CN116199746B - High affinity Trop2 targeting multi-cyclic peptide molecular framework - Google Patents

Abstract

A polycyclic peptide molecular framework targeting Trop2 with high affinity belongs to the field of biotechnology. Using a phage display polycyclic peptide library, ligand screening was performed for human trophoblast surface glycoprotein antigen 2 (Trop2), and a series of polycyclic peptide molecular frameworks that can bind to Trop2 with high affinity were obtained. On this basis, the obtained specific cyclic peptide ligands were further optimized to obtain more polypeptide ligands with drug properties. It can be used to construct various types of polypeptide-drug conjugates to provide targeted recognition motifs for the development of new disease diagnosis and treatment methods. Taking advantage of the orthogonal pairing of the CPPC motif, a polycyclic peptide with obvious oxidation main products and structural rigidity was obtained. Compared with ordinary linear peptides and unit cyclic peptides, the constructed polycyclic peptide has a larger binding interface, a more complex rigid structure, and a better binding affinity with the target.

Description

A multi-cyclic peptide molecular framework for high-affinity targeting of Trop2

Technical Field

The present invention belongs to the field of biotechnology, and in particular relates to a multi-cyclic peptide molecular framework with high affinity targeting Trop2. Based on phage display technology, a series of polypeptide skeletons with high affinity for human trophoblast surface glycoprotein antigen 2 (Trop2) are obtained, and the screening, synthesis, purification and affinity characterization of ternary cyclic peptide molecular ligands that can bind to Trop2 protein with high affinity are carried out.

Background Art

Human trophoblast surface antigen 2 (Trop2) is a cell surface glycoprotein that is overexpressed on a variety of cancer cells, but is lowly expressed or not expressed in normal tissues. Trop2 can regulate signaling pathways such as extracellular regulated protein kinase (ERK), mitogen-activated protein kinase (MAPK), inositol triphosphate (IP ₃ ), and calcium ions, and promote the proliferation and growth of tumor cells through multiple mechanisms. In addition, overexpression of Trop2 can also reduce cell adhesion and promote the metastasis of tumor cells (Michelle Shen, Shiqin Liu, Tanya Stoyanova. The role of Trop2 in prostate cancer: an oncogene, biomarker, and therapeutic target [J]. AmJ Clin Exp Urol, 2021, 9 (1): 73-87.). Based on this, Trop2 has become a molecular marker for clinical detection of tumor malignancy and a tumor treatment target. The development of drugs targeting Trop2 is of great significance for the treatment of various human cancers. Trodelvy is currently the only approved Trop2-targeting antibody-drug conjugate (ADC) for the treatment of triple-negative breast cancer, and has achieved certain therapeutic effects.

There are three common therapeutic drugs: small molecules, peptides, and biomacromolecules. Small molecule drugs have poor specificity and are prone to off-target or cytotoxicity. Biomacromolecule drugs are prone to immunogenicity, while peptide drugs can make up for the defects between the two. In addition, peptides can structurally mimic some features of protein interfaces, and combined with the advantages of their molecular size, they can act on the protein-protein interaction interface. Peptides have good biocompatibility and low biotoxicity, and are very potential drug molecules. Compared with ordinary linear peptides, cyclic peptides have many advantages. Cyclic peptides have higher receptor affinity and selectivity, rigid conformation, low sensitivity to proteolytic enzymes, and high stability.

At present, the development of many drugs starts with peptide ligands as lead compounds. The development of phage display technologies has also provided new technical support for the discovery of peptide ligands for targets, accelerating the development of peptide drugs. The characteristic of phage display technology is that it can quickly and directionally screen phage-displayed peptides that specifically bind to target proteins. At the same time, with the advantages of cyclic peptides, conformationally restricted cyclic peptide libraries can be constructed to screen for macromolecular targets of interest, develop new drug leads and new reagents for chemical biology.

The present invention constructs a series of cyclic peptide ligands that can specifically bind to Trop2 based on phage display technology. These cyclic peptide ligands are simple to synthesize, low in preparation cost, high in affinity and stability, and provide new drug lead molecules for diseases with Trop2 as the therapeutic target.

Summary of the invention

The purpose of the present invention is to provide a multi-cyclic peptide molecular framework for high-affinity targeting of Trop2. This type of polypeptide molecule has three binding rings that interact with the target. Compared with a single cyclic peptide, it has a larger binding interface and can provide better binding affinity and specificity, and is expected to become a drug lead molecule targeting Trop2.

Another object of the present invention is to provide a method for constructing a multi-cyclic peptide molecular framework targeting Trop2 with high affinity.

The third object of the present invention is to provide the use of the above-mentioned multi-cyclic peptide molecular framework in the development of drugs/reagents/drug lead molecules for the treatment and diagnosis of related diseases caused by the Trop2 signaling pathway.

The high-affinity multi-cyclic peptide molecular framework targeting Trop2 is obtained by phage display technology and construction of a secondary peptide library targeting the target protein Trop2. The sequence of the multi-cyclic peptide molecular framework contains the following characteristics:

CPPC(X _a )XW(X _b )EC(X _c )(X _d )(X _e )(X _f )(X _g )C(X _h )(X _i )(X _j )(X _k )(X _l )CPPC;

Wherein, amino acids are all L amino acids, X represents any natural amino acid; ( _Xk ) is D, W, H, T, Y, V, E, S, F, G, M, N, I, preferably D; ( _Xf ) is F, V, L, N, A, D, S, Y, M, I, T, W, preferably F; ( _Xd ) is D, G, S, N, R, T, Q, E, A, preferably G; ( _Xe ) is E, F, L, D, Y, M, preferably E; ( _Xj ) is I, V, L, M, F, R, T, K, preferably V; ( _Xl ) is F, W, G, M, I, Y, L, V, preferably F; ( _Xg ) is any amino acid, preferably N; ( _Xh ) is any amino acid, preferably V; ( _Xc ) is any amino acid, preferably S; ( _Xi) ) is V, A, I, P, L, A, E, T, D, S, F, Y, M, preferably V; (X _a ) is G or S, preferably G; (X _b ) is L or I, preferably L;

D is aspartic acid; W is tryptophan; H is histidine; T is threonine; Y is tyrosine; V is valine; E is glutamic acid; S is serine; F is phenylalanine; G is glycine; M is methionine; N is asparagine; I is isoleucine; L is leucine; A is alanine; R is arginine; K is lysine; P is proline; C is cysteine; Q is glutamine.

Specifically, the ligand sequences of the multi-cyclic peptide molecular framework with high affinity targeting Trop2 include but are not limited to:

CPPCGRWLECDSFTNCWELLTCPPC(peptide 1)

CPPCGAWLECDSFTNCWELLTCPPC(peptide 2)

CPPCGSWIECDSFTNCWELLTCPPC(peptide 3)

CPPCGAWIECDSFTNCWELLTCPPC(peptide 4)

CPPCSEWIECDSFTNCWELLTCPPC(peptide 5)

CPPCGRWLECSDENLCAIIDWCPPC(peptide 6)

CPPCGRWLECYDFNECELIDWCPPC(peptide 7)

CPPCGRWLECWGMFNCQEFQGCPPC(peptide 8)

CPPCGRWLECWTHIDCQFIDWCPPC(peptide 9)

CPPCGRWLECSNDFDDCDYLYFCPPC(peptide 10)

CPPCGRWLECLDEFRCTLIFHCPPC(peptide 11)

CPPCGRWLECYDEFSCEPFWFCPPC(peptide 12)

CPPCGRWLECSDGFTCNAVDFCPPC(peptide 13)

CPPCGRWLECSSPFTCTIFIGCPPC(peptide 14)

CPPCGRWLECSEDFLCDLRTFCPPC(peptide 15).

The method for constructing the high-affinity Trop2-targeting multi-cyclic peptide molecular framework comprises the following specific steps:

1) Screening the specific targeting ligand of Trop2 by using the phage display ternary cyclic peptide library and constructing the phage display peptide library 1;

2) The target protein Trop2 was screened using the phage display peptide library library 1. After three rounds of screening, single clones were selected for sequencing analysis to obtain polypeptide sequences as shown in the sequence listing SEQ ID No. 1 to 5;

3) According to the screening results, the polypeptide peptide 1 with the highest enrichment amount was selected, and a phage display polypeptide library library 2 was constructed according to the polypeptide sequence of polypeptide peptide 1, and the phage display polypeptide library library 2 was used to screen the Trop2 protein; after 3 rounds of screening, phage sequencing analysis was performed to obtain polypeptide sequences as shown in the sequence table SEQ ID No. 6 to 15.

In step 1), the phage display polypeptide library library 1, the polypeptide sequence skeleton from N-terminus to C-terminus is as follows:

CPPC(X) _5-10 CDSFTNCWELLTCPPC(library 1)

Among them, the amino acids are all L-type amino acids, X represents any natural amino acid, and the subscript represents the number of Xs contained.

In step 3), the phage display polypeptide library library 2, the polypeptide sequence skeleton from N-terminus to C-terminus is as follows:

CPPCGRWLEC(X) ₅ C(X) ₅ CPPC(library 2)

Among them, the amino acids are all L-type amino acids, X represents any natural amino acid, and the subscript represents the number of Xs contained.

In step 3), the phage display polypeptide library library 2 is constructed, and the polypeptide sequence skeleton is used as a template, and the polypeptide is fused with the phage pШ protein and displayed on the phage surface to construct a phage library displaying different polypeptide sequences; the phage display polypeptide library library 2 is a secondary library constructed based on the screening results of the phage display polypeptide library library 1 for the target protein Trop2, and the partial conserved sequence obtained by the phage display polypeptide library library 1 for the target protein Trop2 is fixed, and the amino acids of the other two binding loops are randomized.

The high-affinity multi-cyclic peptide molecular framework targeting Trop2 can be used in constructing various types of polypeptide-drug conjugates.

The high-affinity multi-cyclic peptide molecular framework targeting Trop2 can be used as a targeting recognition motif.

The high-affinity Trop2-targeting multi-cyclic peptide molecular framework can be used to develop drugs/reagents/drug lead molecules for the treatment and diagnosis of related diseases caused by the Trop2 signaling pathway.

Compared with the prior art, the present invention has the following technical effects and advantages:

a. The present invention takes advantage of the orthogonal pairing of CPPC motifs to obtain polycyclic peptides with obvious main oxidation products and structural rigidity. Compared with ordinary linear peptides and unit cyclic peptides, the polycyclic peptides constructed by the present invention have a larger binding interface, a more complex rigid structure, and better binding affinity with the target.

b. The polypeptides described in the present invention are all composed of natural amino acids, are simple to synthesize, and can be prepared in large quantities by solid phase synthesis or recombinant expression.

c. The present invention uses phage display technology and constructs a secondary peptide library for the target protein Trop2 to obtain a series of polypeptide frameworks that have high specificity binding to the target protein Trop2. Based on the tendency and conservation of amino acids at different positions, the obtained specific cyclic peptide ligands can be further optimized to obtain more polypeptide ligands with pharmaceutical properties.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the sequencing results of monoclonal clones obtained from the screening of library 1.

Figure 2 shows the top 50 sequences with the highest enrichment in library 2 screening by high-throughput sequencing.

Figure 3 is a chromatogram of peptide 1 after reduction and oxidation purification.

FIG4 is a chromatogram of the reduction and oxidation (after purification) of peptide 6.

FIG5 is a chromatogram of the reduction and oxidation (after purification) of peptide 7.

FIG6 is a chromatogram of the reduction and oxidation (after purification) of peptide 8.

FIG. 7 is a surface plasmon resonance (SPR) curve of peptide 1 (oxidized form).

FIG8 is a surface plasmon resonance (SPR) curve of peptide 6 (oxidized form).

FIG. 9 is a surface plasmon resonance (SPR) curve of peptide 7 (oxidized form).

FIG. 10 is a surface plasmon resonance (SPR) curve of peptide 8 (oxidized form).

FIG. 11 is a schematic diagram of the experimental process of constructing a phage display peptide library through a multi-cyclic peptide molecular framework and screening high-affinity polypeptide ligands.

DETAILED DESCRIPTION

The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

The present invention uses phage display technology and constructs a secondary peptide library targeting the target protein Trop2 to obtain a series of high-affinity multi-cyclic peptide molecular frameworks targeting Trop2. The sequence of the multi-cyclic peptide molecular framework is:

CPPC(X _a )XW(X _b )EC(X _c )(X _d )(X _e )(X _f )(X _g )C(X _h )(X _i )(X _j )(X _k )(X _l )CPPC;

Wherein, amino acids are all L amino acids, X represents any natural amino acid; ( _Xk ) is D, W, H, T, Y, V, E, S, F, G, M, N, I, preferably D; ( _Xf ) is F, V, L, N, A, D, S, Y, M, I, T, W, preferably F; ( _Xd ) is D, G, S, N, R, T, Q, E, A, preferably G; ( _Xe ) is E, F, L, D, Y, M, preferably E; ( _Xj ) is I, V, L, M, F, R, T, K, preferably V; ( _Xl ) is F, W, G, M, I, Y, L, V, preferably F; ( _Xg ) is any amino acid, preferably N; ( _Xh ) is any amino acid, preferably V; ( _Xc ) is any amino acid, preferably S; ( _Xi) ) is V, A, I, P, L, A, E, T, D, S, F, Y, M, preferably V; (X _a ) is G or S, preferably G; (X _b ) is L or I, preferably L;

D is aspartic acid; W is tryptophan; H is histidine; T is threonine; Y is tyrosine; V is valine; E is glutamic acid; S is serine; F is phenylalanine; G is glycine; M is methionine; N is asparagine; I is isoleucine; L is leucine; A is alanine; R is arginine; K is lysine; P is proline; C is cysteine; Q is glutamine.

The high-affinity multi-cyclic peptide molecular framework targeting Trop2 can be constructed by the following method:

1) Screening of Trop2-specific targeting ligands using phage-displayed peptide libraries;

(1) Design and construct a phage display peptide library with different backbones. The backbone of the peptide sequence (from N-terminus to C-terminus) is as follows:

CPPC(X) _5-10 CDSFTNCWELLTCPPC(library 1)

CPPCGRWLEC(X) ₅ C(X) ₅ CPPC(library 2)

Among them, the amino acids are all L-type amino acids, X represents any natural amino acid, and the subscript represents the number of Xs contained.

(2) Using the peptide sequence backbone as a template, the peptide was fused with the phage pШ protein and expressed and displayed on the phage surface to construct a phage library displaying different peptide sequences; the phage display peptide library library 2 was a secondary library constructed based on the screening results of library 1 for the target protein Trop2. Some conserved sequences obtained by screening the phage display peptide library library 1 for the target protein Trop2 were fixed, and the amino acids of the other two binding loops were randomized.

2) The target protein Trop2 was screened using the phage display peptide library library 1. After three rounds of screening, single clones were selected for sequencing analysis to obtain peptide sequences including but not limited to the following peptides 1 to 5 (SEQ ID No. 1 to 5 in the sequence list):

CPPCGRWLECDSFTNCWELLTCPPC(peptide 1)

CPPCGAWLECDSFTNCWELLTCPPC(peptide 2)

CPPCGSWIECDSFTNCWELLTCPPC(peptide 3)

CPPCGAWIECDSFTNCWELLTCPPC(peptide 4)

CPPCSEWIECDSFTNCWELLTCPPC(peptide 5)

3) According to the screening results of phage display peptide library 1 for the target protein Trop2, the peptide 1 with the highest enrichment was selected, and phage display peptide library 2 was constructed according to the polypeptide sequence of polypeptide peptide 1, and the Trop2 protein was also screened; the phage sequencing analysis after the three rounds of screening was performed to obtain the following polypeptide sequences peptide 6 to 15 (SEQ ID No. 6 to 15 in the sequence list):

CPPCGRWLECSDENLCAIIDWCPPC(peptide 6)

CPPCGRWLECYDFNECELIDWCPPC(peptide 7)

CPPCGRWLECWGMFNCQEFQGCPPC(peptide 8)

CPPCGRWLECWTHIDCQFIDWCPPC(peptide 9)

CPPCGRWLECSNDFDDCDYLYFCPPC(peptide 10)

CPPCGRWLECLDEFRCTLIFHCPPC(peptide 11)

CPPCGRWLECYDEFSCEPFWFCPPC(peptide 12)

CPPCGRWLECSDGFTCNAVDFCPPC(peptide 13)

CPPCGRWLECSSPFTCTIFIGCPPC(peptide 14)

CPPCGRWLECSEDFLCDLRTFCPPC(peptide 15)

4) According to the enriched peptide sequences obtained after sequencing, peptide 1, peptide 6, peptide 7 and peptide 8 were synthesized using a solid phase peptide synthesizer, and after oxidative folding, the corresponding oxidation products were characterized by affinity through surface plasmon resonance (SPR) to verify the effectiveness of the screening results.

The present invention uses the advantages of orthogonal pairing of CPPC motifs to construct a phage display ternary cyclic peptide library, and then successfully obtains Trop2-specific cyclic peptide ligands using the liquid phase screening method of phage display technology, and optimizes the affinity of the ligands by constructing a secondary phage polypeptide library to obtain a series of ternary cyclic peptide ligands with high affinity to Trop2. The multi-cyclic peptide ligand can be used to construct various types of polypeptide-drug conjugates, providing targeted recognition motifs for the development of new disease diagnosis and treatment methods. The ternary cyclic peptide ligand molecule can be used to develop drugs/reagents/drug lead molecules for the treatment and diagnosis of related diseases caused by the Trop2 signaling pathway.

The construction of the multi-cyclic peptide molecular framework of the present invention includes: screening Trop2 specific utilization cyclic peptide ligands by using a phage display ternary cyclic peptide library; synthesis, oxidation and affinity characterization of the cyclic peptide ligands with target proteins; and affinity optimization of the cyclic peptide ligands.

Specific embodiments are given below:

Example 1

The library1 phage display peptide library was used to perform three rounds of screening and enrichment of Trop2 protein. Using avidin-coated magnetic beads as solid phase carriers, the biotinylated protein was fixed to the magnetic beads for bio-panning. The magnetic beads with fixed target protein were used as the experimental group, and the magnetic beads were used as the control group. Equal amounts of blocked phages were added to incubate and bind for a period of time, the unbound phages were washed away, and the bound phages were eluted. The ratio of phages eluted from the experimental group to the control group was defined as the enrichment. Whether the screening for the target protein was successful was judged by whether the enrichment was significantly improved. After three rounds of screening, single clones from the experimental group were randomly selected for gene sequencing and peptide sequence analysis. The sequencing results are shown in Figure 1.

The sequence peptide 1 with the largest number of enrichments was selected to construct phage peptide library 2, and the affinity of the ligand for the target protein Trop2 was optimized. The enriched sequences were obtained after three rounds of screening (Figure 2).

Example 2

Peptide synthesis. Peptide 1, peptide 6, and peptide 7 obtained by gene sequencing were synthesized by solid phase synthesis using a fully automatic microwave peptide synthesizer. Using the synthetic strategy of Fmoc-protected amino acids, the synthesis was started from the C-terminus by dehydration condensation. 50 mg of Rink amide MBHA resin was weighed and added to a reactor. 10 mL of N, N-dimethylformamide (DMF) was added to swell for 30 minutes, and then automatic synthesis was started. The removal of the Fmoc protecting group occurred in DMF containing 20% piperidine, and each coupling reaction was carried out by adding an activator (Oxyma and DIC). After the peptide synthesis was completed, it was washed with 10 mL of ether (twice) to obtain a relatively dry peptide resin. After the peptide resin was cut, it was purified by chromatography and freeze-dried into powder. The peptide powder was dissolved in DMSO as a mother liquor, and a certain amount of the peptide mother liquor was taken in a buffer solution containing 20% (v/v) DMSO and 80% (v/v) PB (100mM, pH=7.4), and 10 times excess oxidized glutathione (GSSG) was added to oxidize the peptide. The final concentration of the peptide in the oxidation reaction system was 100μM, and the reaction was carried out in a shaker at 37°C for 6h. The oxidative folding product was separated and purified by analytical high performance liquid chromatography (HPLC), and the product was characterized by mass spectrometry and then freeze-dried. After dissolution, it can be used for binding capacity determination. The oxidative folding chromatograms of peptide 1, peptide 6, peptide 7, and peptide 8 are shown in Figures 3 to 6, respectively.

Example 3

The affinity between peptide 1 and target protein Trop2 was investigated by surface plasmon resonance experiment (SPR). The test used CAP sensor chip and Biacore T200 molecular interaction system. CAP contains a carboxymethylated dextran matrix, on which a single-stranded DNA molecule is fixed. Biotin CAPture reagent (containing complementary single-stranded DNA molecules bound to streptavidin) was injected onto the surface of CAP chip and hybridized with it, and then the biotin-labeled target protein was injected and captured in the experimental group channel, and then the peptide sample to be tested was injected. Different concentrations of peptides (peptide 1: 200~3200nM) were bound to the Trop2 protein on the chip to obtain the corresponding curve (Figure 7), and the corresponding affinity was obtained by fitting.

The affinity of peptide 6, peptide 7, and peptide 8 to the target protein Trop2 was investigated by surface plasmon resonance (SPR) experiment. The test used CM5 sensor chip and Biacore T200 molecular interaction system. There are exposed carboxyl groups on the CM5 chip, which are activated by N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl). The experimental group was injected with Trop2 protein, and its amino group reacted with the activated carboxyl group to couple to the surface of the CM5 chip, and then the peptide sample to be tested was injected. Different concentrations of peptides (peptide 6: 6.25-100nM, peptide 7: 1-16nM, peptide 8: 6.25-100nM) were combined with the Trop2 protein on the chip to obtain the corresponding curves (Figures 8-10), and the corresponding affinity was obtained by fitting.

FIG11 is a schematic diagram showing the experimental process of constructing a phage display peptide library through a multi-cyclic peptide molecular framework and screening high-affinity polypeptide ligands.

In summary, the affinity test of the series of ternary cyclic peptide ligands specifically targeting Trop2 obtained by the present invention and the target protein Trop2 shows that the polypeptides obtained in this way have a high affinity with the target protein. This series of polypeptides are rich in multiple pairs of disulfide bonds, which give them a more complex rigid structure, improve the stability of the polypeptides, and provide a larger interaction interface. At the same time, the affinity of the polypeptide ligands is effectively improved by constructing a secondary library, and the affinity of the ligands is successfully optimized. The affinity of Peptide 6 and Peptide 8 to the target protein is about 10 to 20 nM, and the affinity of Peptide 7 is 1 nM, which provides new drug lead molecules for the development of targeted Trop2 polypeptide therapeutic drugs, and realizes the targeted treatment of tumors with Trop2 as a surface marker.

Claims (4)

1. A high affinity Trop2 targeting multi-cyclic peptide molecular framework, characterized in that it is obtained by phage display technology and construction of a secondary polypeptide library directed against the target protein Trop2, the sequence of said multi-cyclic peptide molecular framework comprising:

CPPCGRWLECDSFTNCWELLTCPPC peptide 1；

CPPCGRWLECSDENLCAIIDWCPPC peptide 6；

CPPCGRWLECYDFNECELIDWCPPC peptide 7；

CPPCGRWLECWGMFNCQEFQGCPPC PEPTIDE 8A 8; or (b)

CPPCGRWLECWTHIDCQFIDWCPPC peptide 9；

D is aspartic acid; w is tryptophan; h is histidine; t is threonine; y is tyrosine; v is valine; e is glutamic acid; s is serine; f is phenylalanine; g is glycine; m is methionine; n is asparagine; i is isoleucine; l is leucine; a is alanine; r is arginine; k is lysine; p is proline; c is cysteine; q is glutamine.

2. Use of the high affinity Trop2 targeting multi-cyclic peptide molecular framework of claim 1 for the construction of polypeptide-drug conjugates.

3. Use of the high affinity Trop2 targeting multi-cyclic peptide molecular framework of claim 1 as a targeting recognition motif.

4. A diagnostic reagent comprising the high affinity Trop2 targeting multi-cyclic peptide molecular framework of claim 1 for use in detecting Trop2 expression levels.