Non-Redundant Roles of T Cell Costimulation Pathways in Inflammatory Arthritis Revealed by Dual Blockade of ICOS and CD28 with Acazicolcept (ALPN-101).

OBJECTIVE: CD28 and inducible T cell costimulator (ICOS) appear to have nonredundant roles in T cell activation and adaptive immunity. We undertook this study to characterize in vitro and in vivo the therapeutic potential of acazicolcept (ALPN-101), an Fc fusion protein of a human variant ICOS ligand (ICOSL) domain designed to inhibit both CD28 and ICOS costimulation, in inflammatory arthritis. METHODS: Acazicolcept was compared in vitro with inhibitors of either the CD28 or ICOS pathways (abatacept and belatacept [CTLA-4Ig], prezalumab [anti-ICOSL monoclonal antibody]) in receptor binding and signaling assays, and in a collagen-induced arthritis (CIA) model. Acazicolcept was also compared in cytokine and gene expression assays of peripheral blood mononuclear cells (PBMCs) from healthy donors or rheumatoid arthritis (RA) or psoriatic arthritis (PsA) patients stimulated with artificial antigen-presenting cells (APCs) expressing CD28 and ICOS ligands*. RESULTS: Acazicolcept bound CD28 and ICOS, prevented ligand binding, and inhibited human T cell functional interactions, matching or exceeding the activity of CD28 or ICOS costimulatory single-pathway inhibitors tested individually or in combination. Acazicolcept administration significantly reduced disease in the CIA model and more potently than abatacept. Acazicolcept also inhibited proinflammatory cytokine production from stimulated PBMCs in cocultures with artificial APCs and demonstrated unique effects on gene expression distinct from those induced by abatacept, prezalumab, or a combination of both. CONCLUSION: Both CD28 and ICOS signaling play critical roles in inflammatory arthritis. Therapeutic agents such as acazicolcept that coinhibit both ICOS and CD28 signaling may mitigate inflammation and/or disease progression in RA and PsA more effectively than inhibitors of either pathway alone.

The engineered CD80 variant fusion therapeutic davoceticept combines checkpoint antagonism with conditional CD28 costimulation for anti-tumor immunity.

Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.

ICOSL+ plasmacytoid dendritic cells as inducer of graft-versus-host disease, responsive to a dual ICOS/CD28 antagonist.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). CD146 and CCR5 are proteins that mark activated T helper 17 (Th17) cells. The Th17 cell phenotype is promoted by the interaction of the receptor ICOS on T cells with ICOS ligand (ICOSL) on dendritic cells (DCs). We performed multiparametric flow cytometry in a cohort of 156 HCT recipients and conducted experiments with aGVHD murine models to understand the role of ICOSL+ DCs. We observed an increased frequency of ICOSL+ plasmacytoid DCs, correlating with CD146+CCR5+ T cell frequencies, in the 64 HCT recipients with gastrointestinal aGVHD. In murine models, donor bone marrow cells from ICOSL-deficient mice compared to those from wild-type mice reduced aGVHD-related mortality. Reduced aGVHD resulted from lower intestinal infiltration of pDCs and pathogenic Th17 cells. We transplanted activated human ICOSL+ pDCs along with human peripheral blood mononuclear cells into immunocompromised mice and observed infiltration of intestinal CD146+CCR5+ T cells. We found that prophylactic administration of a dual human ICOS/CD28 antagonist (ALPN-101) prevented aGVHD in this model better than did the clinically approved belatacept (CTLA-4-Fc), which binds CD80 (B7-1) and CD86 (B7-2) and interferes with the CD28 T cell costimulatory pathway. When started at onset of aGVHD signs, ALPN-101 treatment alleviated symptoms of ongoing aGVHD and improved survival while preserving antitumoral cytotoxicity. Our data identified ICOSL+-pDCs as an aGVHD biomarker and suggest that coinhibition of the ICOSL/ICOS and B7/CD28 axes with one biologic drug may represent a therapeutic opportunity to prevent or treat aGVHD.

Novel Immunomodulatory Proteins Generated via Directed Evolution of Variant IgSF Domains.

Immunoglobulin superfamily member (IgSF) proteins play a significant role in regulating immune responses with surface expression on all immune cell subsets, making the IgSF an attractive family of proteins for therapeutic targeting in human diseases. We have developed a directed evolution platform capable of engineering IgSF domains to increase affinities for cognate ligands and/or introduce binding to non-cognate ligands. Using this scientific platform, ICOSL domains have been derived with enhanced binding to ICOS and with additional high-affinity binding to the non-cognate receptor, CD28. Fc-fusion proteins containing these engineered ICOSL domains significantly attenuate T cell activation in vitro and in vivo and can inhibit development of inflammatory diseases in mouse models. We also present evidence that engineered ICOSL domains can be formatted to selectively provide costimulatory signals to augment T cell responses. Our scientific platform thus provides a system for developing therapeutic protein candidates with selective biological impact for treatments of a wide array of human disorders including cancer and autoimmune/inflammatory diseases.

Biomechanical strain characteristics of soft tissue biceps tenodesis and bony tenodesis.

BACKGROUND: Biomechanical analysis of biceps tenodesis procedures has historically focused on load to failure models. Minimal data exists for the analysis of biomechanical strain properties of the biceps tendon in a sub-failure, physiologic cadaver model. HYPOTHESIS: Tendon strain characteristics are different between bony and soft tissue tenodesis surgery, and the soft tissue tenodesis procedure reproduces a strain pattern more similar to the native biceps tendon. METHODS: Eight fresh frozen cadaver upper extremities were mounted onto a custom device that controls shoulder abduction and rotation. Strain on the tendon was measured using a differential variable reluctance transducer as the arm was moved through cycles of abduction and external rotation. Each arm was mounted once, and all 3 testing procedures were performed on each of the 8 specimens. Statistical analysis was completed using ANOVA, followed by multiple comparisons with Bonferroni correction. RESULTS: The bony tenodesis model placed higher strain on the biceps tendon than the soft tissue tenodesis (p = 0.025). Also, the bony tenodesis model increased the strain on the biceps tendon when compared to the native tendon (p = 0.031). In contrast, the soft tissue tenodesis did not significantly alter strain when compared to the native tendon (p = 0.089). CONCLUSION: The soft tissue tenodesis procedure better maintained the native strain environment when compared to the bony tenodesis using an interference screw. Due to this closer approximation of native biceps tendon biomechanics, the soft tissue procedure may be more preferable clinically than the bony tenodesis. LEVEL OF EVIDENCE: 1, Controlled Laboratory Study.

Neer Award 2012: cerebral oxygenation in the beach chair position: a prospective study on the effect of general anesthesia compared with regional anesthesia and sedation.

BACKGROUND: Devastating neurologic ischemic episodes, such as stroke and deafness, have occurred in patients undergoing shoulder surgery in the beach chair position. We hypothesized that awake patients would be able to avoid significant cerebral deoxygenation events (CDEs) compared with anesthetized patients when procedures were performed in the beach chair position. MATERIALS AND METHODS: Sixty patients underwent elective shoulder surgery in the beach chair position. Thirty patients underwent an interscalene block and monitored sedation (awake group); 30 patients underwent general anesthesia (asleep group). Cerebral oxygenation saturation (Scto2) was measured during the procedure. Scto2 values below critical thresholds were defined as CDEs and treated. RESULTS: Baseline mean arterial pressure and Scto2 values were lower in the asleep group during the operation (P < .0001). A higher incidence of CDEs was seen in the asleep group (56.7% vs 0% awake group), and more CDEs were seen per patient (2.97 in asleep vs 0 awake, P < .0001). Scto2 below a threshold value of 55% was seen in 23.3% in the asleep group vs 3.3% in the awake group. A total of 89 combined desaturation events were documented in the asleep vs 1 in the awake group (P < .0001). CONCLUSIONS: Patients in the beach chair position treated with regional anesthesia and sedation had almost no cerebral desaturation events, unlike patients who had general anesthesia. Avoidance of general anesthesia in the beach chair position may reduce the risk of ischemic neurologic injury.

IL-17RE is the functional receptor for IL-17C and mediates mucosal immunity to infection with intestinal pathogens.

Interleukin 17 receptor E (IL-17RE) is an orphan receptor of the IL-17 receptor family. Here we show that IL-17RE is a receptor specific to IL-17C and has an essential role in host mucosal defense against infection. IL-17C activated downstream signaling through IL-17RE-IL-17RA complex for the induction of genes encoding antibacterial peptides as well as proinflammatory molecules. IL-17C was upregulated in colon epithelial cells during infection with Citrobacter rodentium and acted in synergy with IL-22 to induce the expression of antibacterial peptides in colon epithelial cells. Loss of IL-17C-mediated signaling in IL-17RE-deficient mice led to lower expression of genes encoding antibacterial molecules, greater bacterial burden and early mortality during infection. Together our data identify IL-17RE as a receptor of IL-17C that regulates early innate immunity to intestinal pathogens.

A role for IL-27 in limiting T regulatory cell populations.

IL-27 is a cytokine that regulates Th function during autoimmune and pathogen-induced immune responses. Although previous studies have shown that regulatory T cells (Tregs) express the IL-27R, and that IL-27 inhibits forkhead box P3 upregulation in vitro, little is known about how IL-27 influences Tregs in vivo. The studies presented in this article show that mice that overexpress IL-27 had decreased Treg frequencies and developed spontaneous inflammation. Although IL-27 did not cause mature Tregs to downregulate forkhead box P3, transgenic overexpression in vivo limited the size of a differentiating Treg population in a bone marrow chimera model, which correlated with reduced production of IL-2, a vital cytokine for Treg maintenance. These data identify an indirect role for IL-27 in shaping the Treg pool.

Vstm3 is a member of the CD28 family and an important modulator of T-cell function.

Members of the CD28 family play important roles in regulating T-cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and Tregs. The nectin-family proteins CD155 and CD112 serve as counter-structures for VSTM3, and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well-characterized CD28-CTLA-4-CD80-CD86 network. In the same way that soluble CTLA-4 can be used to block T-cell responses, we show that soluble Vstm3 attenuates T-cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T-cell responses.

Cutting edge: TIGIT has T cell-intrinsic inhibitory functions.

Costimulatory molecules regulate the functional outcome of T cell activation, and disturbance of the balance between activating and inhibitory signals results in increased susceptibility to infection or the induction of autoimmunity. Similar to the well-characterized CD28/CTLA-4 costimulatory pathway, a newly emerging pathway consisting of CD226 and T cell Ig and ITIM domain (TIGIT) has been associated with susceptibility to multiple autoimmune diseases. In this study, we examined the role of the putative coinhibitory molecule TIGIT and show that loss of TIGIT in mice results in hyperproliferative T cell responses and increased susceptibility to autoimmunity. TIGIT is thought to indirectly inhibit T cell responses by the induction of tolerogenic dendritic cells. By generating an agonistic anti-TIGIT Ab, we demonstrate that TIGIT can inhibit T cell responses directly independent of APCs. Microarray analysis of T cells stimulated with agonistic anti-TIGIT Ab revealed that TIGIT can act directly on T cells by attenuating TCR-driven activation signals.

Strain effects of placing an arthroscopic portal through the subscapularis tendon.

HYPOTHESIS: The biomechanical effects of placing a portal through the subscapularis tendon have not been studied. Our hypothesis is that placing a portal through the subscapularis tendon will affect the strain properties of the tendon. MATERIALS AND METHODS: Eight shoulders from deceased donors were dissected to expose the subscapularis musculotendinous unit. The subscapularis muscle was isolated, the arm was locked at neutral (0° abduction, 0° flexion/extension, 0° external rotation/internal rotation), and 3 cables were sutured to the subscapularis musculotendinous junction. Each cable was connected to a static weight. Three differential variable reluctance transducers (DVRTs) from Microstrain were sutured into the subscapularis tendon-superior, inferior, and in line with the proposed 5 o'clock portal. The musculotendinous unit was loaded along its line of action with 3, 9, and then 15 kg. Strain at each DVRT was measured in the native subscapularis tendon at each load level. The same strain measurement was taken after placing and removing a 5-mm suture anchor through the 5 o'clock portal and in the tendon after placing and removing an 8-mm cannula. RESULTS: Penetrating the subscapularis tendon with either a 5-mm suture anchor or an 8-mm cannula does not produce any statistically significant change in strain compared with the native tendon. CONCLUSION: Placing an anchor, or even an 8-mm cannulated portal, does not significantly alter the strain properties of the subscapularis tendon. This lack of effect on the strain characteristics of the subscapularis does not preclude the possibility of clinical effects.

Lck regulates IL-10 expression in memory-like Th1 cells.

The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck(-/-) Th1 skewed cells have normal expression of T-bet and produce IFN-γ at WT levels. However, there is a 3-fold increase in IL-10 producing cells in the mutant cultures. These cells do not have elevated levels of IL-4, GATA3, IL-17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3(+) T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL-10 in the lck(-/-) Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L(+) (naïve) cells are similar to WT cells. Furthermore, this IL-10 expression appears to be dependent on IL-12 and correlates with elevated c-Maf. These data highlight a previously unappreciated role for Lck in regulating IL-10 in Th1 cells.

A role for IL-27p28 as an antagonist of gp130-mediated signaling.

The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.

Lck mediates Th2 differentiation through effects on T-bet and GATA-3.

The Src family kinase Lck has been shown to be crucial in T cell signaling and development. However, its role in Th effector functions is not well understood. Lck has previously been shown to play a role in the cytokine expression of Th2 cells, but the mechanism by which Lck influences Th2 effector functions is unknown. Using a mouse model, we report that Lck is important in regulating the expression of IL-4 in Th2 skewed cells but is not as necessary for the expression of Th2 cytokines IL-5, IL-10, and IL-13. Furthermore, in the absence of Lck, T-bet and GATA-3 expression is aberrant. Moreover, this atypical expression pattern of T-bet and GATA-3 correlates with increased histone 3 acetylation at the Ifng locus and production of the Th1 cytokine IFN-gamma. We find overexpression of GATA-3 restores IL-4 expression in lck(-/-) Th2 cells; this indicates that the decreased IL-4 expression is due in part to reduced amounts of GATA-3. Taken together, these data imply that Lck mediates Th2 differentiation through effects on T-bet and GATA-3.

Engineering of stable bispecific antibodies targeting IL-17A and IL-23.

Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.

IL-17 receptor signaling: ubiquitin gets in on the act.

Engagement of the interleukin-17 (IL-17) receptor complex triggers activation of the transcription factor nuclear factor kappaB (NF-kappaB). A wide array of signaling molecules can contribute to the activation of NF-kappaB, but a number of common themes link the receptors engaged to activate it with the translocation of the active complex to the nucleus; among these is a clear role for ubiquitination. Ubiquitination is essential to the degradation of the inhibitor of NF-kappaB (IkappaB) subunits, which otherwise retain the inactive NF-kappaB complex in the cytosol. However, additional roles for ubiquitination in the assembly of signaling complexes and in enzyme activation are underappreciated aspects of NF-kappaB induction pathways. These roles require a form of ubiquitination biochemically distinct from that which targets proteins for degradation. The identification of Act1, an adaptor protein of the IL-17 receptor, as an E3 ubiquitin ligase capable of initiating this modification provides an impressive connection between the IL-17 receptor complex and pathways that activate NF-kappaB.

The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans.

Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity.

Biomechanical analysis of bursal-sided partial thickness rotator cuff tears.

BACKGROUND: Treatment of partial thickness supraspinatus tendon tears is controversial with no clearly defined treatment algorithms based on severity of tears. This study aims to evaluate the relationship between depth of partial thickness tears and strain. METHODS: Bursal-sided partial thickness tears were created at 1 mm increments in depth at the anterior portion of the supraspinatus tendon to 3/4 tendon width on ten fresh-frozen shoulder specimens. The supraspinatus muscle was dynamically loaded from 0-50N, and strain recorded at both the anterior and posterior portions of the tendon. RESULTS: Strain in the intact posterior portion increased monotonically with tear depth and supraspinatus force. Strain in the torn anterior portion decreased with increasing tear thickness and loading force. At 60% thickness tear, strain was significantly higher (P = 0.023) in the intact posterior portion compared to intact tendon. As the tear thickness exceeded 50% tendon thickness, the strain in the intact tendon rapidly increased nonlinearly. CONCLUSIONS: Biomechanical results herein suggest increasing potential for tear propagation in the transverse plane with increasing depth of tears, and biomechanically supports repairs of grade III (>50% thickness).

Identification of the IL-17 receptor related molecule IL-17RC as the receptor for IL-17F.

The proinflammatory cytokines IL-17A and IL-17F have a high degree of sequence similarity and share many biological properties. Both have been implicated as factors contributing to the progression of inflammatory and autoimmune diseases. Moreover, reagents that neutralize IL-17A significantly ameliorate disease severity in several mouse models of human disease. IL-17A mediates its effects through interaction with its cognate receptor, the IL-17 receptor (IL-17RA). We report here that the IL-17RA-related molecule, IL-17RC is the receptor for IL-17F. Notably, both IL-17A and IL-17F bind to IL-17RC with high affinity, leading us to suggest that a soluble form of this molecule may serve as an effective therapeutic antagonist of IL-17A and IL-17F. We generated a soluble form of IL-17RC and demonstrate that it effectively blocks binding of both IL-17A and IL-17F, and that it inhibits signaling in response to these cytokines. Collectively, our work indicates that IL-17RC functions as a receptor for both IL-17A and IL-17F and that a soluble version of this protein should be an effective antagonist of IL-17A and IL-17F mediated inflammatory diseases.