CC BY
107
E.N. Kosobokova, PhD, Senior Research Worker, the Laboratory of Transgenic Preparations;
V.S. Kosorukov, PhD, Head of the Laboratory of Transgenic Preparations;
A.Yu. Baryshnikov, D.Med.Sc., Professor, Director of Research Institute of Experimental Diagnostics and Tumor Therapy
N.N. Blokhin Russian Cancer Research Center of Russian Academy of Medical Science, Kashirskoe shosse, 24,
Moscow, Russian Federation, 115478
In medicine cytokines play an important role as the immune response modulators. However, biologically active drug application in high doses in oncology is followed by a number of unfavorable side effects resulting in treatment cessation. Target therapy enables to increase the efficiency of cytokine usage, and therefore, reduce the drug doses.
The achievements in genetic engineering and biotechnology led to a growing number of new antibody-cytokine fusion proteins. Such hybrids can have the properties of all components and acquire advantages compared to proteins alone. For example, monoclonal antibodies specific to a particular tumor antigen being fused with cytokines (MAb-C) provide accumulation of cytokines in tumor microenvironment, increase antitumor effect of antibodies and enhancement of the immune response against a tumor. MAb-C with various specificity against a number of tumors have been created in the last twenty years. It was shown on animal models that such fusion proteins being accumulated around a tumor are capable to cause the considerable antitumor response, which in some cases results in complete tumor elimination. The present review describes data on existing models of antibody-cytokine fusion proteins, their technology and application prospects in oncology.
Key words: cytokines; antibodies; fusion proteins; antitumor drugs.
Cytokines consider to be the protein nature mediators, mediator molecules, which take part in intercellular signal transmission. Their main role is to simulate an immune response. A cytokine binding with specific receptors on cytoplasmic membrane activates the mechanism of intercellular signal transmission providing the regulation of a number of genes responsible for the synthesis of both: modulators themselves, and other cytokines, as well as the formation and appearance of cytokine receptors on cell surface [1-3].
By now over 100 various cytokines have been discovered and described [4]. Among these are interleukins (IL), interferons (IFN), colony-stimulating factors (CSF), tumor necrosis factor (TNF), growth factors and chemokines. These mediators exhibit diversified activity, which partially cross. Cytokines are rarely formed separately and rarely act singly. Cytokine system response is of complex network
nature, when the production of one of them has an effect on the formation or activity of others [5-7].
Many cytokines have direct antitumor activity or are the mediators of antitumor immunity [8, 9]. Systemic high-dose cytokine therapy is frequently accompanied by severe side effects, which make further use of cytokines at recommended dosage impossible [10-12]. Target injection of a drug into a tumor partially solves the problem, since cytokine concentration at injection site decreases rapidly. Moreover, frequently tumor or metastases localization make it impossible to carry out a procedure [13, 14].
Other approach is to use gene therapy [15, 16]. In this case a vector carrying gene responsible for cytokine synthesis is injected. The expected result is systemic immune response against tumor [17, 18]. This technology is highly complicated, takes a long time and is expensive, that limits its application.
For contacts: Kosobokova Ekaterina Nicolaevna, phone: 8(495)324-14-59 +7 903-590-05-91; e-mail: [email protected]
102 СТМ J 2013 — vol. 5, No.4 E.N. Kosobokova, V.S. Kosorukov, A.Yu. Baryshnikov
REVIEWS
Tumor-specific monoclonal antibodies genetically fused with cytokines (MAb-C) are an alternative option for intratumoral cytokine accumulation in the concentration sufficient to produce significant antitumor effect without systemic toxicity accompanied. Cytokines can have an effect on both the cell that have bond the antibodies with, and neighboring cells. Moreover, due to the binding to cell surface through antibodies, fusion proteins can simulate cytokine transmembrane form. It has been demonstrated that some cytokines exist both dissolved, and being bind to membrane, and their functions differ. These effects are described, for instance for TNF [19].
As well as antibodies provide cytokine delivery to tumor, they also can be used as antitumor agents by blocking the receptors on the surface of tumor cells [20-24]. About 50% of all antibodies on pharmaceutical market are antitumor, and in 2013 about 10 new agents are expected to appear, which are meant for the treatment of different tumors [25].
Currently, a variety of tumor-specific MAb-C containing different cytokines has been developed and is being under trial (at various phases) [26, 27]. This review summarizes the knowledge on existing fusion proteins, their technologies and prospects for application in oncology.
Fusion proteins technology
There are five classes of human immunoglobulins (antibodies): IgG, IgA, IgM, IgD and IgE, which vary in molecule size, charge, amino acid profile, and carbohydrate status. At the same time there is significant heterogeneity within the limits of an each class. The main structural unit of any class immunoglobulin consists of two similar light and two similar heavy chains held together by disulphide bonds
(See Fig. a). Each light chain consists of one variable (VL) and one constant domain (CL), while a heavy chain — of one variable (VH) and three constant domains (CH1, CH2, CH3).
Variable domains of a heavy and light chain form an immunoglobulin variable region (Fv) providing a specific link with antigen. Antibodies can be split into three fragments using papain protease: two Fab (fragment antigen binding) and one Fc (fragment crystallizable). Fab contains all light chain, VH-domain and CH1, Fc — all other С-domains of a heavy chain. The division occurs in a hinged region (HR) — it is a specific part of a polypeptide chain, which is not a part of domains and genetically not related to them.
Classical technique of monoclonal antibodies production is hybrid technology [28, 29]. The idea lies on the fact that myeloma cells are fused with B-lymphocytes secreting specific antibodies after mice being immunized by an appropriate antigen. Hybrid cells (hybridomas) are capable of unrestricted division and synthesis of target antibodies. Such murine antibodies show antitumor effect blocking the receptors on tumor cells and counteracting fluid-phase ligands, as well as inducing apoptosis [30]. However, Fc-region of murine antibodies is not capable of binding to human effector cells completely that limits their therapeutic potential. Moreover, such an agent is immunogenic, and in patients it causes a response against murine antibodies with their further neutralization and degradation [31,32].
Despite significant target efforts, no adequate approaches have been developed to obtain hybrids based on human cells. Currently, the problem is solved using molecular-genetic approaches [33]. Humanization of murine antibodies is the most common approach. The technique consists in the formation of fusion genetic complexes uniting V-gene of murine monoclonal antibodies and С-genes
Fig. Schematic diagram of IgG molecular structure (a) and examples of existing fusion proteins based on antibodies or mini-antibodies (b). The description is given in the text
Antibody-Cytokine Fusion Proteins СТМ J 2013 — vol. 5, No.4 103
REVIEWS
of human immunoglobulins of a desired isotype. Due to the presence of human constant region, these antibodies have a wide range of biological functions. Rituximab is an example of humanized antibody (Rituxan, MabThera) [3436]. Rituximab recognizes antigen CD20 on the surface of normal and tumor В-cells and induces cell-mediated and complement-mediated cytotoxicity and apoptosis of these tumor cells. There is the more radical approach consisting in fusing hypervariable regions of murine V-genes with genes encoding framework sequence of human V-gene and human С-genes. Such humanized antibodies have the only part remained after mice — a hypervariable region. Trastuzumab (Herceptin) is an example, these antibodies bind to receptor 2 of human epidermal growth factor (HER2). These antibodies are recommended for breast cancer therapy [37-39].
To exclude immunogenicity there is a need to obtain fully human antibodies. For that purpose there were produced transgenic mice, immunoglobulin genome of which was replaced with human ones (of XenoMouse line, Tc-mice). Such mice produce human polyclonal antibodies [40-42].
An alternative of fully antibodies is mini-antibodies with two chains of a single-chain variable antibody region (scFv), a hinged region and CH3-domain or CH2-CH3-domains [4344]. Unlike the first ones, they are characterized by higher elimination rate (10 h instead of 3-4 weeks). Among the disadvantages of mini-antibodies there is the loss of some functions including cell-mediated cytotoxicity, which plays a key role in antitumor mechanism of antibody action [45].
Phage display technology can also be used to receive human antibodies [46, 47]. scFv genes are cloned in phage display vector. scFv fragment expressed on bacteriophage surface has a high degree of relationship with a target antigen.
In the past decade there has been developed a number of MAb-C of various structure and functional significance. Both whole antibodies and their fragments can underlie these recombinant proteins (See Fig. b). The variety of fusion proteins is provided by cytokines used, among them there are both monomers and homodimers or homotrimers, in addition, there are cytokines formed by different polypeptide chains — heterodimers [4].
Currently, recombinant MAb-C present two main forms: F(ab)2/C secreted by mammal cells, and a single-chain FV/C (micro-antibodies) expressed by E. coli. The first one are large molecules, 134-140 kDa, consisting of a pair of light chains and a pair of hybrid heavy chains, each of which includes a variable region CH1, a hinged region and a cytokine. Micro-antibodies, 42-45 kDa, consist of one variable fragment of a heavy chain, one variable fragment of a light chain, and a cytokine. In the early 90-s there was produced MAb-C, in which a cytokine binds to С-terminus of C3-domain of a heavy chain. The researchers demonstrated that in most cases such molecules retain both antibody (capability to bind to antigen), and cytokine functions. This indicates the feasibility of DNA-technology to produce bifunctional proteins.
The use of mammalian cells as expression system for MAb-C (e.g., newborn hamster cell culture) provides posttranslational human specific modifications that increase
biological activity of recombinant immunoglobulins and in vivo complex stability [48].
Plants with temporary expression of recombinant proteins can be one more producer of fully antibodies genetically fused with cytokines [49]. Currently, Trastuzumab analog (Trastuzumab, Herceptin) was produced using this technology [50].
Prospects for MAb-C application in oncology
The pharmaceutical market has no MAb-C approved for anticancer therapy. However, a number of pharmaceuticals are under clinical trials (I-II phases). Preliminary trial data indicate extensive prospects to use recombinant MAb-C as anticancer drugs. Due to a growing number of antibody-cytokine combinations (Table 1) it is impossible to cover all existing variants in the present paper. Let us consider the most developed and advanced variants, in which monoclonal antibodies bind to IL-2, IL-12, GM-CSF and TNF.
MAb-IL-2. IL-2 is known as an immunomodulator of cellular and humoral immunity with an extensive therapeutic potential [51, 52]. This cytokine is capable to inhibit tumor growth inducing apoptosis due to its capability to stimulate macrophages and NK-cells and increase the expression of histocompatibility molecules class II [53]. Е. Ortiz-Sanchez et al. described the main existing MAb-IL-2 and their functional value [26]. First MAb-IL-2 was developed in the early 90-s. They presented IL-2 molecule bound to C-terminus IgG3 specific to dancyl [54]. IgG3-IL-2 could stimulate proliferation of IL-2-dependent murine Т-cells of CTLL-2 line. This fusion protein showed higher (approximately 4 times as high) affinity than recombinant human IL-2, and was more effective at activation of LAK-cells (lymphokine-activated killer). Moreover, IgG3-IL-2 half-life at gastric administration in mice was 7 h that is longer than free IL-2 administration, but shorter than using single IgG3 injection.
There were developed two MAb-IL-2 specific to Id-antigen, one of which included complete IgG1, another — a single chain fragment scFv IgG1 [55]. The researchers showed scFv-IL-2 capacity to bind to antigen to be 30-40 times as low than that of IgG1-IL-2. In addition, scFv-IL-2 is excreted 20 times as fast than IgG1-IL-2. Finally, scFv-IL-2 has no Fc-region necessary to induce antibody-dependent cell-mediated cytotoxicity, and plays a key role in IgG1-IL-2 antitumor activity.
20-30% cases of breast and ovarian cancer show hyperexpression of HER2/neu (erbB2) receptor. The above mentioned Trastuzumab (Herceptin) used in the pharmaceutical market presents monoclonal antibodies to this receptor. Trastuzumab bound to HER2/neu stops cell cycle at G1 phase thus reducing the proliferation of tumor cells. There has been developed anti-HER2/neu IgG3-IL-2 [56]. This fusion protein retains the capability of antibodies to bind to an appropriate receptor, and biological activity similar to recombinant IL-2. There has been studied anti-HER2/neu IgG3-IL-2 effect on murine intestinal tumor cells expressing human HER2/neu. The use of this MAb-C caused significant tumor growth inhibition, while antibodies (anti-HER2/neu IgG3) had no effect.
уттжтжтжтжттттжтжтжтжтттт^ттттжтттт^тттт^тититттттиттг/'
104 СТМ J 2013 — vol. 5, No.4 E.N. Kosobokova, V.S. Kosorukov, A.Yu. Baryshnikov
REVIEWS
Table 1
Summary table on existing fusion proteins based on cytokines and antibodies to antigens presented in the table
Antigens, specificity
Cytokines Lymphoma Depending on an antibody chosen Breast cancer, ovarian cancer, colon cancer Ovarian cancer Hodgkin’s lymphoma Breast cancer, non-Hodgkin’s lymphoma, chronic lymphatic leukemia Melanoma, neuroblastoma, glioblastoma, small-cell carcinoma of lung Carcinomas (including prostate cancer), breast cancer, ovarian cancer, colon cancer Gastrointestinal cancer, bile duct carcinoma Rheumatoid arthrites (D i— О TO с О TO c о (3 І.Е 2 "w <3 5 £ 2 о c .£ 2 о Jz « о TO О To TO О £ “■ TO о c TO о 3 5 о u то c ° 2 8 I to — о *o c c TO TO о To TO TO CO Non-Hodgkin’s lymphoma, chronic lymphatic leukemia TO s о c 'u Із u u u 2 »_ ІІ TO -g ё О TO з « то с с о о о то Е о 5 Adenocarcinoma (epithelial grandular cells), breast cancer
DNC Id HER2/neu TAG-72 CD30 hMHC II GD2 EpCAM CEA ED-A ED-B FAP CD20 TfR MUC-1
IL-2 + + + + + + !! + ! + +
IL-6 +
IL-7 +
IL-10 + +
IL-12 + + + + + !
IL-15 + + +
IL-17
IFN-a + + +
IFN-y + +
GM-CSF + + + +! + + +
TRAIL + +
TNF + + + + !!
FasL + +
Here: GM-CSF — granulocyte macrophage CSF; TRAIL — TNF related apoptosis-inducing ligand; FasL — ligand for Fas membrane molecule; DNC — dancyl chloride (5-(dimethylamino)naphthalene-1-sulphonyl chloride); Id — idiotypic antibodies, analogs or antigen imitators; EpCAM — epithelial cell adhesion molecule; CEA — carcinoembryonic antigen; TfR — transferring receptor; ED-B, ED-A — isoforms of fibronectin; FAP — stromal fibroblast activation protein; ! - phase I clinical trials; !! - phase II clinical trials.
The other MAb-C, anti-erbB2 scFv-Fc-IL-2, was developed on the basis of CH2-CH3-domains of human IgG1 combined with murine scFv specific to HER2/neu. This fusion protein also retained both antibody and cytokine activity [56-58] that was confirmed on Т-cells of CTLL-2 line, as well as on BALB/c mice with induced ovarian cancer, the cells of which are characterized by high HER2/neu expression. Intravenous injection of scFv-Fc-IL2 protein to mice resulted in tumor growth reduction.
Mucin-1 (MUC-1) is a transmembrane protein naturally expressed on the surface of grandular epithelial cells. In carcinomas there is hyperexpression of this protein on tumor cell surface. Thus, MUC-1 can serve as a marker antigen, and specific antibodies can be used for cytokine target delivery in tumor microenvironment. For this purpose scFv of murine MUC-1-specific antibodies was genetically fused with N-terminus of a hinged region of human IgG1 Fc-region, and IL-2 was fused with С-terminus of Fc-region. The obtained fusion protein retained the capability to bind to MUC-1 expressed by human mammary adenocarcinoma cells, as well as biological activity of IL-2 consisting in its
capability to induce the proliferation of CD25+-lymphocytes and activate NK-cells [59].
In Hodgking's lymphoma an increased number of CD30 is secreting on the surface of tumor cells, and antiCD3 antibodies were also used for producing fusion proteins for IL-2 delivery. Recombinant anti-CD30 MAb-IL-2 was produced on the basis of scFv-region of monoclonal anti-CD30 antibody, N-terminus of which was fused with IgG1 hinged region, bound by С-terminus with IL-2. Anti-CD30 MAb-IL-2 was bifunctional, activated Т- and NK-cells, induced in vivo IFN-y production. In vivo studies supported the suggestion that this fusion protein can be used in specific immune therapy in Hodgking's lymphoma [60].
Characteristic marker of non-Hodgkin's lymphoma is CD20. Fusion anti-CD20 MAb-C were fully humanized murine monoclonal antibodies genetically bound to IL-2. In experiments anti-CD20 MAb-IL-2 induced apoptosis of CD20+ Daudi cells (human lymphoma cell line) and retained their capability to bind to a specific receptor and exhibit antibody-dependent cell-mediated cytotoxicity. The produced fusion protein induced antitumor immune
Antibody-Cytokine Fusion Proteins СТМ J 2013 — vol. 5, No.4 105
REVIEWS
response in animal model on SCID mice, which were injected with human lymphoma line cells Daudi Burkitt CD20+ intravenously simulating disseminated lymphoma [61].
MAb-IL-12. IL-12 is a heterodimer protein with molecular mass of 75 kDa [62]. Its main producers are monocytes, macrophages, as well as dendritic cells, neutrophils and lymphocytes. IL-12 activates the proliferation and cytotoxic activity of Т- and NK-cells. The main effect is the stimulation of IFN-y production, which can inhibit tumor growth and increase the expression of МНС class I genes. In addition, IL-12 has anti-angiogenic activity due to the regulation of IFN-y-dependent proteins IP10 and MIG (monokine induced by IFN-y). These cytokines inhibit chemotaxis of endothelial cells and block their differentiation [63].
IL-12 exhibited potential antitumor activity on various animal models as well as was used for the treatment of viral and bacterial infections [64]. However, in clinical studies IL-12 was shown to be less active, and there were severe side effects [65, 66].
In vivo studies demonstrated that fusion anti-HER2/neu MAb-C(IL-12) retains the functions of IL-12 (heparin-binding activity, the capability to induce IFN-y secretion) [26] and IgG (capability to bind to human antigen HER2/neu) [67]. A prolonged administration of anti-HER2/neu MAb-IL-12 in mice was also shown to cause a long-term immune response spreading over other antigens [68].
In order to study MAb-IL-12 anti-angiogenic activity there were developed fusion proteins against human fibronectin ED-B domain (an angiogenesis marker secreted by tumor and endothelial cells). In vitro studies demonstrated that the obtained recombinant protein possessed IL-12 biological activity and antibody specificity. Anti-ED-B MAb-IL-12 capability was shown to inhibit tumor growth in vivo on mice models (colon carcinoma and teratocarcinoma). Moreover, a recombinant protein was localized in tumor microenvironment prior to IL-12. In addition, there were no side effects [69].
The comparison of anti-HER2/neu MAb-IL-12 effect with combined administration of anti-HER2/neu MAb and IL-12, as well as IL-12 monotherapy proved the significance of physical fusion of antibodies with cytokine to increase antitumor activity. The researchers link it to cytokine placement in extracellular matrix of tumor microenvironment [70].
There were also produced anti-antigen CD30 and CEA fusion proteins (See Table 1). In vitro studies demonstrated them to retain both cytokine function (the ability to induce secretion by IFN-y Т- and NK-cells), and antibody function (the ability to bind to antigen) [71, 72]. AS1409 is a fusion protein based on humanized antibodies specific to fibronectin and bound to IL-12. Its first clinical trials indicate the approach safety. In this study maximum tolerated dose was used and effectiveness against metastatic melanoma was demonstrated [73].
MAb-GM-CSF. GM-CSF refers to a group of glycoproteins regulating the proliferation and differentiation of hemopoietic cells [74]. The main field of this cytokine application is prevention of neutropenia and neutropenic complications in patients with highly decreased neutrophils in
blood after cytostatic chemotherapy due to different tumors [75, 76]. In addition, GM-CSF regulates the expression of MHC class II and antigen-presenting capability of APC (antigen-presenting cells). A wide range of action makes this cytokine be potential medication for adjuvant antitumor immune therapy [77].
Systemic GM-CSF injection is accompanied by a number of side effects: fever, shiver, myalgia, loss of appetite, drowsiness, and ostealgia [78, 79]. In order to reduce negative effects and to increase anti-tumor activity, GM-CSF was genetically bound to antibodies against various tumor antigens (See Table 1).
Transferin (Tf) is a glycoprotein transferring iron ions necessary for cell proliferation. Tf receptor's hyperexpression is observed on several types of cancer cells. GM-CSF fused with anti-TfR antibodies was shown on a mice model to demonstrate antitumor activity resulting in growth reduction of murine metastatic hepatic neuroblastoma (NXS2) and lung metastases of murine colon carcinoma (СТ26). The data obtained indicate possible usage of a similar fusion protein to treat patients with malignant tumors characterized by TfR hyperexpression [80].
There were produced chimeric (human/mouse) anti-ganglioside GD2-antobodies. This fusion protein exhibited antigen-dependent cell associated cytotoxicity and complement-dependent cytotoxicity in neuroblastoma cells NMB7 and mononuclear cells isolated from the same patients. In vivo studies showed anti-GD2-GM-CSF to be characterized by higher adhesive and degranulating property compared to antibodies or cytokines. Currently, the products based on anti-GD2-GM-CSF and anti-GD2-IL-2 are under clinical trials [81,82].
Other researchers studied the biological activity of anti-МНС II-GM-CSF. This fusion protein demonstrated the properties of both cytokine (the capability to induce the formation of hemopoietic progenitor cells from bone marrow mononuclear cells), and also antibodies (the capability to bind to tumor cells expressing MHC class II). The studies of biodistribution on a murine xenograft model proved this fusion protein to be capable of selecting specifically human malignant B-cells [83].
Bifunctionality of fusion MAb-C was also demonstrated on anti-ED-B-GM-CSF. This fusion protein exhibited high tumor specificity and capability to reduce significantly tumor growth in experiments on mice 129SvEv with induced teratocarcinoma F9 and adenocarcinoma of colon С51. In addition, an antimetastatic effect was demonstrated on the same models [84].
For the purpose of in-depth study of MAb-C biological activity, effects and mechanisms of action, there were produced murine anti-HER2/neu-GM-CSF [85]. This fusion protein retained the property of cytokine, namely, the ability to stimulate the growth of АВС-З1 line myeloid cells, as well as activate murine macrophage cells J774.2 and enhance antibody-dependent cytokine-mediated lysis in tumor cells. On the other hand, anti-HER2/neu-GM-CSF binds to murine tumor cells СТ26 expressing HER2/neu receptors on its surface, and enhances anti-HER2/neu immune response. An important point is that this fusion protein causes tumor growth rate reduction on those models, on which the use of
уттжтжтжтжттттжтжтжтжтттт^ттттжтттт^тттт^тититттттиттг/'
106 СТМ J 2013 — vol. 5, No.4 E.N. Kosobokova, V.S. Kosorukov, A.Yu. Baryshnikov
REVIEWS
Table 2
Di-cytokine fusion proteins
MAAb-C1C2 Target antigen Target tumor for fusion protein Status References
Anti-EpCAM-humanGM-CSF/IL-2 EpCAM Colon cancer Animal model [97]
Anti-EpCAM-murineGM-CSF/IL-2 EpCAM Gastric cancer In vitro [98]
Anti-HER2/neu-IL-12-IgG3-IL-2 HER2/neu Breast cancer and colon cancer Animal model [68, 99]
Anti-HER2/neu-IL-12-IgG3-GM-CSF HER2/neu Breast cancer and colon cancer Animal model [68, 99]
IL-12-L19-TNF-a ED-B Teratocarcinoma Animal model [100]
KS-IL-12/IL-2 EpCAM Lewis lung carcinoma Animal model [101]
Anti-CD30-IL-12/IL-2) CD30 Hodgkin’s lymphoma Animal model [102]
antibodies only does not provide protection. These findings support once again the potential significance of anti-HER2/ neu-GM-CSF for managing patients with HER2/neu-positive tumors.
MAb-TNF. Tumor necrosis factor is an extracellular multifunctional cytokine produced mainly by monocytes and macrophages [86, 87]. TNF induces apoptosis in endothelial cells in new-formed vessels promoting agents to penetrate into tumor mass and causing hemorrhagic tumor necrosis
[88] . However, systemic injection of this protein is strictly limited by high toxicity consisting in a syndrome like septic shock resulting in multiple organ failure.
In order to reduce a therapeutic dose of high-activity protein, fusion protein L19-TNF was produced, which should provide TNF target delivery in tumor blood vessels
[89] . L19-TNF is anti-B-domain fibronectin L19 antibodies bound to TNF. Preclinical studies on animals demonstrated L19-TNF bifunctionality. In phase I-II of clinical trials there were chosen safe therapeutic doses of the drug. Nonhematological toxicity at such doses was shown to be low, although in rare cases severe myelosuppresion was recorded [90, 91].
Conclusion
To sum up the described data, we can specify the main advantages of applying fusion proteins based on antibodies and cytokines.
1. A recombinant fusion protein is found in tumor microenvironment more quickly than the cytokines alone. The presence of cytokine receptors in normal cells provides competition for fusion protein molecule binding that reduce MAb-C accumulation in the tumor area. Nevertheless, the experiments on animal models and in clinic showed MAb-C concentration in tumor is higher than via systemic injection of free antibodies. MAb-C being concentrated mainly in tumor has less toxic effect on normal organs and tissues. Due to its relationship with specific antigen, cytokine washout period increasesand therapeutic effect is prolonged.
2. A fusion protein possesses higher antitumor activity than combined therapy of antibodies with cytokine, or cytokine in monotherapy (IL-12). Physical combination of two biologically active proteins is likely to provide “correct” spatial positioning of cytokine in extracellular matrix of tumor microenvironment [70]. The represented data indicate
the possibility to reduce an effective dose of cytokine and injection frequency compared to cytokine monotherapy.
Currently, there being studied simplified versions of fusion proteins based on incomplete antibodies, which can ease the product technology. However, they have a number of disadvantages:
lower antigen binding ability;
quicker washout period;
incomplete set of antibody functions (e.g., Fc-region provides induction of antibody-dependent cell-mediated cytotoxic activity).
Over the past decade MAb-C with various functional orientations have been produced and are under clinical trial. One of the directions in this area is the production of antibodies fused with two different cytokines - di-cytokine fusion proteins (Table 2). The source of the idea is the study on the combination of several cytokines, which indicate significant increase of antitumor activity [92-96].
Gene engineering enabled to produce recombinant antibodies using different expression systems — from bacteria to mammalian cells. The production of fusion proteins from plants with temporary expression is being developed intensively. There being searched combined molecules aiming to enhance therapeutic effect. Just a limited number of MAb-C succeeds in passing clinical trials, though their appearance at pharmaceutical market is just a matter of time.
Study Funding and Conflict of interests. The study was not supported by any funds. The authors have no conflict of interest to disclose.
References
1. Miyajima A., Kitamura T., Harada N., Yokota T., Arai K. Cytokine receptors and signal transduction. Annu Rev Immunol 1992; 10: 295-331.
2. Cohen M.C., Cohen S. Cytokine function: a study in biologic diversity. Am J Clin Pathol 1996 May; 105(5): 589-598.
3. Curfs J.H., Meis J.F., Hoogkamp-Korstanje J.A. A primer on cytokines: sources, receptors, effects, and inducers. Clin Microbiol Rev 1997 Oct; 10(4): 742-780.
4. Ketlinskiy S.A., Simbirtsev A.S. Tsitokiny [Cytokine]. Moscow: Foliant; 2008. 552 p.
5. Sun Q., Jones K., McClure B., Cambareri B., Zacharakis B., Iversen P.O., Stomski F., Woodcock J.M., Bagley C.J., D'Andrea R., Lopez A.F. Simultaneous antagonism of interleukin-5, granulocyte-
Antibody-Cytokine Fusion Proteins СТМ J 2013 — vol. 5, No.4 107
REVIEWS
macrophage colony-stimulating factor, and interleukin-3 stimulation of human eosinophils by targeting the common cytokine binding site of their receptors. Blood 1999; 94: 1943-1951.
6. D'Andrea R.J., Gonda T.J. A model for assembly and activation of the GM-CSF, IL-3 and IL-5 receptors: insights from activated mutants of the common beta subunit. Exp Hematol 2000 Mar; 28(3): 231-243.
7. Kotenko S.V., Pestka S. Jak-Stat signal transduction pathway through the eyes of cytokine class II receptor complexes. Oncogene 2000 May 15; 19(21): 2557-2565.
8. Kim-Schulze S., Taback B., Kaufman H.L. Cytokine therapy for cancer. Surg Oncol Clin N Am 2007 Oct; 16(4): 793-818.
9. Tayal V., Kalra B.S. Cytokines and anti-cytokines as therapeutics — an update. Eur J Pharmacol 2008 Jan 28; 579(1-3): 1-12.
10. Leonard J.P., Sherman M.L., Fisher G.L., Buchanan L.J., Larsen G., Atkins M.B., Sosman J.A., Dutcher J.P., Vogelzang N.J., Ryan J.L. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 1997 Oct 1; 90(7): 2541-2548.
11. Schwartz R.N., Stover L., Dutcher J. Managing toxicities of high-dose interleukin-2. Oncology (Williston Park) 2002 Nov; 16(11 Suppl 13): 11-20.
12. Sleijfer S., Bannink M., Van Gool A.R., Kruit W.H., Stoter G. Side effects of interferon-alpha therapy. Pharm World Sci 2005 Dec; 27(6): 423-431.
13. Den Otter W., Jacobs J.J., Battermann J.J., Hordijk G.J., Krastev Z., Moiseeva E.V., Stewart R.J., Ziekman P.G., Koten J.W. Local therapy of cancer with free IL-2. Cancer Immunol Immunothe 2008 Jul; 57(7): 931-950.
14. Johansson A., Hamzah J., Ganss R. Intratumoral TNFa improves immunotherapy. Oncoimmunology 2012 Nov 1; 1(8): 1395-1397.
15. Dranoff G. Cancer gene therapy: connecting basic research with clinical inquiry. J Clin Oncol 1998 Jul; 16(7): 2548-2556.
16. Barar J, Omidi Y. Translational approaches towards cancer gene therapy: hurdles and hopes. Bioimpacts 2012; 2(3): 127-143.
17. Hillman G.G., Slos P., Wang Y., Wright J.L., Layer A., De Meyer M., Yudelev M., Che M., Forman J.D. Tumor irradiation followed by intratumoral cytokine gene therapy for murine renal adenocarcinoma. Cancer Gene Ther 2004 Jan; 11(1): 61-72.
18. Triozzi P.L., Allen K.O., Carlisle R.R., Craig M., LoBuglio A.F., Conry R.M. Phase I study of the intratumoral administration of recombinant canarypox viruses expressing B7.1 and interleukin 12 in patients with metastatic melanoma. Clin Cancer Res 2005 Jun 1; 11(11): 4168-4175.
19. Aggarwal B.B., Gupta S.C., Kim J.H. Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey. Blood 2012 Jan 19; 119(3): 651-665.
20. Weiner L.M., Dhodapkar M.V., Ferrone S. Monoclonal antibodies for cancer immunotherapy. Lancet 2009 Mar 21; 373(9668): 1033-1040.
21. Weiner L.M., Murray J.C., Shuptrine C.W. Antibody-based immunotherapy of cancer. Cell 2012 Mar 16; 148(6): 1081-1084.
22. Galluzzi L., Vacchelli E., Fridman W.H., Galon J., Sautes-Fridman C., Tartour E., Zucman-Rossi J., Zitvogel L., Kroemer G. Trial watch: monoclonal antibodies in cancer therapy. Oncoimmunology
2012 Jan 1; 1(1): 28-37.
23. Shuptrine C.W., Surana R., Weiner L.M. Monoclonal antibodies for the treatment of cancer. Semin Cancer Biol 2012 Feb; 22(1): 3-13.
24. Vacchelli E., Eggermont A., Galon J., Sautes-Fridman C., Zitvogel L., Kroemer G., Galluzzi L. Trial watch: monoclonal antibodies in cancer therapy. Oncoimmunology 2013 Jan 1; 2(1): e22789
25. Reichert J.M. Which are the antibodies to watch in 2013? MAbs
2013 Jan-Feb; 5(1): 1-4.
26. Ortiz-Sanchez E., Helguera G., Daniels T.R., Penichet M.L. Antibody-cytokine fusion proteins: applications in cancer therapy. Expert Opin Biol Ther 2008 May; 8(5): 609-632.
27. Kontermann R.E. Antibody-cytokine fusion proteins. Arch Biochem Biophys 2012 Oct 15; 526(2): 194-205.
28. Kohler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. 1975. J Immunol 2005 Mar 1; 174(5): 2453-2455.
29. Nilsang S., Nehru V., Plieva F.M., Nandakumar K.S., Rakshit S.K., Holmdahl R., Mattiasson B., Kumar A. Threedimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles. Biotechnol Prog 2008 Sep-Oct; 24(5): 1122-1131.
30. Dillman R.O. The history and rationale for monoclonal antibodies in the treatment of hematologic malignancy. Curr Pharm Biotechnol 2001 Dec; 2(4): 293-300.
31. Shawler D.L., Bartholomew R.M., Smith L.M., Dillman R.O. Human immune response to multiple injections of murine monoclonal IgG. J Immunol 1985 Aug; 135(2): 1530-1535.
32. Dillman R.O., Beauregard J.C., Jamieson M., Amox D., Halpern S.E. Toxicities associated with monoclonal antibody infusions in cancer patients. Mol Biother 1988; 1(2): 81-85.
33. Maynard J., Georgiou G. Antibody engineering. Annu Rev Biomed Eng 2000; 2: 339-376.
34. Scheinfeld N. A review of rituximab in cutaneous medicine. Dermatol Online J 2006 Jan 27; 12(1): 3.
35. Barcellini W., Zanella A. Rituximab therapy for autoimmune haematological diseases. Eur J Intern Med 2011 Jun; 22(3): 220-229.
36. Abdulla N.E., Ninan M.J., Markowitz A.B. Rituximab: current status as therapy for malignant and benign hematologic disorders. BioDrugs 2012 Apr 1; 26(2): 71-82.
37. Nahta R., Esteva F.J. Herceptin: mechanisms of action and resistance. Cancer Lett 2006 Feb 8; 232(2): 123-138.
38. Viani G.A., Afonso S.L., Stefano E.J., De Fendi L.I., Soares F.V. Adjuvant trastuzumab in the treatment of her-2-positive early breast cancer: a meta-analysis of published randomized trials. BMC Cancer 2007 Aug 8; 7: 153.
39. Akbarzadeh-Sharbaf S., Yakhchali B., Minuchehr Z., Shokrgozar M.A., Zeinali S. In silico design, construction and cloning of Trastuzumab humanized monoclonal antibody: a possible biosimilar for Herceptin. Adv Biomed Res 2012; 1: 21.
40. Green L.L. Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies. J Immunol Methods 1999 Dec 10; 231(1-2): 11-23.
41. Coughlin M., Lou G., Martinez O., Masterman S.K., Olsen O.A., Moksa A.A., Farzan M., Babcook J.S., Prabhakar B.S. Generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against SARS coronavirus using XenoMouse. Virology 2007 Apr 25; 361(1): 93-102.
42. Jakobovits A., Amado R.G., Yang X., Roskos L., Schwab G. From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice. Nat Biotechnol 2007 Oct; 25(10): 1134-1143.
43. Holliger P., Hudson P.J. Engineered antibody fragments and the rise of single domains. Nat Biotechnol 2005 Sep; 23(9): 1126-1136.
44. Albrecht H., DeNardo S.J. Recombinant antibodies: from the laboratory to the clinic. Cancer Biother Radiopharm 2006 Aug; 21(4): 285-304.
45. Presta L. Antibody engineering for therapeutics. Curr Opin Struct Biol 2003 Aug; 13(4): 519-525.
46. Babaei A., Zarkesh-Esfahani S.H., Gharagozloo M. Production of a recombinant anti-human CD4 single-chain variable-fragment antibody using phage display technology and its expression in Escherichia coli. J Microbiol Biotechnol 2011 May; 21(5): 529-535.
47. Hairul Bahara N.H., Tye G.J., Choong Y.S., Ong E.B., Ismail A., Lim T.S. Phage display antibodies for diagnostic applications. Biologicals 2013 Jul; 41(4): 209-216. Epub 2013 May 3.
48. Cruz H.J., Conradt H.S., Dunker R., Peixoto C.M., Cunha A.E., Thomaz M., Burger C., Dias E.M., Clemente J., Moreira J.L., Rieke E., Carrondo M.J. Process development of a recombinant antibody/ interleukin-2 fusion protein expressed in protein-free medium by BHK cells. J Biotechnol 2002 Jun 26; 96(2): 169-183.
49. Komarova T.V., Skulachev M.V., Zvereva A.S., Schwartz A.M., Dorokhov Y.L., Atabekov J.G. New viral vector for efficient production of target proteins in plants. Biochemistry (Mosc) 2006 Aug; 71(8): 846-850.
50. Komarova T.V., Kosorukov V.S., Frolova O.Y., Petrunia I.V., Skrypnik K.A., Gleba Y.Y., Dorokhov Y.L. Plant-made trastuzumab
уттжтжтжтжттттжтжтжтжтттт^ттттжтттт^тттт^тититттттиттг/'
108 СТМ J 2013 — vol. 5, No.4 E.N. Kosobokova, V.S. Kosorukov, A.Yu. Baryshnikov
REVIEWS
(herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth. PLoS One 2011 Mar 3; 6(3): e17541.
51. Liao W., Lin J.X., Leonard W.J. IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation. Curr Opin Immunol 2011 Oct; 23(5): 598-604.
52. Liao W., Lin J.X., Leonard W.J. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 2013 Jan 24; 38(1): 13-25.
53. Antony G.K., Dudek A.Z. Interleukin 2 in cancer therapy. Curr Med Chem 2010; 17(29): 3297-32302.
54. Harvill E.T., Fleming J.M., Morrison S.L. In vivo properties of an IgG3-IL-2 fusion protein. A general strategy for immune potentiation. J Immunol 1996 Oct 1; 157(7): 3165-3170.
55. Liu S.J., Sher Y.P., Ting C.C., Liao K.W., Yu C.P., Tao M.H. Treatment of B-cell lymphoma with chimeric IgG and single-chain Fv antibody-interleukin-2 fusion proteins. Blood 1998 Sep 15; 92(6): 2103-2112.
56. Penichet M.L., Dela Cruz J.S., Shin S.U., Morrison S.L. A recombinant IgG3-(IL-2) fusion protein for the treatment of human HER2/neu expressing tumors. Hum Antibodies 2001; 10(1): 43-49.
57. Shi M., Xie Z., Feng J., Sun Y., Yu M., Shen B., Guo N. A recombinant anti-erbB2, scFv-Fc-IL-2 fusion protein retains antigen specificity and cytokine function. Biotechnol Lett 2003 May; 25(10): 815-819.
58. Shi M., Zhang L., Gu H.T., Jiang F.Q., Qian L., Yu M., Chen G.J., Luo Q., Shen B.F., Guo N. Efficient growth inhibition of ErbB2-overexpressing tumor cells by anti-ErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo. Acta Pharmacol Sin 2007 Oct; 28(10): 1611-1620.
59. Heuser C., Ganser M., Hombach A., Brand H., Denton G., Hanisch F.G., Abken H. An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells. Br J Cancer 2003 Sep 15; 89(6): 1130-1139.
60. Heuser C., Guhlke S., Matthies A., Bender H., Barth S., Diehl V., Abken H., Hombach A. Anti-CD30-scFv-Fc-IL-2 antibody-cytokine fusion protein that induces resting NK cells to highly efficient cytolysis of Hodgkin's lymphoma derived tumour cells. Int J Cancer 2004 Jun 20; 110(3): 386-394.
61. Gillies S.D., Lan Y., Williams S., Carr F., Forman S., Raubitschek A., Lo K.M. An anti-CD20-IL-2 immunocytokine is highly efficacious in a SCID mouse model of established human B lymphoma. Blood 2005 May 15; 105(10): 3972-3978.
62. Yoon C., Johnston S.C., Tang J., Stahl M., Tobin J.F., Somers W.S. Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12. EMBO J 2000 Jul 17; 19(14): 3530-3541.
63. Vignali D.A., Kuchroo V.K. IL-12 family cytokines: immunological playmakers. Nat Immunol 2012 Jul 19; 13(8): 722-728.
64. Colombo M.P., Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev 2002 Apr; 13(2): 155-168.
65. Lee P., Wang F., Kuniyoshi J., Rubio V., Stuges T., Groshen S., Gee C., Lau R., Jeffery G., Margolin K., Marty V., Weber J. Effects of interleukin-12 on the immune response to a multipeptide vaccine for resected metastatic melanoma. J Clin Oncol 2001; 19: 3836-3847.
66. Lacy M.Q., Jacobus S., Blood E.A., Kay N.E., Rajkumar S.V., Greipp P.R. Phase II study of interleukin-12 for treatment of plateau phase multiple myeloma (E1A96): a trial of the Eastern Cooperative Oncology Group. LeukRes 2009 Nov; 33(11): 1485-1489.
67. Peng L.S., Penichet M.L., Morrison S.L. A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity. J Immunol 1999 Jul 1; 163(1): 250-258.
68. Helguera G., Rodriguez J.A., Daniels T.R., Penichet M.L. Long-term immunity elicited by antibody-cytokine fusion proteins protects against sequential challenge with murine mammary and colon malignancies. Cancer Immunol Immunother 2007 Sep; 56(9): 15071512.
69. Gafner V., Trachsel E., Neri D. An engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting properties. Int J Cancer 2006 Nov 1; 119(9): 2205-2212.
70. Lo K.M., Lan Y., Lauder S., Zhang J., Brunkhorst B., Qin G., Verma R., Courtenay-Luck N., Gillies S.D. huBC1-IL12, an immunocytokine which targets EDB-containing oncofetal fibronectin in tumors and tumor vasculature, shows potent anti-tumor activity in human tumor models. Cancer Immunol Immunother 2007 Apr; 56(4): 447-457.
71. Heuser C., Diehl V., Abken H., Hombach A. Anti-CD30-IL-12 antibody-cytokine fusion protein that induces IFN-gamma secretion of T cells and NK cell-mediated lysis of Hodgkin's lymphoma-derived tumor cells. Int J Cancer 2003 Sep 10; 106(4): 545-552.
72. Makabe K., Asano R., Ito T., Tsumoto K., Kudo T., Kumagai I. Tumor-directed lymphocyte-activating cytokines: refolding-based preparation of recombinant human interleukin-12 and an antibody variable domain-fused protein by additive-introduced stepwise dialysis. Biochem Biophys Res Commun 2005 Mar 4; 328(1): 98-105.
73. Rudman S.M., Jameson M.B., McKeage M.J., Savage P., Jodrell D.I., Harries M., Acton G., Erlandsson F., Spicer J.F. A phase 1 study of AS1409, a novel antibody-cytokine fusion protein, in patients with malignant melanoma or renal cell carcinoma. Clin Cancer Res 2011 Apr 1; 17(7): 1998-2005.
74. Armitage J.O. Emerging applications of recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1998 Dec 15; 92(12): 4491-4508.
75. Itala M., Pelliniemi T.T., Remes K., Vanhatalo S., Vainio O. Long-term treatment with GM-CSF in patients with chronic lymphocytic leukemia and recurrent neutropenic infections. Leuk Lymphoma 1998 Dec; 32(1-2): 165-174.
76. Ravaud A., Chevreau C., Cany L., Houyau P., Dohollou N., Roche H., Soubeyran P., Bonichon F., Mihura J., Eghbali H., Tabah I., Bui B.N. Granulocyte-macrophage colony-stimulating factor in patients with neutropenic fever is potent after low-risk but not after high-risk neutropenic chemotherapy regimens: results of a randomized phase III trial. J Clin Oncol 1998 Sep; 16(9): 2930-2936.
77. Polovinkina V.S., Kosorukov V.S. Rekombinantnyy chGM-KSF v onkologii [Recombinant hGM-CSF in antitumor therapy]. Rossiyskiy bioterapevticheskiy zhurnal — Russian Biotherapy Journal 2009; 8(1): 29-39.
78. Ruef C., Coleman D.L. GM-CSF and G-CSF: cytokines in clinical application. Schweiz Med Wochenschr 1991 Mar 23; 121(12): 397-412.
79. Stern A.C., Jones T.C. The side-effect profile of GM-CSF. Infection 1992; 20 Suppl 2:124-127.
80. Dreier T., Lode H.N., Xiang R., Dolman C.S., Reisfeld R.A. Kang A.S. Recombinant immunocytokines targeting the mouse transferrin receptor: construction and biological activities. Bioconjug Chem 1998 Jul-Aug; 9(4): 482-489.
81. Albertini M.R., Hank J.A., Gadbaw B., Kostlevy J., Haldeman J., Schalch H., Gan J., Kim K., Eickhoff J., Gillies S.D., Sondel P.M. Phase II trial of hu14.18-IL2 for patients with metastatic melanoma. Cancer Immunol Immunother 2012 Dec; 61(12): 2261-2271.
82. Yu A.L., Gilman A.L., Ozkaynak M.F., London W.B., Kreissman S.G., Chen H.X., Smith M., Anderson B., Villablanca J.G., Matthay K.K., Shimada H., Grupp S.A., Seeger R., Reynolds C.P., Buxton A., Reisfeld R.A., Gillies S.D., Cohn S.L., Maris J.M., Sondel P.M. Children's oncology group anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N Engl J Med 2010 Sep 30; 363(14): 1324-1334.
83. Hornick J.L., Khawli L.A., Hu P., Lynch M., Anderson P.M., Epstein A.L. Chimeric CLL-1 antibody fusion proteins containing granulocyte-macrophage colony-stimulating factor or interleukin-2 with specificity for B-cell malignancies exhibit enhanced effector functions while retaining tumor targeting properties. Blood 1997 Jun 15; 89(12): 4437-4447.
84. Kaspar M., Trachsel E., Neri D. The antibody-mediated targeted delivery of interleukin-15 and GM-CSF to the tumor neovasculature inhibits tumor growth and metastasis. Cancer Res 2007 May 15; 67(10): 4940-4948.
85. Dela Cruz J.S., Trinh K.R., Morrison S.L., Penichet M.L. Recombinant anti-human HER2/neu IgG3-(GM-CSF) fusion protein retains antigen specificity and cytokine function and demonstrates antitumor activity. J Immunol 2000 Nov 1; 165(9): 5112-5121.
Antibody-Cytokine Fusion Proteins СТМ J 2013 — vol. 5, No.4 109
REVIEWS
86. Bazzoni F., Beutler B. The tumor necrosis factor ligand and receptor families. N Engl J Med 1996; 334: 1717-1725.
87. Locksley R.M., Killeen N., Lenardo M.J. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 2001; 104: 487-501.
88. van Horssen R., Ten Hagen T.L., Eggermont A.M. TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility. Oncologist 2006 Apr; 11(4): 397-408.
89. Borsi L., Balza E., Carnemolla B., Sassi F., Castellani P., Berndt A., Kosmehl H., Biro A., Siri A., Orecchia P., Grassi J., Neri D., Zardi L. Selective targeted delivery of TNFalpha to tumor blood vessels. Blood 2003 Dec 15; 102(13): 4384-4392.
90. Papadia F., Basso V., Patuzzo R., Maurichi A., Di Florio A., Zardi L., Ventura E., Gonzalez-Iglesias R., Lovato V., Giovannoni L., Tasciotti A., Neri D., Santinami M., Menssen H.D., De Cian F. Isolated limb perfusion with the tumor-targeting human monoclonal antibody-cytokine fusion protein L19-TNF plus melphalan and mild hyperthermia in patients with locally advanced extremity melanoma. J Surg Oncol 2013 Feb; 107(2): 173-179.
91. Spitaleri G., Berardi R., Pierantoni C., De Pas T., Noberasco C., Libbra C., Gonzalez-Iglesias R., Giovannoni L., Tasciotti A., Neri D., Menssen H.D., de Braud F. Phase I/II study of the tumour-targeting human monoclonal antibody-cytokine fusion protein L19-TNF in patients with advanced solid tumours. J Cancer Res Clin Oncol 2013 Mar; 139(3): 447-455.
92. Liebau C., Baltzer A.W., Schmidt S., Roesel C., Karreman C., Prisack J.B., Bojar H., Merk H. Interleukin-12 and interleukin-18 induce indoleamine 2,3-dioxygenase (IDO) activity in human osteosarcoma cell lines independently from interferon-gamma. Anticancer Res 2002 Mar-Apr; 22(2A): 931-936.
93. Khawli L.A., Hu P., Epstein A.L. Cytokine, chemokine, and co-stimulatory fusion proteins for the immunotherapy of solid tumors. Handb Exp Pharmacol 2008; (181): 291-328.
94. Wilke C.M., Wei S., Wang L., Kryczek I., Kao J., Zou W.
Dual biological effects of the cytokines interleukin-10 and interferon-y. Cancer Immunol Immunother 2011 Nov; 60(11): 1529-1541.
95. Peng R.Q., Ding Y., Zhang X., Liao Y., Zheng L.M., Zhang X.S. A pilot study of paclitaxel combined with gemcitabine followed by interleukin-2 and granulocyte macrophage colony-stimulating factor for patients with metastatic melanoma. Cancer Biol Ther 2012 Dec 1; 13(14): 1443-1448.
96. Hombach A.A., Abken H. Targeting two co-operating cytokines efficiently shapes immune responses. Oncoimmunology 2013 Mar 1; 2(3): e23205.
97. Schanzer J.M., Baeuerle P.A., Dreier T., Kufer P. A human cytokine/single-chain antibody fusion protein for simultaneous delivery of GM-CSF and IL-2 to Ep-CAM overexpressing tumor cells. Cancer Immun 2006 Feb 17; 6: 4.
98. Schanzer J.M., Fichtner I., Baeuerle P.A., Kufer P. Antitumor activity of a dual cytokine/single-chain antibody fusion protein for simultaneous delivery of GM-CSF and IL-2 to Ep-CAM expressing tumor cells. J Immunother 2006 Sep-Oct; 29(5): 477-488.
99. Helguera G., Rodriguez J.A., Penichet M.L. Cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal HER2/neu expressing tumors. Mol Cancer Ther 2006 Apr; 5(4): 1029-1040.
100. Halin C., Gafner V., Villani M.E., Borsi L., Berndt A., Kosmehl H., Zardi L., Neri D. Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha. Cancer Res 2003 Jun 15; 63(12): 3202-3210.
101. Gillies S.D., Lan Y., Brunkhorst B., Wong W.K., Li Y., Lo K.M. Bi-functional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer. Cancer Immunol Immunother 2002 Oct; 51(8):449-460.
102. Jahn T., Zuther M., Friedrichs B., Heuser C., Guhlke S., Abken H., Hombach A.A. An IL12-IL2-antibody fusion protein targeting Hodgkin's lymphoma cells potentiates activation of NK and T cells for an anti-tumor attack. PLoS One 2012; 7(9): e44482.
уттттт^тттттттттттттттттттттттттттттттттттттіттттт'
110 СТМ J 2013 — vol. 5, No.4 E.N. Kosobokova, V.S. Kosorukov, A.Yu. Baryshnikov
