CN113444182A

CN113444182A - Fusion protein carrier for targeted delivery of IgG antibody and application thereof

Info

Publication number: CN113444182A
Application number: CN202110694500.XA
Authority: CN
Inventors: 卢晓风; 杨浩; 陶泽; 程惊秋
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2021-09-28
Anticipated expiration: 2041-06-22
Also published as: CN113444182B

Abstract

The invention provides a fusion protein for targeted delivery of IgG antibody, which comprises a functional fragment domain of PDGFR beta specific affinity body, a functional fragment domain of IgG antibody specific affinity body and a trimeric protein domain. The fusion protein has tumor targeting property, can be fused with IgG antibody structural domains, can deliver the IgG antibody to tumor cells in a targeted manner to play an anti-tumor role, and has great application value in anti-tumor drugs.

Description

Fusion protein carrier for targeted delivery of IgG antibody and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a fusion protein carrier for targeted delivery of an IgG antibody and application thereof.

Background

During the development of tumor, some cytokines or receptors are usually highly expressed to promote growth and metastasis. Meanwhile, in order to evade the attack of the immune system, tumors usually express some immunosuppressive molecules, such as immunodetection point proteins, and the like. Thus, blocking the binding of cytokines to their receptors or immunodot proteins to their ligands may exert an anti-tumor effect. The antibody has the advantages of strong specificity, long half-life period, high efficiency and the like, so that the antibody becomes an antitumor drug with great potential. The tumor therapeutic antibodies which have been approved for clinical use are up to several tens of antibodies. However, cytokines and their receptors, which are highly expressed at the tumor site, as well as immune checkpoint proteins, are often also expressed in normal tissues. The systemic administration of antibody drugs may block the associated signaling at non-tumor sites and cause side effects. For example, some immunodetection point proteins are highly expressed in tumor cells. Tumors rely on these immunodetection point proteins to achieve immune escape. The anti-tumor immune response can be recovered by blocking the immunodetection point proteins by using antibodies. However, these immunodetection point proteins are also widely expressed on normal cells and are an important mechanism for the body to avoid autoimmune attack. Therefore, if immunodetection point protein-blocked antibodies are not tumor-targeted, the anti-tumor effect is usually improved only by increasing the dose. In this case, systemic administration of immunodetection point protein-blocked antibodies, while often restoring anti-tumor immune responses, may also over-activate systemic immune responses and cause autoimmune damage (Allenbach et al 2020; Belkhir et al 2017; Dudzinska et al 2020). Therefore, if a therapeutic antibody can be delivered to a tumor site in a targeted manner, the efficiency of the antibody action can be improved and side effects can be reduced.

In order to target therapeutic antibodies to tumor sites, there have been studies to fuse tumor-targeting molecules to antibodies, often by chemical coupling or genetic recombination (Compete et al 2018; Dheily et al 2018; Ishihara J2017). However, the antibody comprises two light chains and two heavy chains, has large molecular weight and complex structure, and is difficult to realize site-specific coupling by a conventional chemical method. If gene fusion is carried out, 4 fusion sites can be selected, and the proper fusion sites need to be screened out one by one, so that the difficulty is high.

If a fusion protein which has tumor targeting and can combine with the IgG antibody can be researched to be used as a targeted delivery carrier of the therapeutic IgG antibody, the anti-tumor therapeutic antibody can be quickly and efficiently enriched on a tumor part under the guidance of the tumor targeting molecules, so that a stronger anti-tumor effect is exerted, and the application prospect is good.

Disclosure of Invention

The invention designs that a tumor-targeting molecule is fused with an IgG antibody binding domain to form a fusion protein which has tumor targeting and can be combined with an antibody, and the fusion protein is used as a targeted delivery carrier of a therapeutic antibody.

The invention provides a fusion protein, which comprises the following parts:

(1) a domain of a functional fragment of a PDGFR β -specific affibody or a domain having a sequence at least 95% homologous thereto;

(2) a domain of a functional fragment of an IgG antibody-specific affibody or a domain having a sequence at least 95% homologous to the functional fragment;

(3) a trimeric protein domain having the sequence shown in SEQ ID No.1 or a domain having a sequence with at least 95% homology with the trimer.

Further, the functional fragment domain of the PDGFR β -specific affibody described in the above section (1) is Z_PDGFRβThe amino acid sequence is shown in SEQ ID NO. 2.

Further, the functional fragment domain of the IgG specific affibody described in the above section (2) is IgBD, and the amino acid sequence thereof is shown in SEQ ID NO. 3.

Further, a linker is provided between the above-mentioned part (1) and part (3).

Further, a linker is provided between the parts (2) and (3).

Further, the above linker is (G4S)₃。

The invention also provides application of the fusion protein as an IgG antibody targeted delivery carrier.

Further, the IgG-class antibody includes: human anti-PD-1 antibody (Nivolumab), anti-PD-L1 antibody (Atezolizumab), anti-CTLA 4 antibody (Iplilimumab), anti-HER 2 antibody (Trastuzumab), anti-CD 20 antibody (Rituximab), or anti-EGFR antibody (Cetuximab).

The invention also provides an anti-tumor medicament which contains the fusion protein and the IgG antibody.

Experimental results show that the fusion protein has tumor targeting property, can be fused with an IgG antibody structure domain, can deliver the IgG antibody to tumor cells in a targeted manner to play an anti-tumor role, and has great application value in anti-tumor drugs.

Linker of the invention (G4S)₃The sequence of (a) is GGGGSGGGGSGGS.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the molecular design (A), recombinant expression and purification (B) of Z-tri.

FIG. 2 shows the molecular design (A) and protein preparation (B) of Z-IgBD-tri.

FIG. 3 is the binding of Z-tri to PDGFR β positive cells.

FIG. 4 is optical in vivo imaging (A) and tissue distribution (B) showing Z-tri enrichment within the tumor.

FIG. 5 is the binding of Z-IgBD-tri to PDGFR β positive cells (A) and PD-L1 antibody (B).

FIG. 6 shows the results of the binding assay of Z-IgBD-tri to human therapeutic IgG class antibodies.

FIG. 7 shows the results of Z-IgBD production and IgG binding capacity analysis.

FIG. 8 shows the results of the preparation of Z-IgBD01-tri and the analysis of its IgG-binding ability.

FIG. 9 shows that Z-IgBD-tri enhances the anti-tumor effect of PD-L1 antibody. A is tumor growth curve, B is tumor body photograph after treatment.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1 preparation of tumor targeting molecule Z-tri

Use of an affibody molecule Z capable of specifically recognizing the platelet-derived growth factor receptor beta_PDGFRβAs a tumor targeting molecule, Z_PDGFRβWith trimerization domain (tri) via (G4S)₃The ligants were fused to construct Z-tri (FIG. 1A).

Z_PDGFRβThe amino acid sequence of (a): AENKFNKELIEAAAEIDALPNLNRRQWNAFIKSLVDDPSQSANLLAEAKKLNDAQAPK

Amino acid sequence of the trimeric domain (tri): GSRNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPA

Based on the amino acid sequence of Z-tri, we designed and entrusted the company to synthesize the gene encoding Z-tri. The obtained gene was cloned into a commercial expression plasmid pQE30 by BamHI and SalI and transformed into E.coli M15 strain. The expression strain is cultured by LB culture medium to logarithmic growth phase, and then 0.1mM isopropyl-beta-D-thiogalactoside is added to induce and culture overnight at 24 ℃. The thalli is collected by centrifugation, and the supernatant is collected after the thalli is broken by high-pressure homogenate. The soluble target protein in the supernatant was purified by Ni-NTA affinity chromatography. As a result, as shown in FIG. 1B, the purified Z-tri appeared as a single band on the electrophoresis gel, indicating that a pure Z-tri was obtained.

Example 2 molecular design and recombinant expression of antibody-targeted delivery vectors

According to the schematic molecular structure shown in FIG. 2A, Z-tri and IgG antibody binding domain IgBD were passed through (G4S)₃The Z-IgBD-tri is constructed by connecting the two components together.

The amino acid sequence of IgBD is: TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTE

We designed and synthesized the gene encoding Z-IgBD-tri based on the amino acid sequence. The gene was inserted into pQE30 and expressed in E.coli M15, and the desired protein was purified by Ni-NTA affinity chromatography. As a result, it was found that purified Z-IgBD-tri showed a single band on the electrophoresis gel (FIG. 2B), indicating that a purified Z-IgBD-tri was obtained.

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 tumor targeting analysis of Z-tri

To test the cell binding capacity of the PDGFR β -specific binding molecule Z-tri, we incubated the tumor cells with the fluorescent dye FAM labeled Z-tri at room temperature, then washed the cells with PBS and analyzed by flow cytometry whether the protein binds to PDGFR β positive cells. The results are shown in fig. 3, and the tumor cell CT26 expresses PDGFR beta, Z-tri and can be combined with the CT26 cell.

To further analyze whether Z-tri was tumor targeted under in vivo conditions, we labeled Z-tri with CF 750. CT26 cells are inoculated to the subcutaneous part of a nude mouse to establish a tumor-bearing model. When the tumor grows to 100-200mm³CF750 labeled Z-tri is injected into mice through tail vein at a dose of 10mg/kg, and the tumor site is scanned by a small animal optical living body imaging system at regular time to observe the enrichment of Z-tri in the tumor. As shown in FIG. 4A, Z-tri enrichment was observed at the tumor site within 4 hours after protein injection. To compare the differences in the distribution of Z-tri in tumors and other tissues and organs, mice were sacrificed after the last in vivo imaging and tumor bodies and vital organs and tissues were removed for simultaneous scanning. As a result, as shown in FIG. 4B, the content of Z-tri in the tumor site and kidney was significantly higher than that in the heart, liver, spleen, lung, small intestine and muscle. Since the kidney is a metabolic organ, the accumulation of Z-tri is mainly due to metabolism.

The above results show that Z_PDGFRβWith trimerization domain (tri) via (G4S)₃After fusion of the linker, Z is not lost_PDGFRβThe Z-tri can be enriched at the tumor part and has tumor targeting.

Experimental example 2 in vitro functional analysis of antibody-targeting delivery vehicle Z-IgBD-tri

Z in Z-IgBD-tri_PDGFRβThe normal function of the IgBD domain is a determinant of its use as an antibody carrier. We first analyzed Z by cell binding experiments_PDGFRβThe function of (c). The results are shown in FIG. 5A, where Z-IgBD-tri was able to bind to PDGFR β positive CT26 tumor cells, indicating that Z in Z-IgBD-tri_PDGFRβThe domains function normally. We analysed the binding of Z-IgBD-tri to an antibody specific for PD-L1(α PD-L1) by gel filtration chromatography. As a result, as shown in FIG. 5B, the product obtained by mixing Z-IgBD-tri with α PD-L1 at an equimolar ratio showed a single protein peak on a gel filtration column having a molecular weight larger than that of IgG. The product of the mixture of Z-tri without IgBD domain and α PD-L1 showed two protein peaks with molecular weights similar to those of Z-tri and α PD-L1 alone on a gel filtration column. It is shown that Z-IgBD-tri forms a complex with alpha PD-L1, while Z-tri cannot bind with alpha PD-L1.

The above results indicate that the IgBD domain in Z-IgBD-tri also functions normally and is capable of binding to the IgG class antibody α PD-L1.

Experimental example 3 binding of antibody-Targeted delivery vector Z-IgBD-tri to other IgG class antibodies

Example 2 demonstrates that Z-IgBD-tri binds to α PD-L1 and targets it to the tumor site to enhance its anti-tumor effect. To further determine whether Z-IgBD-tri could bind other antibodies for use as a targeted delivery vehicle, we examined the binding ability of Z-IgBD-tri to various therapeutic IgG class antibodies by molecular interaction experiments. IgG antibody was first immobilized on the probe via Protein A, and then the probe was inserted into Z-IgBD-tri solutions containing different concentrations (100-1000nM) for binding. FIG. 6 shows that Z-IgBD-tri binds to human anti-PD-1 antibody (Nivolumab), anti-PD-L1 antibody (Atezolizumab), anti-CTLA 4 antibody (Ipilimumab), anti-HER 2 antibody (Trastuzumab), anti-CD 20 antibody (Rituximab), and anti-EGFR antibody (Cetuximab), indicating that Z-IgBD-tri can also be used as a targeted delivery vehicle for the above IgG class antibodies.

Experimental example 4 preparation of Z-IgBD and antibody binding thereto

To demonstrate that Z-IgBD-tri is the optimal molecular form, we fit Z to the schematic structure of FIG. 7A_PDGFRβWith IgBD (G4S)₃The Z-Tri was prepared with Z-IgBD without the Tri domain.

FIG. 7B shows that the Z-IgBD encoding gene was expressed in E.coli by the method described in example 1, and the purified Z-IgBD was shown as a single band on SDS-PAGE gel electrophoresis by Ni-NTA affinity chromatography, indicating that we obtained a purified Z-IgBD.

Further, it was found by molecular interaction analysis that the binding capacity of Z-IgBD to IgG was significantly lower than that of Z-IgBD-tri (FIG. 8C). The Z-IgBD-tri designed and prepared by the invention has obviously stronger IgG antibody binding capacity compared with the Z-IgBD without a trimer structure, and is more suitable for the targeted delivery of the IgG antibody.

Experimental example 5 preparation of Z-IgBD01-tri and antibody binding

There are many domains that can bind to IgG antibodies. The different domains differ from the binding site of the antibody. The binding sites are not suitable and may adversely affect the antibody. Of most concern is whether such domains, when bound to antibodies, cause aggregation and precipitation of the antibodies. Therefore, among the numerous IgG antibody binding domains, it is not easy to screen for the appropriate domain. To illustrate this problem, we selected a domain IgBD01 that is highly homologous to IgBD. The IgBD selected in the embodiment of the invention is combined with an IgG antibody through a Fab segment. IgBD01 binds to IgG antibodies via both Fab and Fc. We constructed Z-IgBD01-tri by substituting the IgBD01 with a highly similar sequence, according to the schematic structure of FIG. 8A, with reference to the molecular design of Z-IgBD-tri.

The amino acid sequence of IgBD01 (SEQ ID NO.4) is: TTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE are provided.

FIG. 8B shows that the gene encoding Z-IgBD01-tri was expressed in E.coli following the procedure described in example 1, and that Z-IgBD01-tri purified by Ni-NTA affinity chromatography showed no single band on SDS-PAGE gel, indicating that we obtained a pure Z-IgBD 01-tri.

To test the effect on the IgG antibodies after binding, the same concentrations of Z-IgBD-tri and Z-IgBD01-tri α were mixed with the same amount of α PD-L1 antibody, and the solution was observed for the presence of precipitate and the absorbance was measured at 340nm using a UV spectrophotometer. If the value is significantly higher than the protein-free solution, aggregation and precipitation of the antibody occurs. As shown in FIG. 8C, the turbidity of the mixture of Z-IgBD-tri and antibody was similar to that of PBS, but the turbidity of the mixture of Z-IgBD01-tri and antibody was much higher than that of the mixture of PBS and Z-IgBD-tri and antibody, indicating that Z-IgBD01-tri would cause the aggregation and precipitation of alpha PD-L1 antibody, while Z-IgBD-tri would cause less risk of antibody aggregation and be more suitable for antibody delivery.

Experimental example 6 analysis of in vivo function of antibody-targeting delivery vehicle Z-IgBD-tri

This test example was carried out on the IgG class antibody. alpha. PD-L1 bound to Z-IgBD-tri by means of Z in the Z-IgBD-tri molecule_PDGFRβThe targeting of the tumor is verified, so that more alpha PD-L1 antibody enters the tumor body to show stronger anti-tumor effect.

To examine the synergistic effect of Z-IgBD-tri on α PD-L1, α PD-L1(10mg/kg) was mixed with the same molar amount of Z-IgBD-tri or Z-tri, respectively, and incubated at room temperature for 30 minutes before injection into the CT26 mouse tumor-bearing model. The drug is administered every other day for three times. Meanwhile, tumor growth was monitored by changes in tumor volume. At the end of the observation, the mice were sacrificed and the tumor was dissected out and photographed. As shown in FIG. 8, the tumors treated with the mixture of Z-IgBD-tri and α PD-L1 (Z-IgBD-tri + α PD-L1) grew significantly slower than those treated with the mixture of Z-tri and α PD-L1 (Z-tri + α PD-L1) (FIG. 8A), and the former treated tumors were observed to be smaller than those treated at the end (FIG. 8B).

The results show that the alpha PD-L1 combined with the Z-IgBD-tri has stronger anti-tumor effect, namely the Z-IgBD-tri can enable more alpha PD-L1 to enter the tumor to play stronger anti-tumor effect compared with the Z-tri, and the Z-IgBD-tri can be used as a carrier for targeted delivery of the IgG antibody alpha PD-L1.

In conclusion, the fusion protein Z-IgBD-tri has tumor targeting property and can be fused with an IgG antibody structure domain, the binding capacity of the fusion protein Z-IgBD-tri and an IgG antibody is obviously superior to that of a Z-IgBD fusion protein without a trimer structure, antibody aggregation is not caused like Z-IgBD01-tri, an IgG antibody can be effectively delivered to tumor cells in a targeted mode to play an anti-tumor role, and the fusion protein Z-IgBD-tri has a huge application value in anti-tumor drugs.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

<120> fusion protein carrier for targeted delivery of IgG antibody and application thereof

<130> GYKH1131-2021P0113176CC

<160> 4

<170> PatentIn version 3.5

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<211> 57

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<213> trimeric Domain tri

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<213> ZPDGFRβ

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Claims

1. A fusion protein comprising the following moieties:

2. The fusion protein of claim 1, wherein the functional fragment domain of the PDGFR β -specific affibody of part (1) is Z_PDGFRβThe amino acid sequence is shown in SEQ ID NO. 2.

3. The fusion protein of claim 1, wherein the functional fragment domain of the IgG-specific affibody of part (2) is IgBD, the amino acid sequence of which is shown in SEQ ID No. 3.

4. The fusion protein of any one of claims 1 to 3, wherein there is a linker between moiety (1) and moiety (3).

5. The fusion protein of any one of claims 1 to 3, wherein there is a linker between moiety (2) and moiety (3).

6. The fusion protein of claim 4 or 5, wherein the linker is (G4S)₃。

7. Use of the fusion protein of any one of claims 1 to 6 as a carrier for targeted delivery of IgG-class antibodies.

8. The use of claim 7, wherein the IgG class antibody comprises: human anti-PD-1 antibody (Nivolumab), anti-PD-L1 antibody (Atezolizumab), anti-CTLA 4 antibody (Iplilimumab), anti-HER 2 antibody (Trastuzumab), anti-CD 20 antibody (Rituximab), or anti-EGFR antibody (Cetuximab).

9. An antitumor agent comprising the fusion protein according to any one of claims 1 to 6 and an IgG antibody.

10. The medicament of claim 9, wherein the IgG-class antibody comprises: human anti-PD-1 antibody (Nivolumab), anti-PD-L1 antibody (Atezolizumab), anti-CTLA 4 antibody (Iplilimumab), anti-HER 2 antibody (Trastuzumab), anti-CD 20 antibody (Rituximab), or anti-EGFR antibody (Cetuximab).