Biological agents targeting beyond TNF-alpha

Biologicals are proteins produced by living organisms to target speciÞ c sites of the inß ammatory cascade, including antibodies against cell surface markers, cytokines and adhesion molecules. The biological agents represent an important addition to the therapies for immuno-inß ammatory conditions and have a great impact on the disease course and quality of life of these patients. However, recent reports of reactivation of TB (tuberculosis) after anti-TNF therapy raised question on their safety. TNF-α plays an important role in the host defense against mycobacterial infection, particularly in granuloma formation and inhibition of mycobacterial dissemination. FDA recommended

Biologicals are proteins produced by living organisms to target speciÞ c sites of the inß ammatory cascade, including antibodies against cell surface markers, cytokines and adhesion molecules. [1] The biological agents represent an important addition to the therapies for immuno-inß ammatory conditions and have a great impact on the disease course and quality of life of these patients. However, recent reports of reactivation of TB (tuberculosis) after anti-TNF therapy raised question on their safety. TNF-α plays an important role in the host defense against mycobacterial infection, particularly in granuloma formation and inhibition of mycobacterial dissemination. [2] FDA recommended a black box for TB on the product labeling of infliximab. [4] Other serious infections reported with etanercept include sepsis secondary to Listeria monocytogenes and Histoplasma capsulatum. [2] Severe disseminated opportunistic infections have been reported in the HIV positive patients. [2] SLE syndrome, demyelinating diseases, neurodegenerative diseases, pancytopenia, cardiovascular diseases, new onset or ß are-up of chronic iridocyclitis, thyroid cancer, hypoglossal nerve paralysis, severe cytomegalovirus pulmonary infection, reactivation of Crohn's disease etc. are the other important adverse effects reported with anti-TNF-α therapy. [4,5] However, the success of TNF blockade clearly indicates that there are various checkpoints in cytokine-mediated inß ammation. Hence, the identiÞ cation and development of molecules targeting such critical 'regulatory cytokines' (beyond TNF) may have the potential to become a novel addition in the armamentarium against various immunoinß ammatory conditions. Hence, in the present article we are reviewing some of these non-TNF-α biological.
Search Methodology: Prominent rheumatology and general/internal medicine journals (MEDLINE, EMBASE, PUBMED between 2000 and were searched for review papers and clinical trials published on drugs targeting inß ammatory cytokines other than TNF-α. All the data was collected and important evidences regarding pharmacology and uses of non-TNF-α biologicals were summarized in the present article. Cytokine networks and their therapeutic targets in clinical practice: [6][7][8] Cytokines are soluble (glyco) proteins, nonimmunoglobulin in nature, released by living cells of the host, which act non-enzymatically in picomolar to nanomolar concentrations through speciÞ c receptors to regulate host cell function. Cytokines are pleiotropic in their biological activities and play pivotal roles in a variety of responses, including the immune response, hematopoiesis, neurogenesis, embryogenesis and oncogenesis. The main types of cytokines are lymphokines, interleukins, monokines, tumor necrosis factors (TNF), interferons, colony-stimulating factors, transforming growth factors, peptide growth factors, heat shock and other stress proteins. Cytokines have been classiÞ ed on the basis of their biological responses into pro-or anti-inß ammatory cytokines, depending on their effects on immunocytes [Table 1]. TNF, interleukin (IL)-1, lymphotoxin, macrophage migration inhibitory factor, resistin, interferon-, granulocyte-macrophage colonystimulating factor, macrophage colony-stimulating factor, Þ broblast growth factor and vascular endothelial growth factor are the proinß ammatory cytokines. Whereas, IL-1Ra, IL-18 binding protein, IL-10, transforming growth factors, IL-11, IL-13, osteoprotegerin, adiponectin, etc are the anti-inß ammatory cytokines. IL-22, oncostatin M etc are equivocal. Cytokines can be released not only by immune cells but also by host tissue cells. Various molecules (anakinra, tocilizumab, atlizumab, abatacept, alefacept, efalizumab, rituximab) targeting these cytokines are in clinical development.
Existing therapeutic targets include the biologicals acting as antagonists of various inß ammatory cytokines Table 1: Cytokines and their role [6][7][8]43,44] Cytokines Function IL-1 Activates APC and CD4+ lymphocytes; affects the differentiation of the B and T-Cells and other immunocompetent cells, takes part in the regulation of productions of other cytokines and GMCSF. IL-2 Stimulates the proliferation and activation of B-Cells and T-Cells. IL-4 Plays a role in the differentiation of TH2, in allergic responses and in the switching of antibody types. IL-5 Stimulates the production and maturation of eosinophils during inß ammation. IL-8 Acts as a chemotactic factor that attracts neutrophils, basophils and T-Cells to sites of inß ammation.

IL-12
A critical linker between the innate immunity and adaptive immunity, capable of TH1 differentiation and IFN-Gamma release by T-Cells and NK cells IL-10 Acts to repress secretion of pro-inß ammatory cytokines. IL-3 Potent activator of the hemopoietic cells. It stimulates NK-Cells and acts as a synergist with IL-4 during the induction of CD4+ lymphocyte activation process. IL-7 Induces apoptosis of tumor cells and causes differentiation of cells from a subgroup of acute myeloblastic leukemia. IL-9 Stimulates the excretion of IL-2, IL-4, IL-6, IL-11 and cytotoxicity of T-killers and NK-Cells, inducing apoptosis.

IL-11
Regulates the functions of B-Cells and T-Cells, induces various killer cells' activities and acts as an autocrine factor for the proliferation of megacaryocytes.

IL-13
Inhibits the proliferation of leukemic pro-B-Cells IL-14 BCGF and the hyper production of this interleukin enables the progression of NHL-B.

IL-15
Increases the antitumor activities of T-killers and NK-Cells, and the production of cytokines CD4+ lymphocytes.

IL-17
Takes part in the regulation of many cytokines and can reinforce the antibody dependant tumor cell destructions.

IL-18
Synergist with IL-12, especially in the induction of IFN-Gamma production and inhibition of angiogenesis.

IL-19
Regulates the functions of macrophages and suppresses the activities of TH1 and TH2.

IL-21
Promotes a high production of T-Cells, fast growth and maturation of NK-Cells and B-Cells population.

IL-22
Similar to IL-10, but does not prohibit the production of pro-inß ammatory cytokines through monocytes.

TNFs
Activates macrophages, inhibits apoptosis of neutrophils and eosinophils, induces vascular endothelial cells to bind to phagocytes, induce proliferation of NK-Cells and stimulate innate and adaptive immune responses. On activation NK cells release IFN-γ.

Resistin
Promote TNF and IL-6 release.
Anakinra: [9][10][11] It is recombinant form of nonglycosylated human IL-1 receptor antagonist expressed in Escherichia coli. Natural IL-1 receptor antagonist is produced by macrophages and activated monocytes in response to various inß ammatory stimuli. Anakinra competitively binds to both type-I and type-II IL-1 receptors, at least partially blocking cellular responses mediated by IL-1-α and IL-1-β. It has a binding afÞ nity similar to IL-1, but it lacks IL-1 agonist activity. Its daily dose is 100 mg/day subcutaneously (SC). Prior hypersensitivity to anakinra or E-coli derived proteins and active infection are the important contraindications with the use of anakinra.
Common adverse reactions reported with it are headache, nausea, diarrhea, sinusitis, erythema, ecchymosis, pruritis at injection site, influenza like symptoms, production of anti-anakinra antibodies, neutropenia, cardiopulmonary arrest and serious infections. Live vaccines should not be administered concurrently with anakinra. It should be used with caution in patients with neutropenia, immuno-suppression, moderate to severe renal impairment, pregnancy or breastfeeding period, and concomitant use of TNF blocking agents.
Clinical Trials: In a dosage ranging multi-center placebo controlled trial patients of Rheumatic arthritis (RA) on 1-2 mg/kg/day of anakinra with MTX 15-25 mg/week achieved more ACR (American College of Rheumatology preliminary criteria for improvement) 20 response than MTX alone at 12 weeks. [12] In another study with 100 mg/day of anakinra in combination with MTX showed more efÞ cacy in retarding radiographic progression than MTX alone. [13] In a two-year prospective, in part retrospective, cohort study drug survival was 78%, 54%, and 14% after 3, 6 and 24 months, respectively. [14] However, National institute of clinical excellence of the united kingdom recommended its use in patients who are not responding to anti-TNFα therapy alone or in patients with juvenile idiopathic arthritis. [12,15] In a clinical trial on 419 patients with moderate-tosevere active RA, who were receiving MTX for six consecutive months, the ACR20 responses at week 12 in the 5 active treatment (0.04, 0.1, 0.4, 1.0, or 2.0 mg/kg of anakinra) plus MTX groups demonstrated a statistically signiÞ cant (P = 0.001) dose-response relationship compared with the ACR20 response in the placebo plus MTX group. [16] In another trial 218 patients received subcutaneous injections of anakinra (30, 75, or 150 mg) once daily. [17] The ACR20 response was 51% at week 24 and 46% at week 48 and this effect was consistent across all dose groups. Anakinra was welltolerated for 76 weeks. [17] Role of anakinra in chronic infantile neurological cutaneous and articular (CINCA) syndrome with a novel missense mutation in exon 4 of the CIAS1 gene (unresponsive to several treatments including prednisolone, immunosuppressants, DMARDs and TNF-blocker inß iximab) has been documented. [18] Anakinra, has a positive impact on both function and quality of life of the patients with RA. [19] However, further clinical studies are needed to establish the additive beneÞ ts of the combination of TNF-α blockade plus IL-1 receptor antagonism in RA.
Abatacept: [20] It is a recombinant fusion protein comprising of the extra-cellular domain of human CTLA4 (cytotoxic T-lymphocyte antigen 4) and a fragment of the Fc domain of human IgG1, which has been modiÞ ed to prevent complement Þ xation. It modulates the CD80 or CD86-CD28 co-stimulatory signal required for full T-cell activation. It is given in a dose of 10 mg/kg by IV infusion (three doses at the interval of two weeks, followed by infusion after every four weeks).
Clinical Trials: In a Phase IIb multi-center international study in RA patients with inadequate response to MTX, ACR 20 response was achieved in 60%, 41.9% and 35.3% patients with abatacept in a dose of 10 mg/kg, 2 mg/kg and placebo respectively after 6 months of the treatment as add on therapy to MTX. [12] In a randomized double blind phase-III trial on patients with active RA refractory to anti-TNF-α therapy, abatacept therapy for 6 months, in addition to at least one DMRDs (disease modifying antirheumatic drugs) produced ACR 20 response rate of 50.4% as compare to 19.5% in the placebo group (P<0.001). [20] At six months, signiÞ cantly more patients in the abatacept group than in the placebo group had a clinically meaningful improvement in physical function (47.3 percent vs 23.3 percent, P<0.001) with incidence of serious infections as 2.3% in both the groups. [20] Alefacept: [2] Alefacept is approved by US food and drug administration (FDA) in January 2003 for treatment in adult patients with moderate to severe chronic plaque psoriasis, who are candidates for systemic therapy or phototherapy. It is a bivalent recombinant fusion protein composed of the Þ rst extra-cellular domain lymphocyte function antigen 3 (LFA-3), fused to the hinge CH2 domain and CH3 domain of human IgG1. The LFA-3 portion of alefacept binds to CD2 receptors on T-cells, thereby blocking their natural interaction with LFA-3. The IgG1 portion of alefacept binds to FcγR on natural killer cells to induce T-cell apoptosis. Its dose is 10-15 mg IM (intramuscular) weekly or 7.5 mg IV (intravenous) weekly and a 12 week course is recommended.
Clinical Trials: In a double-blind RCT (randomized clinical trial) two 12-week courses of once-weekly IV alefacept 7.5 mg and placebo were given and patients were followed for 12 weeks after each course. [21] SigniÞ cantly more patients achieved greater reduction in the PASI (psoriasis area and severity index) than placebo both after Þ rst and second course of therapy. [21] In an international, double-blind, placebocontrolled, RCT, 507 patients with chronic plaque psoriasis, were randomized to receive either 10 mg or 15 mg of alefacept once weekly for 12 weeks, followed by 12 weeks of observation. [22] Thirty three percent and 28% patients achieved 75% reduction in PASI, two weeks after the last dose in 15 mg and 10 mg group respectively. [22] The selective immunomodulatory effect of alefacept against potentially pathogenic T-cells is associated with maintenance of immune function to Þ ght infection and response to vaccinations. [23] It has been reported that it reduces total lymphocyte count and CD4+ and CD8+cell counts. Hence, it is recommended to monitor CD4 counts weekly during therapy.
Efalizumab: [2,3] It is approved by the US FDA in October 2003 for the treatment of psoriasis. It is a recombinant humanized monoclonal IgG1 antibody that binds to CD11a, subunit of leukocyte function-associated antigen 1 (LeFA-1). It interferes with the interaction between LeFA-1 and intercellular adhesion molecules (ICAM-1). By destabilizing the binding of APCs (antigen presenting cells) and T-cells, it reduces the efÞ ciency of initial T-cell activation in lymph nodes. It interferes with the secondary activation of memory-effector T-cells in the target tissues. Its dose is 1 mg/kg (max 200mg) weekly, subcutaneous injection for 12 weeks, following a Þ rst conditioning dose of 0.7 mg/kg.

Clinical trials:
In four large phase III studies in 2000 patients with moderate-to-severe chronic plaque psoriasis, efalizumab (1 mg/kg weekly) produced PASI 75 (>75% in reduction in baseline PASI score) in 27% of patients as compared to 4% patients in placebo group by week 12. [24][25][26][27] Continuation of therapy beyond 12 weeks increased the response rate further in efalizumab group. The relapse of psoriasis was evident after 2 months of discontinuation of therapy with rebound in approximately 5% of the patients, as deÞ ned by ß aring>125% of baseline. Acute flu-like symptoms including headache, chills, fever, nausea and myalgia, an exacerbation of psoriasis after discontinuation of therapy, autoimmune hemolytic anemia, and thrombocytopenia are the common adverse events reported with its use. [2] Rituximab: [28][29][30] Rituximab is a speciÞ c mouse and human chimeric monoclonal antibody. This IgG1 has a long half-life of 76 to 200h and targets the CD20 antigen. The CD 20 antigen is present on pre-B, immature and mature B cells and is important for B-cell activation and proliferation. Binding of rituximab to CD 20 results in complement and antibody-dependent cyto-toxicity (apoptosis) of cells exhibiting this antigen. CD20 is not expressed on stem cells and plasma cells. Hence, depletion of the B-cell subpopulation is transient and does not affect immunoglobulin synthesis. Normal levels of total serum IgG are maintained and antibody levels against HSV (herpes simplex virus) 1/2 and VZV (varicella-zoster virus) are not signiÞ cantly affected after rituximab treatment. Rituximab was the Þ rst therapeutic antibody approved for treating cancer. A supplemental Biological License Application (sBLA) was approved for it in April 2001, adding several new uses related to B-cell non-Hodgkin's lymphoma. In 2006 rituximab in combination with MTX is approved for adult patients with moderately-to-severely active RA, who have had an inadequate response to one or more TNF antagonist therapies.
Clinical Studies: REFLEX, a Phase III clinical study of Rituximab in RA, met its primary endpoint and underpins the FDA's approval. [29] It is also being evaluated in Phase II/III clinical trials for primary progressive and relapsed remitting multiple sclerosis, ANCA-associated vasculitis, systemic lupus erythematosus. [29] In an open label study Rituximab in combination with cyclophosphamide and prednisolone in Þ ve patients of refractory RA showed dramatic and sustained clinical improvement. [31] In a phase III multi-center double blind trial on 161 patients Rituximab in combination with MTX or cyclophosphamide showed more efÞ cacy than MTX alone. [31] Rituximab is the Þ rst treatment for RA that selectively targets immune cells known as CD20-positive B-cells.
Atlizumab: [12,[32][33][34][35] It is a humanized anti-IL-6 receptor monoclonal antibody. It is efÞ cacious in management of RA in a dose of 2-8 mg\kg\dose IV once every two weeks. It is a well-tolerated drug without any increase in antinuclear, anti-DNA or anti-atlizumab antibody. However, increase in blood cholesterol levels has been reported after its use for 24 weeks. Clinical Trial: [12] In a phase I/II double blind RTC, atlizumab (5 mg/kg with MTX single dose) produced ACR20 response in 50% patients of RA as compare to placebo at week two. Improvement was maintained for eight weeks. In a multi-center double blind RCT, 78%, 57% and 11% RA patients achieved ACR20 response after three months of therapy with atlizumab 8 mg/kg, 4 mg/kg and placebo respectively.
Tocilizumab: [36,37] It is a recombinant humanized anti-IL-6R monoclonal antibody. Phase I and II studies of tocilizumab in children with JIA (juvenile idiopathic arthritis), showed significant improvement in the typical symptoms of inflammation and laboratory abnormalities.
AMG714 (previously HuMax-IL15): [6] IL-15 enhances synovial T-cell proliferation and cytokine release and optimizes cognate interactions between T cells and macrophages. IL-15 induces synovial neutrophil activation, granule release from natural-killer cells, activation and migration of endothelial cells and prevents fibroblast apoptosis. AMG714, a fully human IgG1 monoclonal anti-IL-15 antibody, neutralizes soluble and membrane-bound IL-15 in vitro.
Clinical Trials: In a 12-week, dose-ascending, placebocontrolled study, AMG714 (0.5-8 mg/kg) produced signiÞ cant improvement in disease activity in RA patients as compared to placebo. [38] In another dose-Þ nding study 60% of recipients receiving higher doses of AMG714 (160 mg or 240 mg) showed signiÞ cant improvement as compared to lower doses. [6] No signiÞ cant alterations in the levels of circulating leukocyte subsets, including naturalkiller cells and CD8 + memory T cells, were observed.
Alternate approaches to targeting IL-15 include the use of soluble IL-15R-α-derived proteins or antagonistic IL-15-Fc fusion proteins. CRB-15, an IL-15-Fc fusion protein, suppressed delayed-type hypersensitivity and allograft transplant rejection in rodent models. [39] Vitaxin (MEDI-522): [4] It is humanized monoclonal IgG1 antibody that binds to a conformational epitope formed by both the integrin alpha V and beta 3 subunits. Alpha V and beta 3 integrin (vitronectin receptor) is expressed in low levels in most of the normal tissues (intestinal, vascular and smooth muscle cells) and in high levels in bone, mid-menstrual cycle endometrium, placenta, inflammatory sites and invasive tumors. Vitronectin receptors have major role in osteoclast mediated bone resorption, angiogenesis and macrophage dependent inflammation. In RA, activated macrophages are increased in both subchondrial bone and inflamed synovial tissue; whereas, osteoclasts are increased in subchondrial bone at the site of bone erosion and resorption. Hence, antagonists of alpha V and beta 3 integrin have a potential role in the therapeutics of RA. Echistatin is another molecule under development.

Anti Interleukins in Critical Pathological Conditions
Bronchial Asthma: [40,41] Accumulation of eosinophils in the bronchial mucosa of individuals with asthma is considered to be a central event in the pathogenesis of asthma. Mepolizumab is a humanized anti-IL-5 monoclonal antibody. In animal models, airway eosinophil recruitment and airway hyperresponsiveness in response to allergen challenge are reduced by speciÞ c targeting of IL-5. However mepolizumab treatment does not appear to add signiÞ cant clinical beneÞ t in patients with asthma with persistent symptoms despite inhaled corticosteroid therapy.
In a clinically relevant model of chronic allergic asthma in mice neutralizing antibodies to IL-13 effectively suppressed eosinophil recruitment and accumulation of chronic inflammatory cells in the airways. It also partially suppressed changes of airway wall remodeling, including goblet cell hyperplasia/metaplasia and subepithelial Þ brosis, but had limited ability to inhibit airway hyperreactivity (AHR). However, treatment with anti-IFN-markedly suppressed AHR.
Crohn's disease :[43] Crohn's disease is characterized by increased production of IL-12 by antigen-presenting cells in intestinal tissue and interferon-γ and TNF-α by intestinal lymphocytes and macrophages. Anti-IL-12 monoclonal antibody therapy induces clinical response and remission in patients with active Crohn's disease . In a clinical trial 79 patients with active Crohn's disease were randomized to receive seven weekly subcutaneous injections of anti-IL-12 human monoclonal antibody (1 mg/kg body weight or 3 mg/kg body weight) or placebo, in an interrupted (one-month span after Þ rst dose, n = 40) or continuous regimen (n = 39). Continuous weekly therapy with 3-mg/kg anti-interleukin-12 resulted in signiÞ cantly higher response rates at seven weeks compared with placebo.
Acute respiratory distress syndrome: [42] Anti-IL8 antibodies are high in patients of acute respiratory distress syndrome and molecules targeting them can be of potential help in these patients. Septic shock: [44] Patients with septic shock have T cell hyporesponsiveness and immune suppression, which, if persistent, are associated with increased mortality. In the murine cecal ligation and puncture (CLP) model of sepsis, it has been reported that early treatment with the anti-inß ammatory cytokine IL-10 delays the onset of irreversible shock.

Cytokine targets in preclinical development
Interferon (IFN)-γ blockade using a polyclonal anti-IFN-γ antibody has been shown to produce suppression of RA disease activity in a small RCT. [45] Suppression of IL-17, anti-IL-18 antibody and the inhibition of IL-18 secretion via inhibition of caspase 1or antagonism of the proinß ammatory purinergic receptor P2X7 are other approaches under development. [6,[46][47][48][49] Several adipokines (adeponectin) have been shown to modulates TNF-induced inß ammation. [50,51] Resistin is a cysteine-rich secretory protein originally implicated in insulin resistance and atherogenesis. It is expressed at high levels in RA synovial tissues, can promote TNF and IL-6 release. [6,51] Therapeutic targeting of resistin offers the potential to modify not only local inß ammation, but also the systemic insulin resistance that is characteristic of RA and other chronic inß ammatory conditions. A number of biological agents are being studied actively at the present time and it is hoped that they may generate novel therapies for and a greater understanding of immuno-inß ammatory diseases (Table-3). The future for immunomodulatory intervention in rheumatology looks very promising. Greater understanding of the intricacies of the immune response that underlie the disease should continue to yield viable, speciÞ c targets for novel therapies. Advances in biopharmaceuticals should generate treatments that maximize efÞ cacy while minimizing toxicity. These novel therapeutic agents could give new hopes to the clinician truly to modify the disease and achieve tangible improvements in the lives of the patients.