Cytokine Therapies: Novel Approaches for Clinical Indications

Web Sites

The International Society for Interferon and Cytokine Research
A research society dedicated to cytokine research.

The Society for Leukocyte Biology
A research society devoted to the study of the cellular biology of leukocytes and cytokines.

Interferon-Stimulated Gene Database

FDA's Critical Path Initiative
An effort to facilitate the modernization of the scientific process through which a potential human drug, biological product, or medical device is transformed from a discovery into a therapeutic product.

Books

Holland JF, Frei E, eds. 2003. Cancer Medicine, 6th Edition. BC Decker, Hamilton, Ontario.

Holland SM, ed. 2001. Cytokine Therapeutics in Infectious Diseases. Lippincott Williams & Wilkins, Philadelphia.

Ransohoff RM, Benveniste EN. 2006. Cytokines and the CNS. CRC Press, London.

Journal Articles

Raymond P. Donnelly

Donnelly RP, Sheikh F, Kotenko SV, Dickensheets H. 2004. The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain. J. Leukoc. Biol. 76: 314-321.

Junttila IS, Mizukami K, Dickensheets H, et al. 2008. Tuning sensitivity to IL-4 and IL-13: differential expression of IL-4R alpha, IL-13R alpha-1 and the common gamma chain regulates relative cytokine sensitivity. J. Exp. Med. 205: 2595-2608.

Kotenko SV, Gallagher G, Baurin VV, Lewis-Antes A, Shen M, Shah NK, Langer JA, Sheikh F, Dickensheets H, Donnelly RP. 2003. Interferon-lambdas mediate anti-viral protection through a distinct class II cytokine receptor complex. Nat. Immunol. 4: 69-77.

Maher SG, Sheikh F, Scarzello AJ, et al. 2008. IFN-α and IFN-λ differ in their antiproliferative effects and duration of JAK/STAT signaling activity. Cancer Biol. Ther. 7: 1109-1115.

Yoon SI, Logsdon NJ, Sheikh F, et al. 2006. Conformational changes mediate interleukin-10 receptor 2 (IL-10R2) binding to IL-10 and assembly of the signaling complex. J. Biol. Chem. 281: 35088-35096.

Michael T. Lotze

Lee JJ, Lotze MT. 2009. Molecular basis of metastasis. N. Engl. J. Med. 360: 1679.

Lotfi R, Schrezenmeier H, Lotze MT. 2009. Immunotherapy for cancer: promoting innate immunity. Front. Biosci. 14: 818-832.

Moschos SJ, Mandic M, Kirkwood JM, et al. 2008. Focus on FOCIS: interleukin 2 treatment associated autoimmunity. Clin. Immunol. 127: 123-129.

Romo de Vivar Chavez A, de Vera ME, Liang X, Lotze MT. 2009. The biology of interleukin-2 efficacy in the treatment of patients with renal cell carcinoma. Med. Oncol. 26 Suppl 1: 3-12.

Sabatino M., Kim-Schulze S, Panelli MC, et al. 2009. Serum vascular endothelial growth factor (VEGF) and fibronectin predicts clinical response to high-dose interleukin-2 (IL-2) therapy. J. Clin. Oncol. In press.

Steven M. Holland

Haerynck F, Holland SM, Rosenzweig SD, et al. 2008. Disseminated Mycobacterium avium infection in a patient with a novel mutation in the interleukin-12 receptor-beta1 chain. J. Pediatr. 153: 721-722.

Paulson ML, Freeman AF, Holland SM. 2008. Hyper IgE syndrome: an update on clinical aspects and the role of signal transducer and activator of transcription 3. Curr. Opin. Allergy Clin. Immunol. 8: 527-533.

Julian A. Symons

Brideau-Andersen AD, Huang X, Sun SC, et al. 2007. Directed evolution of gene-shuffled IFN-alpha molecules with activity profiles tailored for treatment of chronic viral diseases. Proc. Natl. Acad. Sci. USA 104: 8269-8274.

Crystal T. Mackall

Capitini CM, Cooper LJ, Egeler RM, et al. 2009. Highlights of the First International "Immunotherapy in Pediatric Oncology: Progress and Challenges" Meeting. J. Pediatr. Hematol. Oncol. 31: 227-244.

Dean RM, Fry T, Mackall C, et al. 2008. Association of serum interleukin-7 levels with the development of acute graft-versus-host disease. J. Clin. Oncol. 26: 5735-5741.

Guimond M, Veenstra RG, Grindler DJ, et al. 2009. Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4⁺ T cells. Nat. Immunol. 10: 149-157.

Sportès C, Hakim FT, Memon SA, et al. 2008. Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets. J. Exp. Med. 205: 1701-1714.

William E. Carson III

Gowda A, Roda J, Hussain SR, et al. 2008. IL-21 mediates apoptosis through up-regulation of the BH3 family member BIM and enhances both direct and antibody-dependent cellular cytotoxicity in primary chronic lymphocytic leukemia cells in vitro. Blood 111: 4723-4730.

Mani A, Roda J, Young D, et al. 2008. A phase II trial of trastuzumab in combination with low-dose interleukin-2 (IL-2) in patients (PTS) with metastatic breast cancer (MBC) who have previously failed trastuzumab. Breast Cancer Res. Treat. Dec 3. [Epub ahead of print]

Raig ET, Jones NB, Varker KA, et al. 2008. VEGF secretion is inhibited by interferon-alpha in several melanoma cell lines. J. Interferon Cytokine Res. 28: 553-561.

Skak K, Kragh M, Hausman D, et al. 2008. Interleukin 21: combination strategies for cancer therapy. Nat. Rev. Drug Discov. 7: 231-240.

Zimmerer JM, Lesinski GB, Ruppert AS, et al. 2008. Gene expression profiling reveals similarities between the in vitro and in vivo responses of immune effector cells to IFN-alpha. Clin. Cancer Res. 14: 5900-5906.

Ernest C. Borden

Borden EC. 2007. Augmentation of effects of interferon-stimulated genes by reversal of epigenetic silencing: potential application to melanoma. Cytokine Growth Factor Rev. 18: 491-501.

Borden EC, Sen GC, Uze G, et al. 2007. Interferons at age 50: past, current and future impact on biomedicine. Nat. Rev. Drug Discov. 6: 975-990.

Cheriyath V, Glaser KB, Waring JF, et al. 2007. G1P3, an IFN-induced survival factor, antagonizes TRAIL-induced apoptosis in human myeloma cells. J. Clin. Invest. 117: 3107-3117.

Taylor KL, Leaman DW, Grane R, et al. 2008. Identification of interferon-beta-stimulated genes that inhibit angiogenesis in vitro. J. Interferon Cytokine Res. 28: 733-740.

Ahmad A. Tarhini

Kirkwood JM, Tarhini AA. 2009. Biomarkers of therapeutic response in melanoma and renal cell carcinoma: potential inroads to improved immunotherapy. J. Clin. Oncol. Apr 13. [Epub ahead of print]

Kirkwood JM, Tawbi HA, Tarhini AA, Moschos SJ. 2009. Does pegylated interferon alpha-2b confer additional benefit in the adjuvant treatment of high-risk melanoma? Nat. Clin. Pract. Oncol. 6: 70-71.

Kirkwood JM, Tarhini AA, Panelli MC, et al. 2008. Next generation of immunotherapy for melanoma. J. Clin. Oncol. 26: 3445-3455.

Tarhini AA, Kirkwood JM, Gooding WE, et al. 2008. A phase 2 trial of sequential temozolomide chemotherapy followed by high-dose interleukin 2 immunotherapy for metastatic melanoma. Cancer 113: 1632-1640.

Tarhini AA, Stuckert J, Lee S, et al. 2008. Prognostic significance of serum S100B protein in high-risk surgically resected melanoma patients participating in Intergroup Trial ECOG 1694. J. Clin. Oncol. 27: 38-44.

Abraham Abuchowski

Abuchowski A, McCoy JR, Palczuk NC, et al. 1977. Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. J. Biol. Chem. 252: 3582-3586.

Kenneth A. Dawson / Anna Salvati

Dawson KA, Salvati A, Lynch I. 2009. Nanotoxicology: nanoparticles reconstruct lipids. Nat. Nanotechnol. 4: 84-85.

Larry W. Moreland

Kremer JM, Genant HK, Moreland LW, et al. 2008. Results of a two-year followup study of patients with rheumatoid arthritis who received a combination of abatacept and methotrexate. Arthritis Rheum. 58: 953-963.

Mease PJ, Reich K; Alefacept in Psoriatic Arthritis Study Group. 2009. Alefacept with methotrexate for treatment of psoriatic arthritis: open-label extension of a randomized, double-blind, placebo-controlled study. J. Am. Acad. Dermatol. 60: 402-411.

Moreland LW, Curtis JR. 2008. Systemic nonarticular manifestations of rheumatoid arthritis: focus on inflammatory mechanisms. Semin. Arthritis Rheum. Nov 18. [Epub ahead of print]

Saag KG, Teng GG, Patkar NM, et al; American College of Rheumatology. 2008. American College of Rheumatology 2008 recommendations for the use of nonbiologic and biologic disease-modifying antirheumatic drugs in rheumatoid arthritis. Arthritis Rheum. 59: 762-784.

Lothar Steidler

Frossard CP, Steidler L, Eigenmann PA. 2007. Oral administration of an IL-10-secreting Lactococcus lactis strain prevents food-induced IgE sensitization. J. Allergy Clin. Immunol. 119: 952-959.

Neirynck S, Steidler L. 2006. Delivery of therapeutic proteins through Lactococcus lactis. Biotechnol. Genet. Eng. Rev. 22: 253-266.

Steidler L, Rottiers P. 2006. Therapeutic drug delivery by genetically modified Lactococcus lactis. Ann. NY Acad Sci. 1072: 176-186.

Waeytens A, Ferdinande L, Neirynck S, et al. 2008. Paracellular entry of interleukin-10 producing Lactococcus lactis in inflamed intestinal mucosa in mice. Inflamm. Bowel Dis. 14: 471-479.

Richard Ransohoff

Rudick RA, Ransohoff RM. 2008. Biomarkers for interferon response in MS: are we there yet? Neurology 70: 1069-1070.

Raphaela Goldbach-Mansky

Glaser RL, Goldbach-Mansky R. 2008. The spectrum of monogenic autoinflammatory syndromes: understanding disease mechanisms and use of targeted therapies. Curr. Allergy Asthma Rep. 8: 288-298.

Goldbach-Mansky R, Dailey NJ, Canna SW,et al. 2006. Neonatal-onset multisystem inflammatory disease responsive to interleukin-1beta inhibition. N. Engl. J. Med. 355: 581-592.

Goldbach-Mansky R, Shroff SD, Wilson M, et al. 2008. A pilot study to evaluate the safety and efficacy of the long-acting interleukin-1 inhibitor rilonacept (interleukin-1 Trap) in patients with familial cold autoinflammatory syndrome. Arthritis Rheum. 58: 2432-2442.

Ryan JG, Goldbach-Mansky R. 2008. The spectrum of autoinflammatory diseases: recent bench to bedside observations. Curr. Opin. Rheumatol. 20: 66-75.

M. Virginia Pascual

Bosca I, Coret F, Valero C, et al. 2008. Effect of relapses over early progression of disability in multiple sclerosis patients treated with beta-interferon. Mult. Scler. 14: 636-639.

Pascual V, Allantaz F, Patel P, et al. 2008. How the study of children with rheumatic diseases identified interferon-alpha and interleukin-1 as novel therapeutic targets. Immunol. Rev. 223: 39-59.

Rönnblom L, Pascual V. 2008. The innate immune system in SLE: type I interferons and dendritic cells. Lupus 17: 394-399.

Neil Stahl

Hoffman HM, Throne ML, Amar NJ, et al. 2008. Efficacy and safety of rilonacept (interleukin-1 Trap) in patients with cryopyrin-associated periodic syndromes: results from two sequential placebo-controlled studies. Arthritis Rheum. 58: 2443-2452.

Allen Radin

Radin AI, Kim HT, Grant BW, et al.; Eastern Cooperative Oncology Group. 2003. Phase II study of alpha2 interferon in the treatment of the chronic myeloproliferative disorders (E5487): a trial of the Eastern Cooperative Oncology Group. Cancer 98: 100-109.

Radin AI, Buckley P, Duffy TP. 1991. Interferon therapy for agnogenic myeloid metaplasia complicated by immune hemolytic anemia. Hematol. Pathol. 5: 83-88.

Howard A. Young

Hodge DL, Yang J, Buschman MD, et al. 2009. IL-l5 enhances proteasomal degradation of Bid in normal lymphocytes: implications for large granular lymphocyte leukemias. Cancer Res. 69: 3986-3994.

Rodriguez-Galan MC, Reynolds D, Graciela Correa S, et al. 2009. Co-expression of IL-18 strongly attenuates IL-12-induced systemic toxicity through a rapid induction of IL-10 without affecting its anti-tumor capacity. J. Immunol. in press (July 1).

Young HA, Ortaldo J 2006. Cytokines as critical co-stimulatory molecules in modulating the immune response of natural killer cells. Cell Res. 16: 20-24.

Sponsors

Presented by:

This activity is supported by an educational donation provided by:

• Abbott Laboratories
• Amgen Inc.
• Celgene Corporation
• Centocor Research and Development, Inc.

This conference has been made possible through the generous support of the following organizations:

• BioLegend
• PBL InterferonSource

Can better animal models be developed to study the action of cytokines?

What can be learned from "human translational data" that comes from studying populations that have inborn cytokine defects?

How can the scientist-clinician collaboration be improved so that information flows more freely from bedside to bench in addition to the reverse direction?

What is the role of the microbiome in modulating the immune system?

How can cytokine formulations be improved so that a one-size-fits-all approach doesn't have to be used?

How can cytokines or cytokine antagonists be delivered more directly to the tissues or organs where they would be most effective?

Can gene therapy be used for site-specific delivery?

How can researchers enhance the half-life of cytokines without increasing the risk of adverse effects?

Is development of antibodies to a cytokine always a bad thing? How can the immunogenicity of cytokines be minimized?

How can sample collection and cytokine measurements be better standardized so that researchers can compare results from clinical studies conducted at different sites?

Highlights

• Adding polyethylene glycol to molecules can greatly improve their half-life in the body and enhance other pharmacologically important properties such as solubility.
• Nanoparticles might facilitate targeted delivery of cytokines.
• Cytokines can be delivered to the gut using genetically engineered bacteria, providing a viable delivery mechanism for the treatment of inflammatory bowel disease.
• Targeted delivery can be enhanced when cytokines are attached to PEG and targeting antibodies via a "dock-and-lock" system.

Introduction to delivery and pharmacokinetic problems

Though cytokine therapeutics have defined biological activities that could contribute to disease amelioration or cure, they pose two major challenges: first, they have very rapid in vivo kinetics, and second, they can induce a multitude of effects, given the broad expression of cytokine receptors on many different cells in the body. Cytokines are rapidly eliminated, both by receptor-mediated uptake as well as by enzymatic inactivation. The very short half-lives of these agents significantly reduces their efficacy. Increasingly the stability of these biological agents would allow these cytokine drugs to be given less frequently and at lower doses.

Because these therapies are identical to naturally occurring cytokines, they have multiple effects on cells of the immune system, as well as non-immune system cells, leading to unwanted toxicities. A more selective means of drug delivery might help reduce many of the toxic and undesirable side effects of these agents.

Researchers have developed several mechanisms for extending the half-life of cytokines and targeting them to specific organs or regions in the body.

Polyethylene glycol (PEG) extends the half-life of cytokines

The addition of polyethylene glycol (PEG) to proteins can greatly improve their half-life in the body and enhance other pharmacologically important properties such as their solubility. Pegylation, as it is now called, was pioneered by Abraham Abuchowski of Prolong Pharmaceuticals, while a PhD student at Rutgers University. It is now a standard approach to prolonging in vivo activity of biological therapeutics in the pharmaceutical industry.

Pegylation has a number of benefits. It is non-toxic and enhances the circulating life of drugs thereby reducing the number of doses and the frequency of dosing. Adding PEG causes molecules to become more water-soluble because PEG binds water readily, creating a hydrodynamic shell that doesn't seem to interfere with binding to receptors.

Numerous methods have been developed to attach PEG to protein and non-protein molecules. However, Abuchowski noted that PEG must be added in a way as to preserve the biological activity of the protein drug, making the addition of PEG to a protein as much an art as a science.

Nanoparticles for targeted delivery of cytokines

Nanoparticles are another promising innovation that may help improve the bioactivity of cytokines, said Anna Salvati of the Centre for BioNano Interactions School of Chemistry and Chemical Biology, University College Dublin. These synthetic spheres are just tens of nanometers in diameter.

Nanoparticles represent a novel way to introduce bioactive proteins into compartments in the body that are difficult to reach. Due to their small size, they can diffuse through the blood brain barrier, enter cells, and even enter the nuclei of cells. Placing drugs into nanoparticles protects them from degradation, thereby increasing the half-life, permitting lower doses, and decreases potential toxicity.

The challenge is to create particles that can transport drugs to targets where they will release their contents. These researchers created nanosized beads and coated them with proteins that can selectively target and bind to cell-surface proteins or be taken up by cells where they interact with intracellular proteins and deliver their cargo.

Cytokine delivery via food-grade bacteria

One novel delivery system enables oral delivery of therapeutic peptides and proteins via genetically engineered bacteria. Pioneered by Lothar Steidler and his team at ActoGeniX NV in Belgium, these noninvasive, noncolonizing food-grade bacteria secrete bioactive proteins and peptides into the gastrointestinal tract. These ActoBiotics are a type of bacteria called Lactococcus lactis that are engineered in such a way as to prevent survival outside of the body.

The ActoBiotics can deliver proteins in the gastrointestinal tract.

The company has used this novel delivery method to deliver recombinant human IL-10 to the gut for the successful treatment of inflammatory bowel disease (IBD). This cytokine was tested in the past for treatment of IBD, but it was given intravenously, and with its short half life, probably did not reach the critical target area, the mucosal lining of the gut. In the initial studies performed in the mid-1990s, parenteral administration of IL-10 at high concentrations proved to be toxic but the use of Lactococcus to deliver IL-10 orally now provides an improved method to deliver this cytokine to the gut, where it suppresses inflammation.

Improved targeting and bioavailability using the dock-and-lock (DNL) method

The dock-and-lock method uses a dimerization and docking domain module (DDD2) and an anchoring module (AD2) locked together by a disulfide bond.

Ken Chang of Immunomedics described a method that can be adapted to deliver cytokines via site-specific conjugation of molecules using naturally occurring protein-protein interactions. The "dock-and-lock" method allows researchers to design and construct novel compounds that are multispecific, multifunctional, and multivalent. It can be used to attach polyethylene glycol (PEG) molecules to cytokines or attach cytokines to antibodies for targeted delivery to specific tissues in the body.

Highlights

• IL-2 induces complete remission in 8% to 10% of renal cell carcinoma cancer patients.
• Cytokines activate several pathways that help the body destroy tumor cells through apoptosis and inhibit formation of new blood vessels around the tumor.
• Researchers are gaining a better understanding of how cytokines activate downstream genes that in turn kill tumor cells.

A fresh look at IL-2

The antiproliferative properties of cytokines are mediated via a number of mechanisms. They can be anti-angiogenic, activate immune system cells, and induce expression of genes that are antiproliferative through mechanisms not yet fully understood. Cytokines can also induce apoptosis, a type of programmed cell death. IL-2, for example, can activate cytotoxic T-lymphocytes (CTLs) that secrete a protein called perforin. Perforin pokes holes in the cell membrane and allows preformed proteins called granzymes to enter and induce apoptosis.

IL-2 raised great hopes in the 1990s of providing a treatment or perhaps even a cure for cancer, said Michael T. Lotze of the University of Pittsburgh Cancer Institute. The cytokine causes a durable 8% to 10% remission rate in patients with melanoma and renal cell carcinoma.

In many ways IL-2 is a success story. However, the small percentage of patients in which it demonstrates success is not sufficiently robust. It is thus important to discover the mechanism by which this therapy works in the small subset of responders. A recent study by Howard Kaufman's lab looked at responsiveness and non-responsiveness to IL-2 therapy and found that individuals with high levels of the growth factor VEGF prior to therapy were less likely to respond compared to patients with low concentrations of VEGF in their bloodstream. Similarly fibronectin was high in non-responders but low in responders. These levels could perhaps be used to prospectively identify patients who will respond to IL-2.

Digesting the situation

Another way to understand the low rate of success with IL-2 therapy is to examine it in the context of the cell's response to growth factors and stress. Cancer is essentially a metabolic disorder because the body's metabolism supports tumor formation, said Lotze. The immune system is complicit because it tolerates formation of the tumor and surrounding new blood vessels. Researchers have known for some time that apoptosis is disabled during tumor formation. Lotze believes that a competing cell death process called autophagy occurs when cells are under survival stress. He further noted that VEGF and HMGB1 are important switches, modulating apoptosis and autophagy.

To improve the remission rate of IL-2, a better understanding of how IL-2 works is essential. In addition to induction of apoptosis, IL-2 activates NK cells and macrophages, promotes Th1 cell activity, and induces proliferation of B cells. IL-2 therapy for cancer is also associated with induction of autoimmunity, which, in the setting of IFN-α treatment appears correlated with clinical benefit. "The failure or success of our cytokines is tied closely to our understanding of the science," said Lotze.

Finding the mechanism of cytokine action

Ahmad Tarhini of the University of Pittsburgh Cancer Institute described the clinical and immunological basis of IFN-α therapy in melanoma patients. Melanoma is a highly curable cancer if diagnosed and treated in the early stages of development, but is usually fatal if allowed to metastasize. Some spontaneous regressions occur, suggesting that the innate immune system can fight the disease, perhaps through the involvement of T cells, macrophages and NK cells. The presence of T-cell infiltrates is a positive prognostic marker and the presence of T-cell infiltrates within regional nodal metastasis predict benefit from IFN-α2 therapy.

Tarhini and his colleagues are attempting to discover biomarkers that indicate who will respond effectively to IFN-α treatment. The researchers found that patients with high pretreatment levels of certain pro-inflammatory cytokines (IL-1-α, β; MIP-1-α, β; IL-6; TNF-α) were more likely to have relapse-free survival. Survival after IFN-α treatment was greater in patients who developed autoimmunity (often seen as vitiligo or autoimmune thyroiditis in melanoma patients).

Ernest Borden of the Cleveland Clinic underscored the need to understand the mechanisms that occur downstream of interferon binding to its cell surface receptor. One common downstream signaling pathway is JAK/STAT1 phosphorylation. A drug called stibogluconate (SSG), a small molecular weight molecule, can enhance JAK/STAT1 phosphorylation and increase the activity of therapeutic cytokines such as IFN-α. In combination with IFN-α2, stibogluconate is now being studied in a phase 1 trial in melanoma patients.

Borden indicated that to implement the therapeutic potential of interferons, we must gain a better understanding of the regulation and function of the more than 300 genes induced by this cytokine. Some of these genes are pro-apoptotic, such as TRAIL and XAF1 whereas genes such as G1P3 (ISG 6-16) can silence apoptosis. Many other IFN-inducible genes are immune-modulating or anti-angiogenic.

Understanding the function of interferon-stimulated genes (ISGs) can help researchers define and overcome resistance mechanisms and drug-related toxicities, and lead to improved ways to enhance anti-tumor activity through modification of signaling. "One needs to be open," said Borden, "to new ideas as to how to utilize cytokines as to their ultimate impact."

IL-21 in addition to monoclonal antibodies for the treatment of cancer

A relative newcomer in the cytokine field, IL-21, is also being tested as a cancer therapy agent. This cytokine is secreted by activated CD4⁺ T cells and Natural Killer (NK) cells. It helps regulate immunoglobulin production and isotype switching by B cells, and has activating effects on macrophages.

IL-21 has demonstrated anticancer properties in mouse studies as well as in early phase clinical trials and has structural homology to other cytokines including IL-2, said William E. Carson III of Ohio State University. In mice, the cytokine mediates anti-tumor effects in models of melanoma, renal cell carcinoma, colon adenocarcinoma, breast cancer, and other tumors. The anti-tumor effects appear to be mediated by NK cells and CD8+ T cells.

Researchers tested IL-21 in combination with trastuzumab (Herceptin, Genentech), a monoclonal antibody that inhibits the growth of Her2/Neu positive tumors and mediates antibody-dependent cellular cytotoxicity (ADCC). They found that IL-21 enhances NK cell-mediated ADCC and cytokine production in conjunction with treatment with monoclonal antibodies rituximab (Rituxan, Genentech) and trastuzumab. "Cytokines can be an extra kick to monoclonal antibody therapy," said Carson.

Lessons learned

Since the clinical trials of the 1990s, numerous technological advances have occurred that could revive the potential of these cytokine therapies as viable drug candidates. These include advances in drug delivery platforms and ways to prolong the in vivo half-life of the therapeutic proteins. Biomarkers could now be used to prospectively identify patients who are more likely to respond to therapy. The analytical tools that exist today, including microarray technology for genes and proteins, could help identify better biomarkers of cytokine-mediated pharmacodynamic activities.

Basic investigations into cytokine biology will also improve the therapeutic potential of these proteins. Normally cytokines interact with receptors to initiate downstream signaling cascades that turn on or off key genes that mediate biological activities. Researchers are studying how to determine what genes or groups of genes mediate the effects of cytokines.

Some cytokines that failed clinical trials in the 1990s might be worth reexamining. A promising but all but abandoned cytokine is IL-12. This cytokine was explored for treatment of some infectious diseases and cancer but was found to be highly toxic and largely ineffective as a mono-therapeutic agent in several clinical trials. However, IL-12 might be more effective as an anti-cancer agent if administered at lower, less toxic concentrations together with other anti-cancer drugs or cytokines.

Highlights

• Endogenous cytokines are involved in inflammation and mediate the immune responses characteristic of autoimmune diseases.
• Monoclonal antibodies that block the activity of cytokines, either by blocking cytokine receptors or neutralizing cytokines' activity, hold great promise for the treatment of autoimmune diseases such as lupus, inflammatory bowel disease, and psoriasis.
• The study of rare autoimmune and inflammatory disorders could provide researchers with knowledge of how cytokines participate in a variety of immune-mediated diseases.
• Gene expression profiling could help researchers understand how cytokines play a role in depressing or exacerbating the inflammation associated with certain autoimmune disorders.
• New methods for targeting and delivering cytokine therapeutics have the potential to improve therapeutic utility and reduce toxicity.

TNF-α inhibitors and rheumatoid arthritis

Cytokines act as messengers to activate or suppress the immune system, so they represent promising therapeutic agents for many diseases that involve the immune system. These diseases include inflammatory diseases characterized by an overproduction of inflammatory cytokines such as IFN-γ or TNF-α, and autoimmune diseases, which are characterized by hyper-immune system responses directed against the body's own proteins or cells.

The treatment of many immune-related disorders has been improved greatly by the discovery of cytokine inhibitors. These include cytokine receptor constructs that can bind to specific cytokines and monoclonal antibodies that target specific cytokines.

Rheumatoid arthritis is a classic example of an inflammatory disease where cytokines play a prominent role. In the joint, when arthritic antigens are present they cause the activation of T cells that in turn produce cytokines such as TNF-α, IL-1, IL-6, IFN-γ, and others. These cytokines in turn mediate the activation of tissue-destroying metalloproteinases, activation of vascular adhesion molecules that recruit lymphocytes, macrophages, and other immune system cells to the joints. A consequence of these events is the activation of B cells which produce auto-antibodies. If unchecked, these processes can lead to progressive joint destruction.

The progression of rheumatoid arthritis.

For many patients, rheumatoid arthritis affects systems other than the joints. Patients can suffer from cardiovascular disease, chronic pulmonary obstructive disease (COPD), blood disorders, neurological symptoms, pulmonary effects, and ocular problems.

Larry Moreland at the University of Pittsburgh reviewed ways to target TNF-α, a cytokine involved in rheumatoid arthritis (RA). TNF-α inhibitors have been shown to decrease symptoms, slow disease progression, and improve quality of life in many RA patients.

Five biological agents that inhibit TNF-α are currently approved for use in the USA. Three are monoclonal antibodies (mAbs) against TNF-α and two are receptor constructs that act by binding TNF-α and facilitating its clearance from the body.

TNF-α appears to play a central role in disease activity in roughly three-quarters of all RA patients. It may be that the remaining 25% of patients develop neutralizing antibodies to anti-TNF-α agents after being treated with one or more of these anti-TNF agents for some period of time. Comparative studies to accurately quantify the incidence of anti-TNF antibodies in RA patients treated with different anti-TNF agents have not yet been reported. Finally, the use of contemporary cytokine profiling platforms could provide very useful information regarding which patients will respond most favorably to anti-TNF therapies.

Blocking IL-12 and IL-23 as a therapy for autoimmune diseases

Psoriasis is an inflammatory disease characterized by the rapid growth of skin cells. Researchers have found that T cells become activated and produce cytokines that spur the growth of skin cells as well as inflammation. One such cytokine is IL-12, which acts to promote development of Th1 cells that in turn produce IFN-γ. IL-23 acts preferentially to promote development of Th17 cells that in turn produce a variety of pro-inflammatory cytokines, such as IL-6, IL-17, and IL-22.

Ustekinumab (Stelara) is a human monoclonal antibody that binds the p40 subunit of IL-12 and IL-23 and prevents these cytokines from binding to the IL-12Rβ1 receptor and subsequent signaling. The drug is marketed in Canada and Europe for moderate to severe plaque psoriasis and is currently under review by the FDA for use in the U.S., said Michael Elliott of Centocor Inc.

By blocking both IL-12 and IL-23, ustekinumab inhibits inflammatory cell infiltration, normalizes cytokine expression in the skin, reduces epidermal hyperplasia, and promotes the maintenance of normal skin dendritic cell populations. The drug does not appear to affect circulating Th1, Th2, Treg, or NK cell populations, although there may be a trend to reductions in circulating Th17 cells.

Ustekinumab may also be useful as a treatment for psoriatic arthritis, and Crohn's disease, although these are unapproved indications. However it did not show efficacy against multiple sclerosis (MS).

Interferon-β in autoimmune disease

One cytokine that is often very effective in treatment of MS is interferon-β, said Richard Ransohoff of the Cleveland Clinic Foundation. Treatment with recombinant human IFN-β is partially effective against MS but it is expensive, inconvenient, has many side effects, and doesn't work for every patient. Nevertheless until its discovery, there were very few treatments options for the disease.

Researchers would like to be able to predict which patients will respond to interferon therapy. To find predictive markers, Ransohoff and his colleagues evaluated gene expression profiles to see if individuals treated with IFN-β had varied responses to this cytokine. The researchers used microarrays of a set of interferon-stimulated genes (ISGs) to examine changes in gene expression levels following IFN-β treatment.

Regulation of IFN-α

They found that neither the magnitude nor the stability of the biological response to IFN-β was responsible for the difference in responsiveness among patients. They concluded that the specific gene or combination of genes induced or repressed by the treatment must be responsible. Their group and others have recently identified a number of genes that are associated with MS, and they are currently exploring how these genes respond to IFN-β treatment.

Another team that is performing gene expression profiling to learn more about how cytokines influence diseases is that of Virginia Pascual of the Baylor Institute for Immunology Research. They are looking for gene signatures associated with Systemic Lupus Erythematosus (SLE) that can serve as biomarkers for diagnosis and assessment of lupus disease activity. The diagram above shows the regulation of interferon-α, which is implicated in inflammation and could be a target for SLE therapy.

IL-1 in autoinflammatory disease

Research in rare genetic diseases can help expand our understanding of inflammatory processes in more common diseases, explained Raphaela Goldbach-Mansky of the Translational Autoinflammatory Disease Section at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS). Three such rare diseases are collectively called cryopyrin-associated periodic syndromes (CAPS). Familial cold autoinflammatory syndrome (FCAS) involves cold-induced attacks of fever, neutrophilic urticaria, conjunctivitis, and joint pain, lasting 12 to 24 hours then resolving. Muckle Wells syndrome (MWS) is a more severe and persistent disease that is not cold-induced and involves fever, neutrophilic urticaria, joint pain, progressive hearing loss, and amyloidosis. The third disorder is neonatal onset multi-system inflammatory disease (NOMID), involving the same symptoms as well as bony overgrowth of the knees, organ damage, and mental retardation.

Cryopyrin-associated periodic syndromes (CAPS).

All three of these diseases appear to be mediated by the proinflammatory cytokine, IL-1. A recombinant human IL-1 receptor antagonist, approved in 2000 (anakinra, KineretTM, Amgen) for the treatment of rheumatoid arthritis, provides a potential treatment for these diseases as well. Blocking IL-1 using anakinra in patients with NOMID resulted in immediate resolution of the skin rash. Their symptoms returned when IL-1 inhibitors were withdrawn. In patients with NOMID, blocking IL-1 helped restore hearing and vision in some patients but had no effect in others. However, IL-1 inhibitors did not help prevent the growth of bony lesions in the knees. Early diagnosis and treatment with an IL-1 inhibitor such as anakinra can reduce and perhaps prevent the development of organ specific damage and disability.

IL-1β in autoinflammatory disease

Rilonacept traps IL-1 and prevents it from binding to cell surface receptors.

IL-1β plays a role in several inflammatory diseases, including RA, CAPS, and gout, said Neil Stahl of Regeneron Pharmaceuticals. Rilonacept (Arcalyst, Regeneron) a drug that is approved for treating CAPS and now is in phase 3 trials for gout, is a receptor-Fc fusion protein that traps and removes IL-1. It is highly specific and has a very high affinity for IL-1β. Rilonacept and IL-1β form a complex that prevents the biological activity of the cytokine.

If the rate of rilonacept-IL-1β complex removal from plasma is known, then it may be possible to calculate how much IL-1β is being made in different disease states. Stahl and his colleagues found that IL-1β levels are highest in CAPS (FCAS) followed by gout and then RA. Normal healthy volunteers appear to have very low IL-1β synthesis.

The researchers concluded that studying IL-1β:Rilonacept complex levels may prove useful in identifying IL-1β-"driven" diseases and in identifying diseases that are more responsive to IL-1 inhibition. Regeneron scientists, including Allen Radin, are exploring the use of Rilonacept for the treatment of chronic gouty arthritis, a rare subset of gout that is resistant to traditional gout therapeutics.