RU2725493C2 - System for stable expression of tumor-associated antigens based on lentiviral vector - Google Patents

Abstract

FIELD: immunology.SUBSTANCE: disclosed is a lentiviral DNA-based system for stable expression of tumor-associated antigens, followed by transfer into the endosome through a system of the MHC II complex.EFFECT: present invention can find further application in stimulating the immune system to recognize tumor cells.3 cl, 3 dwg, 1 tbl

Description

The invention relates to the field of biotechnology. It can be used for expression of tumor-associated antigens with their subsequent incorporation into the endosome through the MHC II histocompatibility complex system.

About 7 million people die every year from malignant neoplasms in the world. According to the WHO, oncological diseases are on the second place in frequency of occurrence, and in the first place in some industrialized regions. Traditionally, until recently, the main treatment methods were considered: surgical, cytotoxic chemotherapy and radiation therapy.

Advances in immunological science in recent years have led to the creation of fundamentally new approaches and methods for the treatment of cancer.

So, more than 40 years ago it was shown that many tumors are immunogenic. However, cytotoxic T-lymphocytes are not activated when cancer cells and their specific antigens appear and are not able to secrete cytokines, which they secrete when they encounter foreign antigens. Therefore, one of the strategies for controlling tumors is to stimulate the immune system to recognize tumor cells.

In particular, attempts were made to create individual antitumor vaccines (A.Yu. Baryshnikov, Principles and Practice of Cancer Vaccine Therapy, Bulletin SB RAMS, No. 2 (112). 2004, pp. 59-63).

Currently, considerable attention is paid to the use of dendritic cells and preparations (vaccines) based on them.

Dendritic cells are professional antigen-presenting cells (APCs) and play a key role in the formation of the primary immune response. An important feature of dendritic cells is their ability to capture various antigens from the environment through pinotomy and endocytosis. After antigen uptake, dendritic cells migrate to the lymphoid organs, where antigenic determinants are presented in the context of the main histocompatibility complex (MHC) to various populations of T-lymphocytes. There are two types of receptors for the major histocompatibility complex. MHC I receptors are expressed in all nuclear cells of the body, implementing a “friend or foe” recognition mechanism and presenting endogenous antigens, such as viral or cancer antigens. In this case, the binding of the receptor and antigen occurs in the endoplasmic reticulum. MHC II receptors are expressed by antigen-presenting cells and other types of blood cells, such as B cells, and present exogenous antigens absorbed as a result of endocytosis. Unlike MHC I, the binding of MHC II and antigen occurs in the region of the endosome. In the case of MHC I, the presentation of a foreign antigen, for example, a viral peptide, activates CD8 + cytotoxic T-lymphocytes and leads to an immediate immune response. The result is the destruction of the infected cell. The presentation of a foreign exogenous peptide by MHC II complex molecules activates both CD8 + cytotoxic T-lymphocytes and activation of CD4 + helper T-lymphocytes, which leads to the emergence of an “immune memory” mechanism. Such a mechanism is characterized by the emergence of a population of B-lymphocytes that produce specific antibodies and populations of more specific cytotoxic T-lymphocytes aimed at a foreign antigen.

A known method of inducing a high level of CD4 + and CD8 + T-cell response and specific types of cytokines in order to achieve a high level of protection and therapeutic activity due to immunogenic HER2-specific polyepitope constructs containing CTL and / or Th epitopes and optimized spacer sequences. Designs can be used to produce antigen-presenting cells, for example, dendritic cells (DC), to present the desired epitopes to lymphocytes (application EA 201290813 (A1), publ. 2013-04-30).

A known method of expression of polyepitope tumor-associated antigens in dendritic cells capable of stimulating specific cytotoxic cells based on the use of recombinant plasmid DNA pCI-UB-POLYEPI, which contains 11 epitopes of tumor-associated antigens of colorectal cancer, has a size of 6355 bp (RF patent 2507265, publ. 02.20.2014). This source can be indicated as the closest analogue prototype.

The objective of the invention is to create a system that can include specific antigenic determinants through the complex system of MHC II, which can significantly expand the possibilities of using dendritic cells of the patient, and thereby increase the effectiveness of anticancer therapy.

The problem is solved by a DNA construct based on a lentiviral vector for the expression of tumor-associated antigens with their subsequent transport to the endosome through the MHC complex system P.

The proposed plasmid lentiviral constructs include the sequence of the invariant domain of the CD74 / Ii complex of the MHC II complex, as the leading signal region attached to the target polypeptides (antigens). CD74 / Ii acts as a signal sequence directing the transport of the MHC II complex to the endosomal compartment.

The technical result of the invention is the creation of a universal lentiviral system that allows you to transport a wide range of fused protein antigens to the endosome, where they will be cleaved and presented by molecules of the MHC II complex.

Unlike the case with the complex of MHC I, MHC II prevents the attachment of endogenous peptide molecules in the region of rough EPR (endoplasmic reticulum). CD74 / Ii also acts as a signal sequence directing the transport of the MHC II complex to the endosomal compartment. In the endosome, MHC II undergoes proteolytic cleavage, as a result of which the Ii domain is cleaved and the already proteolized exogenous peptides (antigens) are attached. Thus, the use of the CD74 / Ii sequence as the leading signal region attached to the target polypeptides (antigens) expressed from plasmid DNA or from the dendritic cell genome will allow such fusion proteins to be transported to the endosome, where they will be cleaved and presented by MHC II complex molecules .

According to the invention, a wide range of tumor-associated antigens can be expressed, for example: SBA, TGF-PRII (colorectal carcinoma), TAG-72 (prostate carcinoma), HPV E6, E7 (cervical carcinoma), BING-4, SSX-2, TRP -1 / -2 (melanoma), Cyclin-B1, EphA3, Her2 / neu (multi cancer), Ep-CAM (breast cancer), prostate-specific antigen (prostate cancer), BRCA1 / 2 (breast and ovarian carcinoma), MUC1 (ductal carcinoma), Ig, TSJA (lymphoma), etc.

To deliver the sequences of antigens fused to the signal sequences of CD74 / Ii chains into antigen-presenting dendritic cells, the lentiviral transfer technique of the target sequences was used.

The use of lentiviral vectors has several significant advantages compared to such approaches as transfection transfer of plasmid DNA or electroporation of DNA cells with constructs:

- lentiviral vectors are able to quickly integrate into the genome of the target cell, ensuring stable transgene expression;

- lentiviral vectors allow cloning rather long DNA sequences up to 10 kb, while maintaining a high level of packaging of viral particles and their transduction ability;

- lentiviral vectors have a wide tropism due to the introduction of typing proteins into the envelope of viral particles. This property allows you to significantly increase the transduction ability of the virus for a certain type of cell, which is extremely important in working with hardly transfected primary blood cell cultures;

- lentiviral vectors make it possible to deliver DNA even to hardly transfected, non-dividing cells, including primary differentiated cells, such as dendritic cells and lymphocytes.

As the main genetic framework forming the lentiviral vector system, we used the previously constructed universal plasmid lentiviral DNA construct (Fig. 1). The image illustrates the scheme of the plasmid lentiviral DNA construct pLA-CMV-ELuc-puro.

The composition of this plasmid includes a number of key genetic elements:

- AmpR - ampicillin resistance sequence necessary for amplification of plasmid DNA in cells of a bacterial strain of E. coli;

- HIV 5 'and 3' LTR - long terminal repeats of retroviruses (in particular, human immunodeficiency virus), ensure the integration of the sequence of the virus genome into the genome of the host cell;

- CPPT (central polypurine tract) - the central polypurine tract, promotes the incorporation of virus DNA into the genome of mitotically inactive cells;

- CMV - promoter region, provides a stable and high level of expression of the 3 'underlying DNA sequence;

- NLuc - section of the N-terminal region of luciferase, at the 5 'and 3' ends contains a number of consensus regions recognized by restriction-modification enzymes;

- WPRE (Woodchuck hepatitis virus Posttranscriptional Regulatory Element) - an enhancer region that enhances the expression of target proteins.

The presentation of tumor-associated antigens mediated by the molecules of the main histocompatibility complex II directly depends on the effective transfer of the newly-expressed antigen to the region of the endosomal compartment. Due to the fact that one of the main regulators of the transport of MHC II to the endosome is CD74 / Ii protein, which is used as a signal sequence directing the antigen to the endosome. However, most of the polypeptide sequence serves to form a special spatial structure of the protein necessary for its interaction with the MHC II molecule. Only the small functional area that can be used separately is responsible for the transport of the CD74 itself.

Moreover, several variants of even shorter sequences responsible for the transfer of CD74 / Ii-MHC II may be included in this N-terminal domain. To determine the signal sequence leading to the best loading in MHC II, several variants of the signal peptides of the CD74 / Ii receptor were tested.

For amplification, a series of primers was used (table). The image illustrates primer sequences for amplification of various signal regions of the N-terminal domain of an invariant chain.

The sequence diagram of the CD74 protein with marked portions of signal peptides of endosomal localization is shown in FIG. 2

As the most optimal option, an elongated version of the Ii key signal section was selected (hereinafter referred to as "Ii key long"). As a tumor antigen, the ERBB2 protein (HER2 / neu) is used. The ERBB2 receptor is one of the highly specific oncological markers, due to the fact that increased amplification and expression of this receptor is characteristic of a number of aggressive types of breast cancer and some other types of malignant tumors. The receptor also ERBB2 is one of the largest specific tumor antigens, therefore, the successful presentation of the antigenic determinants of such a molecule will make it possible to judge the success of loading the remaining tumor-associated antigens.

Thus, a lentiviral DNA construct was obtained that contained the sequence of the tumor-associated antigen ERBB2 / HER2 and the sequence of the signal region of the CD74 gene, which ensures the transport of this antigen to the endosome (Fig. 3). The image illustrates the lentiviral plasmid DNA construct of pLA-CMV-Ii-key-long-ERBB2 containing the sequence of the Ii key long signal region. This design is one of the preferred, but not limiting the invention options.

The invention is illustrated by the following graphic material:

FIG. 1 - Scheme of the plasmid lentiviral DNA construct pLA-CMV-ELuc-puro

FIG. 2 - The sequence diagram of the protein CD74 with marked sections of the signal peptides of endosomal localization

FIG. 3 - Scheme of lentiviral plasmid DNA construct pLA-CMV-Ii-key-long-ERBB2 containing the sequence of the signal region Ii key long.

Tab. - Primer sequences for amplification of various signal regions of the N-terminal domain of an invariant chain.

SEQ ID NO: 1 CD74 N-terminal portion

Nucleotide sequence

ATGGATGACCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCATCCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTGTACCAGCAGCAGGGCCGGCTGGACAAACTGACAGTCACCTCCCAGAACCTGCAGCTGGAGAACCTGCGCATGAAGCTTCCCAAGCCTCCCAAGCCTGTGAGCAAGATGCGCATGGCCACCCCGCTGCTGATGCAGGCGCTGCCCATGGGAGCCCTGCCCCAGGGGCCCATGCAGAATGCCACCAAGTATGGCAACATGACAGAGGACCATGTGATGCACCTGCTCCAGAATGCTGACCCCCTGAAGGTGTACCCGCCACTGAAGGGGAGCTTCCCGGAGAACCTGAGACACCTTAAGAACACCATGGAGACCATAGACTGGAAGGTCTTTGAGAGCTGGATGCACCATTGGCTCCTGTTTGAAATGAGCAGGCACTCCTTGGAGCAAAAGCCCACTGACGCTCCACCG

Amino Acid Sequence

MDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPHLKLKFLPLKLKFLFNLFHLFLFNLFLFNFLFNLFLFNFLFLFNLFLFNFLFLFNFLFLFNFLFLFNFLFNFLFNFLFNFLFNFLFNFLFINF

SEQ ID No. 2 Ii-ER signal sequence

Nucleotide sequence

ATGGATGACCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGC

Amino Acid Sequence

MDDQRDLISNNEQLPMLGRRPGAPESKCSR

SEQ ID No. 3 Ii key signal sequence

Nucleotide sequence

CTGCGCATGAAG

Amino Acid Sequence

Lrmk

SEQ ID No. 4

Ii key long (extended signal sequence)

Nucleotide sequence

CTGCGCATGAAGCTTCCCAAGCCTCCCAAG

Amino Acid Sequence

LRMKLPKPPK

SEQ ID No. 5 CLIP signal sequence

Nucleotide sequence

CCTGTGAGCAAGATGCGCATGGCCACCCCGCTGCTGATGCAGGCG

Amino Acid Sequence

PVSKMRMATPLLMQA

Claims (5)

1. A system based on a lentiviral DNA construct for stable expression of tumor-associated antigens and their subsequent transport to the endosome through the MHC II complex system, characterized in that it contains the nucleotide sequence of SEQ ID No. 4 encoding the invariant domain of the MHC II complex CD74 / Ii as the leading signal region necessary for transporting the protein to the endosome, attached to the nucleotide sequence encoding the target polypeptide sequence of the tumor-associated antigen. 2. The system based on the lentiviral DNA construct according to claim 1, characterized in that the target polypeptide sequence of the tumor-associated antigen is a tumor-associated antigen sequence ERBB2 / HER2. 3. The system based on the lentiviral DNA construct according to claim 1 or 2, characterized in that for the amplification of the sequence of the invariant domain of the complex MHC II CD74 / Ii use primers selected from: SEQ ID No. 6 AGAGAGGAATTCCATGCTGCGCATGAAGC SEQ ID No. 7 AGAGAGTCTAGACTTGGGAGGCTTGGGAAGCTTCATG

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