CN117587028A

CN117587028A - Polynucleotide expression cassette and plasmid encoding anti-VEGF antibody or antigen binding fragment

Info

Publication number: CN117587028A
Application number: CN202311414827.2A
Authority: CN
Inventors: 柯潇; 罗爽; 郑强
Original assignee: Chengdu Hongji Biotechnology Co ltd
Current assignee: Chengdu Hongji Biotechnology Co ltd
Priority date: 2023-10-27
Filing date: 2023-10-27
Publication date: 2024-02-23

Abstract

The present invention relates to the use of antibodies or antigen binding fragments encoding anti-VEGF in polynucleotide expression cassettes, plasmids containing the polynucleotide expression cassettes, host cells, recombinant viruses, and compositions thereof.

Description

Polynucleotide expression cassette and plasmid encoding anti-VEGF antibody or antigen binding fragment

Technical Field

The present invention relates to the field of genetic engineering/biomedical technology, and in particular, to a polynucleotide expression cassette encoding an anti-VEGF antibody or antigen binding fragment, a plasmid, a host cell, a recombinant virus containing the polynucleotide expression cassette, and compositions, uses and therapeutic methods thereof.

Background

The formation of new blood vessels is a key place for the development and spread of many diseases. Many diseases of the eye involve angiogenesis, including age-related macular degeneration (AMD), retinal Vein Occlusion (RVO), diabetic Retinopathy (DR), and pathological myopia, among others. VEGF is a highly specific vascular endothelial cell growth factor and has the effects of promoting vascular permeability increase, extracellular matrix denaturation, vascular endothelial cell migration, proliferation, angiogenesis and the like. VEGF exerts its biological effects after binding to 3 tyrosine kinase receptors, namely vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, VEGFR-3). The extra-membrane portion of VEGFR has approximately 750 amino acid residues and consists of 7 Ig domains that resemble immunoglobulin structures. When the receptor is activated by VEGF, intracellular tyrosine kinase groups phosphorylate and cause a series of signalling, ultimately triggering angiogenesis. Studies have shown that the crystal structure of the VEGFR-1 extracellular region shows that the second Ig domain is the region that binds to the ligand. The third Ig domain of VEGFR-2 plays a role in the specificity of ligand binding.

VEGF is widely distributed in various tissues in humans and animals, and normal ocular retinal pigment epithelial cells, vascular endothelial cells and pericytes produce lower levels of VEGF. Many studies have demonstrated that pathological neovascular ocular disease can be induced if VEGF is overexpressed. Due to the importance of VEGF signaling on angiogenesis, blocking VEGF or VEGF receptors to inhibit angiogenesis has important therapeutic effects on angiogenesis-related diseases including cancer, retinal vascular disease, etc. Over the past decade, some anti-VEGF drugs for the treatment of ocular neovascular eye diseases have been born, such as pipadatinib (Macugen), bevacizumab (Avastin), ranibizumab (Lucentis), albesiex (Eylea), and the like. Although these drugs are clinically effective, due to their short half-life, current anti-VEGF antibodies or antigen-binding fragments require repeated intravitreal administration, which places a burden on both the physician and patient, and thus gene therapy is a desirable therapeutic regimen.

Gene therapy achieves therapeutic effects by introducing exogenous anti-VEGF genes into target cells via appropriate vectors (e.g., adenoviruses) and by effectively expressing the corresponding agents. It aims to achieve long-term maintenance of drug efficacy through sustained expression of drugs to significantly improve physiological and psychological burden of patients. However, a significant challenge of gene therapy is how to make exogenous genes sufficiently highly express functional proteins or antibodies in target cells, which becomes critical to the success or failure of gene therapy.

Disclosure of Invention

Based on the above objects, an aspect of the present invention is to provide a polynucleotide expression cassette comprising: 1) An expression regulatory element;

2) Coding sequences encoding anti-VEGF antibodies or antigen-binding fragments.

In some embodiments, the anti-VEGF antibody or antigen-binding fragment heavy chain variable region of the invention comprises SEQ ID NO:1, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2, and a polypeptide sequence of the same. In some embodiments, the anti-VEGF antibody or antigen-binding fragment heavy chain of the invention comprises SEQ ID NO:3, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide sequence of the polypeptide.

The present invention includes coding sequences encoding the above anti-VEGF antibodies or antigen binding fragments, which are operably linked to expression regulatory elements. In some embodiments, the anti-VEGF antibody or antigen-binding fragment thereof comprises SEQ ID NO:5, and a nucleotide sequence described in seq id no. In encoding the expressed anti-VEGF antibody or antigen-binding fragment thereof, the heavy and light chains are linked by an F2A peptide.

In other embodiments, the coding sequence of the anti-VEGF antibody or antigen-binding fragment of the invention is codon optimized, e.g., the coding sequence is modified by methods known in the art, e.g., using codons that are expressed at high frequencies in the target cell instead of codons that are expressed at low frequencies, so that the coding sequence of the invention has increased expression in the target cell relative to the original codon, and the expression level of the encoded product in the target cell can be increased.

The polynucleotide expression cassettes of the invention also include expression control elements, e.g., the expression control elements are promoters operably linked to coding sequences. Promoters include promoter sequences or functional fragments thereof. The promoter is specific for eukaryotic cells or mammalian cells. The promoter is selected from the group consisting of promoters of: beta-actin promoter (CBA), cytomegalovirus promoter (CMV), elongation factor 1 alpha promoter (EF 1 alpha), MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, opsin promoter, artificial splice promoter (CASS), pMNTC promoter. Preferably, the promoter comprises a CBA promoter, a CMV promoter, a CAG promoter or an EF1 a promoter. In certain embodiments.

The expression regulatory element of the present invention may further include a polyadenylation signal (polyA). Polyadenylation signals protect the mRNA from exonuclease attack and are important for transcription termination, export of mRNA from the nucleus and translation. Polyadenylation signals comprise a plurality of consecutive adenosine monophosphates, typically containing AAUAAA repeats. The polyadenylation signal of the present invention is located downstream of the coding sequence encoding the anti-VEGF antibody or antigen-binding fragment. In some embodiments, the polyadenylation signal comprises monkey vacuolated virus 40 (SV 40), human Growth Hormone (HGH), bovine Growth Hormone (BGH), or β -globin.

The expression regulatory element of the present invention may further include an intron, which is located after the promoter. In some cases, an intron may refer to any sequence that may be transcribed but not translated. In some cases, an intron may refer to any sequence that is transcribed and removed from the mature RNA transcript in a cell. The intron is selected from PI intron, VH4 intron, SV40 intron, chi intron, U12 intron, RHD intron, etc.

In certain embodiments, the polynucleotide expression cassettes of the invention flank functional adenovirus Inverted Terminal Repeats (ITRs) at the 5 'and 3' ends. Functional adenovirus Inverted Terminal Repeats (ITRs) refer to ITR sequences as used for integration, replication and packaging of AAV virions. The inverted terminal repeat is an adeno-associated viral ITR of serotype selected from the group consisting of AAV1ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9ITR, AAV10 ITR, AAV11 ITR, and AAV12 ITR.

In some embodiments, the polynucleotide expression cassette comprises an RNA export signal downstream of the coding sequence encoding the anti-VEGF antibody or antigen-binding fragment and upstream of the polyadenylation region. The RNA output signal is illustratively selected from the group consisting of a human hepatitis b virus posttranscriptional element (HPRE) sequence and a woodchuck hepatitis virus posttranscriptional element (WPRE) sequence. Preferably, the RNA export signal is selected from woodchuck hepatitis virus post-transcriptional elements (WPREs).

In some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':

1)AAV 5'ITR；

2) A promoter region comprising a CBA promoter sequence;

3) A coding sequence region encoding an anti-VEGF antibody or antigen-binding fragment operably linked to a promoter sequence;

4) A WPRE transcriptional regulatory element;

5) An SV40 polyadenylation signal sequence;

6)AAV 3'ITR。

in some preferred embodiments, the polynucleotide expression cassette of the invention comprises SEQ ID NO:6, and a nucleotide sequence described in the following.

1)AAV 5'ITR；

2) An EF1 alpha promoter sequence;

3) A coding sequence encoding an anti-VEGF antibody or antigen-binding fragment operably linked to a promoter sequence;

4) A WPRE transcriptional regulatory element;

5) An SV40 polyadenylation signal sequence;

6)AAV 3'ITR。

in some preferred embodiments, the polynucleotide expression cassette of the invention comprises SEQ ID NO: 7.

1)AAV 5'ITR；

2) A promoter region comprising a CMV promoter sequence;

4) A WPRE transcriptional regulatory element;

5) BGH polyadenylation signal sequence;

6)AAV 3'ITR。

in some preferred embodiments, the polynucleotide expression cassette of the invention comprises SEQ ID NO:8, and a nucleotide sequence described in seq id no.

1)AAV 5'ITR；

2) An EF1 alpha promoter sequence;

2) A coding sequence encoding an anti-VEGF antibody or antigen-binding fragment operably linked to a promoter sequence;

3) An SV40 polyadenylation signal sequence;

4)AAV 3'ITR。

in some preferred embodiments, the polynucleotide expression cassette of the invention comprises SEQ ID NO: 9.

1)AAV 5'ITR；

2) An EF1 alpha promoter sequence;

4) A WPRE transcriptional regulatory element;

5) HGH polyadenylation signal sequence;

6)AAV 3'ITR。

1)AAV 5'ITR；

2) A CMV promoter sequence;

4) A WPRE transcriptional regulatory element;

5) An SV40 polyadenylation signal sequence;

6)AAV 3'ITR。

1)AAV 5'ITR；

2) A CMV promoter sequence;

4) A WPRE transcriptional regulatory element;

5) HGH polyadenylation signal sequence;

6)AAV 3'ITR。

1)AAV 5'ITR；

2) A CBA promoter sequence;

4) A WPRE transcriptional regulatory element;

5) BGH polyadenylation signal sequence;

6)AAV 3'ITR。

1)AAV 5'ITR；

2) An EF1 alpha promoter sequence;

4) A WPRE transcriptional regulatory element;

5) BGH polyadenylation signal sequence;

6)AAV 3'ITR。

1)AAV 5'ITR；

2) CAG promoter sequence;

4) A WPRE transcriptional regulatory element;

5) An SV40 polyadenylation signal sequence;

6)AAV 3'ITR。

1)AAV 5'ITR；

2) An LCR-L promoter sequence;

4) A WPRE transcriptional regulatory element;

5)3’UTR；

6) An SV40 polyadenylation signal sequence;

7)AAV 3'ITR。

in some embodiments, the polynucleotide expression cassettes of the invention may also include Kozak sequences. The Kozak sequence is located between the promoter and the coding sequence of the anti-VEGF antibody or antigen-binding fragment.

It is another object of the present invention to provide a plasmid comprising the polynucleotide expression cassette of the present invention.

It is another object of the present invention to provide a host cell transfected with a polynucleotide expression cassette or plasmid of the present invention. In some embodiments, the host cell is a mammalian cell, a yeast cell, a bacterial cell, or an insect cell.

In another aspect of the invention, there is provided a vector for gene delivery comprising the nucleotide cassette of the invention. In some embodiments, the gene delivery vector is a recombinant virus comprising: capsid proteins and polynucleotide expression cassettes according to the invention. In some embodiments, the recombinant virus is a recombinant adeno-associated virus (rAAV), wherein the recombinant adeno-associated virus comprises an AAV capsid protein and a polynucleotide cassette of the invention. In some embodiments, the capsid protein is a wild-type AAV capsid protein, illustratively, the AAV serotype capsid protein is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12. In other embodiments, the AAV capsid protein is a variant AAV capsid protein. In one embodiment, the AAV capsid variant is a variant of AAV 2.

In another aspect, the invention provides a pharmaceutical composition. The pharmaceutical composition comprises the polynucleotide expression cassette, plasmid or recombinant virus of the invention, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a recombinant virus described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is prepared as a formulation for intravitreal injection, subretinal injection, suprachoroidal injection, intravenous injection, intratumoral injection, or intramuscular injection.

In another aspect, the invention provides the use of a polynucleotide expression cassette, plasmid or recombinant virus according to the invention in the manufacture of a medicament for the treatment of a disease associated with VEGF. In certain embodiments, the VEGF-related disease is ocular neovascular disease. In certain embodiments, the ocular neovascular disorder is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration of very low vision, choroidal neovascularization secondary to pathological myopia.

Another aspect of the invention is to provide a method of treating a VEGF-related disease in a mammalian subject, the method comprising delivering to the eye a therapeutically effective amount of a recombinant virus or pharmaceutical composition comprising a polynucleotide expression cassette of the invention. In certain embodiments, the VEGF-related disease is ocular neovascular disease. In certain embodiments, the ocular neovascular disease is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidMembranous vasculopathy, wet age-related macular degeneration of very low vision, is secondary to choroidal neovascularization of pathological myopia. In some embodiments, the recombinant viruses or pharmaceutical compositions of the invention are administered by intravitreal injection, subretinal injection, suprachoroidal injection, intravenous injection, intratumoral injection, or intramuscular injection. In some embodiments, the method of treatment comprises administering 10 ⁶ To 10 ¹⁶ vg, preferably 10 ⁸ To 10 ¹³ vg, more preferably 10 ⁹ To 10 ¹² vg to the eye.

Drawings

FIG. 1 provides a schematic representation of a recombinant plasmid in which an exemplary polynucleotide expression cassette is positioned between Inverted Terminal Repeats (ITRs) of adeno-associated virus serotype 2 (AAV 2)

Detailed Description

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. And the following examples are not representative of all or only of the following experiments in which the present invention was developed.

Example 1: construction of plasmids

Construction using standard recombinant DNA cloning techniques or general molecular biology techniques produced a series of polynucleotide expression cassettes containing various combinations of regulatory elements and coding sequences (table 1). Recombinant plasmids comprising the polynucleotide expression cassettes described in table 1 were then constructed and cloned in e.coli using conventional DNA recombination and cloning techniques for subsequent preparation of recombinant AAV viruses. FIG. 1 shows a schematic representation of a recombinant plasmid expression cassette of the invention, wherein an exemplary polynucleotide expression cassette is positioned between Inverted Terminal Repeats (ITRs) of adeno-associated virus serotype 2 (AAV 2).

As represented in the schematic representation of the recombinant plasmid of FIG. 1, each cassette is positioned between the Inverted Terminal Repeats (ITRs) of adeno-associated virus serotype 2 (AAV 2), cassettes 1-4 comprising, in order from 5 'to 3', a 5'ITR, a promoter, a coding sequence encoding an anti-VEGF antibody (the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1, the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3), a WPRE transcriptional regulatory element (or not) and a 3' ITR. The main composition of each polynucleotide expression cassette is shown in table 1.

TABLE 1 Polynucleotide expression cassettes

Then respectively constructing plasmids based on the polynucleotide expression cassettes 1-4, respectively named No.1, no.2, no.3 and No.4, wherein the polynucleotide expression cassettes respectively comprise SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9.

Example 2: protein expression in 293 cells transiently transfected with plasmids

To assess the expression profile of each polynucleotide expression cassette or plasmid in vitro, each recombinant construct was transfected into HEK293F cells in suspension using transfection reagents. The specific method comprises the following steps: selecting the appropriate cell number (no more than P15 generation), and carefully adding 10ml DMEM (10% fbs) medium 2h before plasmid transfection without disturbing the cell monolayer; then pass through Lipofectamine ^TM 2000, specifically comprising: 2. Mu.g of the plasmid containing the target gene (with endotoxin removed) was added to 1mL of serum-free medium, and 5. Mu.L of LiP2000 was added to 1mL of serum-free medium; standing for 5min respectively; then lightly mixing and standing for 15min; adding the mixed solution obtained in the previous step into a cell culture dish, uniformly distributing the mixed solution at each position when adding, slightly and uniformly mixing the whole culture solution after finishing, and culturing at 37 ℃ and 5% CO 2; after 6h of transfection, carefully remove the medium and change to fresh medium (10% FBS,1% P/S), continue culturing for 48-72 h, collect cells and cell supernatant; cell lysates containing expressed proteins were prepared by repeated freeze thawing 3 times, by ELIThe SA measured the protein content and the results are shown in Table 2.

TABLE 2

Claims

1. A polynucleotide expression cassette, comprising:

1) An expression regulatory element;

2) A coding sequence encoding an anti-VEGF antibody or antigen-binding fragment thereof; the anti-VEGF antibody or antigen-binding fragment thereof heavy chain variable region comprises SEQ ID NO:1, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2, the amino acid sequence is described in the specification; preferably, the anti-VEGF antibody or antigen-binding fragment thereof heavy chain comprises SEQ ID NO:3, the light chain comprises the amino acid sequence of SEQ ID NO:4, and a polypeptide sequence of the polypeptide.

2. The polynucleotide expression cassette of claim 1, wherein the anti-VEGF antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:5, and a nucleotide sequence described in seq id no.

3. The polynucleotide expression cassette of claim 1, wherein the polynucleotide expression cassette comprises flanking Inverted Terminal Repeats (ITRs); preferably, the ITR is an adeno-associated virus ITR of serotype selected from the group consisting of AAV1ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9ITR, AAV10 ITR, AAV11 ITR and AAV12 ITR.

4. The polynucleotide expression cassette of claim 1, wherein the polynucleotide expression cassette comprises, in order from 5 'to 3':

1)AAV 5'ITR；

2) A promoter region comprising a CBA promoter sequence;

4) A WPRE transcriptional regulatory element;

5) An SV40 polyadenylation signal sequence;

6) AAV 3' itrs; or alternatively

The polynucleotide expression cassette comprises, in order from 5 'to 3':

1)AAV 5'ITR；

2) A promoter region comprising a CMV promoter sequence;

4) A WPRE transcriptional regulatory element;

5) BGH polyadenylation signal sequence;

6)AAV 3'ITR。

5. the polynucleotide expression cassette of claim 1, wherein the polynucleotide expression cassette comprises the sequence of SEQ ID NO: 6. SEQ ID NO:8, and a nucleotide sequence described in seq id no.

6. A plasmid comprising the polynucleotide expression cassette of any one of claims 1-5.

7. A host cell comprising the polynucleotide expression cassette of any one of claims 1-5 or the plasmid of claim 6; preferably, the host cell is a mammalian cell, a yeast cell, a bacterial cell or an insect cell.

8. A recombinant virus comprising a capsid protein and the polynucleotide expression cassette of any one of the preceding claims 1-5; preferably, the recombinant virus is a recombinant adeno-associated virus; preferably, the capsid protein is a serotype capsid protein selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or a variant of any of the foregoing.

9. A pharmaceutical composition comprising the polynucleotide expression cassette of any one of claims 1-5, or the plasmid of claim 6, or the recombinant virus of claim 8 and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is an intravitreal injection, subretinal injection, intracoronary injection, intravenous injection, intratumoral injection or intramuscular injection.

10. Use of the polynucleotide expression cassette of any one of claims 1-5, or the plasmid of claim 6, or the recombinant virus of claim 8, in the manufacture of a medicament for the treatment of a VEGF-related disease, preferably an ocular neovascular disease; more preferably, the ocular neovascular disease is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration of very low vision, choroidal neovascularization secondary to pathological myopia.