Grigory A. Efimov

Cell-type–restricted anti-cytokine therapy: TNF inhibition from one pathogenic source

Significance Anti-cytokine therapy has revolutionized the treatment of autoimmune diseases. However, recent data suggest that cytokines, in particular TNF, produced by various cell types may play distinct and sometimes opposite roles in the inflammatory responses. In certain autoimmune diseases TNF produced by monocytes and macrophages plays a pathogenic role, whereas TNF produced by T cells may be protective. In addition, T-cell–derived TNF is indispensable for resistance to infections, such as tuberculosis. To demonstrate that cell-type–restricted anti-cytokine therapy may be advantageous, we generated bispecific antibodies that neutralize TNF produced by myeloid cells. Cell-targeted inhibition of TNF is more effective than systemic TNF ablation in protecting mice from TNF-mediated hepatotoxicity. This provides a rationale for the development of novel anti-TNF agents. Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type–restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)—a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine–induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.

Tumor necrosis factor, lymphotoxin and cancer

Initially TNF has been discovered as an anti‐tumor factor, but it is now considered as one of the universal effectors of innate signaling implicating its key role in host defense and inflammation. Other physiological functions of TNF are primarily linked to organization of lymphoid tissues. TNF can exert deleterious effects on the organism when its local or systemic concentrations exceed certain levels. This is the main reason for the failure of TNF therapy in oncology. Moreover, in certain experimental models TNF to TNFRp55 signaling axis was found to play a pro‐tumorigenic role. On the other hand, anti‐TNF therapy proved to be beneficial in rheumatic and other autoimmune diseases. Taking into consideration the pivotal function of TNF in the immune system, it is obvious that such therapy cannot be entirely free of adverse effects including suppression of host defense and, possibly, predisposition to lymphomas. Lymphotoxins alpha and beta are the two related cytokines that exist in distinct trimeric forms which can signal through TNFR I and TNFR II, as well LTbetaR receptors, depending on the composition of the trimer. These signals have important functions in the development and homeostasis of the immune system. Importantly, there is a recently uncovered link between the LTalpha/LTbeta to LTbetaR signaling axis and cancer. Here we review the current status of the field with the focus on one particular issue: are TNF and lymphotoxins intrinsically anti‐cancer or pro‐tumorigenic.

Tumor Necrosis Factor and the consequences of its ablation in vivo

Although TNF has been discovered due to anti-tumor activity, its physiological functions are different. Current knowledge places TNF downstream of many receptors of innate immunity, implying its primary role in host defense and inflammation. When overproduced systemically or locally, TNF may exert deleterious effects on the organism. Anti-TNF therapy is highly efficient in several autoimmune and inflammatory diseases. However, due to TNF unique beneficial functions in immune system, such therapy cannot be entirely free of adverse effects. We review the current status of the field with the focus on drugs and strategies used for TNF ablation in vivo.

Modalities of Experimental TNF Blockade In Vivo: Mouse Models

TNF was originally discovered due to its potent anti-tumor activity in mice but later emerged as one of immune mediators with critical non-redundant functions in health and disease. TNF-deficient mice fail to mount protective responses against intracellular bacteria, such as Listeria or Mycobacteria, partly due to defective bactericidal granuloma formation or its structural maintenance. TNF is also critical for the structure of peripheral lymphoid tissues. On the other hand, pathogenic overproduction of TNF was implicated in several autoimmune diseases, and therapies based on systemic blockade of cytokine signaling are being widely used in clinic. We are using panels of engineered mice with specific inactivation or enhancement of TNF signaling to uncover the fine balance between beneficial and deleterious physiologic functions of TNF. In particular, we have generated mice allowing us to define the source (type of immunocyte) and molecular form (soluble versus membrane-bound) of TNF with non-redundant specific roles in the structural organization of lymphoid tissues, as well as in pathologies, such as EAE or septic shock. Additionally, we have developed novel “humanized” mouse models allowing side-by-side comparison of the effects of clinically used drugs. One of our goals is to design safer modalities of anti-TNF therapy.

Can we design a better anti‐cytokine therapy?

Cytokine neutralization is successfully used for treatment of various autoimmune diseases and chronic inflammatory conditions. The complex biology of the two well‐characterized proinflammatory cytokines TNF and IL‐6 implicates unavoidable consequences when it comes to their global blockade. Because systemic cytokine ablation may result in unwanted side effects, efforts have been made to develop more specific cytokine inhibitors, which would spare the protective immunoregulatory functions of a given cytokine. In this article, we review current research and summarize new strategies for improved anti‐TNF and anti‐IL‐6 biologics, which specifically target only selected parts of the signaling cascades mediated by these ligands.

VHH-Based Bispecific Antibodies Targeting Cytokine Production

Proinflammatory cytokines, such as TNF, IL-6, and IL-1, play pathogenic roles in multiple diseases and are attractive targets for biologic drugs. Because proinflammatory cytokines possess non-redundant protective and immunoregulatory functions, their systemic neutralization carries the potential for unwanted side effects. Therefore, next-generation anti-cytokine therapies would seek to selectively neutralize pathogenic cytokine signaling, leaving normal function intact. Fortunately, the biology of proinflammatory cytokines provides several such opportunities. Here, we discuss various applications of bispecific antibodies targeting cytokines with specific focus on selective TNF neutralization targeted directly to the surface of specific populations of monocytes and macrophages. These bispecific antibodies combine an anti-TNF VHH with VHHs or scFvs directed against abundant surface molecules on myeloid cells and serve to limit the bioavailability of TNF produced by these cells. Such reagents may become prototypes of a novel class of anti-cytokine biologics.

Interleukin-6: From molecular mechanisms of signal transduction to physiological properties and therapeutic targeting

Interleukin-6 (IL-6)--one of the most important pro-inflammatory cytokines that has a broad spectrum of immunoregulatory properties. Molecular mechanisms of signal transduction of IL-6 and its receptor, which were previously established, have recently been supplemented with a concept of trans-signaling. Selective inhibition of this signaling cascade would allow to modulate the pathological effects of IL-6. Methods of reverse genetics have helped to establish the physiological functions of IL-6 in normal state and in various diseases, including neoplasias. Therapeutic inhibitors of IL-6 or its receptor are already used for the treatment of several autoimmune diseases, however, systemic inhibition inevitably also neutralizes the protective functions of this cytokine. It is expected that in the future systemic therapy will be replaced by more specific and effective approaches that take into account the peculiarities of molecular signaling pathways in target cells and differences in the function of IL-6, depending on the cell source.

Making anti-cytokine therapy more selective: Studies in mice

Graphical abstract Figure. No caption available. HighlightsDysregulated cytokine signaling is often associated with the development of autoimmunity.Systemic cytokine ablation is used to treat autoimmune diseases, but is not free of side effects.Selective cell‐type or receptor‐specific cytokine inhibition may prove more effective over systemic approach. ABSTRACT Cytokines are involved in a wide range of functions shaping the normal immune response, yet inflammatory changes in the immune system due to dysregulated cytokine signaling may lead to the induction of autoimmunity. Cytokine inhibitors have revolutionized the treatment of many autoimmune diseases in recent years. Systemic cytokine ablation, however, is often associated with the development of adverse side effects and some patients simply do not respond to therapy. TNF, IL‐1 and IL‐6 are the best characterized proinflammatory cytokines considered as the main therapeutic targets for the treatment of several autoimmune and inflammatory diseases. But can anti‐cytokine therapy become more selective and thus more efficient? This mini‐review discusses several recently emerging paradigms and summarizes current experimental attempts to validate them in mouse studies.

In Silico Analysis of the Minor Histocompatibility Antigen Landscape Based on the 1000 Genomes Project

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is routinely used to treat hematopoietic malignancies. The eradication of residual tumor cells during engraftment is mediated by donor cytotoxic T lymphocytes reactive to alloantigens. In a HLA-matched transplantation context, alloantigens are encoded by various polymorphic genes situated outside the HLA locus, also called minor histocompatibility antigens (MiHAs). Recently, MiHAs have been recognized as promising targets for post-transplantation T-cell immunotherapy as they have several appealing advantages over tumor-associated antigens (TAAs) and neoantigens, i.e., they are more abundant than TAAs, which potentially facilitates multiple targeting; and unlike neoantigens, they are encoded by germline polymorphisms, some of which are common and thus, suitable for off-the-shelf therapy. The genetic sources of MiHAs are nonsynonymous polymorphisms that cause differences between the recipient and donor proteomes and subsequently, the immunopeptidomes. Systematic description of the alloantigen landscape in HLA-matched transplantation is still lacking as previous studies focused only on a few immunogenic and common MiHAs. Here, we perform a thorough in silico analysis of the public genomic data to classify genetic polymorphisms that lead to MiHA formation and estimate the number of potentially available MiHA mismatches. Our findings suggest that a donor/recipient pair is expected to have at least several dozen mismatched strong MHC-binding SNP-associated peptides per HLA allele (116 ± 26 and 65 ± 15 for non-related pairs and siblings respectively in European populations as predicted by two independent algorithms). Over 70% of them are encoded by relatively frequent polymorphisms (minor allele frequency > 0.1) and thus, may be targetable by off-the-shelf therapeutics. We showed that the most appealing targets (probability of mismatch over 20%) reside in the asymmetric allele frequency region, which spans from 0.15 to 0.47 and corresponds to an order of several hundred (213 ± 47) possible targets per HLA allele that can be considered for immunogenicity validation. Overall, these findings demonstrate the significant potential of MiHAs as targets for T-cell immunotherapy and emphasize the need for the systematic discovery of novel MiHAs.

Humanization of Murine Monoclonal anti-hTNF Antibody: The F10 Story

Tumor necrosis factor (TNF) is a proinflammatory cytokine implicated in pathogenesis of multiple autoimmune and inflammatory diseases. Anti-TNF therapy has revolutionized the therapeutic paradigms of autoimmune diseases and became one of the most successful examples of the clinical use of monoclonal antibodies. Currently, anti-TNF therapy is used by millions of patients worldwide. At the moment, fully human anti-TNF antibody Adalimumab is the best-selling anti-cytokine drug in the world. Here, we present a story about a highly potent anti-TNF monoclonal antibody initially characterized more than 20 years ago and further developed into chimeric and humanized versions. We present comparative analysis of this antibody with Infliximab and Adalimumab.