RU2627215C1 - Recombinant target toxin specific to her2 receptor expressing cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: recombinant targeted toxin, specific for HER2 receptor expressing cells is proposed. The recombinant target toxin comprises a HER2-specific targeting module represented by the HER2-specific darpin DARPin class coding sequence and a toxic module in the form of a fragment of pseudomonade exotoxin A ETA interconnected by a flexible hydrophilic 16-amino acid linker represented by the sequence of SEQ ID NO: 1, oligogistidine sequence His6 and KDEL sequence at the C-terminus of the molecule. The recombinant target toxin is represented by the sequence of SEQ ID NO: 2.

EFFECT: increased toxic effect on the HER2 receptor expressing cells and increased effectiveness of targeted therapy.

2 cl, 6 dwg

Description

The present invention relates to the field of medicine and biochemistry, relates to recombinant targeted toxin specific for cells expressing the HER2 receptor, which can be used as a therapeutic agent for targeted therapy of tumors overexpressing the HER2 receptor, characterized by an increased risk of metastasis and resistance to chemotherapy.

Well-studied tumor markers are proteins of the epidermal growth factor receptor family, HER1 and HER2 / neu, the overexpression of which is characteristic of a number of carcinomas (breast, prostate, lung, ovary, bladder, etc.). The difference in the level of expression of these proteins in normal and tumor cells makes them promising targets for targeted delivery of therapeutic and / or diagnostic agents [Polyanovsky OL, Lebedenko EN, Deev SM // Biochemistry. 2011. T. 77. S. 289-311].

The development of new methods and approaches for the treatment of cancer today is one of the most relevant and actively developing areas of biology and medicine. A leading trend in this area is a personalized approach based on determining the molecular genetic characteristics of the tumor for each particular case and choosing the optimal treatment method based on the information received. Although the main methods of treating solid tumors in the clinic are still surgical intervention followed by chemo- or radiation therapy, due to the progress in studying the molecular basis of carcinogenesis in recent decades, targeted (targeted) therapy has become increasingly important. The cornerstone of this approach is monoclonal antibodies that bind predominantly to tumor cells that express a specific marker specific to them and cause their death.

Since such antibodies are not effective enough as stand-alone drugs, they are conjugated to highly effective toxic molecules; such compounds are called immunotoxins. Despite the fact that in preclinical trials, immunotoxins show a pronounced specific cytotoxic effect on tumor cells, only two of these drugs, trastuzumab emtansine (Kadcyla = TD-M1) and brentuximab vedotin (Adcetris), are approved for clinical use.

Over the past decades, hundreds of monoclonal antibodies to various antigens have been created and studied in preclinical studies, dozens of them have reached the stage of clinical trials, individual monoclonal antibodies have been approved for use as medicines [Deev S.M. Modern technologies for creating non-natural antibodies for clinical use. / Deev S.M., Lebedenko E.N. // Acta Naturae 2009.Vol. 1. S.32-50; Bronte G. Monoclonal antibodies for the treatment of non-haematological tumors: update of an expanding scenario. / Bronte G., Sortino G., Passiglia F., Rizzo S., Lo Vullo F., Galvano A., Bazan V., Rolfo C., Russo A. // Expert Opin Biol Ther 2014. P. 1-15 ].

The lack of effectiveness of "unloaded" antibodies in relation to targets prompted researchers to conjugate antibodies with other effector molecules. In this case, monoclonal antibodies can be used as a means of targeted delivery of toxic agents to the tumor. Such compounds are called immunoconjugates. Immunoconjugates are usually composed of three main components: a monoclonal antibody, a therapeutic agent, and a linker that binds the antibody to a therapeutic agent [Cao Y. Single-chain antibody-based immunotoxins targeting Her2 / neu: design optimization and impact of affinity on antitumor efficacy and off-target toxicity. / Cao Y., Marks J.D., Huang Q., Rudnick S.I., Xiong C., Hittelman W.N., Wen X., Marks J.W., Cheung L.H., Boland K., Li C., Adams G.P., Rosenblum M.G. // Mol Cancer Ther 2012. V. 11, No. 1. P. 143-53; Kreitman R.J. Immunotoxins for targeted cancer therapy. [text] / Kreitman R.J. // The AAPS Journal 2006. V. 8, No. 3. P. E532-E551]. The immunoconjugate constructed in this way can serve for targeted delivery of various therapeutic agents to tumor cells, including radioisotopes, toxins, interleukins, enzymes, activating drugs, etc. [Deev S.M. Modern technologies for creating non-natural antibodies for clinical use. / Deev S.M., Lebedenko E.N. // Acta Naturae 2009.Vol. 1. S. 32-50].

Immunoglobulins or fragments thereof, which have high specificity and binding affinity for the corresponding antigens, are most often used as a guiding module. However, over the past few years, several alternative variants of non-immunoglobulin-guiding molecules have appeared - ligands of specific receptors that recognize peptides, affibodies, DARPins, etc.

Chemically synthesized or natural (bacterial, plant or animal) toxins most often act as a therapeutic agent. In the arsenal of researchers today are auristatins, maytansinoids, calicheamicins and protein toxins: ricin, pseudomonas and diphtheria toxins [Oldham R.K. Principles of Cancer Biotherapy. NY: Springer Science. 2009. 780 p.].

Currently, only two immunotoxin-based drugs have been approved for clinical use - Brentuximab vedotin and trastuzumab emtansine, which are used to treat certain lymphomas and breast cancer [Dosio F. Immunotoxins and Anticancer Drug Conjugate: Role of the Linkage between Components. / Dosio F., Brusa P., Cartel L. // Toxins 2011. V. 3, No. 7. P. 848-883]. This is largely due to the fact that despite the fact that in preclinical trials, immunotoxins show a pronounced specific cytotoxic effect on tumor cells, progress in the field of targeted therapy of tumors is inhibited by three main problems. First, the tumor has genetic heterogeneity associated with the genotype of each individual patient and the extreme instability of the genome of tumor cells, which leads to the emergence of tumor resistance to these drugs [Al-Lazikani B. Combinatorial drug therapy for cancer in the post-genomic era. / Al-Lazikani B., Banerji U., Workman P. // Nat Biotech 2012. V. 30, No. 7. P. 679-692]. Secondly, immunotoxins exhibit significant immunogenicity, due to their origin, usually bacterial, plant or fungal. In addition, preparations based on immunotoxins, like other antitumor drugs, do not penetrate deep enough into the tumor tissue. This is usually associated with a number of reasons, among which are: (1) the abnormal structure of the vascular bed of the tumor and the absence of normal lymphatic drainage, which leads to an increase in interstitial pressure in the tumor parenchyma and prevents convection transport of nanosized molecules and particles from vessels located mainly on the periphery of the tumor, to the deeper lying cells of the tumor parenchyma; (2) tight intercellular contacts of tumor cells; (3) fibrosis of tumor tissue. It is important to understand that when using immunotoxins, increased single or multiple dosing of therapeutic drugs, although it leads to their more uniform distribution in the tumor volume, is not acceptable due to their immunogenicity [Marcucci F. How to improve exposure of tumor cells to drugs - Promoter drugs increase tumor uptake and penetration of effector drugs. / Marcucci F., Corti A. // Advanced Drug Delivery Reviews 2012. V. 64, No. 1. P. 53-68]. A consistent or comprehensive solution to these problems is the basis of all modern developments in the development of targeted oncotherapy.

For example, an immunotoxin is known that contains a guiding module in the form of a coding sequence of a darpin class protein (DARPin) specific for the EpCAM protein and a toxic module in the form of a pseudomonas exotoxin A ETA fragment interconnected by a GS linker (“The Promise of DARPins for Site-Specific Drug Conjugation & Pharmacokinetic Optimization)), Fabian Brande, 02.15.2016).

The amino acid sequences of the regions of the EpCAM-specific darpin molecule providing affinity for the EpCAM protein differ from the sequences of the corresponding regions of the HER2-specific darpin molecule. The use of an EpCAM-specific darpin, which does not have specificity for the HER2 / neu receptor, as a guiding module excludes the possibility of its use for targeted therapy of tumors that overexpress HER2 receptor.

The closest in technical essence and the achieved result to the present invention is a recombinant immunotoxin specific for cells expressing the HER2 receptor, protected by patent RU No. 2576232 C1, cl. A61K 39/395, A61P 35/00, publ. 02/27/2016, adopted for the closest analogue (prototype).

The recombinant immunotoxin of the prototype contains a guiding module in the form of an scFv antibody, comprising the variable domains of the light and heavy chains of a monoclonal humanized HER2-specific antibody linked by a linker, and a toxic module in the form of a pseudomonas exotoxin A ETA fragment. In this case, the guide module is represented by the 4D5scFv antibody, which is connected to the ETA toxic module by a flexible hydrophilic hinge-like linker. At the C-terminus of the molecule, the KDEL sequence is performed. The use of this recombinant immunotoxin can increase toxicity to cells expressing the HER2 receptor, and, therefore, therapeutic efficacy.

The objective of the invention is the creation of a new recombinant targeted toxin specific for cells expressing the HER2 receptor.

The technical result from the use of the present invention is to enhance the toxic effects on cells expressing the HER2 receptor, and to increase the effectiveness of targeted therapy.

This object is achieved in that the recombinant targeted toxin specific for cells expressing the HER2 receptor contains an HER2-specific targeting module represented by the coding sequence of an HER2-specific protein of the darpin class DARPin and a toxic module in the form of a pseudomonas exotoxin A ETA fragment interconnected a flexible hydrophilic 16-amino acid linker represented by the sequence SEQ ID NO: 1, the His6 oligohistidine sequence and the KDEL sequence at the C-terminus of the molecule, when the recombinant targeted toxin volume is represented by the sequence of SEQ ID NO: 2; the gene encoding the recombinant targeted toxin is under the control of the T7 promoter.

In FIG. Figure 1 shows the genetic design of the recombinant targeted toxin containing HER2-specific darpin and a fragment of the pseudomonas exotoxin A (DARPin-ETA), where: T7 is the plasmid regulatory element is the T7 promoter, DARPin is the coding sequence of the HER2-specific darpin, H is the coding sequence of the hydrophilic linker, ETA - coding sequence of a fragment of pseudomonas exotoxin A, His6 - coding sequence of polyhistidine oligopeptide, K - KDEL sequence.

In FIG. 2 shows photographs reflecting the results of electrophoretic analysis of the obtained purified preparation of recombinant targeted toxin containing HER2-specific darpin and a fragment of pseudomonas exotoxin A, where: A - electrophoresis in 12% SDS page; B - Western blot analysis; M is a protein marker of molecular weight.

In FIG. Figure 3 presents graphs reflecting the results of an analysis of the affinity of a recombinant targeted toxin containing HER2-specific darpin and a fragment of pseudomonas exotoxin A. Kinetics of the interaction of DARPin-ETA with the extracellular domain of the HER2 receptor, obtained using the surface plasmon resonance method at various receptor densities on the chip and various concentrations DARPin-ETA.

In FIG. 4 presents the results of immunocytochemical analysis of the binding of DARPin-ETA protein to SKOV3ip cells, where: A - cells incubated with

immunotoxin; B - cells incubated with phosphate-buffered saline as a control.

In FIG. Figure 5 shows graphs reflecting the results of the analysis of the relative viability of HER2-positive SKBR-3 cells (red line and inverted triangle) and HeLa (black line and triangle), and HER2-negative CHO cells (blue line and circle) after their treatment with recombinant protein DARPin -ETA for 72 hours. Error bars are represented by standard error of the mean.

In FIG. 6 is a growth chart of SKBR-3 HER2-overexpressing xenograft tumor, where: gray columns are in groups of control animals that received phosphate-buffered saline injections, blue columns are in groups of animals that received a 5 × 5 μg DARPin-ETA dose, green columns - in groups of animals that received a dose of DARPin-ETA of 5 × 10 μg, red columns - in groups of animals that received a dose of DARPin-ETA of 4 × 20 μg. Days of DARPin-ETA injection are indicated by arrows. Error bars are represented by the standard error of the mean. "*" - p <0.05 (comparison with the control group according to the Dunnet criterion, n = 4-6).

A genetically encoded HER2-specific targeted (directed) toxin, DARPin-ETA is a recombinant protein in which two independent functional modules of a protein nature - directing and toxic - are combined into a single polypeptide chain using a flexible hydrophilic linker (16 a.a.).

The gene encoding the targeted toxin is under the control of the T7 promoter, which is the promoter of the T7 bacteriophage RNA polymerase. After transformation of the producer strain cells with a vector containing the target protein gene, the lac operon inducer isopropylthiogalactoside (IPTG), a synthetic analogue of lactose that is not metabolized by cells, is added to the culture medium. Under these conditions, T7 phage RNA polymerase is synthesized, after which the expression of the target gene is triggered.

The directing module, DARPin, is an HER2-specific darpin class scaffold. DARPin is characterized by high affinity for the HER2 receptor (equilibrium dissociation constant 3.8 nM), high stability in a wide range of conditions, small size (18 kDa), and ease of production in large quantities in bacterial producers.

The toxic module, ETA, is a fragment of the natural exotoxin A from Pseudomonas aeruginosa (Pseudomonas aeruginosa) containing domains II, Ib and III of the natural toxin, which ensure efficient intracellular transport of the toxin into the cytoplasm and the toxic effect of ADP-ribosylation of eukaryotic elongation factor 2 (eET2) . The natural directing domain I of exotoxin A has been replaced by the DARPin directing protein necessary for our purposes.

The directing and toxic modules were connected by a flexible hydrophilic 16-amino acid linker represented by the sequence SEQ ID NO: 1, which is a derivative of the hinge region of mouse IgG3 immunoglobulin. This linker can form a distance of 2.5-2.7 nm between the sections of the protein molecule that it connects, allowing the two protein domains not to experience steric difficulties and maintain their functional properties.

Recombinant protein DARPin-ETA contains a polyhistidine sequence (“tag”) of 6 histidine residues necessary for purification of the target protein from a bacterial lysate by metal chelate affinity chromatography on Ni2 + -NTA-sepharose. The KDEL sequence, necessary for the manifestation of the toxic effect of pseudomonas exotoxin on the target cell, was also introduced at the C-terminus of the obtained molecule.

In vitro and in vivo experiments, it was confirmed that the resulting recombinant protein with high affinity binds to the recombinant extracellular domain of the HER2 receptor, selectively inhibits the growth of HER2-positive cells in culture, and also inhibits the growth of xenograft HER2-overexpressing tumors in vivo. Thus, DARPin-ETA is a HER2-specific targeted toxin.

A genetic engineering construct was created encoding the recombinant protein DARPin-ETA, allowing it to be produced in high quantities in E. coli E. coli cells (Fig. 1).

A procedure was developed and optimized for the isolation and purification of the corresponding recombinant protein. Protein was produced in E. coli cells of strain BL21 and isolated by metal chelate affinity and ion exchange chromatography.

Electrophoretic analysis of the obtained recombinant DARPin-ETA protein in 12% PAG under denaturing conditions showed that the obtained purified protein preparation was homogeneous and did not contain impurities detected by Coomassi blue G-250 staining when applying the sample with overload (Fig. 2A). The authenticity of the obtained recombinant protein was confirmed by Western blot using antihistidine monoclonal antibodies (Fig 2B). The molecular weight of the obtained DARPin-ETA protein according to the results of electrophoresis and Western blot coincided with the calculated (58.1 kDa).

The high affinity of the obtained DARPin-ETA protein for the extracellular domain of the HER2 receptor was shown by surface plasmon resonance using a BIAcore 3000 optical biosensor (GE Healthcare, USA). The recombinant extracellular domain of HER2 was covalently immobilized on the surface of a CM5 carboxymethyldextran chip with a density of 1200 and 2200 cu Targeted toxin DARPin-ETA was used at concentrations of 0.3 and 0.1 μM. The value of the equilibrium dissociation constant (KD), which is a quantitative measure of affinity, was calculated using the BIAevaluation 3.0 Software program based on the obtained sensograms (Fig. 3).

It was shown that the KD of the DARPin-ETA protein was 10.8 nM, which is consistent with previously published values for the free DARPin protein (3.8 nM), which is the directing module of the targeted target toxin. Thus, the directing protein DARPin retains high affinity for the HER2 receptor in the constructed recombinant protein DARPin-ETA.

The ability of the obtained DARPin-ETA protein to bind to tumor cells expressing the HER2 receptor has been shown.

To confirm the specificity of binding of the obtained immunotoxin to the HER2 receptor, SKOVip human ovarian adenocarcinoma cell culture was used, which overexpresses the corresponding receptor on the cell surface. A suspension of cells with a concentration of 2 × 104 cells / ml was plated on 96-well plates (Corning) and cultured overnight. Cells were fixed with formaldehyde and incubated with DARPin-ETA protein solution (10 μg / ml). The cells were then sequentially incubated with 6x-His Epitope Tag Antibody monoclonal antibodies (HIS.H8) (Thermo Scientific, cat. # MA1-21315) specific for the oligohistidine sequence at the C-terminus of the DARPin-ETA protein and HRP horseradish polyclonal antibodies with HRP horseradish peroxidase Goat Anti-Mouse Ig (BD Pharmingen, cat. # 554002). Cells were stained using diaminobenzidine as a substrate for peroxidase.

Cells were detected using an Axiovert 200 inverted microscope (Zeiss) using a 40x magnification lens. After incubation of SKOVip cells overexpressing HER2 / neu on the outer surface of the cytoplasmic membrane with DARPin-ETA protein in micrographs, a characteristic brown staining along the edge of the cells is noticeable (Fig. 4A), significantly different from the background (Fig. 4B).

The specific cytotoxic effect of the obtained targeted toxin on tumor cells expressing the HER2 receptor was shown.

We used SKBR-3 human breast adenocarcinoma cells (ATCC catalog number - NTV-30), HER2 receptor overexpressing, HeLa human cervical carcinoma cells (ATCC catalog number - CCL-2) with low HER2 / neu expression (~ 1 × 104 molecules per cell) and control cells of the Chinese hamster CHO ovary (ATCC catalog number - CCL-61) not expressing this receptor. Cells were incubated for 72 hours at 37 ° C and 5% CO2 in a growth medium containing various concentrations of DARPin-ETA protein. At the end of the incubation, cell viability was assessed using a standard MTT assay.

It was shown that the obtained HER2-specific protein DARPin-ETA significantly reduces the viability of HER2-positive SKBR-3 and HeLa cells, with almost no effect on HER2-negative CHO cells (Fig. 5). In this case, the revealed cytotoxic effect correlates with the expression level of the HER2 receptor: the IC50 value (concentration of DARPin-ETA, which causes a 50% decrease in cell viability relative to the control) for SKER-3 HER2-expressing cells and HeLa cells with low expression was 0.1 pM and 37 PM, respectively.

The antitumor effect of the DARPin-ETA protein against the HER2 hyperexpressing xenograft tumor has been shown.

To obtain an experimental model of a human tumor, nude immunodeficient mice were subcutaneously inoculated with a suspension of SKBR-3 cells in an amount of 10 million cells per animal. Tumor growth was monitored based on the volume of tumor nodes. Upon reaching a tumor size of 100 mm ^{3, the} animals were divided into groups according to the administered dose of DARPin-ETA: five-time administration of 5 μg, five-time administration of 10 μg or four-time administration of 20 μg of DARPin-ETA (intravenous).

A marked slowdown of tumor growth was detected in response to the administration of DARPin-ETA, while this effect was dose-dependent (Fig. 6). With the introduction of DARPin-ETA protein at a dose of 4 × 20 μg, no tumor growth was observed until the end of the observation, while the calculated value of the inhibition coefficient of tumor growth was 94%.

The high affinity of the DARPin-ETA protein for the HER2 receptor provides a stronger cytotoxic effect of the recombinant targeted toxin on tumor cells expressing the HER2 receptor, as well as a greater antitumor effect with respect to the HER2 hyperexpressing xenograft tumor compared to the recombinant immunotoxin of the prototype.

Thus, the use of HER2-specific darpin as a directing module provides an increase in the toxic effect on cells expressing the HER2 receptor and an increase in the effectiveness of targeted therapy.

Claims (2)

1. The recombinant targeted toxin specific for HER2 receptor expressing cells contains an HER2-specific targeting module represented by the coding sequence of an HER2-specific protein of the darpin class DARPin and a toxic module in the form of a pseudomonas exotoxin A ETA fragment interconnected by a flexible hydrophilic 16- the amino acid linker represented by the sequence SEQ ID NO: 1, the His6 oligohistidine sequence and the KDEL sequence at the C-terminus of the molecule, with the recombinant targeted toxin pr dstavlen sequence SEQ ID NO: 2. 2. The recombinant targeted toxin according to claim 1, characterized in that the gene encoding the recombinant targeted toxin is under the control of the T7 promoter.

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