We have demonstrated that caspase-1-deficient (caspase-1

Fractalkine (CX3CL1) is expressed on injured endothelial cells and is a potent chemoattractant and adhesion molecule for macrophages carrying the fractalkine receptor (CX3CR1). The aim of this study was to investigate the role of CX3CL1, and its ligand CX3CR1, in ischemic acute renal failure (ARF) in mice. On immunoblotting, CX3CL1 protein expression in the kidney increased markedly in ischemic ARF. On immunofluorescence staining, the intensity of CX3CL1 staining in blood vessels was significantly more prominent in ischemic ARF compared with controls. A specific anti-CX3CR1 antibody (25 microg i.p. 1 h before induction of ischemia) was functionally and histologically protective against ischemic ARF. CX3CR1 is predominantly expressed on macrophages. Macrophage infiltration in the kidney in ischemic ARF was significantly decreased after anti-CX3CR1 antibody treatment. To determine the role of macrophages in ischemic ARF, macrophages in the kidney were depleted using liposomal-encapsulated clodronate (LEC). LEC resulted in significant functional and histological protection against ischemic ARF. In summary, in ischemic ARF, 1) there is upregulation of CX3CL1 protein in the kidney, specifically in blood vessels; 2) CX3CR1 inhibition using a specific antibody is partially protective and is associated with reduced macrophage infiltration in the kidney; and 3) macrophage depletion in the kidney is protective.

Inflammatory mechanisms contribute to cisplatin-induced acute renal failure (CisARF). Our first aim was to determine renal macrophage infiltration in CisARF. A more than 2-fold increase in CD11b-positive macrophages in the kidney on day 2 preceded the increase in blood urea nitrogen (BUN) and serum creatinine (SCr). Our next aim was to determine the chemoattractant for macrophage infiltration in CisARF. Fractalkine (CX 3 CL1) is expressed on activated endothelial cells and is a potent chemoattractant for macrophages that express its receptor (CX 3 CR1). Immunoblotting showed that whole-kidney CX 3 CL1 expression on days 1, 2, and 3 after cisplatin administration was increased. On immunofluorescence, the intensity of renal endothelial staining of CX 3 CL1 in blood vessels was significantly increased on day 2. Circulating von Willebrand factor (vWF), a measure of systemic endothelial injury, was increased on day 2. Next we determined whether macrophages played an injurious role in CisARF. Macrophages were depleted with injections of liposome-encapsulated clodronate (LEC). LEC resulted in a decrease in renal CD11b-positive macrophages on day 3. However, LEC-treated mice were not protected from CisARF on day 3. To determine the role of CX 3 CR1, both a specific anti-CX 3 CR1 antibody and CX 3 CR1

Background: Inflammation is thought to play a role in ischemic acute kidney injury (AKI). We have demonstrated that macrophage and dendritic cell depletion, using liposome-encapsulated clodronate (LEC), is protective against ischemic AKI. Methods: To determine whether macrophages or dendritic cells or both play a role in ischemic AKI, we performed ischemic AKI in CD11b-DTR mice that have a diphtheria toxin (DT)-induced depletion of CD11b cells (macrophages) and CD11c-DTR mice that have a DT-induced depletion of CD11c cells (dendritic cells). Results: While LEC-treated animals had a significant functional protection from AKI, CD11b-DTR and CD11c-DTR mice were not protected against AKI despite a similar degree of renal macrophage and dendritic cell depletion. Proinflammatory cytokines are known to play a role in ischemic AKI. To determine the possible reasons for the lack of protection in CD11b-DTR and CD11c-DTR mice compared to LEC-treated mice, 32 cytokines/chemokines were measured in these mice. Of the cytokines/chemokines measured, IL-6, MCP-1, GMCSF, IL-1β and CXCL1 (also known as IL-8 in humans or KC in mice) showed significant differences in the LEC-treated, CD11b-DTR and CD11c-DTR mice. MCP-1 and CXCL1 (known mediators of AKI), and also GMCSF and IL-1β were increased in AKI and decreased in LEC-treated AKI but not AKI in CD11b-DTR or CD11c-DTR mice. Conclusions: These findings suggest that LEC-mediated protection from AKI is not simply mediated by depletion of renal macrophage or dendritic cell subpopulations. Protection against AKI in LEC-treated compared to CD11b-DTR or CD11c-DTR mice may be partially explained by differences in proinflammatory cytokine profiles.

IL-18 function is neutralized in IL-18 binding protein transgenic (IL-18BP Tg) mice. First, we determined whether IL-18BP Tg mice are protected against ischemic acute kidney injury (AKI). Ischemic AKI was induced by bilateral renal pedicle clamping. IL-18BP Tg mice were functionally and histologically protected against ischemic AKI as determined by blood urea nitrogen, serum creatinine, and acute tubular necrosis score. We have demonstrated that the injurious effect of IL-18 in the kidney is independent of neutrophils and lymphocytes. Thus the effect of IL-18 inhibition on renal macrophage infiltration was determined. The number of macrophages was significantly reduced in IL-18BP Tg compared with wild-type kidneys. To determine the cytokines and chemokines that are dependent on IL-18, we performed flow cytometry based assays. Multiple chemokines/cytokines, IL-3, IL-6, IL-15, IL-18, leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1 were significantly increased in AKI vs. sham kidneys. Only CXCL1 (also known as KC or IL-8) was significantly increased in AKI vs. sham kidneys and significantly reduced in IL-18BP Tg AKI vs. wild-type AKI kidneys. To determine whether macrophages are the source of CXCL1 in the kidney, we depleted macrophages with liposomal encapsulated clodronate. CXCL1 was significantly decreased in macrophage-depleted vs. control AKI mice. In summary, in ischemic AKI in mice, 1) IL-18BP Tg mice are functionally and histologically protected, 2) macrophage infiltration in the kidney and CXCL1 are significantly reduced in IL-18BP Tg mice, and 3) macrophage depletion significantly reduces CXCL1 in the kidney. In conclusion, protection against ischemic AKI in IL-18BP Tg mice is associated with less macrophage infiltration and less production of CXCL1 in the kidney.

We previously reported in ischemic acute kidney injury (AKI) in mice that caspase-1-mediated production of interleukin-18 (IL-18) is pathogenic and that macrophage depletion by liposome-encapsulated clodronate (LEC) is protective. Therefore, our aim was to determine whether macrophages are a source of IL-18 in ischemic AKI in mice. On immunofluorescence staining of the outer stripe of outer medulla, the number of macrophages double stained for CD11b and IL-18 was significantly increased in AKI and significantly decreased by LEC. Adoptive transfer of RAW 264.7 cells, a mouse macrophage line that constitutively expresses IL-18 mRNA, reversed the functional protection against AKI in both LEC-treated wild-type and caspase-1 -/- mice. To test whether IL-18 in macrophages is necessary to cause AKI, we adoptively transferred macrophages in which IL-18 was inhibited. Peritoneal macrophages isolated from wild-type mice, IL-18 binding protein transgenic (IL-18 BP Tg) mice, and IL-18 -/- mice were used. IL-18 BP Tg mice overexpress human IL-18 BP and exhibit decreased biological activity of IL-18. Adoptive transfer of peritoneal macrophages from wild-type as well as IL-18 BP Tg and IL-18 -/- mice reversed the functional protection against AKI in LEC-treated mice. In summary, adoptive transfer of RAW cells, that constitutively express IL-18, reverses the functional protection in macrophage-depleted wild-type and caspase-1 -/- mice with AKI. However, adoptive transfer of peritoneal macrophages in which IL-18 function was inhibited also reverses the functional protection in macrophage-depleted mice. In conclusion, IL-18 from adoptive transfer of macrophages is not sufficient to cause ischemic AKI.

BackgroundProbody® therapeutics are antibody prodrugs designed to be activated by tumor-associated proteases. This conditional activation restricts antibody binding to the tumor microenvironment, thereby minimizing ‘off-tumor’ toxicity. Here, we report the phase 1 data from the first-in-human study of CX-072 (pacmilimab), a Probody immune checkpoint inhibitor directed against programmed death-ligand 1 (PD-L1), in combination with the anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) antibody ipilimumab.MethodsAdults (n=27) with advanced solid tumors (naive to PD-L1/programmed cell death protein 1 or CTLA-4 inhibitors) were enrolled in the phase 1 combination therapy dose-escalation portion of this multicenter, open-label, phase 1/2 study (NCT03013491). Dose-escalation pacmilimab/ipilimumab followed a standard 3+3 design and continued until the maximum tolerated dose (MTD) was determined. Pacmilimab+ipilimumab was administered intravenously every 3 weeks for four cycles, followed by pacmilimab administered every 2 weeks as monotherapy. The primary objective was identification of dose-limiting toxicities and determination of the MTD. Other endpoints included the rate of objective response (Response Evaluation Criteria In Solid Tumors v.1.1).ResultsTwenty-seven patients were enrolled in pacmilimab (mg/kg)+ipilimumab (mg/kg) dose-escalation cohorts: 0.3+3 (n=6); 1+3 (n=3); 3+3 (n=3); 10+3 (n=8); 10+6 (n=6); and 10+10 (n=1). Dose-limiting toxicities occurred in three patients, one at the 0.3+3 dose level (grade 3 dyspnea/pneumonitis) and two at the 10+6 dose level (grade 3 colitis, grade 3 increased aspartate aminotransferase). The MTD and recommended phase 2 dose was pacmilimab 10 mg/kg+ipilimumab 3 mg/kg administered every 3 weeks. Pacmilimab-related grade 3–4 adverse events (AEs) and grade 3–4 immune-related AEs were reported in nine (33%) and six (22%) patients, respectively. Three patients (11%) discontinued treatment because of AEs. The overall response rate was 19% (95% CI 6.3 to 38.1), with one complete (anal squamous cell carcinoma) and four partial responses (cancer of unknown primary, leiomyosarcoma, mesothelioma, testicular cancer). Responses lasted for >12 months in four patients.ConclusionsThe MTD and recommended phase 2 dose of pacmilimab (10 mg/kg)+ipilimumab (3 mg/kg) every 3 weeks is active and has a favorable tolerability profile.

4130 Background: Glucocorticoid receptor (GR) pathway activation has been linked with chemotherapy resistance (CTR). RELA (formerly CORT125134, Corcept Therapeutics), a potent selective GR modulator, in combination with paclitaxel reduced CTR and enhanced activity against tumor growth in preclinical models of solid tumors. Methods: Patients (pts) with advanced solid tumors, ≤3 prior lines of cytotoxic therapy, ECOG status 0-1, and adequate marrow function received RELA (100, 150, or 200mg) + NP (60, 80, or 100mg/m2). Once daily RELA was given either continuously (CON) or intermittently (INT) (day before, of, and after NP). NP was dosed weekly for 3 of 4 weeks (wks) of a 28-day cycle. Prior NP therapy was allowed. Results: 72 pts have been enrolled [mean age 60 (range 18-81), mean number of prior therapies 3, prior taxane (TXN) treatment 54/72 (75%)]. 61 pts received ≥1 dose of RELA. Grade ≥3 AE ≥10% for CON: neutropenia (6/43, 14%); INT: neutropenia (6/18, 33%), anemia (2/18, 11%), and mucosal inflammation (2/18, 11%). Prophylactic G-CSF became mandatory in later cohorts. Recommended Phase 2 Dose: RELA 100mg-CON/150mg-INT + NP 80mg/m2 (exposures similar to NP 100mg/m2 due to CYP3A4 inhibition by RELA). Disease control (DC) > 24 wks was noted in 5/27 (19%) PDAC pts: 3 PR, 2 SD (27-50 wks). 3 pts achieved benefit despite progression on prior TXN with time to progression (TTP) 1.9-3.6x longer than prior TXN. 4/13 (31%) OvCA pts had DC > 24 wks: 1 CR, 1 PR, 2 SD (33-54+ wks). 1 pt had TTP 4.4x longer than prior TXN. 3 additional PRs were observed: acinar pancreatic cancer, TTP 31 wks (4.4x prior TXN); vulvar SCC HPV+, TTP 55 wks (3.9x prior TXN); cholangiocarcinoma, DC 29+ wks. Expression of GR-regulated genes involved in inflammation, apoptosis, and CTR distinguished pts with DC from pts without DC, providing proof of mechanism. Conclusions: RELA+NP resulted in durable disease control in pts with metastatic PDAC, OvCA, and other solid tumors, including those that have progressed on prior TXN. TTP was often several-fold longer than previously achieved on TXN therapy. Toxicities are manageable with prophylaxis for neutropenia. Further evaluation in OvCA NCT03776812, PDAC, and others are planned. Clinical trial information: NCT02762981.