Jacqueline Benson

Adoptive Immunotherapy of Experimental Autoimmune Encephalomyelitis Via T Cell Delivery of the IL-12 p40 Subunit

CD4+ T cells are believed to play a central role in the initiation and perpetuation of autoimmune diseases such as multiple sclerosis. In the murine model for multiple sclerosis, experimental autoimmune encephalomyelitis, pathogenic T cells exhibit a Th1-like phenotype characterized by heightened expression of proinflammatory cytokines. Systemic administration of “regulatory” cytokines, which serve to counter Th1 effects, has been shown to ameliorate autoimmune responses. However, the inherent problems of nonspecific toxicity limit the usefulness of systemic cytokine delivery as a potential therapy. Therefore, we used the site-specific trafficking properties of autoantigen-reactive CD4+ T cells to develop an adoptive immunotherapy protocol that provided local delivery of a Th1 cytokine antagonist, the p40 subunit of IL-12. In vitro analysis demonstrated that IL-12 p40 suppressed IFN-γ production in developing and effector Th1 populations, indicating its potential to modulate Th1-promoted inflammation. We have previously demonstrated that transduction of myelin basic protein-specific CD4+ T cells with pGC retroviral vectors can result in efficient and stable transgene expression. Therefore, we adoptively transferred myelin basic protein-specific CD4+ T cells transduced to express IL-12 p40 into mice immunized to develop experimental autoimmune encephalomyelitis and demonstrated a significant reduction in clinical disease. In vivo tracking of bioluminescent lymphocytes, transduced to express luciferase, using low-light imaging cameras demonstrated that transduced CD4+ T cells trafficked to the central nervous system, where histological analysis confirmed long-term transgene expression. These studies have demonstrated that retrovirally transduced autoantigen-specific CD4+ T cells inhibited inflammation and promoted immunotherapy of autoimmune disorders.

Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease.

CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.

Distinct Roles of IL-23 and IL-17 in the Development of Psoriasis-Like Lesions in a Mouse Model

Psoriasis is an inflammatory disease with dynamic interactions between the immune system and the skin. The IL-23/Th17 axis plays an important role in the pathogenesis of psoriasis, although the exact contributions of IL-23 and IL-17 in vivo remain unclear. K5.Stat3C transgenic mice constitutively express activated Stat3 within keratinocytes, and these animals develop skin lesions with histological and cytokine profiles similar to those of human plaque psoriasis. In this study, we characterized the effects of anti-mouse IL-17A, anti-mouse IL-12/23p40, and anti-mouse IL-23p19 Abs on the development of psoriasis-like lesions in K5.Stat3C transgenic mice. Treatment with anti–IL-12/23p40 or anti–IL-23p19 Abs greatly inhibited 12-O-tetradecanoylphorbol-13-acetate–induced epidermal hyperplasia in the ears of K5.Stat3C mice, whereas the inhibitory effect of an anti–IL-17A Ab was relatively less prominent. Treatment with anti–IL-12/23p40 or anti–IL-23p19 Abs markedly lowered transcript levels of Th17 cytokines (e.g., IL-17 and IL-22), β-defensins, and S100A family members in skin lesions. However, anti–IL-17A Ab treatment did not affect mRNA levels of Th17 cytokines. Crossing IL-17A–deficient mice with K5.Stat3C mice resulted in partial attenuation of 12-O-tetradecanoylphorbol-13-acetate–induced lesions, which were further attenuated by anti–IL-12/23p40 Ab treatment. FACS analysis of skin-draining lymph node cells from mice that were intradermally injected with IL-23 revealed an increase in both IL-22–producing T cells and NK-22 cells. Taken together, this system provides a useful mouse model for psoriasis and demonstrates distinct roles for IL-23 and IL-17.

Suppression of Ongoing Disease in a Nonhuman Primate Model of Multiple Sclerosis by a Human-Anti-Human IL-12p40 Antibody

IL-12p40 is a shared subunit of two cytokines with overlapping activities in the induction of autoreactive Th1 cells and therefore a potential target of therapy in Th1-mediated diseases. We have examined whether ongoing disease in a nonhuman primate model of multiple sclerosis (MS) can be suppressed with a new human IgG1κ Ab against human IL-12p40. Lesions developing in the brain white matter were visualized and characterized with standard magnetic resonance imaging techniques. To reflect the treatment of MS patients, treatment with the Ab was initiated after active brain white matter lesions were detected in T2-weighted images. In placebo-treated control monkeys we observed the expected progressive increase in the total T2 lesion volume and markedly increased T2 relaxation times, a magnetic resonance imaging marker of inflammation. In contrast, in monkeys treated with anti-IL-12p40 Ab, changes in the total T2 lesion volume and T2 relaxation times were significantly suppressed. Moreover, the time interval to serious neurological deficit was delayed from 31 ± 10 to 64 ± 20 days (odds ratio, 0.312). These results, in a disease model with high similarity to MS, are important for ongoing and planned trials of therapies that target IL-12 and/or IL-23.

Ustekinumab: Lessons Learned from Targeting Interleukin‐12/23p40 in Immune‐Mediated Diseases

Interleukin (IL)‐12 and IL‐23 are related cytokines that have been implicated in the pathogenesis of several immune‐mediated disorders. IL‐12 and IL‐23 are heterodimers made up of a common p40 subunit complexed to unique p35 (IL‐12) or p19 (IL‐23) subunits. Ustekinumab is a human monoclonal antibody that specifically binds the p40 subunit of IL‐12/23. Ustekinumab prevents IL‐12 and IL‐23 from binding their cell surface receptor complexes, thereby blocking the T helper (Th) 1 (IL‐12) and Th17 (IL‐23) inflammatory pathways. Here, we discuss the preclinical and human translational data supporting a role for IL‐12/23 in the pathogenesis of immune‐mediated disorders, and how that rationale was challenged in the clinic during the course of the ustekinumab development program in several indications including psoriasis, psoriatic arthritis, Crohns disease, and multiple sclerosis. We review the key efficacy and safety data in each of these immune‐mediated diseases and compare and contrast the safety lessons learned from IL‐12/23 genetically‐deficient mice and humans in context of the overall clinical trial experience with ustekinumab.

Development of the IL-12/23 antagonist ustekinumab in psoriasis: past, present, and future perspectives

The development of ustekinumab as a first‐in‐class anti‐interleukin (IL) 12/23p40 therapeutic agent for psoriasis represents an important example of modern and rational drug design and development. Psoriasis is a chronic, systemic, immune‐mediated skin disorder with considerable clinical, psychosocial, and economic burden. Ustekinumab is a human monoclonal antibody (mAb) that binds the p40 subunit common to IL‐12 and IL‐23, key cytokines in psoriasis pathogenesis. The therapeutic mAb was developed using human gamma‐1 immunoglobulin (IgG)‐expressing transgenic mice, which created a molecule with endogenous IgG1 biologic properties and low immunogenicity. Ustekinumab was well tolerated in clinical studies and yielded rapid, significant, and sustained efficacy plus improved quality of life/work performance and reduced depression/anxiety. Its pharmacologic properties afford the most convenient dosing regimen among approved biologics, representing a significant advancement in the treatment of moderate to severe psoriasis. Ustekinumab also holds promise for other immune‐mediated disorders with significant unmet need.

Structural basis for the dual recognition of IL-12 and IL-23 by ustekinumab.

Interleukin (IL)-12 and IL-23 are heterodimeric proinflammatory cytokines that share a common p40 subunit, paired with p35 and p19 subunits, respectively. They represent an attractive class of therapeutic targets for the treatment of psoriasis and other immune-mediated diseases. Ustekinumab is a fully human monoclonal antibody (mAb) that binds specifically to IL-12/IL-23p40 and neutralizes human IL-12 and IL-23 bioactivity. The crystal structure of ustekinumab Fab (antigen binding fragment of mAb), in complex with human IL-12, has been determined by X-ray crystallography at 3.0 Å resolution. Ustekinumab Fab binds the D1 domain of the p40 subunit in a 1:1 ratio in the crystal, consistent with a 2 cytokines:1 mAb stoichiometry, as measured by isothermal titration calorimetry. The structure indicates that ustekinumab binds to the same epitope on p40 in both IL-12 and IL-23 with identical interactions. Mutational analyses confirm that several residues identified in the IL-12/IL-23p40 epitope provide important molecular binding interactions with ustekinumab. The electrostatic complementarity between the mAb antigen binding site and the p40 D1 domain epitope appears to play a key role in antibody/antigen recognition specificity. Interestingly, this structure also reveals significant structural differences in the p35 subunit and p35/p40 interface, compared with the published crystal structure of human IL-12, suggesting unusual and potentially functionally relevant structural flexibility of p35, as well as p40/p35 recognition. Collectively, these data describe unique observations about IL-12p35 and ustekinumab interactions with p40 that account for its dual binding and neutralization of IL-12 and IL-23.

The Thymus Plays a Role in Oral Tolerance in Experimental Autoimmune Encephalomyelitis

The oral administration of myelin proteins has been used for the successful prevention and treatment of experimental autoimmune encephalomyelitis (EAE). We questioned whether the thymus was involved in oral tolerance. In this study, euthymic myelin basic protein (MBP) TCR transgenic mice are protected from EAE when fed MBP but are not protected when thymectomized. Similarly, in a cell transfer system, T cell responses to OVA measured in vivo were suppressed significantly only in the OVA-fed euthymic mice but not in the thymectomized mice. We observed that the absence of the thymus dramatically enhanced the Th1 response. We explored three alternatives to determine the role of the thymus in oral tolerance: 1) as a site for the induction of regulatory T cells; 2) a site for deletion of autoreactive T cells; or 3) a site for the dissemination of naive T cells. We found that Foxp3+CD4+CD25+ T cells are increased in the periphery but not in the thymus after Ag feeding. These CD4+CD25+ T cells also express glucocorticoid-induced TNFR and intracellular CTLA4 and suppress Ag-specific proliferation of CD4+CD25− cells in vitro. The thymus also plays a role in deletion of autoreactive T cells in the periphery following orally administered MBP. However, thymectomy does not result in homeostatic proliferation and the generation of memory cells in this system. Overall, the oral administration of MBP has a profound effect on systemic immune responses, mediated largely by the generation of regulatory T cells that act to prevent or suppress EAE.

Development of the IL-12/23 antagonist ustekinumab in psoriasis: past, present, and future perspectives - an update: Ustekinumab development as psoriasis treatment

Since the original publication of the article “Development of the IL‐12/23 antagonist ustekinumab in psoriasis: Past, present and future perspectives” in March 2011 (see Appendix), 1 there have been several new publications and developments of note. A number of new reports from the ustekinumab psoriasis clinical development program have been published. The analysis of efficacy and safety in the PHOENIX 1 long‐term extension demonstrated that continuous stable maintenance dosing of ustekinumab was generally well tolerated and sustained durable efficacy through up to three years of treatment. 2 Pooled safety data from the phase 2 and phase 3 global trials showed that the safety profile of long‐term continuous ustekinumab treatment through up to three years 3,4 and four years 5 of follow‐up was favorable and comparable to what has been reported previously in the shorter‐term ustekinumab psoriasis studies. 6–8 This represents the greatest exposure and longest follow‐up of psoriasis patients treated with a biologic published to date. Additional phase 3 trials in Asian populations demonstrated similar high levels of efficacy and favorable safety profiles in Japanese, 9,10 Korean, 11,12 and Taiwanese 11,12 patients as those observed in trials conducted in mostly White populations in North America and Europe. 6–8 These data support the positive benefit:risk profile and consistency of response to ustekinumab over years of usage, and in multiple ethnic groups. Results from up to five years of treatment with ustekinumab in the long‐term extensions of the phase 3 trials, and the efficacy, safety, and effect on quality of life in Chinese patients will be available in 2012. In addition to clinical trials of ustekinumab for the treatment of psoriasis, 24‐week data from one phase 3 study of ustekinumab for the treatment of psoriatic arthritis has recently been presented 13 and another study is ongoing. A Phase 2b trial in Crohns disease has also been presented, 14 and three phase 3 studies in Crohns disease are currently in progress.

Rapid Depletion of Peripheral Antigen-Specific T Cells in TCR-Transgenic Mice After Oral Administration of Myelin Basic Protein

In myelin basic protein (MBP)-specific TCR-transgenic (Tg) mice, peripheral T cells express the Vα2.3/Vβ8.2-Tg TCR, demonstrate vigorous proliferative responses to MBP in vitro, and can exhibit experimental autoimmune encephalomyelitis (EAE) within 5 days of pertussis toxin injection. We explored the effects of oral administration of MBP on the cellular trafficking of the MBP-specific TCR-Tg cells and the ability of oral MBP to protect Tg mice from EAE. Tg mice were fed MBP, OVA or vehicle and sacrificed at various times after feeding. An immediate and dramatic decrease in Vα2.3/Vβ8.2+-Tg cells was observed in the periphery within 1 h after feeding. By 3 days after feeding, the percentage of Tg cells increased to near control levels, but decreased again by 10 days. When MBP or vehicle-fed Tg mice were challenged for EAE at this point, disease was severe in the vehicle-fed mice and reduced in the MBP-fed mice over the 40-day observation period. In vitro studies revealed a biphasic pattern of MBP proliferative unresponsiveness and an induction of Th1 cytokines. Immunohistochemical staining showed that the number of Tg cells found in the intestinal lamina propria increased dramatically as the number of Tg cells in the periphery decreased. There was no apparent proliferation of Tg cells in the lamina propria, indicating that Tg cells trafficked there from the periphery. Taken together, these results suggest that T cell trafficking into the site of Ag deposition acts to protect the TCR-Tg mouse from EAE.