RU2552609C1 - Method of obtaining system of directed delivery of protein molecules (oncolytic proteins) into tumour cells based on activated lymphocytes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.

EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.

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Description

The invention relates to the field of medicine and biotechnology.

Modern methods of anti-cancer therapy are based on the application of an integrated approach using a combination of agents aimed at certain features of the biological properties of tumor cells. As knowledge of tumor cells is deepened and the most universal mechanisms that distinguish cancer cells from normal are clarified, it becomes possible to develop more effective, specific and harmless biological therapy agents for the body. In contrast to insufficiently specific chemotherapeutic agents, biological methods of therapy are called upon to use natural activities and mechanisms to detect and destroy cancer cells.

Currently, researchers are attracting more and more attention to the prospects of using approaches to the treatment of malignant neoplasms based on the use of biomolecules - proteins that have a selective inhibitory or even toxic effect on tumor cells, while not exerting similar effects on normal cells. Such approaches can also include the use of specially designed antibodies against tumor-specific epitopes that inhibit tumor growth either by blocking the receptors necessary for active proliferation of the tumor, or by direct cytotoxic effect of the toxin covalently coupled to the antibody. Another group of protein molecules that have no less potential for use in therapy are oncolytic proteins - molecules that, when introduced into a tumor cell, initiate a cascade of proapoptotic signals, depending on pathological signaling pathways that are characteristic of transformed cells, but not normal.

The main advantage of protein molecules compared to traditionally used cytotoxic chemical compounds is that their mechanism of action is aimed at the fundamental features of cancer cells, which provides extremely high selectivity of their action, combined with low toxicity to the body and the absence of side effects. Due to this, a strong cumulative effect from the effects of several drugs on the tumor can be achieved without the complications commonly observed with anticancer therapy.

Recently, more and more works describe the inactivation of the tumor suppressor p53 as one of the main causes of cancer. As you know, p53 is a key component responsible for triggering protective signaling pathways in response to DNA damage in mammalian cells. Accumulation of damage in the cell’s genetic apparatus leads to the activation of the p53 protein, which results in stopping the cell cycle and DNA replication, with severe damage, programmed cell death or apoptosis occurs. Thus, a mechanism works that can prevent and remove potentially oncogenic cells, and mutation or loss of the p53 gene leads to the accumulation of many DNA damage, degeneration of the cell and, ultimately, to cancer. In about 50% of all malignant tumors, the p53 gene mutation is detected. Therefore, studies devoted to the search for alternative, p53-independent apoptotic pathways are of great scientific and applied value. Recently it was found that apoptosis induced by Apoptin has such a mechanism.

Apoptin (a product of the VP3 gene of the chicken infectious anemia virus genome) is a proline-rich protein with a molecular weight of 13.6 kDa, consisting of 121 amino acid residues.

Of great interest is the ability of apoptin to selectively induce apoptosis of transformed cells of birds and mammals, while not affecting normal, non-transformed cells. Based on the data obtained, we can speak of 70 different tumor lines that are sensitive to apoptin. Apoptin has a nuclear localization region located in the C-terminal region of the protein between 80-121 amino acid residues, and a nuclear export region located in the Ν-terminal region, between 33 and 46 amino acid residues. Many studies have shown that nuclear localization of apoptin in tumor cells is necessary for the induction of apoptosis, whereas in normal cells, the protein is localized mainly in the cytoplasm, where it is unstable and rapidly degrades.

As already mentioned, VP3 is capable of causing the death of only cancer cells, while not affecting normal cells. This selectivity can be explained by nucleo-cytoplasmic transfer. Experimental data showed that the nuclear export of apoptin in normal cells is determined by a unique signal sequence (nuclear export sequence) with which CRT1 exportin interacts, thus ensuring the constant removal of protein from the nucleus. The reverse transfer does not occur in cancer cells due to the phosphorylation of 108 threonine (108 Thr) in the nuclear export sequence, which blocks this interaction with CRM1. Thus, Thr-108 phosphosylation seems to be one of the key events necessary for the accumulation of apoptin in the nucleus and apoptin-induced apoptosis. A further, more detailed study of this phenomenon showed that apoptin phosphorylation is not a prerequisite for its tumor-specific accumulation in the nucleus. Thus, the replacement of 107 and 108 threonine with alanine residues, and further verification of the subcellular localization of mutant apoptin in MCF-7 cells and normal cells did not reveal any changes. This protein still accumulated in the nuclei of tumor cells and subsequently led to apoptosis. This suggests the existence of other mechanisms of apoptin accumulation in the nuclei of tumor cells.

The medical use of oncolytic proteins, including apoptin, has so far been complicated by the low efficiency of the used methods of delivering biological preparations to the tumor sites. Another problem is the delivery of protein directly to the tumor cell, because unlike small chemical molecules, proteins lack the ability to penetrate through the plasma membrane. A significant limitation is the immunogenicity of proteins and the rapid formation of antibodies, which, over time, can completely neutralize the activity of the oncolytic protein. In order for oncolytic proteins to become an effective means of antitumor therapy, it is necessary to find ways to effectively solve these problems: to determine modifications of the protein structure necessary for its unhindered passage through the plasma membrane and to develop fundamentally new means of targeted delivery of such therapeutic agents to tumor cells. Such delivery methods should, on the one hand, protect the protein from the action of antibodies, and on the other hand, bring the release of the active protein closer to the location of the tumor cell.

Currently, attempts are being made to adapt apoptin, as the most promising oncolytic protein, for clinical use.

Possessing high selectivity of action against tumor cells, apoptin has good prospects for use in the treatment of cancer. In order to ensure the delivery of apoptin to tumor cells, it is necessary to either introduce a genetic construct expressing apoptin into these cells or modify the structure of apoptin, giving it the ability to penetrate into tumor cells through the plasma membrane. The indicated first delivery method is used in anticancer therapy using oncolytic viruses, while the second delivery method allows apoptin to be used in a variety of ways: both traditionally, by introducing synthetic recombinant apoptin, and using biological delivery vehicles.

Medical prospects for the use of apoptin and oncolytic proteins, in general, depend on the development of means for the delivery of recombinant proteins to the tumor. Parenteral administration of the synthesized protein will not allow creating a local high concentration in the tumor and carries the risk of immunizing the patient with the introduced protein or the development of allergic reactions. It is far from always possible to inject directly into the tumor, in addition, such an injection can cause a number of complications. The optimal use of oncolytic proteins would be the presence in the tumor of producer cells of these proteins. In this case, the effect would be long, and the concentration of the drug - maximum in the tumor area. To achieve this, recombinant viral vectors assembled on the basis of lentivirus or adenovirus are usually used.

Lentivirus-based vectors are described, for example, in Ye Feng et al., Construction of three recombinant lentiviruses containing apoptin driven by survivin promoter of different lengths and comparison of their tumor suppressing efficency in vitro, Journal of Third Military Medical University, 2012-01. Lentiviral vectors will allow the introduction and constitutive expression of recombinant apoptin in tumor cells and surrounding tissues. Such expression will be longer, while the risk of immunization against the virus practically does not arise, since it is not able to replicate. However, the nature of the integration of viral DNA into the genome carries the risk of damage to it and, as a consequence, additional malignancy of the tumor.

Patent RU 2252255 is known, which describes recombinant adenoviral vectors containing apoptin for the treatment of cancer, which are obtained by cotransfection into the helper cell line 911 of the adapter plasmids p.AMb-VP3 (in the case of expression of the VP3 protein) or plasmids pMAb-VP2 (in the case of protein expression VP2) and plasmid DNA JM17. Plasmids pAMb-VP3 carry the apoptin gene in the 5′-3 ′ orientation, the expression of which is carried out under the control of the adenovirus major late promoter. Plasmid DNA JM17 contains complete adenovirus DNA with the exception of the E1 and E3 regions. The pMAb-VP2 plasmids carry the apoptin gene, with 2 point mutations within the coding region.

A gene delivery vector capable of inducing apoptosis in a cell, including a DNA fragment encoding a protein having apoptin-like activity, while apoptosis is induced in cancer cells and to a lesser or no extent in normal diploid non-transformed / non-cancerous cells, the gene delivery vector being independently infectious vector and is an adenovirus.

A significant drawback of this method is the gradual immunization of the patient to a specific adenovirus strain.

The patent RU 2492238 C1 is known, which relates to recombinant plasmid DNA pGEM-Puro-DS-Apo carrying a synthetic apoptin gene of 363 bp in size. with a specific nucleotide sequence flanked by fragments of the genome of smallpox vaccine virus having a size of 6298 bp, a molecular weight of 4.19 mDa, and recombinant strains of smallpox vaccine virus producing apoptin of the chicken anemia virus. When using this method, gradual immunization of the patient will also be observed.

The described disadvantages necessitate the development of a new, non-viral, method for the delivery and expression of recombinant proteins in the tumor

Known US patent 5656465, relating to methods of gene delivery in vivo. The use of non-integral viral vectors having low replicative efficacy for inserting a gene into a cell, such as a lymphocyte or tumor cell, has been described, is the preferred system for converting such cells for use in somatic cell therapy or gene therapy. Genes are inserted into target cells, in particular into a target cell, which is lymphocytes that penetrate into tumor tissue. It is noted that the genes encoding TNF, TGF-α, TGF-β, hemoglobin, interleukin-1 and the like, as well as biologically active muteins of these proteins are of interest. The possibility of targeted apoptin delivery is not mentioned.

The objective of the present invention is to develop a new means of targeted delivery of apoptin to tumor cells, which allows, on the one hand, to protect the protein from the action of antibodies, and on the other hand, to bring the release of the active protein closer to the location of the tumor cell.

Effect: increase the ability of apoptin to penetrate into tumor cells and have an oncolytic effect in relation to all tumor cells. Such therapy can be used in combination with other antitumor drugs, which will reduce the duration of treatment and reduce side effects.

The problem is solved by a method of obtaining a system for the directed delivery of apoptin into tumor tissues. The method is characterized in that apoptin is modified by fusion with the secretory signal of lactotransferrin and transduction signal (ST-CTP-apoptin), a lentiviral construct expressing ST-CTP-apoptin is obtained, this construct is used to produce recombinant lentiviral particles defective in replication and bearing modified apoptin, which are then introduced into autotransplanted lymphocytes that penetrate into the tumor tissue.

Autotransplanted lymphocytes penetrating into tumor tissues (TIL, tumor infiltrating lymphocytes) are proposed as a means of delivery and producer of oncolytic proteins. Lymphocytes can be obtained during surgical removal of the tumor or in the process of obtaining a biopsy. Lymphocytes isolated from peripheral blood activated by culturing in the presence of interleukin-2 can also be used. Such cultivation increases the tropism of lymphocytes to the tumor, and, after being introduced back into the bloodstream, a substantial part of the lymphocytes is localized in the tumor tissue.

The main difference of the invention is the use of oncotoxic lymphocytes as a carrier and producer of apoptin - is new and not described in the literature. Such an approach will allow combining the oncolytic effect of apoptin with the antitumor effect of cell and immunotherapy of malignant neoplasms. The difference from the known solution is also that it describes constructs that do not integrate into the genome of the vector, while the claimed lentiviral construct is integrated.

To solve the problem, signal peptides were developed that secreted the expression of the expressed oncolytic protein from the cell and subsequent penetration into the surrounding cells due to the protein domain, which allows penetration through the plasma membrane. The invention uses the property of cytotoxic lymphocytes to penetrate and accumulate in the tumor. Expression of the secreted oncolytic protein by lymphocyte will allow the release of active therapeutic molecules directly at the location of the cancer cells and reduce the likelihood of neutralization of the active protein with antibodies.

The possibility of carrying out the invention is demonstrated by the following examples.

Example 1. Determination of the optimal variant of recombinant apoptin with maximum oncolytic activity

The cytoplasmic transduction domain (CTP) and secretory signal are integrated into apoptin cDNA by PCR, and the resulting sequence is cloned into a lentiviral vector. A series of constructs was created containing various variants of the STR domain (4 variants, a fragment of the HIV TAT protein (PTD domain) and 3 synthetic peptides), and various variants of the ST peptide (5 variants, 3 of which are synthetic, based on the predictions of the NMM -algorithm for determining the effectiveness of a transducting peptide, and 2 variants are taken from naturally occurring proteins, cystatin C and lactotransferrin). A series of obtained constructs was introduced into producer cells, and the most effective variant was selected from them. For this, the filtered medium in which the producers were cultured treated cells of the SAOS2 and RKO tumor lines, and a comparative assessment of the cytotoxicity of each protein variant was performed. This approach allows us to determine the functional activity of the protein under conditions close to real ones. As a control construct, as well as for visual assessment of the efficiency of secretion and uptake of a modified protein, a set of producers secreting a fluorescent protein fused to the tested STR and ST domains was obtained.

A series of genetic engineering constructs was created aimed at the expression of various variants of the oncolytic protein apoptin and red fluorescent tagRFP protein. Designs expressing wild apoptin (wt-apoptin), apoptin fused to secretion signals (ST-apoptin) derived from lactoferrin proteins, cystatin C, as well as three synthetic ST signals obtained based on the predictions of the NMM algorithm were created determining the effectiveness of the secretory peptide. Also, constructs expressing apoptin fused to cytoplasmic transduction signals were obtained — one variant representing a fragment of the HIV TAT protein (PTD domain) and three synthetic peptides. In addition to constructs expressing apoptin fused separately with secretion and cytoplasmic transduction signals (5 and 4 pieces, respectively), expressor vectors containing all variants of apoptin fused with both secretory and transduction signals simultaneously were obtained.

All apoptin-expressing constructs were introduced by the method of lentiviral transfection into the tumor cell cultures of the SAOS2 and RKO lines, and a comparative assessment of the cytotoxicity of each protein variant was performed. For this, the obtained recombinant lentiviruses, after concentration with polyethylene glycol, had a titer determined (by real-time RT-PCR method), and then, after infection of cell cultures with an equal number of viral particles, it was determined in which case cytotoxicity is maximal. In fact, various variants of apoptin showed less cytotoxicity than wild apoptin (as expected), but some variants, including apoptin fused with the secretory signal of lactotransferrin and the transducer signal of PTD, showed a practically unchanged level of cytotoxicity.

In parallel with these experiments, cells of the SAOS2 and RKO lines were transfected with tagRFP-containing constructs. After selection and sorting, as a result of which 90-95% of the cells in the culture became tagRFP-expressing, the cells were mixed in a 1/1 ratio with untransfected SAOS2 and RKO, respectively. After co-culturing for 24 hours, the comparative fluorescence of two populations of co-cultured cells was changed on a cytometer. Based on the enhancement of the characteristic fluorescence of tagRFP in untransfected adhesives, conclusions were drawn on the effectiveness of a particular pair of secretory and transductor peptides. As a result of the experiments, it turned out that the pair of secretory signal of lactotransferrin and transduction signal PTD has the highest efficiency among the tested options.

The sequence of apoptin with secretory and transductor peptides of SEQ ID NO 1 is shown in FIG. 1. An example of an expression construct containing apoptin with a signal peptide is shown in FIG. 2.

Thus, the most effective structure was determined by which oncotoxic proteins can be introduced into tumor cells - ST-CTP-apoptin. The expression of ST-CTP-apoptin in a small part of the tumor cells, or cells of the tissue adjacent to it, leads to the long-term presence of high concentrations of apoptin in the intercellular space of the tumor.

Example 2. Cultivation and activation of lymphocytes in model animals, lines of athymic mice

A tumor was implanted in homozygotes of athymic mice; in parallel, cells of a mononuclear fraction were isolated from peripheral blood in heterozygous animals of the same line. Lymphocytes were transduced with the tagRFP lentiviral expressor and cultured under various activating conditions (phytohemagglutinin, IL-2, IL-4, GM-CSF, lectins). After accumulating a sufficient number of activated lymphocytes, they were introduced into athymic mice with inoculated tumors. Then, the removed tumor was analyzed on a flow cytometer in order to determine the amount of fluorescent lymphocytes contained in it.

Example 3. Comparative study of antitumor efficacy

A lentiviral construct expressing ST-CTP apoptin was introduced into the lymphocytes in parallel with the control lentiviral construct. Cells were subjected to identical cultivation conditions and introduced into animals with inoculated tumors. The antitumor efficacy of ST-CTP-apoptin was evaluated in vivo by measuring the dynamics of tumor development in mice that received apoptin-expressing and control lymphocytes expressing fluorescent protein. The introduction of lymphocytes expressing apoptin leads to a significantly higher efficiency of this method with respect to the panel of human tumor cells cultured in athymic mice.

Claims (1)

A method of obtaining a system for the targeted delivery of apoptin into tumor tissues, comprising obtaining a lentiviral construct expressing modified apoptin, characterized in that the modified apoptin is apoptin fused to the secretory signal of lactotransferrin and transduction signal (ST-CTP-apoptin) and having the sequence SEQ ID NO 1 shown in FIG. 1, a lentiviral construct expressing ST-CTP apoptin having the element arrangement shown in FIG. 2, followed by production of recombinant lentiviral particles defective in replication and carrying modified apoptin, which are then introduced into T-lymphocytes (TILs) obtained by surgical removal of the tumor or in the process of obtaining a biopsy that have the ability to penetrate into tumor tissue, followed by autotransplantation specified lymphocytes to a specified patient.

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