N, N, N ', N' -tetramethyl-O- (1H-benzotriazole) urea hexafluorophosphate),

DIEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide),

DCM (dichloromethane), acetonitrile, 2-Chlorotrityl Chloride Resin, piperidine, 2% hydrazine hydrate TFA,

TIS (triisopropylsilane), EDT (triisopropylsilane), nitrogen, anhydrous ether, ninhydrin, KCN.

The above reagents are all purchased from the market.

Solid phase polypeptide synthesis operation steps:

Swelling of resin

0.6g of AM Resin with a degree of substitution of 0.4mmol/g was weighed into a reaction tube, DCM (15ml/g) was added, and the mixture was shaken for 30min.

Two, and then the first amino acid

Filtering off solvent by sand core, adding 3 times mol of Fmoc-L-Lys (Dde) -OH amino acid, adding 3 times mol of DIEA and 3 times mol of HBTU, finally adding a small amount of DMF to dissolve, oscillating for 1h, and alternately washing for 6 times by DMF and DCM.

Third, deprotection

15ml of 20% piperidine DMF solution (15ml/g) was added for 5min, removed and 15ml of 20% piperidine DMF solution (15ml/g) was added for 15min.

fourth, detection

the piperidine solution is pumped out, dozens of particles of resin are taken out, washed with ethanol for three times, added with ninhydrin, KCN and phenol solution respectively in a drop manner, heated at the temperature of 105 ℃ and 110 ℃ for 5min, and turned dark blue is a positive reaction.

And washing twice with DMF (10ml/g), twice with methanol (10ml/g) and twice with DMF (10 ml/g). (hereinafter referred to as "washing 6 times")

Sixthly, connecting with a second amino acid

The solvent was filtered off by suction through sand core, 3 times molar Fmoc-L-Asp (Otbu) -OH amino acid was added, 3 times molar DIEA, 3 times molar HBTU were added, finally a small amount of DMF was added for dissolution, shaken for 1h and washed with DMF and DCM alternately for 6 times.

Deprotection: adding 15ml 20% piperidine DMF solution (15ml/g), removing, adding 15ml 20% piperidine DMF solution (15ml/g), 15 min;

And (3) detection: the piperidine solution is pumped out, dozens of particles of resin are taken out, washed with ethanol for three times, added with ninhydrin, KCN and phenol solution respectively in a drop manner, heated at the temperature of 105 ℃ and 110 ℃ for 5min, and turned dark blue is a positive reaction.

Seven, connecting with a third amino acid

Filtering off the solvent by sand core, adding 3 times mol of Fmoc-L-Gly-OH amino acid, then adding 3 times mol of DIEA and 3 times mol of HBTU, finally adding a small amount of DMF to dissolve, oscillating for 1h, and alternately cleaning for 6 times by DMF and DCM.

Eight, connecting with fourth amino acid

Filtering off the solvent by sand core, adding 3 times mol of Fmoc-L-Pro-OH amino acid, then adding 3 times mol of DIEA and 3 times mol of HBTU, finally adding a small amount of DMF to dissolve, oscillating for 1h, and alternately cleaning for 6 times by DMF and DCM.

And (3) detection: the piperidine solution is pumped out, dozens of particles of resin are taken out, washed with ethanol for three times, and added with ninhydrin, KCN and phenol solution. One drop, heating at 105 ℃ and 110 ℃ for 5min, and turning dark blue to be positive reaction.

Nine, connecting other amino acids in sequence

According to the two-to-four-step operation principle, when the fifth and sixth amino acids are sequentially added, the seventeenth amino acid synthesis raw materials are sequentially connected from right to left, so that the amino acid sequence connected from left to right is Gly Ser Ser Lys Ser Pro Ser Lys Lys Lys Lys Lys Lys ProGly Asp Lys sequence.

myristic acid

Filtering off solvent by sand core, adding myristic acid 3 times mol, adding DIEA 3 times mol, HBTU 3 times mol, adding small amount of DMF for dissolving, oscillating for 1h, and alternately cleaning with DMF and DCM for 6 times.

Eleven, removal of side chain Dde protecting group

adding 15ml of 2% hydrazine hydrate for reaction for 15min, removing the added 15m of 22% hydrazine hydrate for reaction for 15min, and washing for 6 times. Taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solution one drop at a time, heating the mixture at the temperature of 105 ℃ and 110 ℃ for 5min, and taking a dark blue color as a positive reaction.

Twelve, connecting nitrine acetic acid NHS active ester:

Filtering off the solvent through a sand core, adding 3 times of mol of NHS active ester, then adding 3 times of mol of DIEA, 3 times of mol of HBTU, and finally adding a small amount of DMF to dissolve, oscillating for 1h, and alternately cleaning for 6 times by using DMF and DCM; taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solution one drop at a time, heating the mixture at the temperature of 105 ℃ and 110 ℃ for 5min, and taking a dark blue color as a positive reaction.

Thirteen, cleavage of the polypeptide derivative from the resin

Preparing cutting fluid: TFA (trifluoroacetic acid) 94.5%; 2.5 percent of water; 2.5 percent of EDT; 1% of TIS;

And (3) putting the resin into a flask or a centrifuge tube, shaking the resin and the cutting fluid at a constant temperature for 120min according to the proportion of 10 ml/g.

Fourteen, blow-dry washing

And (3) carrying out suction filtration, collecting filtrate, adding diethyl ether, carrying out chromatography by using diethyl ether to obtain the polypeptide derivative, filtering, washing a filter cake by using diethyl ether for six times, volatilizing the filter cake at normal temperature, and blowing residual diethyl ether in the filter cake by using nitrogen to obtain a crude product of the polypeptide derivative.

Fifteen, purifying the crude product of the polypeptide derivative by HPLC

the method comprises the following specific operation steps:

(1) putting 200mg of the crude polypeptide derivative product into a vessel, dissolving with 2-5ml of 50% acetonitrile water solution, and performing ultrasonic treatment for 2 min; (2) the solution was filtered through a 0.45um filter.

(3) analysis conditions were as follows: taking 3ul of the solution (crude polypeptide derivative solution) obtained in the step 2, wherein the mobile phase is water and acetonitrile, performing gradient elution for 30min, balancing HPLC with an initial gradient for 5min, and then injecting a sample, wherein the initial gradient is water 95%, the acetonitrile is 5%, the final ratio is water 5%, and the acetonitrile is 95%.

The method is suitable for separating and purifying crude polypeptide derivatives through exploration and identification of analysis conditions.

(4) Preparation: and (5) preparing a sample injection for the dissolved sample. Preparative HPLC equilibration for 10min, initial gradient water 95%, acetonitrile 5%, end gradient water 25%, acetonitrile 75%, gradient time 40min, collected samples from the detector.

(5) and (3) identification: the collected samples were sampled for purity and MS identification.

through conventional purity and MS identification, the polypeptide derivative synthesized by the Fmoc solid-phase polypeptide derivative is a target polypeptide derivative sequence, the MS is 1815.72, and the purity reaches 97.1%.

sixthly, freeze-drying the purified solution under the conventional conditions to obtain a finished product, wherein the finished product is the polypeptide derivative which is white powder.

seventhly, storing the polypeptide derivative at the temperature of 20 ℃ below zero. The obtained polypeptide derivative is named as a 17-sequence polypeptide derivative.

The polypeptide derivative obtained in this example has the following structural formula by sequencing:

Example 2 Synthesis of polypeptide derivatives

The peptide chain is completed by utilizing Fmoc solid phase polypeptide synthesis technology, and the purity is higher than 95% through HPLC detection.

the synthesis starting materials and the reagents are as in example 1.

Fmoc-L-Asp (Otbu) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Lys (Boc) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Gly-OH, Gill Biochemical (Shanghai) Ltd

Fmoc-Pro-OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-Ser (Tbu) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Lys (Dde) -OH, Gill Biochemical (Shanghai) Co., Ltd

Cyclooctyne NHS active ester:

solid phase polypeptide synthesis operation steps:

Swelling of resin

Weigh 1.0g of AM Resin with a degree of substitution of 0.4mmol/g, place the Resin in a reaction tube, add DCM (18ml/g), and shake for 50min.

two, and then the first amino acid

the solvent was filtered off by suction through sand core, 3 times molar Fmoc-L-Lys (Dde) -OH amino acid was added, 3 times molar DIEA, 3 times molar HBTU were added, finally a small amount of DMF was added for dissolution, shaken for 1.5h, and washed with DMF and DCM alternately for 6 times.

Third, deprotection

Fourth, detection

Sixthly, connecting with a second amino acid

seven, connecting with a third amino acid

Eight, connecting with fourth amino acid

Nine, connecting other amino acids in sequence

The twenty-first amino acid synthesis starting material is added sequentially when the fifth and sixth amino acid are followed by the twenty-second amino acid according to the two-to four-step operating principle, and the amino acid sequence connected from left to right is the lysprolysglsetthrysserpro serlylyslylyslylyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslyslysasp lyslys.

Docosahexaenoic acid

Filtering off solvent by sand core, adding 3 times mol of docosahexaenoic acid, adding 3 times mol of DIEA and 3 times mol of HBTU, adding a small amount of DMF, dissolving, oscillating for 1h, and alternately washing with DMF and DCM for 6 times.

Eleven, removal of side chain Dde protecting group

Adding 20ml of 2% hydrazine hydrate for reaction for 25min, removing the added 20ml of 22% hydrazine hydrate for reaction for 25min, and washing for 6 times. Taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solution one drop at a time, heating the mixture at the temperature of 105 ℃ and 110 ℃ for 5min, and taking a dark blue color as a positive reaction.

twelve, grafting alkynyl: filtering off the solvent from the alkynyl active ester in the formula (10) by using a sand core, adding 3 times of mol of alkynyl under the protection of nitrogen, adding 3 times of mol of DIEA and 3 times of mol of HBTU, finally adding a small amount of DMF for dissolving, oscillating for 1h, and alternately cleaning for 6 times by using DMF and DCM; taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solution one drop at a time, heating the mixture at the temperature of 105 ℃ and 110 ℃ for 5min, and taking a dark blue color as a positive reaction.

Thirteen, cleavage of the polypeptide derivative from the resin

Fourteen, blow-dry washing

Fifteen, purifying the crude product of the polypeptide derivative by HPLC

The method comprises the following specific operation steps:

(1) Putting 200mg of the crude polypeptide derivative product into a vessel, dissolving with 2-5ml of 50% acetonitrile water solution, and performing ultrasonic treatment for 2 min;

(2) The lysate was filtered through a 0.45um filter.

(3) And (3) analysis: taking 3ul of the solution obtained in the step 2, wherein the mobile phase is water and acetonitrile, performing gradient elution for 30min, balancing HPLC by using an initial gradient for 5min, and then injecting a sample, wherein the initial gradient is 95% of water, the initial gradient is 5% of acetonitrile, and the final ratio is 5% of water and 95% of acetonitrile.

Through purity and MS identification, the polypeptide derivative synthesized by the Fmoc solid-phase polypeptide derivative is a target polypeptide derivative sequence, the MS is 1919.23, and the purity reaches 96.4%.

Seventhly, storing the polypeptide derivative at the temperature of 20 ℃ below zero. The obtained polypeptide derivative is named as 20-sequence polypeptide derivative.

By sequencing, the polypeptide derivative obtained in this example has the following structural formula:

EXAMPLE 3 polypeptide derivative Synthesis

the synthesis starting materials and the reagents are as in example 1.

Fmoc-L-Asp (Otbu) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Lys (Boc) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Gly-OH, Gill Biochemical (Shanghai) Ltd

Fmoc-Pro-OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-Ser (Tbu) -OH, Gill Biochemical (Shanghai) Co., Ltd

Fmoc-L-Lys (Dde) -OH, Gill Biochemical (Shanghai) Co., Ltd

Solid phase polypeptide synthesis operation steps:

Swelling of resin

Two, and then the first amino acid

third, deprotection

15ml of 20% piperidine DMF solution (15ml/g) was added for 5min, the piperidine solution was removed and 15ml of 20% piperidine DMF solution (15ml/g) was added for 15min.

Fourth, detection

Sixthly, connecting with a second amino acid

Deprotection: 15ml of 20% piperidine DMF solution (15ml/g) was added thereto for 5min, and the piperidine solution was removed therefrom, and 15ml of 20% piperidine DMF solution (15ml/g) was added thereto for 15min, and the piperidine solution was removed therefrom.

Seven, connecting with a third amino acid

deprotection: adding 15ml of 20% piperidine DMF solution (15ml/g), draining the piperidine solution for 5min, adding 15ml of 20% piperidine DMF solution (15ml/g), and draining the piperidine solution for 15 min;

Eight, connecting with fourth amino acid

Nine, connecting other amino acids in sequence

The amino acid sequence linked from left to right was the sequence prolysglylys serserprolysserlylys lyslys lys rosglyasplys, which was sequentially linked from right to left according to the two-to four-step procedure followed by the fifth and sixth.

Lauric acid

The solvent was filtered off by suction through sand core, 3 times molar lauric acid was added, 3 times molar excess DIEA, 3 times molar HBTU were added, finally a small amount of DMF was added to dissolve, shaking for 1h, washing with DMF and DCM alternately 6 times.

Eleven, removal of side chain Dde protecting group

Twelve, grafting Cys:

Filtering off the solvent through a sand core, adding Cys with the molar weight of 3 times under the protection of nitrogen, adding DIEA with the molar weight of 3 times and HBTU with the molar weight of 3 times, finally adding a small amount of DMF to dissolve, oscillating for 1h, and alternately cleaning for 6 times by using DMF and DCM; taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solution one drop at a time, heating the mixture at the temperature of 105 ℃ and 110 ℃ for 5min, and taking a dark blue color as a positive reaction.

Thirteen, cleavage of the polypeptide derivative from the resin

Fourteen, blow-dry washing

fifteen, purifying the crude product of the polypeptide derivative by HPLC

The method comprises the following specific operation steps:

according to conventional purity and MS identification, the polypeptide derivative synthesized by the Fmoc solid-phase polypeptide derivative is a target polypeptide derivative sequence, the MS is 2251.63, and the purity reaches 97.7%.

Seventhly, storing the polypeptide derivative at the temperature of 20 ℃ below zero. The obtained polypeptide derivative is named as 22 sequence polypeptide derivative.

Example 4 antibody modification

NHS esters (N-hydroxysuccinimide esters) are the most common amino-reactive active esters. Since amino groups are very common in proteins and polypeptides, amino group modification is one of the most common of biomacromolecule modifications. Other oligonucleotides, DNA and saccharides containing amino groups can be modified with amino groups to label groups such as fluorescent probes.

The reaction of NHS esters with amino groups is very sensitive to pH, and too low or too high a pH can affect the reaction: at low pH, the amino group is protonated and cannot be modified; the modification efficiency is greatly reduced when the NHS ester is hydrolyzed too fast at high pH; the optimum pH for the modification reaction is between 8.3 and 8.5.

The modification reaction is usually carried out in water, if the NHS ester-linked functional group is not easily soluble in water, DMSO and DMF can be added to aid dissolution, the pH value is adjusted to 8.3-8.5, and care is taken that no amino group can be contained in DMF.

The antibody modification procedure is as follows:

(1) the amount of NHS ester used was calculated

the amount of NHS ester [ mg ] ═ 8 of the amount of charged amino molecules [ mg ]. of the molar mass of NHS ester [ Da ]/of charged amino molecules [ Da ].

8 indicates excess NHS ester, an experimental value for monoamino labeling, applicable to most proteins and polypeptides. However, in some particular cases more or less NHS esters may be required, depending on the structure of the protein, the solvent and the solubility conditions. The molar mass of a substance is numerically equal to the molecular weight of the substance.

The amount of amino-bearing molecules [ mg ] refers to the amount of mono-amino-labeled substance, and if multiple amino groups are to be labeled on a protein or polypeptide, the amount of NHS ester is multiplied.

(2) Determining the volume of the reaction System

The reaction of the NHS ester with the amino group can be carried out at different volumes. However, when the reaction volume is small (10-20. mu.L), it is preferred to use a higher concentration (1-10 mg of amino group-bearing substance per ml).

(3) the NHS ester was dissolved in 1/10 reaction volumes of DMF or DMSO, with amino-free DMF being preferred. The fully dissolved NHS ester solution can be stored at-20 ℃ for 1-2 months.

(4) The amino group bearing molecule was dissolved in 9/10 reaction volume buffer and the pH was adjusted to 8.3-8.5.

The pH is of utmost importance, and the pH of 0.1M sodium bicarbonate buffer and 0.1M phosphate buffer substantially meet the requirements. In addition, the buffer is prevented from containing free amino groups.

When large scale labeling (several hundred mg of NHS ester was labeled), care was taken to acidify the solution by hydrolysis of the NHS ester. It is necessary to observe the change of pH value at any time or to use a buffer at a high concentration.

(5) And (3) adding the NHS ester solution prepared in the step (3) into the reactant solution in the step (4), fully stirring, and monitoring the reaction by HIC-HPLC. The reaction is carried out on ice overnight or at room temperature for more than 4 h.

(6) Purification of the reactants

Macromolecular gel filtration is the most commonly used method. Precipitation and chromatography may also be used. Organic impurities (e.g. N-hydroxysuccinimide, NHS esters, acids produced by hydrolysis) are easily isolated. For proteins and nucleic acids, ethanol and acetone precipitation purification can also be used.

Antibodies used in the examples, Her2 antibody, EGFR antibody, 5T4 antibody, VEGFR antibody, CD19 antibody, CD20 antibody, RGD, were all purchased from the market.

preparation of modification product of NHS active ester and trastuzumab (Her2)

At room temperature of 5-40 ℃, preferably 25 ℃, 5mg/ml trastuzumab Her2 solution (trastuzumab is dissolved in phosphate buffer PBS, the concentration range of the trastuzumab is 2-35mg/ml), 8 times molar amount (relative to trastuzumab) of active ester solution (dissolved in DMF or DMSO, the volume is not more than 20% of that of the PBS) of formula (10) is dripped into the solution, the reaction is slightly stirred or shaken, HIC-HPLC monitors the reaction, and after the reaction is finished, the excessive small molecular NHS active ester hydrolysate is removed by ultrafiltration. The modification was loaded onto a hydrophobic chromatography column, equilibrated with 0.75M ammonium sulfate solution, eluted with 25mM ammonium sulfate solution, and the eluates were combined and replaced with PBS to give trastuzumab (Her2) modification.

Preparation of modifier of NHS active ester and 5T4 monoclonal antibody

At room temperature of 5-40 ℃, preferably 25 ℃, 5mg/ml5T4 monoclonal antibody solution (5T4 monoclonal antibody is dissolved in phosphate buffer PBS, the concentration range is 2-35mg/ml), 8 times molar amount (relative to the monoclonal antibody) of active ester solution (dissolved in DMF or DMSO, the volume is not more than 20% of the PBS) of formula (11) is dripped in, the reaction is stirred slightly or shaken, HIC-HPLC monitors the reaction, and after the reaction is finished, the excessive small molecular NHS active ester hydrolysate is removed by ultrafiltration. The modifier is loaded into a hydrophobic chromatographic column, the hydrophobic chromatographic column is equilibrated by 0.75M ammonium sulfate solution and then eluted by 25mM ammonium sulfate solution, and eluent is combined and replaced by PBS to obtain the 5T4 monoclonal antibody modifier.

Preparation of modifier of NHS active ester and VEGFR monoclonal antibody

Dripping 8 times molar amount (relative to the monoclonal antibody) of active ester solution (dissolved in DMF or DMSO, the volume does not exceed 20 percent of that of PBS) of formula (12) into 5-40 ℃, preferably 25 ℃, 5mg/ml VEGFR monoclonal antibody solution (VEGFR monoclonal antibody is dissolved in phosphate buffer PBS, the concentration range is 2-35mg/ml) at room temperature, slightly stirring or shaking the reaction, monitoring the reaction by HIC-HPLC, and removing excessive small molecular NHS active ester hydrolysate by ultrafiltration after the reaction is finished. And loading the modifier into a hydrophobic chromatographic column, balancing with 0.75M ammonium sulfate solution, eluting with 25mM ammonium sulfate solution, combining eluates, and replacing with PBS to obtain VEGFR monoclonal antibody modifier.

Preparation of modifier of NHS active ester and EGFR monoclonal antibody

at room temperature of 5-40 ℃, preferably 25 ℃, 5mg/ml of EGFR monoclonal antibody solution (EGFR monoclonal antibody is dissolved in phosphate buffer PBS, the concentration range of the EGFR monoclonal antibody is 2-35mg/ml), 8 times of molar amount (relative to the monoclonal antibody) of active ester solution (which is dissolved in DMF or DMSO, the volume is not more than 20% of the PBS), slightly stirring or shaking for reaction, HIC-HPLC monitoring the reaction, and after the reaction is finished, ultra-filtering to remove excessive small molecular NHS active ester hydrolysate. And loading the modifier into a hydrophobic chromatographic column, balancing with 0.75M ammonium sulfate solution, eluting with 25mM ammonium sulfate solution, combining eluates, and replacing with PBS to obtain EGFR monoclonal antibody modifier.

Preparation of modifier of NHS active ester and CD19 monoclonal antibody

Dripping 8 times molar amount (relative to the monoclonal antibody) of active ester solution (dissolved in DMF or DMSO, the volume is not more than 20% of PBS) of formula (14) into phosphate buffer PBS (CD19 monoclonal antibody is dissolved in PBS, the concentration range is 2-35mg/ml) at room temperature of 5-40 ℃, preferably 25 ℃, and ultrafiltering to remove excessive small molecular NHS active ester hydrolysate after the reaction is finished, wherein the volume of the active ester solution is not more than 20% of that of the PBS by stirring or shaking slightly. And loading the modifier into a hydrophobic chromatographic column, balancing with 0.75M ammonium sulfate solution, eluting with 25mM ammonium sulfate solution, combining eluates, and replacing with PBS to obtain the CD19 monoclonal antibody modifier.

Modification preparation of NHS active ester and CD20 monoclonal antibody

dripping 8 times molar amount (relative to the monoclonal antibody) of active ester solution (dissolved in DMF or DMSO, the volume is not more than 20% of that of PBS) of formula (azido NHS active ester) into 5-40 ℃, preferably 25 ℃, 5mg/ml CD20 monoclonal antibody solution (CD20 monoclonal antibody is dissolved in phosphate buffer PBS, the concentration range is 2-35mg/ml) at room temperature, slightly stirring or shaking for reaction, monitoring the reaction by HIC-HPLC, and removing excessive small molecular NHS active ester hydrolysate by ultrafiltration after the reaction is finished. And loading the modifier into a hydrophobic chromatographic column, balancing with 0.75M ammonium sulfate solution, eluting with 25mM ammonium sulfate solution, combining eluates, and replacing with PBS to obtain the CD20 monoclonal antibody modifier.

Note that: when NHS active ester is coupled with antibody, pH has obvious influence on the coupling reaction, preferably pH is 6.8-8.5, preferably pH is 7.5-8.4, more preferably pH is 8.1-8.3, such as 8.1, 8.2, 8.3.

EXAMPLE 5 reaction of polypeptide derivatives with antibodies to form polypeptide derivative-antibody complexes

Dissolving the polypeptide derivative synthesized in example 1 with dimethyl sulfoxide to prepare a solution with a concentration of 40-50mg/ml, adding 1.5mg of the synthesized polypeptide derivative into 10ml of D-PBS (Dulbecco's phospate Buffered Saline) with a pH of 7.4, fully mixing uniformly, adding 1.5mg of trastuzumab modifier (Her2), reacting at 0 ℃ for 24h, after the reaction is finished, using an ultrafiltration tube with 20-100Kd, concentrating the solution, removing unbound polypeptide derivative, finally concentrating to 80-120 μ l, and detecting the concentration of the polypeptide derivative-antibody complex by a BCA method.

In this embodiment, under the condition that other reaction conditions are not changed, the trastuzumab modifier (Her2) may be replaced by any one of 5T4 monoclonal antibody modifier, VEGFR monoclonal antibody modifier, EGFR monoclonal antibody modifier, CD19 monoclonal antibody modifier, and CD20 monoclonal antibody modifier, and a corresponding polypeptide derivative-antibody complex may also be obtained.

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 1 with trastuzumab modifier is named as a 17-sequence polypeptide derivative-Her 2 antibody complex;

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 1 with the 5T4 monoclonal antibody modifier is named as a 17-sequence polypeptide derivative-5T 4 antibody complex;

the polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 1 with the VEGFR monoclonal antibody modifier is named as a 17-sequence polypeptide derivative-VEGFR antibody complex;

The polypeptide derivative-antibody compound obtained by reacting the polypeptide derivative obtained in the example 1 with the EGFR monoclonal antibody modifier is named as a 17-sequence polypeptide derivative-EGFR antibody compound;

the polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 1 with the monoclonal antibody modifier CD19 is named as a 17-sequence polypeptide derivative-CD 19 antibody complex;

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 1 with the monoclonal antibody modifier CD20 was named as a 17-sequence polypeptide derivative-CD 20 antibody complex.

EXAMPLE 6 reaction of polypeptide derivatives with antibodies to form polypeptide derivative-antibody complexes

Dissolving the polypeptide derivative synthesized in example 2 with dimethyl sulfoxide to prepare 45-50mg/ml, adding 5mg of the synthesized polypeptide derivative into 10ml of D-PBS (Dulbecco's phospate Buffered Saline) with pH 7.4, fully mixing, adding 1mg of trastuzumab modifier (Her2), reacting at 37 ℃ for 2h, concentrating and replacing liquid by using an ultrafiltration tube with 100Kd after the reaction is finished, removing unbound polypeptide derivative, finally concentrating to 80-120 μ l, and detecting the concentration of the polypeptide derivative-antibody complex by a BCA method.

under the condition that other reaction conditions are not changed, the trastuzumab modifier (Her2) can be replaced by any one of 5T4 monoclonal antibody modifier, VEGFR monoclonal antibody modifier, EGFR monoclonal antibody modifier, CD19 monoclonal antibody modifier and CD20 monoclonal antibody modifier, and a corresponding polypeptide derivative-antibody compound can also be obtained.

the polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 2 with trastuzumab modifier is named as 20-sequence polypeptide derivative-Her 2 antibody complex;

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in the example 2 with the 5T4 monoclonal antibody modifier is named as 20-sequence polypeptide derivative-5T 4 antibody complex;

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 2 with the VEGFR monoclonal antibody modifier is named as 20-sequence polypeptide derivative-VEGFR antibody complex;

The polypeptide derivative-antibody compound obtained by reacting the polypeptide derivative obtained in the example 2 with the EGFR monoclonal antibody modifier is named as a 20-sequence polypeptide derivative-EGFR antibody compound;

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 2 with the monoclonal antibody modifier CD19 is named as 20-sequence polypeptide derivative-CD 19 antibody complex;

the polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 2 with the monoclonal antibody modifier CD20 was named 20-sequence polypeptide derivative-CD 20 antibody complex.

EXAMPLE 7 reaction of polypeptide derivatives with antibodies to form polypeptide derivative-antibody complexes

Dissolving the polypeptide derivative synthesized in example 3 with dimethyl sulfoxide to prepare 45-50mg/ml, adding 10mg of the synthesized polypeptide derivative into 10ml of D-PBS (Dulbecco's phospate Buffered Saline) with pH 7.4, fully mixing, adding 1mg of trastuzumab modifier (Her2), reacting at 50 ℃ for 1h, concentrating and replacing the solution by using an ultrafiltration tube with 100Kd after the reaction is finished, removing unbound polypeptide derivative, finally concentrating to 80-120 μ l, and detecting the concentration of the polypeptide derivative-antibody complex BCA by using a method.

The polypeptide derivative-antibody compound obtained by reacting the polypeptide derivative obtained in the example 3 with trastuzumab modifier is named as 22-sequence polypeptide derivative-Her 2 antibody compound;

the polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in the example 3 with the 5T4 monoclonal antibody modifier is named as a 22-sequence polypeptide derivative-5T 4 antibody complex;

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 3 with the VEGFR monoclonal antibody modifier is named as a 22-sequence polypeptide derivative-VEGFR antibody complex;

the polypeptide derivative-antibody compound obtained by reacting the polypeptide derivative obtained in the example 3 with the EGFR monoclonal antibody modifier is named as a 22-sequence polypeptide derivative-EGFR antibody compound;

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in the example 3 with the CD19 monoclonal antibody modifier is named as a 22 sequence polypeptide derivative-CD 19 antibody complex;

The polypeptide derivative-antibody complex obtained by reacting the polypeptide derivative obtained in example 3 with the monoclonal antibody modifier CD20 was named as 22-sequence polypeptide derivative-CD 20 antibody complex.

Each of the polypeptide derivatives of examples 5 to 7 was reacted with each of the antibodies to produce the corresponding polypeptide derivative-antibody complex, and the concentration of each polypeptide derivative-antibody complex was measured by the BCA method, and the results are shown in Table 1.

Table 1: the BCA method detects the concentration of polypeptide derivative-antibody complex: (Unit mg/ml)

the numerical meanings in table 1 are illustrative:

0.174 indicates that the concentration of the 17 sequence polypeptide derivative-Her 2 antibody complex was 0.174 mg/ml;

0.121 represents a 20 sequence polypeptide derivative-5T 4 antibody complex concentration of 0.121 mg/ml;

the meaning of other numbers can be deduced analogously.

From the detection results, it is known that: the concentration exceeds the use concentration of the antibody in the reaction, the binding rate of the polypeptide derivative and the antibody is high, and a polypeptide derivative-antibody compound is well formed for the preparation of subsequent experimental design.

Example 8: polypeptide derivatives binding to cells

identification of binding of synthetic polypeptide derivatives (17-sequence polypeptide derivatives) to cells by live cell imaging

NK cells, NKT cells, α β T cells, γ δ T cells were diluted to a certain concentration, and then plated onto cell culture plates of 18 × 18mm 6-well plates coated with lysine. Cells were cultured for 12 hours in a growth plate, and then washed 2 times with 1ml of PBS, and then 5. mu.M of a synthetic polypeptide derivative (17-sequence polypeptide derivative) was added thereto, and co-cultured for 5 hours, and then washed 3 times with PBS. The plates were then placed in a custom designed chamber, using L-15 medium without phenol red (Invitrogen), and then viewed with a Nikon Ti-Eclipse confocal inverted microscope. The 17 sequence polypeptide derivative binds to cells as shown in FIG. 1.

Example 9: polypeptide derivative-antibody complex binding to cells (NK cells)

Binding the polypeptide derivative-antibody complex obtained in example 5 or example 6 or example 7 to a cell, respectively, wherein the antibody includes but is not limited to Her2 antibody, EGFR antibody, 5T4 antibody, VEGFR antibody, CD19 antibody, CD20 antibody; cells include, but are not limited to, NK cells, γ δ T cells, α β T cells, NKT cells, and the like.

collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; EasySep from Stemcell corporation was used^TMhuman NK Cell Enrichment Kit, NK cells sorted according to Kit instructions, and NK cells cultured using X-vivo15 medium (Loza) with the amount of 17 sequence polypeptide derivative-5T 4 antibody complex being 15ug polypeptide derivative-antibody complex/1X 10⁶And after incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and resuspending the cells by using an X-Vivo15 culture medium for later use.

Example 10: polypeptide derivative-antibody complex binding to cells (γ δ T cells)

collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; NK Cells were sorted according to the kit instructions using a Gamma Delta T Cells Robust expansion kit from Fujian StemEry Bio-techCo., Ltd, and cultured using RMPI 1640 containing 10 vt% FBS, and the amount of 17-sequence polypeptide derivative-Her 2 antibody complex was 15ug of polypeptide derivative-antibody complex/1X 10⁶After incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and using an X-Vivo15 culture medium to resuspend the cells for later use.

example 11: polypeptide derivative-antibody Complex binding to cells (α β T cells)

Collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; EasySep from Stemcell corporation was used^TMHuman (α β) TCell Enrichment Kit, sorting α β T cells according to Kit instructions, culturing the α β T cells using X-vivo15 medium (Loza), the amount of 17 sequence polypeptide derivative-EGFR antibody complex being 15ug polypeptide derivative-antibody complex/1 × 10⁶and after incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and resuspending the cells by using an X-Vivo15 culture medium for later use.

Example 12: polypeptide derivative-antibody complexes binding to cells (NKT cells)

Collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; NKT cells were sorted according to the Kit instructions using CD3+ CD56+ NKT Cell Isolation Kit from Miltenyi Biotec, and cultured in T503 medium containing 10% (by volume) of autologous plasma in an amount of 15ug of polypeptide derivative-antibody complex/1X 10⁶After incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and using an X-Vivo15 culture medium to resuspend the cells for later use.

Example 13: polypeptide derivative-antibody complex binding to cells (NK cells)

Collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; EasySep from Stemcell corporation was used^TMHuman NK Cell Enrichment Kit, sorting NK cells according to Kit instructions, culturing NK cells using X-vivo15 medium (Loza), the amount of 20 sequence polypeptide derivative-Her 2 antibody complex being 15ug polypeptide derivative-antibody complex/1X 10⁶And after incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and resuspending the cells by using an X-Vivo15 culture medium for later use.

example 14: polypeptide derivative-antibody complex binding to cells (γ δ T cells)

Collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; the gamma delta T Cells were sorted using the gamma delta T Cells Robust expansion kit from Fujian StemEry Bio-techCo., Ltd, according to the kit instructions, and cultured using RMPI 164(Loza) containing 10 vt% FBS, and the amount of 20-sequence polypeptide derivative-EGFR antibody complex was 15ug of polypeptide derivative-antibody complex/1X 10⁶And after incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and resuspending the cells by using an X-Vivo15 culture medium for later use.

Example 15: polypeptide derivative-antibody Complex binding to cells (α β T cells)

Collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; EasySep from Stemcell corporation was used^TMhuman (alpha beta) TCell Enrichment Kit, sorting alpha beta T cells according to Kit instructions, culturing the alpha beta T cells using X-vivo15 medium (Loza), the amount of 20 sequence polypeptide derivative-5T 4 antibody complex is 15ug polypeptide derivative-antibody complex/1X 10⁶Individual cells, incubated at 37 ℃ 2And h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at the temperature of 400 Xg and 20 ℃, adding 10ml of D-PBS for resuspending the cells after the centrifugation is finished, washing once at the temperature of 400 Xg and 20 ℃, centrifuging for 5min, adding 10ml of D-PBS for resuspending the cells again, washing again at the temperature of 400 Xg and 20 ℃, centrifuging for 5min, and resuspending the cells by using an X-Vivo15 culture medium for later use.

Example 16: polypeptide derivative-antibody complexes binding to cells (NKT cells)

Collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; NKT cells were sorted according to the Kit instructions using CD3+ CD56+ NKT Cell Isolation Kit from Miltenyi Biotec, and cultured in T503 medium containing 10% (by volume) of autologous plasma in an amount of 15ug of polypeptide derivative-antibody complex/1X 10 of VEGFR antibody complex⁶After incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and using an X-Vivo15 culture medium to resuspend the cells for later use.

example 17: polypeptide derivative-antibody complex binding to cells (NK cells)

Collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; EasySep from Stemcell corporation was used^TMHuman NK Cell Enrichment Kit, NK cells sorted according to Kit instructions, cultured with X-vivo15 medium (Loza), and the amount of 22 sequence polypeptide derivative-CD 19 antibody complex was 15ug polypeptide derivative-antibody complex/1X 10⁶Incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, 400 Xg and 20 DEG Cand centrifuging for 5min, and using X-Vivo15 culture medium to resuspend the cells for later use.

Example 18: polypeptide derivative-antibody complex binding to cells (γ δ T cells)

collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; gamma delta T Cells were sorted according to the kit instructions using a Gamma delta T Cells Robust expansion kit from Fujian StemEry Bio-techCo., Ltd, and cultured using RMPI 164 containing 10 vt% FBS, and the amount of 22 sequence polypeptide derivative-Her 2 antibody complex was 15ug of polypeptide derivative-antibody complex/1X 10⁶After incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and using an X-Vivo15 culture medium to resuspend the cells for later use.

Example 19: polypeptide derivative-antibody Complex binding to cells (α β T cells)

Collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; EasySep from Stemcell corporation was used^TMHuman (alpha beta) TCell entity Kit, sorting alpha beta T cells according to the Kit instructions, culturing the alpha beta T cells using X-vivo15 medium (Loza), the amount of 22 sequence polypeptide derivative-5T 4 antibody complex is 15ug polypeptide derivative-antibody complex/1X 10⁶after incubating the cells at 37 ℃ for 2h, collecting the cells into a 15ml sterile centrifuge tube, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells after the centrifugation is finished, washing once, centrifuging for 5min at 400 Xg and 20 ℃, adding 10ml of D-PBS to resuspend the cells, washing again, centrifuging for 5min at 400 Xg and 20 ℃, and using an X-Vivo15 culture medium to resuspend the cells for later use.

Example 20: polypeptide derivative-antibody complexes binding to cells (NKT cells)

collecting peripheral blood from healthy volunteers using a heparin anticoagulant vacuum collection tube, and separating Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Paque PLUS (GE Healthcare) density gradient; NKT cells were sorted according to the Kit instructions using CD3+ CD56+ NKT Cell Isolation Kit from Miltenyi Biotec, NKT cells were cultured in T503 medium containing 10% (volume percentage) of autologous plasma, the amount of 22-sequence polypeptide derivative-VEGFR antibody complex was 15ug of polypeptide derivative-antibody complex/1 × 106 cells, after incubation at 37 ℃ for 2h, the cells were collected in a 15ml sterile centrifuge tube, centrifuged at 400 × g and 20 ℃ for 5min, after centrifugation was completed, 10ml of D-PBS was added to resuspend the cells, washed once, 400 × g and 20 ℃, centrifuged for 5min, 10ml of D-PBS was added to resuspend the cells, washed again, 400 × g and 20 ℃, centrifuged for 5min, and the cells were resuspended in X-Vivo15 medium for use.

the polypeptide derivative-antibody complexes were bound to immune cells to prepare corresponding immune cell-polypeptide derivative-antibody complexes according to the experimental principles and procedures for preparation of polypeptide derivative-antibody complexes and immune cells of examples 8-19, all of which were designed in the group of table 2 of example 20.

wherein the polypeptide derivative comprises: 17 sequence polypeptide derivative, 20 sequence polypeptide derivative and 22 sequence polypeptide derivative.

Wherein the antibody comprises: her2 antibody, EGFR antibody, 5T4 antibody, VEGFR antibody, CD19 antibody, CD20 antibody.

wherein the immune cells comprise: NK cells, γ δ T cells, α β T cells, NKT cells.

Example 21: detection of distribution of polypeptide derivative-antibody complexes on immune cell surface

setting and preparing experimental groups:

Polypeptide derivative-antibody complex group: preparation methods various polypeptide derivative-antibody complexes were prepared according to the methods of examples 8 to 19;

Group of polypeptide derivatives: the preparation was carried out according to the principles and operating methods of the groups of examples 8 to 19, with the difference that: the amount of the polypeptide derivative is 15 ug/1 × 10⁶the other operations are the same;

Antibody group: the preparation was carried out according to the principles and operating methods of the groups of examples 8 to 19, with the difference that: the amount of antibody was 15ug antibody/1X 10⁶The other operations were the same for each cell.

(1) detection of distribution of 17-sequence polypeptide derivative-5T 4 antibody complex on NK cell surface by Confocal fluorescence microscope

After the 17-sequence polypeptide derivative-5T 4 antibody complex was bound to NK cells, goat anti-human IgG FITC antibody (Beijing China fir bridge Biotech Co., Ltd.) was added, and the distribution of the 17-sequence polypeptide derivative-5T 4 antibody complex on the NK cell surface was examined by Confocal fluorescence microscopy, as shown in FIG. 2, the 17-sequence polypeptide derivative-5T 4 antibody complex was bound to the NK cell surface at almost 100%.

(2) Flow cytometry for detecting distribution of 17 sequence polypeptide derivative-5T 4 antibody complex on NK cell surface

After the 17-sequence polypeptide derivative-5T 4 antibody complex is combined with NK cells, a goat anti-human IgG FITC antibody (Beijing China fir bridge Biotechnology Co., Ltd.) is added, the distribution of the 17-sequence polypeptide derivative-5T 4 antibody complex on the surface of the NK cells is detected by flow cytometry, as shown in FIG. 3, the combination of the 17-sequence polypeptide derivative-5T 4 antibody complex and the NK cells is detected by flow cytometry, the combination of the 17-sequence polypeptide derivative and the NK cells is shown in FIG. 3-a, the combination rate of the 5T4 antibody and the NK cells is very low or not combined, the combination rate of the 17-sequence polypeptide derivative-5T 4 antibody complex and the NK cells is shown in FIG. 3-c, the combination rate of the 17-sequence polypeptide derivative-5T 4 antibody complex and the blank control are shown in FIG. 3-d, and the experimental results show that most of the 17-sequence polypeptide derivative-5T 4, Almost all bound to the NK cell surface.

The binding rate of each of the polypeptide derivative-antibody complexes of examples 8-19 on the cell surface was statistically determined by flow cytometry, as shown in Table 2.

Table 2: detecting the binding rate of the polypeptide derivative-antibody complex on the cell surface by a flow cytometer: (unit:%)

The meanings given in table 2 are illustrative:

for example, it can be seen from table 2:

The surface binding rate of the 17 sequence polypeptide derivative-Her 2 antibody complex to NK cells is 98.3%;

The surface binding rate of 20 sequence polypeptide derivative-Her 2 antibody complex and NK cells is 97.9%;

The surface binding rate of the 22 sequence polypeptide derivative-Her 2 antibody complex to NK cells is 98.5%;

The binding rate of other polypeptide derivative-antibody complexes to the surface of immune cells can be similarly derived from Table 2.

Example 22: NK cell-17 sequence polypeptide derivative-5T 4 antibody compound remarkably enhances killing activity of NK cells on non-small cell lung cancer cells H-1975

Setting and preparing experimental groups:

Immune cell-polypeptide derivative-antibody complex group: preparation methods various polypeptide derivative-antibody complexes were prepared according to the methods of examples 8 to 19, and various polypeptide derivative-antibody complexes were added at 15 ug/1X 10 of polypeptide derivative-antibody complex in the treatment of immunocytes⁶The cells were subjected to the same procedure to obtain various types (immunocyte-polypeptide derivative-antibody complex group);

immune cell group: the preparation was carried out according to the principles and operating methods of the groups of examples 8 to 19, with the difference that: when the immune cells are treated, the antibody or the polypeptide derivative is not added, and other operations are the same, so that an immune cell group is obtained;

Immune cell-antibody group: the preparation was carried out according to the principles and operating methods of the groups of examples 8 to 19, with the difference that: when used with immunocytes, the amount of the added antibody is 15ug antibody/1X 10⁶And (4) obtaining the immune cell-antibody group by using the cells and performing the same other operations.

staining non-small cell lung cancer H-1975 cells with 5. mu.M CFSE, incubating the NK cell-17 sequence polypeptide derivative-5T 4 antibody complex or NK cell-5T 4 antibody group or PBS and H-1975 cells at a ratio of 20:1 at 37 ℃ for 4H, adding 1 mug/ml PI dye, the CFSE + PI double positive cell is dead cell, as shown in FIG. 4, the killing rate of simple NK cells to H-1975 is more than 40%, the killing rate of NK cells-5T 4 antibody group to H-1975 is 60%, the killing rate of the NK cell-17 sequence polypeptide derivative-5T 4 antibody compound to H-1975 reaches 98%, the 17-sequence polypeptide derivative-5T 4 antibody compound can remarkably enhance the target killing activity of NK cells on cancer cells, and almost 100% kills H-1975 cells.

Example 23: NK cell-17 sequence polypeptide derivative-5T 4 antibody complex remarkably inhibits growth of non-small cell lung cancer cell H-1975 transplanted tumor

will be 5X 10⁶h-1975 cells were inoculated on hind limbs of BALB/c nude mice until tumors grew to 100mm³When the tumor growth condition is observed, the mice are divided into four groups (10 mice in each group), PBS, NK cells, an NK cell-5T 4 antibody group and an NK cell-17 sequence polypeptide derivative-5T 4 antibody compound are respectively injected through tail veins, the injection is performed once every week and is performed four times in total, and the tumor growth condition is observed, as shown in figures 5a, 5b and 5c, the NK cell-17 sequence polypeptide derivative-5T 4 antibody compound can obviously enhance the inhibition on the growth of H-1975 cell transplantable tumors. And the killing and tumor growth inhibiting effects of the NK cell-17 sequence polypeptide derivative-5T 4 antibody compound are sequentially stronger than those of the NK cell, the NK cell-5T 4 antibody group and the like, so that the targeting of the NK cell-17 sequence polypeptide derivative-5T 4 antibody compound to cancer cells is enhanced, and the targeting of immune cells can be enhanced by the 17 sequence polypeptide derivative-5T 4 antibody compound.

Example 24: NK cell-20 sequence polypeptide derivative-5T 4 antibody complex remarkably enhances killing activity of NK cells on breast cancer cells HCC1954

The CFSE of 5 mu M is used for staining breast cancer HCC1954 cells, the NK cell-20 sequence polypeptide derivative-5T 4 antibody complex or the NK cell-5T 4 antibody group or the NK cell-C1 (positive control) or the NK cell and the HCC1954 cells are incubated for 4 hours at 37 ℃ according to the ratio of 20:1, 1 mu g/ml of PI dye is added, the cells which are double positive to the CFSE + PI are dead cells, and as shown in figure 6, the killing activity of the CFSE + PI targeted killing activity (killing rate 58.9%) of the NK cells is remarkably enhanced by the 20 sequence polypeptide derivative-5T 4 antibody complex, and is remarkably higher than the killing activity of the NK cell-5T 4 antibody complex (killing rate 19.2%) or the NK cell-C1 (positive control) (killing rate 28.9%) or the killing activity of the NK cells (killing rate 23.6%) on the HCC1954 cells.

Example 25: gamma delta T cell-20 sequence polypeptide derivative-Her 2 antibody compound obviously enhances the killing activity of gamma delta T cells on breast cancer cells HCC1954

The breast cancer HCC1954 cells are stained by using 5 mu M CFSE, after a gamma delta T cell-20 sequence polypeptide derivative-Her 2 antibody complex or a gamma delta T cell-Her 2 antibody group or a gamma delta T cell-C1 (positive control) or a gamma delta T cell or PBS and the HCC1954 cells are incubated for 4 hours at 37 ℃ according to the ratio of 20:1, 1 mu g/ml of PI dye is added, and cells with double positive CFSE and PI are dead cells, as shown in figure 7, the targeted killing activity of the gamma delta T cells (the killing rate is 70.1%) can be remarkably enhanced by the 20 sequence polypeptide derivative-Her 2 antibody complex, and the killing activity of the gamma delta T cells is remarkably higher than that of the gamma delta T cell-Her 2 antibody group (the killing rate is 37.6%) or the gamma delta T cell-C1 (positive control) (the killing rate is 18.3%) or the killing rate of the gamma delta T cells (the killing rate is 35.1%) on the HCC1954 cells.

Example 26: gamma delta T cell-22 sequence polypeptide derivative-Her 2 antibody compound remarkably enhances the killing activity of gamma delta T cells on cancer cells NCI-N87

cancer NCI-N87 cells are stained by 5 mu M CFSE, after gamma delta T cell-22 sequence polypeptide derivative-Her 2 antibody complex or gamma delta T cell-Her 2 antibody group or gamma delta T cells or PBS and NCI-N87 cells are incubated for 4 hours at 37 ℃ according to the ratio of 20:1, 1 mu g/ml of PI dye is added, cells with CFSE + PI double positive are dead cells, and as shown in figure 8, 22 sequence polypeptide derivative-Her 2 antibody complex can remarkably enhance the killing activity of gamma delta T cells (killing rate of 66.5%) and is remarkably higher than that of the gamma delta T cell-Her 2 antibody group (killing rate of 55.6%) or gamma delta T cells (killing rate of 37.6%) on NCI-N87 cells.

Example 27: killing activity of immune cell-polypeptide derivative-antibody complex on cancer cell

According to the experimental process of example 21, cancer cells were stained with 5 μ M CFSE, the immune cell-polypeptide derivative-antibody complex and cancer cells were incubated at 37 ℃ for 4h at a ratio of 20:1, and then 1 μ g/ml PI dye was added, cells that were both CFSE + PI positive were dead cells, the immune cell-polypeptide derivative-antibody complex was able to significantly enhance the target killing activity of various immune cells against various cancer cells, and the killing activity of the immune cell-polypeptide derivative-antibody complex formed by different immune cells was different for each cancer cell, as shown in table 3, the experimental data of the killing activity of the immune cell-polypeptide derivative-antibody complex against cancer cells were shown in statistics.

The killing activity of the immunocyte-polypeptide derivative-antibody complexes of examples 8 to 19 above against breast cancer (BT-474, MCF-7), lung cancer (H-1975), colorectal cancer (HCT116, SW620), liver cancer (SMMC-7721), glioma (KALS-1), pancreatic cancer (SW1990), ovarian cancer (SKOV3), prostate cancer (PC-3), kidney cancer (Ketr-3), melanoma (SKMel-23), sarcoma (Saos-2), myeloma (RPMI-8226), leukemia (K562), and gastric cancer (NCI-N87) was statistically shown in Table 3.

TABLE 3 killing activity of immune cell-polypeptide derivative-antibody Complex against cancer cells (unit:%)

TABLE 3

In the detection experiment aiming at the killing activity of each immune cell-polypeptide derivative-antibody compound on each cancer cell in table 3, an immune cell-antibody group and an immune cell group are designed in the experiment as a control group, wherein the statistical results of each experimental group of NK cell-polypeptide derivative-5T 4 antibody compound, NK cell-polypeptide derivative-Her 2 antibody compound and gamma delta T cell-polypeptide derivative-Her 2 antibody compound are shown; in the test and detection of the other immune cell-polypeptide derivative-antibody complex groups in table 3, the detection results of the killing activity of each cancer cell of each control immune cell-antibody group and each immune cell group show that the killing activity of the immune cell-antibody group ranges from about 15% to 45%, the killing activity of each cancer cell of the immune cell is about 6% to 40%, and the killing activity of the immune cell-antibody group and the immune cell group is smaller than that of the corresponding immune cell-polypeptide derivative-antibody complex, so that the targeting killing effect of the immune cell-polypeptide derivative-antibody complex in the invention can be seen.

In summary, the following embodiments can be obtained: the killing performance of the immune cell-polypeptide derivative-antibody compound on cancer cells and the effect of inhibiting tumor growth are both stronger than the killing performance of the immune cells and the immune cell-antibody group on the cancer cells and the effect of inhibiting the tumor growth. The targeting of the immune cell-polypeptide derivative-antibody compound is enhanced, and the polypeptide derivative-antibody compound has the function of enhancing the targeting of the immune cell.

Sequence listing

<110> Anhui Ruida health industries, Ltd

<120> polypeptide, polypeptide derivative, polypeptide-antibody compound, preparation and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Artificial sequence

<400> 1

Gly Ser Ser Leu Ser Pro Ser Leu Leu Leu Leu Leu Leu Pro Gly Ala

1 5 10 15

Leu

<210> 2

<211> 20

<212> PRT

<213> Artificial sequence

<400> 2

Leu Pro Leu Gly Ser Thr Leu Ser Pro Ser Leu Leu Leu Leu Leu Leu

1 5 10 15

Pro Gly Ala Leu

20

<210> 3

<211> 22

<212> PRT

<213> Artificial sequence

<400> 3

Pro Leu Gly Leu Leu Leu Ser Pro Leu Ser Pro Ser Leu Leu Leu Leu

1 5 10 15

Leu Leu Pro Gly Ala Leu

20

Claims

1. The polypeptide is any one of amino acid sequences shown in (I) and (II):

(I) has the sequence of SEQ ID NO: 1;

2. The polypeptide of claim 1, wherein said substitution is a K substitution in the amino acid sequence to R, and/or an S substitution in the amino acid sequence to T, and wherein the proportion of K in the polypeptide after substitution is 40-47%, and wherein the amino acid proximal to the carboxy terminus of the polypeptide is lysine.

3. The polypeptide of claim 1, wherein the addition is of 1, 2, 3, 4, or 5 amino acids.

4. The polypeptide of claim 1, wherein the amino acid sequence shown in (II) is any one of the amino acid sequences shown in (III) or (IV):

(iii) has SEQ ID NO: 2;

(iv) has the sequence of SEQ ID NO: 3.

5. The polypeptide derivative is any one of amino acid sequences shown in (V), (VI), (VII) and (VIII):

(V)

(VI)

Wherein,

The R is₀Is alkynyl, any one of formula (1) or formula (2);

the R is₁Is alkyl or alkenyl.

6. The polypeptide derivative of claim 5, wherein the substitution is R for K and/or T for S in the amino acid sequence, and the ratio of K in the substituted polypeptide derivative is 40-47%, and the amino acid near the carboxy-terminal end of the polypeptide derivative is lysine.

7. the polypeptide derivative of claim 5, wherein the addition is of 1, 2, 3, 4 or 5 amino acids.

8. the polypeptide derivative according to claim 5, wherein the amino acid sequence represented by (VII) is any one of formula (3) and formula (4), and the amino acid sequence represented by (VIII) is any one of formula (5) and formula (6),

Wherein,

The R is₀Is alkynyl, any one of formula (1) or formula (2);

The R is₁Is alkyl or alkenyl.

9. The polypeptide derivative according to any one of claims 5 to 8,

10. A polypeptide derivative-antibody complex, wherein said polypeptide derivative-antibody complex has the general formula:

Polypeptide derivative-R₂；

The polypeptide derivative is the polypeptide derivative of any one of claims 5-9;

The R is₂is an antibody.

11. The polypeptide derivative-antibody complex of claim 10, wherein R is₂Is any one of Her2 antibody, EGFR antibody, 5T4 antibody, VEGFR antibody, CD19 antibody and CD20 antibody.

12. A cellular complex, characterized in that said cellular complex has the general formula:

immunocyte-polypeptide derivative-R₂；

The R is₂is an antibody.

13. the cellular complex of claim 12, wherein the immune cell is any one of NK cell, NKT cell, α β T cell, γ δ T cell, and R is₂Is Her2 antibodyany one of an EGFR antibody, a 5T4 antibody, a VEGFR antibody, a CD19 antibody, and a CD20 antibody.

14. The preparation method of the polypeptide derivative-antibody compound is characterized in that the polypeptide derivative is dissolved, buffer solution is added, mixing is carried out, and NHS active ester modified antibody is added for reaction, so as to obtain the polypeptide derivative-antibody compound.

15. The method for preparing the polypeptide derivative-antibody complex according to claim 14, wherein the mass ratio of the polypeptide derivative to the NHS active ester-modified antibody is 0.5-10:1, the reaction time is 0.5-24h, and the reaction temperature is 0-50 ℃.

16. The method for preparing the polypeptide derivative-antibody complex according to claim 15, wherein the mass ratio of the polypeptide derivative to the NHS active ester-modified antibody is 2.5:1, the reaction time is 2 hours, the reaction temperature is 37 ℃, the buffer solution is D-PBS, and the solvent for dissolving the polypeptide derivative is dimethylsulfoxide.

17. A polypeptide derivative-antibody complex produced by the production method according to any one of claims 11 to 16.

18. Use of the polypeptide according to any one of claims 1 to 4, the polypeptide derivative according to any one of claims 5 to 9, the polypeptide derivative-antibody complex according to claim 10 or 11 or 17, or the cell complex according to claims 12 to 13 for the preparation of an antitumor medicament.

19. Use of the polypeptide of any one of claims 1 to 4, the polypeptide derivative of any one of claims 5 to 9, the polypeptide derivative-antibody complex of claim 10, 11 or 17, and the cell complex of claims 12 to 13 for the preparation of an antitumor medicament, wherein the tumor comprises any one or more of breast cancer, lung cancer, colorectal cancer, liver cancer, glioma, pancreatic cancer, ovarian cancer, prostate cancer, renal cancer, melanoma, sarcoma, myeloma, leukemia, and gastric cancer.