One important distinction between many tumor cell types and normal cells consists in the translocation of a number of intracellular proteins, in particular the 70 kDa heat shock protein (HSP70), to the surface of the plasma membrane. It has been demonstrated that such surface localization of HSP70 on tumor cells is recognized by cytotoxic effectors of the immune system, which increases their cytolytic activity. The mechanisms behind this interaction are not fully clear; however, the phenomenon of surface localization of HSP70 on cancer cells can be used to develop new approaches to antitumor immunotherapy. At the same time, it is known that the presence of HSP70 on a cells surface is not a universal feature of cancer cells. Many types of tumor tissues do not express membrane-associated HSP70, which limits the clinical potential of these approaches. In this context, targeted delivery of exogenous HSP70 to the surface of cancer cells with the aim of attracting and activating the cytotoxic effectors of the immune system can be considered a promising means of antitumor immunotherapy. Molecular constructs containing recombinant mini-antibodies specific to tumor-associated antigens (in particular, antibodies specific to HER2/neu-antigen and other markers highly expressed on the surface of a wide range of cancer cells) can be used to target the delivery of HSP70 to tumor tissues. In order to assess the feasibility and effectiveness of this approach, recombinant constructs containing a mini-antibody specific to the HER2/ neu-antigen in the first module and HSP70 molecule or a fragment of this protein in the second module were developed in this study. Strong selective interaction between the modules was ensured by a cohesive unit formed by the barnase:barstar pair, a heterodimer characterized by an unusually high constant of association. During testing of the developed constructs in in vitro models the constructs exhibited targeted binding to tumor cells expressing the HER2/neu antigen and the agents had a significant stimulating effect on the cytotoxic activity of NK cells against the respective cancer cells.

Одним из принципиальных отличий многих типов опухолевых клеток от клеток нормальных тканей является транслокация на поверхность плазматической мембраны ряда внутриклеточных протеинов, в частности белка теплового шока 70 кДа (HSP70). Продемонстрировано, что такая необычная поверхностная локализация HSP70 распознается цитотоксическими эффекторами иммунной системы и приводит к усилению их цитолитического действия на данные клетки-мишени. Механизмы этого взаимодействия до конца не раскрыты, однако феномен поверхностной локализации HSP70 на раковых клетках может быть использован для разработки новых подходов к противоопухолевой иммунотерапии. В то же время известно, что присутствие HSP70 на клеточной поверхности не является универсальным признаком раковых клеток. Многие разновидности опухолевых тканей не экспрессируют мембрано-ассоциированные HSP70, что ограничивает потенциальные клинические возможности указанных подходов. В связи с этим в качестве одного из перспективных направлений противоопухолевой иммунотерапии можно рассматривать направленную доставку на поверхность раковых клеток экзогенных HSP70 с целью привлечения и активации цитотоксических эффекторов иммунной системы. Для осуществления адресной доставки HSP70 в опухолевые ткани можно использовать молекулярные конструкции, содержащие рекомбинантные мини-антитела к опухолеассоциированным антигенам, в частности, к HER2/neu-антигену и другим маркерам, высоко экспрессирующимся на поверхности широкого спектра раковых клеток. С целью оценки возможности и эффективности применения указанного подхода, в данном исследовании были созданы двухмодульные рекомбинантные конструкции, содержащие в первом модуле мини-антитела к HER2/neuантигену, а во втором модуле - молекулу HSP70 или ее фрагмент. Избирательное взаимодействие белковых модулей обеспечивалось состыковочным звеном, образуемым входящей в состав молекулярной конструкции парой барназа:барстар - гетеродимером с уникально высокой константой взаимодействия. Результаты, полученные при тестировании указанных препаратов в моделях in vitro, продемонстрировали селективное связывание разработанных конструкций с опухолевыми клетками, экспрессирующими HER2/neu-антиген, и достоверное стимулирующее действие этих препаратов на цитотоксическую активность клеток-эффекторов иммунной системы по отношению к соответствующим клеткам-мишеням.

MICA is a stress-induced protein that, as a rule, is not expressed in healthy tissues, but appears in large amounts on the surface of cells undergoing malignant transformation. In humans, this protein can either initiate antitumor immune response, or facilitate tumor cells for their escape of destruction. After shedding from tumor cell surface, soluble MICA enters blood circulation and contributes to decreased activity of effector cells, due to inactivation of NKG2D receptor. The aim of our study was to determine differences in circulating sMICA concentrations in sera of patients with different types of malignant lymphomas, and to evaluate the impact of sMICA upon NKG2D-positive cytotoxic lymphocytes. In experimental models with C1R-MICA cells, the MICA shedding was shown to occur from the surface of cultured tumor cells into the extracellular space. A reduced NKG2D expression dependent on sMICA concentration in the culture medium was demonstrated by flow cytometry in peripheral mononuclear cells, thus suggesting a role of serum sMICA in suppression of antitumor immune response. The sMICA detection was performed in patients with various types of B- or T-cell non-Hodgkin’s lymphomas by means of enzyme-linked immunosorbent assay. The groups of patients with increased sMICA content were identified and compared with the control group. Minimal amounts of serum sMICA were registered in the control group, with the median of 20 pg/ml. In a combined group of patients with various B-cell lymphomas, an increase in circulating sMICA amounts was shown, at the levels of more than 6.5 times exceeding the control values. The highest values of sMICA were recorded among the patients with chronic lymphocytic leukemia, diffuse large cell lymphoma, and multiple myeloma. Maximal sMICA levels among the investigated groups of patients were observed in the group of patients with T-cell anaplastic lymphoma (median of 574 pg/ml). The study provides preliminary evidence for a suppressive effect of different (immuno)chemotherapy components, in particular, rituximab and radiation therapy, upon serum sMICA contents in the lymphoma patients. Thus, elevated serum sMICA levels in patients with hematological malignancies may be considered as an additional criterion for application of the antitumor therapy. sMICA monitoring at different stages of cytostatic treatment may be useful in order to evaluate its efficiency.

MICA is a stress-induced protein that, as a rule, is not expressed in healthy tissues, but appears in large amounts on the surface of cells undergoing malignant transformation. In humans, this protein can either initiate antitumor immune response, or facilitate tumor cells for their escape of destruction. After shedding from tumor cell surface, soluble MICA enters blood circulation and contributes to decreased activity of effector cells, due to inactivation of NKG2D receptor. The aim of our study was to determine differences in circulating sMICA concentrations in sera of patients with different types of malignant lymphomas, and to evaluate the impact of sMICA upon NKG2D-positive cytotoxic lymphocytes. In experimental models with C1R-MICA cells, the MICA shedding was shown to occur from the surface of cultured tumor cells into the extracellular space. A reduced NKG2D expression dependent on sMICA concentration in the culture medium was demonstrated by flow cytometry in peripheral mononuclear cells, thus suggesting a role of serum sMICA in suppression of antitumor immune response. The sMICA detection was performed in patients with various types of B- or T-cell non-Hodgkin’s lymphomas by means of enzyme-linked immunosorbent assay. The groups of patients with increased sMICA content were identified and compared with the control group. Minimal amounts of serum sMICA were registered in the control group, with the median of 20 pg/ml. In a combined group of patients with various B-cell lymphomas, an increase in circulating sMICA amounts was shown, at the levels of more than 6.5 times exceeding the control values. The highest values of sMICA were recorded among the patients with chronic lymphocytic leukemia, diffuse large cell lymphoma, and multiple myeloma. Maximal sMICA levels among the investigated groups of patients were observed in the group of patients with T-cell anaplastic lymphoma (median of 574 pg/ml). The study provides preliminary evidence for a suppressive effect of different (immuno)chemotherapy components, in particular, rituximab and radiation therapy, upon serum sMICA contents in the lymphoma patients. Thus, elevated serum sMICA levels in patients with hematological malignancies may be considered as an additional criterion for application of the antitumor therapy. sMICA monitoring at different stages of cytostatic treatment may be useful in order to evaluate its efficiency.