CN114085290A

CN114085290A - Antibody prodrug, preparation method and application thereof

Info

Publication number: CN114085290A
Application number: CN202010861303.8A
Authority: CN
Inventors: 王皓; 陶静; 张大鹏
Original assignee: Taizhou Mabtech Pharmaceutical Co ltd
Current assignee: Taizhou Mabtech Pharmaceutical Co ltd
Priority date: 2020-08-25
Filing date: 2020-08-25
Publication date: 2022-02-25

Abstract

The invention provides a targeting antibody prodrug, which consists of A, B, C parts, wherein part A is a protein with targeting property, part C is an antibody/antibody receptor fusion protein with therapeutic action, part A is connected with part C through part B, and part A is connected with an antigen binding region of the antibody of part C through the protein with targeting property.

Description

Antibody prodrug, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and discloses an antibody prodrug, a preparation method and application thereof, and more particularly discloses a targeting antibody prodrug, a preparation method and application thereof.

Background

Tumor is a new organism formed by local tissue proliferation under the action of various tumorigenic factors, and the tumor cells can generate metabolic change different from normal cells, and can adapt to the change of metabolic environment through the conversion between glycolysis and oxidative phosphorylation. Similarly, some autoimmune and other diseases may also respond to local or systemic inflammatory/metabolic abnormalities.

In the treatment of tumors, except for conventional operations, radiotherapy, chemotherapy, biological treatment and traditional Chinese medicine treatment, different targeted treatment technologies can be used for targeted treatment aiming at different targets of tumors at organ tissues and molecular level. The target of the molecular targeted therapy is a malignant phenotype molecule aiming at tumor cells, acts on channels such as specific cell receptors for promoting tumor growth and survival, signal conduction and the like, and regulates neovascularization and cell cycle, thereby realizing the anti-tumor effect of inhibiting the growth of the tumor cells or promoting apoptosis. Also in the treatment of autoimmune diseases or other local or systemic abnormalities, targeted therapy plays an increasingly important role.

The use of antibodies in targeted therapy of tumors or autoimmune diseases is the most common, and the existing drugs for the targeted therapy of main molecules can be divided into: having a targeted Epidermal Growth Factor Receptor (EGFR) blocker, such as Cetuximab; monoclonal antibodies directed against certain specific cell markers, such as Herceptin; tyrosine kinase receptor inhibitors; anti-tumor angiogenesis inhibitors, such as Bevacizumab; monoclonal antibodies against immune checkpoint proteins, such as Nivolumab, pembrolizumab, Atezolizumab, anti-CD 47 antibodies, and the like; monoclonal antibodies against the inflammatory factor TNF, such as Infliximab, Adalilimumab, Golimumab, and the like.

Antibodies are a class of immunoglobulins that specifically bind to antigens, and natural antibody classes include IgA, IgD, IgE, IgG, IgM, etc., where antibodies used for tumor therapy are often of the IgG class, which have four subclasses IgG1, IgG2, IgG3, IgG 4. Among them, IgG1 and IgG3 can efficiently activate complement, bind to Fc receptors on macrophages and NK cells, and cause ADCC, and some antibodies also cause CDC.

ADCC (antibody-dependent cell-mediated cytotoxicity) is an antibody-dependent cell-mediated cytotoxicity, which means that the Fab end of an antibody is combined with an epitope of a virus-infected cell or tumor, and the Fc end of the antibody is combined with the FcR on the surface of a killer cell (NK cell, macrophage and the like) to mediate the killer cell to directly kill a target cell.

Cdc (complementary dependent cytotoxicity) complement dependent cytotoxicity, MAC (membrane attack complex) formation on the surface of target cells after the complement system is activated, leading to target cell lysis. The complement system can also cause damage to body tissues or cells, which can lead to hypersensitivity and autoimmune diseases.

In the process of targeted therapy of diseases such as tumor or autoimmune diseases by using antibody drugs, the marker antigen for targeted therapy of tumor is also expressed in normal cells or tissues, so when the Fab end of the antibody binds to the corresponding epitope expressed by tumor or to the inflammatory factor at the site of inflammation, it also binds to the corresponding epitope expressed by normal cells or the corresponding factor at the site of normal tissues, and ADCC, CDC or neutralization is generated after binding, and the binding to the epitope expressed by tumor kills tumor cells, but the binding to the epitope expressed by normal cells kills normal cells by mistake, and the binding to the factor at the site of inflammation can reduce the inflammatory reaction at the site of inflammation, and the binding to the factor at the site of normal cells can neutralize the normal physiological function of the factor (such as reducing the immunity of the organism), it has adverse effects on normal cells, tissues or the whole body, and further has side effects.

Conventionally, in order to solve the side effects due to ADCC effect or CDC effect, IgG2 type antibody is generally used as a therapeutic antibody, and although the IgG2 type antibody does not cause ADCC effect or CDC effect, the effect of tumor killing is reduced, and there is a conflict between the two. There is no suitable solution for the neutralizing or killing effect of normal factors or antigen production.

Probody technology is an "antibody precursor" or "antibody-based prodrug" technology, Probody is a completely recombinant monoclonal antibody, the antigen binding site of which is blocked by blocking peptide masking technology, remains inactive (inert) in healthy tissues, and is cleaved by specific proteases such as tumor or inflammation in tumor micro-or inflammatory environments to be selectively activated, exposing the antigen binding site.

Probody medicine aims at utilizing unique conditions of tumor micro or inflammation and other environment, raising targeting property of antibody to tumor inflammation part and reducing medicine activity and side effect to normal tissue.

However, the Probody drug lacks targeting property before entering the inflammatory site of tumor tissue, and only when the drug contacts tumor specific protease in the tumor microenvironment, the drug is digested by enzyme, and the subsequent drug therapy effect can be exerted.

Disclosure of Invention

Part A has targeting protein for blocking the antigen/ligand binding site of part C, part A is the complete antibody, antigen binding fragment, single chain antibody, receptor/ligand protein binding fragment or other functional protein with targeting; the antibody with the part C having the therapeutic effect is a complete antibody, an antigen binding fragment or a single-chain antibody, an antibody receptor fusion protein; part a is linked to part C via part B, which is a linker peptide containing a substrate for tumour or inflammation specific protease cleavage.

The invention provides a targeting antibody prodrug, wherein part A has targeting proteins, part C has therapeutic antibody/antibody receptor fusion protein with binding performance with tumor-expressed factors or inflammatory factors, under the targeting action of part A, the targeting protein can be quickly and accurately positioned to tumor cells or relatively enriched to inflammatory sites, after positioning, in the microenvironment or inflammatory environment of tumors, tumor-specific protease or inflammatory site-specific protease acts on a connecting peptide part B, part A and part C are cut and separated, the antigen or ligand binding site of part C is exposed, part C is combined with tumor cells or inflammatory site second antigen or inflammatory factors, ADCC effect, CDC effect, neutralization effect or activation/inhibition of local immunity and other functions are generated, the tumor cells are further killed, the tumor cells are subjected to anti-tumor therapy, and the like, Or neutralizing inflammatory factors or changing immune functions, and has therapeutic effects on tumor, autoimmune diseases, etc.

The target antibody prodrug provided by the invention has targeting property of part A, enzyme cutting property of tumor specificity or inflammation specificity enzyme and curative property of part C; under the guidance of tumor or inflammation part targeting of part A with targeting protein, the targeting antibody prodrug is positioned in tumor or inflammatory tissue, and the connecting peptide of the targeting protein of part A and part C antibody is enzyme-cut by specific enzyme in tumor microenvironment or inflammatory environment, so that the antigen binding region or ligand binding domain of part C antibody with therapeutic action is exposed and combined with tumor or inflammatory specific molecules, thereby achieving the purpose of treating tumor or inflammatory diseases.

The target antibody prodrug provided by the invention can achieve a treatment effect by using a lower dose of antibody medicine in the treatment process of tumors or inflammatory diseases, greatly improves the targeting property of the tumor or inflammatory disease treatment effect medicine, and reduces adverse effects and toxic and side effects of the tumor or inflammatory disease treatment effect medicine on normal cells, tissues or systems.

The protein with targeting property of the part A is a complete antibody, an antigen binding fragment, a single-chain antibody, a receptor/ligand protein binding fragment or other functional proteins of a targeting tumor specific factor, such as an anti-EGFR complete antibody, an antigen binding fragment, a single-chain antibody and a VEGF receptor; or an anti-VEGF whole antibody, antigen binding fragment, single chain antibody, VEGF Trap; or an anti-IgE intact antibody, an antigen-binding fragment, a single chain antibody; or is an anti-HER 2 whole antibody, antigen-binding fragment, single chain antibody, anti-IL 17A whole antibody, antigen-binding fragment, single chain antibody, TNF beta-binding whole antibody, antigen-binding fragment, single chain antibody, receptor; whole antibodies, antigen-binding fragments, single chain antibodies, sirpa, etc., that bind CD 47. Preferably, the first antibody is an anti-VEGF whole antibody, an antigen-binding fragment, a single chain antibody, a VEGF Trap.

The part C of the invention is a complete antibody, an antigen binding fragment and a single-chain antibody or an antibody receptor fusion protein with the function of treating tumors or inflammatory diseases, such as an anti-EGFR complete antibody, an antigen binding fragment, a single-chain antibody and an EGFR receptor; or an anti-VEGF whole antibody, antigen binding fragment, single chain antibody, VEGF Trap; or an anti-IgE intact antibody, an antigen-binding fragment, a single chain antibody; or is an anti-HER 2 whole antibody, antigen-binding fragment, single chain antibody; or anti-TNF α whole antibody, antigen binding fragment, single chain antibody, TNF receptor, etc., preferably the second antibody is an anti-EGFR whole antibody, antigen binding fragment, single chain antibody, EGFR receptor domain.

The connecting peptide connecting the part A and the part C of the invention contains tumor or inflammation specificity enzyme digestion substrates, such as plasmin substrate, caspase substrate, Matrix Metalloenzyme (MMP) substrate and the like, preferably plasmin substrate, preferably urokinase type plasmin substrate or Matrix Metalloenzyme (MMP) substrate.

The amino acid sequence and length of the part B can influence the effect of the antibody prodrug, the part B is too long, the part A cannot play a role in blocking the binding activity of the part C, the part B is too short, the enzyme cutting site is not sufficiently exposed, the part A cannot be cut off, and the activity of the part C is difficult to play.

The invention discloses a targeting antibody prodrug, which consists of A, B, C parts, wherein part A is a protein with targeting property, part C is an antibody/antibody receptor fusion protein with therapeutic action, part A is connected with part C through part B, and part A is connected with an antigen binding region of the antibody of part C through the protein with targeting property.

The prodrug of the targeting antibody disclosed by the invention has a molecular structure shown in figure 1, figure 2, figure 3 and figure 4.

The targeting antibody prodrug, wherein the protein with targeting property of the part A is a targeting complete antibody, an antigen binding fragment, a single-chain antibody, a receptor/ligand protein binding fragment or other functional proteins.

The targeting antibody prodrug, wherein the part A targeting protein is an anti-VEGF complete antibody, an anti-VEGF single-chain antibody, a VEGF antigen binding fragment and a VEGF Trap.

The variable region of the light chain of the anti-VEGF single-chain antibody of the targeting antibody prodrug has an amino acid sequence shown by SEQ ID NO. 2, and the variable region of the heavy chain has an amino acid sequence shown by SEQ ID NO. 4.

The targeting antibody precursor drug as described above, the antibody with the part C having the therapeutic effect is a complete antibody, an antigen binding fragment or a single-chain antibody,

the targeting antibody prodrug is characterized in that the antibody with the part C having the treatment effect is an anti-EGFR whole antibody, wherein the light chain of the anti-EGFR whole antibody has an amino acid sequence shown in SEQ ID NO. 6, and the heavy chain of the anti-EGFR whole antibody has an amino acid sequence shown in SEQ ID NO. 8.

The targeting antibody prodrug has the structure shown in the specification, wherein the part B is a connecting peptide containing a substrate cleaved by tumor or inflammation specific protease, and the part B has a structure shown in the specification of (GS) n + cleavage site + (GS) n'.

The targeting antibody prodrug as described above, wherein the substrate for the tumor-or inflammation-specific protease cleavage is a plasmin substrate, a caspase substrate, or a matrix metalloenzyme substrate. The preferred specific enzyme cutting substrate is urokinase-type plasmin substrate, and the connecting peptide containing the urokinase-type plasmin substrate has an amino acid sequence shown in ASLSGRSDNHGSAS.

The target antibody prodrug, preferably the substrate for specific protease digestion is a substrate of matrix metalloenzyme, and the connecting peptide containing the substrate of matrix metalloenzyme has an amino acid sequence of GSGPLGVRGSGS.

The invention also discloses a pharmaceutical composition, which comprises an effective dose of the targeting antibody prodrug and a pharmaceutically acceptable excipient.

The invention also discloses an application of the targeting antibody prodrug, the application of the targeting antibody prodrug or the composition containing the targeting antibody prodrug in preparing anti-tumor drugs or preparing inflammation treatment drugs, and the combination use of the targeting antibody prodrug or the composition containing the targeting antibody prodrug and other tumor treatment drugs or other inflammation treatment drugs.

Drawings

FIG. 1, Structure 1 schematic of a prodrug of a targeting antibody;

FIG. 2, Structure 2 schematic of a prodrug of a targeting antibody;

FIG. 3, Structure 3 of a prodrug of a targeting antibody;

FIG. 4, Structure 4 schematic of a prodrug of a targeting antibody;

figure 5, free oligosaccharide profile of targeting antibody prodrug CMAB 0301;

FIG. 6, detection map of binding of targeting antibody prodrug CMAB0301 to VEGF-165 (ELISA method);

FIG. 7, graph of the affinity detection of the targeting antibody prodrug CMAB0301 with VEGF-165 (SPR method);

FIG. 8, detection map of binding of targeting antibody prodrug CMAB0301 to EGFR (ELISA method);

FIG. 9, graph of the affinity detection of the targeting antibody prodrug CMAB0301 with EGFR (SPR method);

FIG. 10 shows the detection pattern (ELISA) of the binding between the target antibody prodrug CMAB0301 and VEGF-165 after the digestion;

FIG. 11 shows the binding detection pattern (SPR method) of the target antibody prodrug CMAB0301 and VEGF-165 after the enzyme cleavage;

FIG. 12 shows the detection pattern (ELISA method) of the binding of the target antibody prodrug CMAB0301 to EGFR after enzyme cleavage;

FIG. 13 shows the binding detection pattern (SPR method) of the target antibody prodrug CMAB0301 after enzyme cleavage and EGFR;

FIG. 14, microscopic observation of the binding staining pattern of the targeting antibody prodrug CMAB0301 to tumor tissue;

FIG. 15, microscopic observation of the binding staining pattern of the targeting antibody prodrug CMAB0301 to paracancerous tissues;

figure 16, tumor suppression effect profile of the target antibody prodrug CMAB0301 in a preventative tumor model;

figure 17, tumor suppression effect profile of the target antibody prodrug CMAB0301 in therapeutic tumor model.

Detailed Description

The following examples are further illustrative of the present invention and should not be construed as limiting thereof.

In the embodiments of the present invention, the detailed description of the basic experimental techniques and methods in the biotechnology field, such as the techniques and methods of plasmid construction and expression vector construction, is not included; for example, transfection of host cells with expression vectors encoding protein drug genes, and the like, the basic and well-known techniques will not be described in detail in the present invention.

Example 1: construction of Targeted antibody prodrugs

A part A of the protein gene sequence with targeting property, such as VEGF single-chain antibody gene sequence, is synthesized by using a genetic engineering technology, a light chain variable region has a nucleotide sequence shown by SEQ ID NO. 1 and an amino acid sequence shown by SEQ ID NO. 2, and a heavy chain variable region has a nucleotide sequence shown by SEQ ID NO. 3 and an amino acid sequence shown by SEQ ID NO. 4.

The gene sequence of partial C antibody with therapeutic action is synthesized by using genetic engineering technology, such as EGFR complete antibody gene sequence, its light chain has the nucleotide sequence shown in SEQ ID NO. 5 and amino acid sequence shown in SEQ ID NO. 6, and its heavy chain has the nucleotide sequence shown in SEQ ID NO. 7 and amino acid sequence shown in SEQ ID NO. 8.

The linker peptide portion B, which links part a to part C, has an amino acid sequence shown at ASLSGRSDNHGSAS, or an amino acid sequence shown at GSGPLGVRGSGS.

And the connecting peptide part B which is formed by connecting the part A and the part C comprises a substrate of urokinase type plasminogen activator or a matrix metalloenzyme substrate, when the connecting peptide is in a tumor microenvironment or an inflammation microenvironment, the connecting peptide is interacted with the urokinase type plasminogen activator or the matrix metalloenzyme, the connecting peptide is subjected to enzyme digestion and hydrolysis, so that an antigen/ligand binding site of the part C antibody/antibody receptor fusion protein with the therapeutic effect is exposed, and the part C is combined with a tumor specific molecule or an inflammation specific factor to play the tumor therapeutic effect or the inflammation therapeutic effect of the part C.

Preparing an expression vector containing the gene sequence, transfecting CHO host cells, culturing the host cells, expressing target protein, separating and purifying.

Example 2: cell culture, expression and purification of targeting antibody prodrug

And (3) performing serum-free culture, expression and purification on the CHO host cell transfected with the targeting antibody prodrug to obtain the targeting antibody prodrug CMAB 0301.

Part A of the targeting antibody prodrug CMAB0301 is a VEGF single-chain antibody of which the light chain variable region has an amino acid sequence shown by SEQ ID NO. 2 and the heavy chain variable region has an amino acid sequence shown by SEQ ID NO. 4; part B is a connecting peptide containing a urokinase type plasminogen activation substrate and has an amino acid sequence shown in ASLSGRSDNHGSAS; part C is an EGFR complete antibody of which the light chain has an amino acid sequence shown by SEQ ID NO. 6 and the heavy chain has an amino acid sequence shown by SEQ ID NO. 8.

The target antibody precursor medicine CMAB0301 adopts the methods of basic culture and supplementary culture to culture and express CHO host cell containing CMAB0301 target antibody precursor medicine gene sequence. Wherein the basal medium is according to CHOM-B01 or CHOM-B03; the supplement culture medium is as follows: CHOM-S01 or CHOM-S04, basic culture medium CHOM-B01 or CHOM-B03, and supplementary culture medium CHOM-S01 or CHOM-S04 are all purchased from Shanghai Mitaijuno biotechnologies GmbH.

Preferably, the basic culture medium CHOM-B01 is supplemented with culture medium CHOM-S04.

Cell expansion: a) and (3) recovering working cells: putting the working cells in water bath at 37 ℃ for thawing; using 5ml of basic culture medium, carrying out centrifugation and liquid change, inoculating the medium into a 100 ml-150 ml shake flask, and adding 10-15 ml of basic culture medium into the flask; and placing the shake flask in a carbon dioxide shaking table for shaking culture at the temperature of 37 ℃ and the speed of 70-170 rpm, wherein the concentration of carbon dioxide is 2-10%. b) Seed amplification: injecting into a basic culture medium, setting the temperature at 35-39 ℃ and the pH value at 6.6-7.1, regulating and controlling the pH value by using carbon dioxide and sodium carbonate solution, and swingingThe speed is 25-30 rpm, waves are shaken and uniformly mixed, the surface aeration flow is 0.1-0.8L/min, the dissolved oxygen is set to be 30-60%, and glucose is supplemented to 3-5 g/L every day. The density of viable cells after inoculation is not less than 0.3 × 10⁶cell/ml, the inoculation ratio is 1: 2-1: 8, the cell viability is required to be more than 90%, no abnormality is caused by microscopic examination, and the next stage is transferred according to the ratio of 1: 2-1: 8.

Perfusion culture: transferring the cells after the cell amplification into a 500L cell culture reactor to make the initial cell density not less than 0.3 × 10⁶cell/ml. The temperature is 35-39 ℃, the temperature control range is adjusted according to the requirement of the culture process, and the optimal temperature is 35.5-36.5 ℃; adjusting pH to 6.6-7.2, preferably pH 6.6-6.8, with CO₂Regulating pH with sodium carbonate solution; stirring at 80-150 rpm; surface layer air, deep layer microbubble ventilation including air, oxygen and carbon dioxide; 30-60% of dissolved oxygen, preferably 30-40%; supplementing 3-5 g/L of glucose every day.

And (3) supplementary culture: after the perfusion culture is carried out for 48-72 hours, adjusting the temperature of a cell culture reactor to 32-34 ℃, preferably 33 ℃, adjusting the pH to 7.0-7.3, preferably 7.0-7.1, and adding a supplementary culture medium and a glucose solution; adding a supplementary culture medium according to the cell density until harvesting, wherein the daily addition amount is 1-5% of the initial tank feeding volume, and culturing for 8-10 days at a low temperature.

And (3) after 8-10 days of culture expression, collecting cell culture solution, clarifying the cell culture solution by deep filtration to remove cells and fragments thereof, reducing the biological load by primary membrane filtration to obtain culture supernatant containing the CMAB0301 antibody, and purifying the culture supernatant.

Collecting the host cell culture supernatant obtained by the above culture method, and performing affinity chromatography, ion exchange chromatography and other purification steps to obtain targeting antibody prodrug CMAB0301

The results of LC-MS free oligosaccharide saccharide type detection of the obtained targeting antibody prodrug CMAB0301 are shown in fig. 5, and it is apparent from the results of the detection that the sialylation type contained in IgG, which does not contain mice, of the targeting antibody prodrug CMAB0301 obtained by the above culture method using the above basal medium and supplementary medium is Neu5Gc (NGNA) and does not contain α -1,3 galactose.

Example 3: detection of in vitro activity of target antibody prodrug

The targeting antibody prodrug CMAB0301 is obtained by the method, and the combination and affinity detection of VEGF and EGFR are carried out.

(1) VEGF binding assay (ELISA):

recombinant VEGF165 antigen (1. mu.g/ml) was coated on 96-well elisa plates overnight at 4 ℃ with 100. mu.l per well; blocking the ELISA plate with PBS containing 10% BSA at 37 ℃ for 1 h; adding CMAB0301 diluted in multiple times in sequence from high concentration, incubating at 37 deg.C for 1 h, and washing with PBS containing 0.1% Tween-20 for three times; adding horseradish peroxidase-conjugated goat anti-human F (ab') 2 secondary antibody (1:1,000), and incubating for 30 min; adding TMB developing solution, developing for 10 min, stopping reaction with 1M sulfuric acid, and measuring absorbance value (OD value) at 450 nm with enzyme-labeling instrument.

The binding force of the targeting antibody prodrug CMAB0301 to VEGF165 antigen is preliminarily determined by indirect ELISA. As shown in fig. 6, the targeting antibody prodrug CMAB0301 has stronger binding ability to VEGF165 antigen, and we calculate the EC50 value of the targeting antibody prodrug CMAB0301 as: 0.028 mu g/mL, the result shows that the targeting antibody prodrug CMAB0301 has the binding force with VEGF165 antigen.

(2) VEGF affinity assay (SPR):

anti-human IgG (Fc) is coupled to the surface of a CM5 chip, targeting antibody prodrug CMAB0301 is captured, VEGF165 with sequentially increased concentration is detected, the binding kinetic parameters of the targeting antibody prodrug CMAB0301 and VEGF165 are evaluated, and regeneration treatment is carried out between samples.

Results were collected using a Biacore instrument and analyzed using a Biacore Evaluation software for analysis of data, see figure 7 for results, and the Ka, Kd and Kd parameters were evaluated, as detailed in table 1 below.

TABLE 1 table of affinity assay results of CMAB0301 and VEGF-165 (SPR method)

Batch number	Ka（1/Ms）	Kd（1/s）	KD（M）
				CMAB0301	6.942 × 10⁵	6.287 × 10^-5	9.057 × 10^-11

(3) EGFR binding assay (ELISA):

coating the recombinant EGFR-extracellular domain (ECD) antigen (0.5 mu g/ml) on a 96-well enzyme label plate, standing overnight at 4 ℃, and adding 100 mu l of antigen into each well; sealing the enzyme label plate for 1 h by 10% of skimmed milk powder at 37 ℃; adding CMAB0301 diluted in multiple times in sequence from high concentration, incubating at 37 deg.C for 1 h, and washing with PBS containing 0.1% Tween-20 for three times; adding horseradish peroxidase-conjugated goat anti-human F (ab') 2 secondary antibody (1:1,000), and incubating for 30 min; adding TMB developing solution, developing for 10 min, stopping reaction with 1M sulfuric acid, and measuring absorbance value (OD value) at 450 nm with enzyme-labeling instrument.

The binding capacity of CMAB0301 to the extracellular domain (ECD) of the antigen EGFR was preliminarily determined by indirect ELISA. As shown in fig. 8, CMAB0301 shows a certain binding capacity, and the calculated EC50 value is: 0.15. mu.g/mL.

(4) EGFR affinity assay (SPR):

the equilibrium dissociation constant (KD) of the antibody was determined using Biacore T200 to assess the affinity of the antibody; the results are shown in FIG. 9.

Anti-human IgG Fc fragment antibody was diluted to 20. mu.g/ml with 10 mM sodium acetate and coated on a Biacore chip CM 5; capturing 2.6 mu g/ml of antibodies to be detected (MAB 0301, a first antibody, a second antibody and enzyme-digested CMAB 0301) by using a chip coated with an anti-human IgG Fc segment; injecting EGFR extracellular region protein with different concentrations, analyzing a sensing map by utilizing BIAevaluation software, performing data fitting on an experimental result by adopting a 1:1 Langmuir combination model, and determining dissociation and combination constants of an antibody; the equilibrium dissociation constant (KD) = dissociation constant (KD)/binding constant (Ka) of the antibody was calculated.

TABLE 2 table of the results of affinity assay of CMAB0301 with EGFR (SPR method)

Batch number	Ka（1/Ms）	Kd（1/s）	KD（M）
				After enzyme digestion CMAB0301	2.241×10⁵	2.840×10^-4	1.267×10^-9

Example 4: detection of activity of target antibody prodrug after in vitro enzyme digestion

The targeting antibody prodrug CMAB0301 is obtained by the method, and after the cleavage by urokinase type plasminogen activator (uPA), the binding activity of the targeting antibody prodrug with VEGF and EGFR is detected.

(1) VEGF binding assay (ELISA):

recombinant VEGF165 antigen (1. mu.g/ml) was coated on 96-well elisa plates overnight at 4 ℃ with 100. mu.l per well; blocking the ELISA plate with PBS containing 10% BSA at 37 ℃ for 1 h; adding enzyme-digested CMAB0301 which is sequentially diluted in multiple proportions from high concentration, incubating for 1 h at 37 ℃, and washing for three times by PBS containing 0.1% Tween-20; adding horseradish peroxidase-conjugated goat anti-human F (ab') 2 secondary antibody (1:1,000), and incubating for 30 min; adding TMB developing solution, developing for 10 min, stopping reaction with 1M sulfuric acid, and measuring absorbance value (OD value) at 450 nm with enzyme-labeling instrument.

The binding capacity of the enzyme-cleaved CMAB0301 to VEGF165 antigen was preliminarily determined by indirect ELISA. As shown in FIG. 10, CMAB0301 has no significant binding ability to VEGF165 antigen after cleavage.

The experimental result shows that the CMAB0301 is mainly bound with the VEGF165 antigen by the antibody 1, and the binding ability with the VEGF165 antigen is lost after the antibody 1 is removed by enzyme digestion.

(2) VEGF affinity assay (SPR):

coupling Anti-human IgG (Fc) on the surface of a CM5 chip, capturing CMAB0301 after enzyme digestion, detecting VEGF165 with sequentially increased concentration, and evaluating the binding kinetic parameters of the CMAB0301 and the VEGF165 after enzyme digestion. Regeneration treatment is performed between samples.

As a result, when the sample was collected by using a Biacore instrument, it was found that the affinity of cleaved CMAB0301 to VEGF was extremely low (as shown in FIG. 11), and no affinity data could be detected, indicating that the anti-VEGF single-chain antibody had been completely cleaved.

(3) EGFR binding assay (ELISA):

coating the recombinant EGFR-extracellular domain (ECD) antigen (0.5 mu g/ml) on a 96-well enzyme label plate, standing overnight at 4 ℃, and adding 100 mu l of antigen into each well; sealing the enzyme label plate for 1 h by 10% of skimmed milk powder at 37 ℃; adding enzyme-digested CMAB0301 which is sequentially diluted in multiple proportions from high concentration, incubating for 1 h at 37 ℃, and washing for three times by PBS containing 0.1% Tween-20; adding horseradish peroxidase-conjugated goat anti-human F (ab') 2 secondary antibody (1:1,000), and incubating for 30 min; adding TMB developing solution, developing for 10 min, stopping reaction with 1M sulfuric acid, and measuring absorbance value (OD value) at 450 nm with enzyme-labeling instrument.

The binding force of the enzyme-digested CMAB0301 to the EGFR extracellular domain (ECD) is preliminarily determined by using indirect ELISA.

As shown in FIG. 12, CMAB0301 shows stronger binding capacity after cleavage. We calculated EC50 values as: 0.014. mu.g/mL. Thus, CMAB0301 shows about 10 times higher antigen binding after cleavage than before cleavage. The above results show that antibody 1 added by us can significantly reduce the binding of CMAB0301 to antigen, while CMAB0301 shows complete binding activity to EGFR after enzyme cleavage activation. This result preliminarily indicates the property of CMAB0301 that can be activated by enzymatic cleavage.

(4) EGFR affinity assay (SPR):

the equilibrium dissociation constant (KD) of the antibody was determined using Biacore T200 to assess the affinity of the antibody, and the results are shown in fig. 13.

TABLE 3 affinity assay result table of cleaved CMAB0301 and EGFR (SPR method)

Batch number	Ka（1/Ms）	Kd（1/s）	KD（M）
				CMAB0301	8.825×105	7.111×10^-5	8.058×10^-9

Example 5: indirect in vivo activation of Targeted antibody precursors in human tissue sections

To validate the targeted selective activation of CMAB0301 in clinical conditions, we performed in situ enzyme activity assays using frozen tissue sections from human colon cancer (CRC). The test process is as follows: preparing colon cancer tissue and paracancer mucosa tissue slices. These tissue sections were incubated with CMAB0301 and antibody 2 for 1.5 hours at room temperature. The unbound components are then washed away. Incubation was then performed with goat anti-human HRP reagent for 1 hour, and then unbound fraction was washed away. Finally, the color reaction is carried out by using a DAB reagent, and the color reaction is observed by using a microscope after the mounting.

First, tumor tissues of colon cancer patients with positive EGFR staining were screened by immunohistochemical staining, and the screened tissues were incubated with antibody 2 or CMAB 0301. To confirm the protease-activatable properties of CMAB0301, frozen tumor tissue was pretreated with a broad spectrum protease inhibitor. In contrast to CMAB0301, which had a positive signal in non-pretreated tissue, its binding activity was inhibited in pretreated tumor tissue (fig. 14). However, pre-treatment did not affect the binding of anti-EGFR antibodies. Furthermore, pre-incubation with unlabeled high concentration (100 μ g/ml) of panitumumab inhibited CMAB0301 binding, indicating that the binding site of EGFR was blocked (fig. 14). As shown in fig. 15, the binding signal of the anti-EGFR antibody to the paracancerous colonic mucosal tissue was higher than CMAB 0301.

Statistical analysis showed that in paracancerous normal mucosal tissue, the proportion of cells staining positive for Pan-is approximately 63% + -15.3%, whereas cells staining positive for CMAB0301 is only approximately 5% + -1.4%. Thus, while only observed in a limited number of samples, CMAB0301 has significantly lower binding activity to paracancerous tissues than anti-EGFR antibodies. These results lay the foundation for further evaluation of the targeted binding toxicity of CMAB0301 to normal skin tissues.

Example 6: therapeutic efficacy of Targeted antibody prodrugs in murine tumor models

To evaluate the therapeutic efficacy of CMAB0301, we performed the establishment of a prophylactic and therapeutic mesoflux model by first subcutaneously inoculating 1 × 106 a431 cells in the right back of 6-8 week old female nude mice. After the cells were inoculated, they were randomly divided into 3 groups of 6 nude mice each, and the specific grouping was as shown in Table 6-1.

a. For therapeutic tumor models: when the tumor volume after cell inoculation reaches about 150 mm3 on average, the antibody is injected intraperitoneally 2 times a week for four weeks. Tumor volume and mouse body weight were measured every two days and tumor growth curves were plotted as the mean size of each group of tumors versus time.

b. For prophylactic tumor models: on the day following tumor cell injection, intraperitoneal injections of antibody were started 2 times per week for three weeks. Tumor volume and mouse body weight were calculated for each two-day measurement and tumor growth curves were plotted as mean size of each group versus time.

TABLE 4 grouping of tumor-bearing mice

Group of	Dosage form
		Human IgG (control group)	50 mg/kg
Antibody 214	50 mg/kg
		CMAB0301	50 mg/kg

In both models described above, the tumor size is calculated as: length by width 2 by 0.5. Rats were euthanized by carbon dioxide asphyxiation. Animal experiments were conducted in accordance with the relevant institutional guidelines.

The experimental results show that: the effect was equivalent to antibody 2 in either a prophylactic model, i.e., treatment with CMAB0301 1 days after tumor cell inoculation (figure 16) or a therapeutic model, i.e., treatment after tumor formation (figure 17). Thus, the tumor suppressive effect of CMAB0301 is consistent with the previously demonstrated target activation profile.

Sequence listing

<110> Taizhou Mibo Taike pharmaceutical Co., Ltd

<120> antibody prodrug, preparation method and application thereof

<130> 20200327

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gagatcgtga tgacccagag ccccagcacc ctgagcgcca gcgtgggcga cagggtgatc 60

atcacctgcc aggccagcga gatcatccac agctggctgg cctggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacctg gccagcaccc tggccagcgg cgtgcccagc 180

aggttcagcg gcagcggcag cggcgccgag ttcaccctga ccatcagcag cctgcagccc 240

gacgacttcg ccacctacta ctgccagaac gtgtacctgg ccagcaccaa cggcgccaac 300

ttcggccagg gcaccaagct gaccgtg 327

<210> 2

<211> 109

<212> PRT

<213> Artificial sequence (Artificial sequence)

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Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Glu Ile Ile His Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Tyr Leu Ala Ser Thr

85 90 95

Asn Gly Ala Asn Phe Gly Gln Gly Thr Lys Leu Thr Val

100 105

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<211> 360

<212> DNA

<213> Artificial sequence (Artificial sequence)

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gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcaccg ccagcggctt cagcctgacc gactactact acatgacctg ggtgaggcag 120

gcccccggca agggcctgga gtgggtgggc ttcatcgacc ccgacgacga cccctactac 180

gccacctggg ccaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc cggcggcgac 300

cacaacagcg gctggggcct ggacatctgg ggccagggca ccctggtgac cgtgagcagc 360

<210> 4

<211> 120

<212> PRT

<213> Artificial sequence (Artificial sequence)

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Gly Phe Ile Asp Pro Asp Asp Asp Pro Tyr Tyr Ala Thr Trp Ala

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Gly Gly Asp His Asn Ser Gly Trp Gly Leu Asp Ile Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser

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<212> DNA

<213> Artificial sequence (Artificial sequence)

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aagcttgccg ccaccatgga ttttcaggtg cagattttca gcttcctgct aatcagtgcc 60

tcagtcataa tatccagagg agacatccag atgacccagt ctccatcctc cctgtctgca 120

tctgtaggag acagagtcac catcacttgc caggcgagtc aggacatcag caactattta 180

aattggtatc agcagaaacc agggaaagcc cctaaactcc tgatctacga tgcatccaat 240

ttggaaacag gggtcccatc aaggttcagt ggaagtggat ctgggacaga ttttactttc 300

accatcagca gcctgcagcc tgaagatatt gcaacatatt tctgtcaaca ctttgatcat 360

ctcccgctcg ctttcggcgg agggaccaag gtggagatca aacgtactgt ggctgcacca 420

tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc ctctgttgtg 480

tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc 540

ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac 600

agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc 660

tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa caggggagag 720

tgttgagaat tc 732

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<211> 214

<212> PRT

<213> Artificial sequence (Artificial sequence)

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu

85 90 95

Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 7

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<212> DNA

<213> Artificial sequence (Artificial sequence)

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caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagc agtggtgatt actactggac ctggattcgg 120

cagtccccag ggaagggact ggagtggatt ggacacatct attacagtgg gaacaccaat 180

tataacccct ccctcaagag tcgactcacc atatcaattg acacgtccaa gactcagttc 240

tccctgaagc tgagttctgt gaccgctgcg gacacggcca tttattactg tgtgcgagat 300

cgagtgactg gtgcttttga tatctggggc caagggacaa tggtcaccgt ctcttcagct 360

agcaccaagg gcccatccgt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagctgccc tgggctgcct ggtcaaggac tacttccccg aacccgtgac cgtgtcttgg 480

aactctggcg ccctgaccag cggcgtgcac accttccctg ctgtcctcca gtcctctgga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcctgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagcgtgt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccc tgcccagcac ctgaactcct ggggggacca 720

tccgtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccat gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccaa gggaagagca gtacaacagc 900

acatacagag tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agcccagaga accacaggtg tacaccctgc ccccatccag ggacgagctg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagcccgag aacaactaca agaccacacc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcctg ctccgtgatg catgaggctc tgcacaacca ctacacccag 1320

aagagcctct ccctgtctcc cggtaaa 1347

<210> 8

<211> 449

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

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

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

Claims

1. A targeting antibody precursor drug is composed of A, B, C three parts, wherein part A is a targeting protein, part C is an antibody/antibody receptor fusion protein with therapeutic effect, part A is connected with part C through part B, and part A has the antigen/ligand binding region of the targeting protein connected with part C.

2. The targeted antibody prodrug of claim 1, wherein the protein to which moiety a targets is a full antibody, an antigen binding fragment, a single chain antibody, a receptor/ligand protein binding fragment, or other functional protein to which targeting is directed.

3. The targeted antibody prodrug of claim 2, wherein the protein targeting moiety a is an anti-VEGF whole antibody, an anti-VEGF single chain antibody, a VEGF antigen binding fragment, a VEGF Trap.

4. The targeted antibody prodrug of claim 3, wherein the light chain variable region of the anti-VEGF single chain antibody has the amino acid sequence shown in SEQ ID NO. 2, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 4.

5. The targeted antibody prodrug of claim 1, wherein the antibody/antibody receptor fusion protein having a therapeutic effect of moiety C is a whole antibody, an antigen-binding fragment, a single chain antibody or an antibody receptor fusion protein.

6. The targeted antibody prodrug of claim 5, wherein the antibody with therapeutic effect of the moiety C is an anti-EGFR whole antibody.

7. The targeted antibody prodrug of claim 6, wherein the light chain of the anti-EGFR whole antibody has the amino acid sequence shown in SEQ ID NO. 6 and the heavy chain has the amino acid sequence shown in SEQ ID NO. 8.

8. The targeted antibody prodrug of claim 1, wherein the moiety B is a linker peptide comprising a substrate for tumor-or inflammation-specific protease cleavage.

9. The targeted antibody prodrug of claim 8, wherein the tumor-or inflammation-specific protease cleavage substrate is a plasmin substrate, a caspase substrate, or a matrix metalloenzyme substrate.

10. The targeted antibody prodrug of claim 9, wherein the specific cleavage substrate is a urokinase-type plasmin substrate.

11. The targeted antibody prodrug of claim 10, wherein the linker peptide comprising a urokinase-type plasmin substrate has the amino acid sequence of ASLSGRSDNHGSAS.

12. The targeted antibody prodrug of claim 9, wherein the specific protease cleavage substrate is a matrix metalloenzyme substrate.

13. The targeted antibody prodrug of claim 12, wherein the linker peptide comprising a substrate for a matrix metalloenzyme has an amino acid sequence of GSGPLGVRGSGS.

14. A pharmaceutical composition comprising an effective dose of the targetable antibody prodrug of claims 1-13 and a pharmaceutically acceptable excipient.

15. Use of a prodrug of a targeting antibody according to claims 1 to 13 for the manufacture of an anti-tumor medicament or a medicament for the treatment of inflammation.

16. Use of a targeting antibody prodrug of claim 14 in the preparation of an anti-tumor drug or a drug for the treatment of inflammation.

17. Use of a targetable antibody prodrug according to claims 1-13 in combination with other tumor therapy agents or other inflammation therapy agents.

18. Use of a targeting antibody prodrug according to claim 14 in combination with other tumor treating agents or other inflammatory treating agents.