Resolution of organ dysfunction as a predictor of long-term survival in necrotizing soft tissue infections: Analysis of the AB103 Clinical Composite Endpoint Study in Necrotizing Soft Tissue Infections trial and a retrospective claims database-linked chart study.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) are an acute surgical condition with high morbidity and mortality. Timely identification, resuscitation, and aggressive surgical management have significantly decreased inpatient mortality. However, reduced inpatient mortality has shifted the burden of disease to long-term mortality associated with persistent organ dysfunction. METHODS: We performed a combined analysis of NSTI patients from the AB103 Clinical Composite Endpoint Study in Necrotizing Soft Tissue Infections randomized-controlled interventional trial (ATB-202) and comprehensive administrative database (ATB-204) to determine the association of persistent organ dysfunction on inpatient and long-term outcomes. Persistent organ dysfunction was defined as a modified Sequential Organ Failure Assessment (mSOFA) score of 2 or greater at Day 14 (D14) after NSTI diagnosis, and resolution of organ dysfunction defined as mSOFA score of 1 or less. RESULTS: The analysis included 506 hospitalized NSTI patients requiring surgical debridement, including 247 from ATB-202, and 259 from ATB-204. In both study cohorts, age and comorbidity burden were higher in the D14 mSOFA ≥2 group. Patients with D14 mSOFA score of 1 or less had significantly lower 90-day mortality than those with mSOFA score of 2 or higher in both ATB-202 (2.4% vs. 21.5%; p < 0.001) and ATB-204 (6% vs. 16%: p = 0.008) studies. In addition, in an adjusted covariate analysis of the combined study data sets D14 mSOFA score of 1 or lesss was an independent predictor of lower 90-day mortality (odds ratio, 0.26; 95% confidence interval, 0.13-0.53; p = 0.001). In both studies, D14 mSOFA score of 1 or less was associated with more favorable discharge status and decreased resource utilization. CONCLUSION: For patients with NSTI undergoing surgical management, persistent organ dysfunction at 14 days, strongly predicts higher resource utilization, poor discharge disposition, and higher long-term mortality. Promoting the resolution of acute organ dysfunction after NSTI should be considered as a target for investigational therapies to improve long-term outcomes after NSTI. LEVEL OF EVIDENCE: Prognostic/epidemiology study, level III.

A Novel Immune Modulator for Patients With Necrotizing Soft Tissue Infections (NSTI): Results of a Multicenter, Phase 3 Randomized Controlled Trial of Reltecimod (AB 103).

BACKGROUND AND OBJECTIVE: Reltecimod, a CD 28 T-lymphocyte receptor mimetic, inhibits T-cell stimulation by an array of bacterial pathogens. A previous phase 2 trial demonstrated improved resolution of organ dysfunction after NSTI. We hypothesized that early administration of reltecimod would improve outcome in severe NSTI. METHODS: Randomized, double-blind, placebo-controlled trial of single dose reltecimod (0.5 mg/kg) administered within 6 hours of NSTI diagnosis at 65 of 93 study sites. Inclusion: surgical confirmation of NSTI and organ dysfunction [modified Sequential Organ Failure Assessment Score (mSOFA) score ≥3]. Primary analysis was modified Intent-to-Treat (mITT), responder analysis using a previously validated composite endpoint, necrotizing infection clinical composite endpoint, defined as: alive at day 28, ≤3 debridements, no amputation beyond first operation, and day 14 mSOFA ≤1 with ≥3 point reduction (organ dysfunction resolution). A prespecified, per protocol (PP) analysis excluded 17 patients with major protocol violations before unblinding. RESULTS: Two hundred ninety patients were enrolled, mITT (Reltecimod 142, Placebo 148): mean age 55 ±â€Š15 years, 60% male, 42.4% diabetic, 28.6% perineal infection, screening mSOFA mean 5.5 ±â€Š2.4. Twenty-eight-day mortality was 15% in both groups. mITT necrotizing infection clinical composite endpoint success was 48.6% reltecimod versus 39.9% placebo, P = 0.135 and PP was 54.3% reltecimod versus 40.3% placebo, P = 0.021. Resolution of organ dysfunction was 65.1% reltecimod versus 52.6% placebo, P = 0.041, mITT and 70.9% versus 53.4%, P = 0.005, PP. CONCLUSION: Early administration of reltecimod in severe NSTI resulted in a significant improvement in the primary composite endpoint in the PP population but not in the mITT population. Reltecimod was associated with improved resolution of organ dysfunction and hospital discharge status.

Microbiota differences between commercial breeders impacts the post-stroke immune response.

Experimental reproducibility between laboratories is a major translational obstacle worldwide, particularly in studies investigating immunomodulatory therapies in relation to brain disease. In recent years increasing attention has been drawn towards the gut microbiota as a key factor in immune cell polarization. Moreover, manipulation of the gut microbiota has been found effective in a diverse range of brain disorders. Within this study we aimed to test the impact of microbiota differences between mice from different sources on the post-stroke neuroinflammatory response. With this rationale, we have investigated the correlation between microbiota differences and the immune response in mice from three commercial breeders with the same genetic background (C57BL/6). While overall bacterial load was comparable, we detected substantial differences in species diversity and microbiota composition on lower taxonomic levels. Specifically, we investigated segmented filamentous bacteria (SFB)-which have been shown to promote T cell polarization-and found that they were absent in mice from one breeder but abundant in others. Our experiments revealed a breeder specific correlation between SFB presence and the ratio of Treg to Th17 cells. Moreover, recolonization of SFB-negative mice with SFB resulted in a T cell shift which mimicked the ratios found in SFB-positive mice. We then investigated the response to a known experimental immunotherapeutic approach, CD28 superagonist (CD28SA), which has been previously shown to expand the Treg population. CD28SA treatment had differing effects between mice from different breeders and was found to be ineffective at inducing Treg expansion in SFB-free mice. These changes directly corresponded to stroke outcome as mice lacking SFB had significantly larger brain infarcts. This study demonstrates the major impact of microbiota differences on T cell polarization in mice during ischemic stroke conditions, and following immunomodulatory therapies.

Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition.

Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.

Regulatory T cells ameliorate intrauterine growth retardation in a transgenic rat model for preeclampsia.

Preeclampsia is a multisystemic syndrome during pregnancy that is often associated with intrauterine growth retardation. Immunologic dysregulation, involving T cells, is implicated in the pathogenesis. The aim of this study was to evaluate the effect of upregulating regulatory T cells in an established transgenic rat model for preeclampsia. Application of superagonistic monoclonal antibody for CD28 has been shown to effectively upregulate regulatory T cells. In the first protocol (treatment protocol), we applied 1 mg of CD28 superagonist or control antibody on days 11 and 15 of pregnancy. In the second protocol (prevention protocol), the superagonist or control antibody was applied on days 1, 5, and 9. Superagonist increased regulatory T cells in circulation and placenta from 8.49±2.09% of CD4-positive T cells to 23.50±3.05% and from 3.85±1.45% to 23.27±7.64%, respectively. Blood pressure and albuminuria (30.6±15.1 versus 14.6±5.5 mg/d) were similar in the superagonist or control antibody-treated preeclamptic group for both protocols. Rats treated with CD28 superagonist showed increased pup weights in the prevention protocol (2.66±0.03 versus 2.37±0.05 g) and in the treatment protocol (3.04±0.04 versus 2.54±0.1 g). Intrauterine growth retardation, calculated by brain:liver weight ratio, was also decreased by the superagonist in both protocols. Further analysis of brain development revealed a 20% increase in brain volume by the superagonist. Induction of regulatory T cells in the circulation and the uteroplacental unit in an established preeclamptic rat model had no influence on maternal hypertension and proteinuria. However, it substantially improved fetal outcome by ameliorating intrauterine growth retardation.

A Novel Drug for Treatment of Necrotizing Soft-Tissue Infections: A Randomized Clinical Trial.

IMPORTANCE: Necrotizing soft-tissue infections (NSTI) have high morbidity and mortality rates despite aggressive surgical debridement and antibiotic therapy. AB103 is a peptide mimetic of the T-lymphocyte receptor, CD28. We hypothesized that AB103 will limit inflammatory responses to bacterial toxins and decrease the incidence of organ failure. OBJECTIVES: To establish the safety of AB103 in patients with NSTI and evaluate the potential effects on clinically meaningful parameters related to the disease. DESIGN, SETTING, AND PARTICIPANTS: A prospective, randomized, placebo-controlled, double-blinded study was performed in 6 academic medical centers in the United States. Participants included adults with NSTI. Of 345 patients screened, 43 were enrolled for the intent-to-treat analysis, and 40 met criteria for the modified intent-to-treat analysis; 15 patients each were included in the high-dose and low-dose treatment arms, and 10 in the placebo arm. INTERVENTION: Single intravenous dose of AB103 (0.5 or 0.25 mg/kg) within 6 hours after diagnosis of NSTI. MAIN OUTCOMES AND MEASURES: Change in the Sequential Organ Failure Assessment score within 28 days, intensive care unit-free and ventilator-free days, number and timing of debridements, plasma and tissue cytokine levels at 0 to 72 hours, and adverse events. RESULTS: Baseline characteristics were comparable in the treatment groups. The Sequential Organ Failure Assessment score improved from baseline in both treatment groups compared with the placebo group at 14 days (change from baseline score, -2.8 in the high-dose, -2 in the low-dose, and +1.3 in the placebo groups; P = .04). AB103-treated patients had a similar number of debridements (mean [SD], 2.2 [1.1] for the high-dose, 2.3 [1.2] for the low-dose, and 2.8 [2.1] for the placebo groups; P = .56). There were no statistically significant differences in intensive care unit-free and ventilator-free days or in plasma and tissue cytokine levels. No drug-related adverse events were detected. CONCLUSIONS AND RELEVANCE: AB103 is a safe, promising new agent for modulation of inflammation after NSTI. Further study is warranted to establish efficacy. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01417780.

Potent control of acute graft-versus-host disease lethality after immunization with a novel DNA vaccine targeted to B7-1/CD28 costimulatory signaling pathway.

A major challenge associated with allogeneic hematopoietic stem cell transplantation is effective prevention and/or attenuation of symptoms associated with acute graft-versus-host disease (aGVHD) that can result from a failure of either host and/or donor CD4(+)CD25(+) regulatory T (Tr) and CD8(+)CD28 suppressor T (Ts) cells to dampen immunopathogenic responses mediated by alloreactive donor CD4(+)CD28(+) Th1 (Th1) and CD8(+)CD28(-) Tc1 (Tc1) cell-mediated inflammatory processes. Considering the crucial role of CD28/B7-1 costimulatory signal pathway in development, activation, differentiation, and function of these T subsets, we developed a targeted DNA vaccine encoding the Pseudomonas exotoxin-A and the B7-1 molecule that could act as both an antagonist to Th1-mediated and Tc1-mediated responses while concomitantly acting as an agonist stimulating Tr-mediated and Ts-mediated responses as a strategy for reducing aGVHD-associated lethality. A single intramuscular injection with this vaccine significantly increased Tr and Ts levels in a murine aGVHD model. In addition, immunized mice presented with significantly diminished Th1-cytokines interferon-γ and interleukin-2 response and a moderately upregulated Th2-cytokine interleukin-10 and Th3-cytokine transforming growth factor-ß response. More importantly, vaccination significantly reduced aGVHD, measured by significantly extended mean survival times, decreased mean weight loss, recovery of peripheral leukocyte numbers, disease presentation associated with mild-moderate histopathologic changes, a balanced Th1-Th2-Th3-cytokine responses and functional Tr-mediated and Ts-mediated regulatory/suppressor mechanisms at levels more potent than observed in animals treated with cyclosporine A+methotrexate. Our data first provide the proof-of-principal that B7-1-PE40KDEL targeted DNA vaccine represents a prophylactic approach for reducing alloreactive Th1-mediated and Tc-mediated aGVHD lethality by CD28/B7-1 axis.

Rejection of syngeneic colon carcinoma by CTLs expressing single-chain antibody receptors codelivering CD28 costimulation.

A new strategy to improve the therapeutic utility of redirected T cells for cancer involves the development of novel Ag-specific chimeric receptors capable of stimulating optimal and sustained T cell antitumor activity in vivo. Given that T cells require both primary and costimulatory signals for optimal activation and that many tumors do not express critical costimulatory ligands, modified single-chain Ab receptors have been engineered to codeliver CD28 costimulation. In this study, we have compared the antitumor potency of primary T lymphocytes expressing carcinoembryonic Ag (CEA)-reactive chimeric receptors that incorporate either TCR-zeta or CD28/TCR-zeta signaling. Although both receptor-transduced T cell effector populations demonstrated cytolysis of CEA(+) tumors in vitro, T cells expressing the single-chain variable fragment of Ig (scFv)-CD28-zeta chimera had a far greater capacity to control the growth of CEA(+) xenogeneic and syngeneic colon carcinomas in vivo. The observed enhanced antitumor activity of T cells expressing the scFv-CD28-zeta receptor was critically dependent on perforin and the production of IFN-gamma. Overall, this study has illustrated the ability of a chimeric scFv receptor capable of harnessing the signaling machinery of both TCR-zeta and CD28 to augment T cell immunity against tumors that have lost expression of both MHC/peptide and costimulatory ligands in vivo.

Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway.

We have explored the role of an activation-induced T cell molecule, 4-1BB (CDw137), in the amplification of tumor immunity by retrovirus-mediated transduction of the 4-1BB ligand (4-1BBL) into tumor cells. Mice inoculated with P815 tumor cells expressing 4-1BBL developed a strong cytotoxic T lymphocyte (CTL) response and long-term immunity against wild-type tumor. The optimal effect of 4-1BBL in CTL stimulation required B7-CD28 interaction since blockade of this interaction by antibodies down-regulated the expression of 4-1BB on T cells and decreased CTL activity. Furthermore, co-expression of 4-1BBL and B7-1 in the poorly immunogenic AG104A sarcoma enhanced the induction of effector CTL and the rejection of the wild-type tumor while neither 4-1BBL nor B7-1 single transfectants were effective, suggesting a synergistic effect between the 4-1BB and the CD28 co-stimulatory pathways. Our results underscore the importance of the 4-1BB T cell stimulation pathway in the amplification of an antitumor immune response.