Full size IgG antibodies (Mw ~ 150 kDa) are Y-shaped multidomain proteins consisting of two heavy (_H) chains and two light (_L) chains, each of which contain a constant (C) and variable (V) region. Antigen binding is mediated in a bivalent fashion by V_H and V_L domains located on the two Fab (V_HV_LC_H1C_L) arms of the antibody. The remaining constant domains (C_H2C_H3) form the Fc region, which recruits effector functions. The bivalent nature of antibodies contributes to high affinity binding of the antigen and provides long retention times at the target site. IgGs typically display a long serum half-life (t_1/2) of several days, which is mainly mediated by interaction of the Fc region with neonatal Fc receptors (FcRn) [9,10]. Binding to FcRn allows the transport of IgGs within and across cells and salvages them from default lysosomal degradation [11]. The role of this interaction in mediating prolonged antibody half-life is underlined by the finding, that IgGs with lower binding affinity to FcRn are more rapidly cleared. A number of genetic modifications as well as isotype-specific Fc properties have been explored to modify FcRn binding and serum half-life of IgG antibodies [12,13]. Tissue penetration of IgGs has been found to be slow and heterogeneous. In particular in solid tumors, which display elevated interstitial pressure, the large IgG molecule shows limited extravasation and diffusion capacity [14].

To the other extreme, single-chain Fvs (scFvs; Mw ~ 28 kDa) represent the smallest format, which retains the antigen-binding affinity, but not avidity, of the parental antibody. They consist of a variable V_H and a variable V_L domain fused by a flexible polypeptide linker. The low molecular weight of scFvs has shown to improve tissue penetration compared to IgGs [15,16]. However, rapid clearance of the small fragments combined with reduced retention property due to the monovalent nature of the antigen interaction leads to low absolute accumulation (8–10 fold lower than IgG) at the target site [17].

A number of multivalent antibody formats (i.e., diabody, SIP, scFvFc) of intermediate size have been engineered to profit from longer target-site retention than scFvs, as well as deeper tissue penetration compared to full size IgGs [8,18]. For example, reducing the linker length of the scFv to 5 residues or less, promotes the formation of stable scFv homodimers, called diabodies (Mw ~ 55k Da) [19]. This bivalent format displays high tumor-to-blood ratios with an increased absolute tumor accumulation compared to scFv of the same antigen specificity.

The clinically most advanced immunocytokines are recombinant fusion proteins in which the antibody moiety is used in the full IgG or in diabody format (Figure 2).

Immunocytokines have been reported to display a significantly reduced serum half-life compared to their corresponding naked antibody [20]. The IgG-IL2 fusion protein hu14.18-IL2, for example, displayed a serum half-life of ~4 h in melanoma and neuroblastoma patients [21]. In comparison, the serum half-life of the diabody L19 was 2–3 h when fused to IL2 [22]. In either case this represents a significant decrease of blood clearance of IL2 alone (t_1/2 ~ 30–45 min) [20].

In some cases the cytokine moiety of immunocytokines can significantly interfere with targeting. One example is the immunocytokine L19-IFNγ, which targeted tumors in mice lacking IFNγ receptors, but not in wild-type mice [23].

Enrichment of cytokines at the target site is mediated by the antibody-antigen interaction. Recruiting effector functions is crucial for the therapeutic effect of immunocytokines and is mediated by binding of the cytokine moiety to the correspondent cytokine receptors on effector cells. For example, binding of the IL2 moiety of immunocytokines to the IL2 receptor stimulates the expansion and activation of immune cells such as cytotoxic T cells and natural killer (NK) cells. Newly generated antibody fusion proteins are validated for functional cytokine signaling.

In addition, immunocytokines can activate antigen dependent cellular cytotoxicity (ADCC). The Fc region of IgG-based fusion proteins mediates ADCC by binding Fcγ receptors (FcγR) on the surface of immune effector cells, such as NK cells. IL2 immunocytokines have shown that IL2 enhances the ADCC function of antibodies [7,24,25]. Recent findings describe the formation of activating immune synapses (AIS) between FcR-deficient NK cells and tumor cells mediated by IL2 immunocytokines. These functional AIS were formed by the interaction of the IL2 component of the immunocytokine and IL2 receptors on effector cells [26,27], demonstrating that IL2 immunocytokines can initiate direct target cell lysis even without Fc involvement.

Epithelial cell adhesion molecule (EpCAM) is a well-described cell surface antigen for a broad range of carcinomas, such as breast, colorectal and pancreatic carcinomas. EpCAM overexpression is considered to be a marker of tumor initiating cells and is involved in cancer metastasis. However, the selectivity of EpCAM is limited by its expression in healthy epithelia [28,29].

GD2 disialoganglioside is expressed on the cell surface of neuroectodermal tumors, such as melanoma and neuroblastoma and soft tissue sarcomas [21,30,31]. In the healthy body, GD2 expression is restricted to neurons and peripheral pain fibers, and thereby displays a high degree of selectivity [32].

Progression of cancer or immune diseases involves the formation of a specific disease environment, which involves tissue remodeling, angiogenesis and the recruitment of stromal and immune cells [33,34]. Disease environments provide a number of well accessible target structures.

Newly formed blood vessels are particularly attractive targets for antibody-mediated delivery approaches. Vascular antigens are readily reached via the blood stream and angiogenesis is involved in the disease progression of most cancers as well as numerous inflammatory diseases [35]. Our group has extensively studied the in vivo performance of vascular targeting antibodies [36]. Splice isoforms of fibronectin and of tenascin-C represent some of the best-characterized markers of newly formed blood vessels [37]. Specifically, the alternatively-spliced extra-domains EDA and EDB of fibronectin, as well as the extra-domain A1 of tenascin-C, are virtually undetectable in normal adult tissues, but are strongly expressed at sites of physiological and pathogenic angiogenesis. The human monoclonal antibodies F8, L19 and F16 specifically recognize the EDA and EDB domains of fibronectin and the A1 domain of tenascin-C, respectively, and have shown selective accumulation at the disease site in a broad range of cancers and inflammatory disorders in animal models as well as in patients [38,39,40,41].

An alternative approach, termed target necrosis therapy (TNT), targets necrotic tissue, which is present primarily in the hypoxic environment of solid tumors and is absent in the healthy body. The monoclonal antibody NHS76 recognizes DNA / histone complexes, which have been demonstrated to be exposed by necrotic cells in the tumor core as well as metastases [42,43].

The identification of novel disease markers is of great importance for the development of targeted therapies. This process is driven mainly by comparative transcriptomic [44] and proteomic technologies, which have rapidly advanced in the past years [44,45,46]. For example, in vivo labeling followed by proteomic analysis has led to the identification a number of vascular accessible antigens [47,48], some of which are expressed in a broad range of diseases [39,49], while others are more restricted to specific malignancies [50].

Tumor formation and progression is a complex process involving a multitude of changes within both cancer cells and the body’s defense mechanisms. The tumor microenvironment harbors cytokines and other inflammatory mediators, which influence immunosurveillance, the growth of cancer cells, tissue remodeling and angiogenesis [33]. Anti-inflammatory factors, mainly IL10 and TGFβ, interfere with the cell-mediated immune response against cancer cells and promote tumor development. However, pro-inflammatory signals and chronic inflammation also play a crucial role in tumor growth and progression [51,53].

The development of immunocytokines for cancer therapies has so far focused on the delivery of pro-inflammatory cytokines, which mediate the infiltration of leukocytes into the tumor mass and promote an antitumor immune response. The most promising results were reported for fusion proteins with interleukin 2 (IL2), interleukin 12 (IL12) and tumor necrosis factor (TNF).

Chronic inflammatory diseases, such as rheumatoid arthritis, psoriasis, endometriosis, atherosclerosis or inflammatory bowel disease, are characterized by persistent inflammation mediated by complex interactions between immune cells and the disease microenvironment. These commonly both activate the immune system and interfere with pathways involved in the resolution of inflammation [34,52].

Immunocytokines based on the anti-inflammatory cytokine interleukin 10 (IL10) have been tested for applications in rheumatoid arthritis, psoriasis and endometriosis.

A number of preclinical studies have been performed to explore the combinatorial effect of immunocytokines with other therapeutic agents, such as chemotherapeutics, monoclonal antibodies and other biologicals.

Most chemotherapeutics that are used for cancer therapy are traditionally considered to be immunosuppressive by killing or inhibiting the progression of dividing cells, including lymphocytes. But accumulating evidence indicates that cytotoxic drugs and radiotherapy also indirectly and directly effect the immune system to contribute to tumor regression [79].

For example, preclinical combination experiments with F8-IL2 [80] and KS-IL2 [81] showed that paclitaxel, when administered prior to the immunocytokine, enhances the antibody-targeted delivery of IL2 to the disease site and promotes a synergistic anti-cancer effect in different cancer models. Encouraging results were also observed for the combination of F16-IL2 and temozolomide in an ortothopic glioblastoma mouse model, demonstrating the effective delivery of cytokines to a pathological disorder in the brain [82]. In this case simultaneous administration of the immunocytokine and the chemotherapeutic agent led to complete eradication of lymphoma xenografts [57].

Radiofrequency ablation (RFA) followed by KS-IL2 treatment enhanced the antitumor effect and survival, compared to mice treated with RFA or KS-IL2 alone, in a murine colon adenocarcinoma model [83].

Some antibody-cytokine fusions (in particular IL2- and TNF-based immunocytokines) display vasoactive properties and can therefore also enhance the uptake of therapeutic agents at disease site, if administered as a pretreatment [84]. In general, immunocytokines and conventional chemotherapeutic agents are promising combination partners because they typically do not exhibit overlapping limiting toxicities. But the ideal combination partners, dose regimen and also administration sequence have to be elucidated for different disease settings.

The combined use of therapeutic antibodies and cytokines has been shown to potentiate the ADCC, due to an increase of effector cells [85,86]. And in fact, a synergistic therapeutic effect was observed for F8-IL2 and the monoclonal antibody sunitinib in mouse models of renal cell carcinomas [87]. A comparable effect was described for the combination of L19-IL2 and rituximab in a mouse model of B-cell lymphoma [57].

In addition, the combination of different immunocytokines, such as L19-IL12 and L19-TNF, demonstrated enhanced therapeutic benefit compared to the administration of each immunocytokine alone [88]. In accordance with these findings, bifunctional antibody cytokine fusion proteins (KS-IL2/IL12) displayed synergistic effects in vivo [89].

Some clinical results of combination therapies with immunocytokines and chemotherapeutic agents have recently been reported. The combination of KS-IL2 with cyclophosphamide and the combination of F16-IL2 with doxorubicin or with paclitaxel are being studied in phase Ib clinical trials in patients with solid tumors [90,91]. And the combined use of L19-IL2 (at the same dose applied as monotherapy) with dacarbazine showed an excellent tolerability profile and led to objective responses in 8/29 patients and a complete response in one patient in a phase Ib trial in patients with metastatic melanoma. This combination therapy is currently being evaluated in a randomized phase II trial [92].