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B7-H4 protein is frequently overexpressed in ovarian cancer. Here, we engineered T cells with novel B7-H4-specific chimeric antigen receptors (CARs) that recognized both human and murine B7-H4 to test the hypothesis that B7-H4 CAR T cell therapy can be applied safely in preclinical models. B7-H4 CAR T cells specifically secreted IFN-γ and lysed B7-H4(+) targets. In vivo , B7-H4 CAR T cells displayed antitumor reactivity against B7-H4(+) human ovarian tumor xenografts. Unexpectedly, B7-H4 CAR T cell treatment reproducibly showed delayed, lethal toxicity 6–8 weeks after therapy. Comprehensive assessment of murine B7-H4 protein distribution uncovered expression in ductal and mucosal epithelial cells in normal tissues. Postmortem analysis revealed the presence of widespread histologic lesions that correlated with B7-H4(+) expression, and were inconsistent with graft versus host disease. Lastly, expression patterns of B7-H4 protein in normal human tissue were comparable to distribution in mice, advancing our understanding of B7-H4. We conclude that B7-H4 CAR therapy mediates control of cancer outgrowth. However, long-term engraftment of B7-H4 CAR T cells mediates lethal, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. This model system provides a unique opportunity for preclinical evaluation of safety approaches that limit CAR-mediated toxicity after tumor destruction in vivo .

To evaluate the utility of serum (HE4) as a marker for high risk disease in patients with endometrial cancer (EC).Preoperative serum HE4 levels were measured from a cohort of 75 patients surgically treated for EC. Cases were compared to matched controls without a history of cancer. HE4 levels were analyzed as a function of primary tumor diameter, grade, stage and histological subtype. Wilcoxon rank-sum test, ROC curve, Spearman rank correlation coefficient and contingency tables were used for statistical analyses.Stage distribution was as follows: 49 stage I, 2 stage II, 20 stage III, 4 stage IV. Type I EC was present in 54 patients, type II in 21. Median HE4 was significantly elevated in both types I and II EC compared to controls (P0.001 and P=0.019, respectively). There was significant correlation between type I EC, median HE4, deep myometrial invasion (MI) (50%, P0.001) and primary tumor diameter (PTD) (2 cm, P=0.002). Low risk patients (type I, MI ≤ 50% and PTD ≤ 2 cm) had significantly lower median HE4 compared to all other type I EC patients (P0.01). In comparison to prior investigations, HE4 (cutoff of 8 mfi) was more sensitive than CA125 in detecting advanced stage disease.Our data suggest that HE4 is elevated in a high proportion of EC patients, is correlated with PTD and MI, and is more sensitive than CA125 in EC. These observations suggest potential utility of HE4 in the preoperative prediction of high risk disease and the necessity for definitive surgical staging.

Objectives. The serum tumor marker CA 125 is elevated in most clinically advanced ovarian carcinomas. Because these elevations may precede clinical detection by a year or more, CA 125 is potentially useful for early detection as part of an ovarian cancer screening program. However, CA 125 is often not elevated in clinically detected cancer and is frequently elevated in women with benign ovarian tumors. CA 125 may be more useful in conjunction with one or more other tumor biomarkers. Additional markers could play a role if, when used with CA 125, they identify some carcinomas missed by CA 125 (i.e., they improve sensitivity), rule out false positives (i.e., improve specificity), or are able to detect the same cancers earlier. Methods. We have evaluated a composite marker (CM) that combines CA 125 and a previously described soluble mesothelin related (SMR) marker in sera from 52 ovarian cancer cases, 43 controls with benign ovarian tumors, and 220 normal risk controls who participated in a screening program, including 25 healthy women having two serum samples collected 1 year apart. CA 125, SMR, and CM were evaluated for their ability to identify clinical disease and for their temporal stability, which assesses their ability to obtain even greater sensitivity when used in a longitudinal screening program. Results. CM has the best sensitivity, with specificity equal to CA 125. Importantly, CM has temporal stability at least as high as CA 125. Conclusion. The CM may outperform CA 125 alone in a longitudinal screening program as well as in a diagnostic setting.

B7-H4 is a negative immune regulatory molecule frequently overexpressed on the surface of cancer cells and immunosuppressive tumor-associated macrophages (TAMs). Ligation of B7-H4 with an unknown receptor on activated T cells results in inhibited effector function via cell cycle arrest, decreased proliferation and reduced IL-2 production. B7-H4 expression levels in ovarian cancer inversely correlate with patient survival, making B7-H4 an attractive candidate for therapeutic intervention. However, trials of B7-H4 targeted therapy have not been conducted and the biodistribution of B7-H4 protein in mice or humans is not well understood.Here, we tested the hypothesis that safe and effective B7-H4 targeting can be achieved using T cells genetically redirected with chimeric antigen receptors (CARs) against B7-H4 in a preclinical model of human ovarian cancer. Four independent anti-B7-H4 CAR lentivirus constructs were generated using anti-B7H4 single chain variable fragments (scFv) with affinities ranging from low to high, which were transduced into primary human T cells. Transduced T cells efficiently expressed CARs on their cell surface, and each CAR was capable of binding both human and mouse recombinant B7-H4 protein with distinct binding patterns; two CARs (3#68 and 3#54) had high capacity for rhB7-H4 protein binding, another had low binding capacity (56), and the last had virtually none (26). In co-culture assays, high binding B7-H4 CAR T cells secreted Th1 cytokines and specifically lysed human B7-H4+ ovarian cancer cells. In an immunodeficient mouse model, transferred B7-H4 CAR T cells persisted after infusion and exerted efficient anti-tumor activity against subcutaneous B7-H4+ human ovarian tumor xenografts.Unexpectedly, B7-H4 CAR T cell treated mice reproducibly showed signs of delayed onset, lethal toxicity emerging 6-8 weeks after therapy. Postmortem analysis revealed widespread immunopathology in nearly all tissue analyzed, with marked infiltration and destruction of peripheral nerve bundles and perivasculature regions that was inconsistent with non-specific graft versus host disease (GVHD). Immunopathology was evident in both tumor-bearing and healthy mice administered B7-H-4 CAR T cells, and mice administered control CAR T cells experienced tumor regression but without concomitant toxicity, ruling out a generalizable GVHD effect.Our results indicate that CAR T cell-based targeting of B7-H4 can mediate control of B7-H4+ cancer outgrowth in vivo, however, long-term engraftment of B7-H4 CAR T cells mediates lethal on-target, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. Accordingly, B7-H4 CAR T cells represent an important preclinical model system for the evaluation of safety approaches that limit CAR-mediated toxicity whilst maintaining potent anti-tumor activity in vivo.

Objective Maternal morbidity and/or mortality (MM) is increased in pyelonephritis and influenza. Alterations in the immune response could account for the increase MM. We sought to determine whether the immune response is functionally different during pregnant and nonpregnant (NP) states. Study Design Mouse model of systemic and localized inflammation was used. Maternal serum was assessed for expression of T-helper cell type 1 and 2 cytokines. Maternal spleens were harvested for immunohistochemistry. Results Systemic administration of lipopolysaccharides resulted in no mortality to NP mice compared with 88% in preterm and 100% in term mice. A potent cytokine response was present in both NP and pregnancy. Systemic inflammation in pregnancy results in increased CD8 and CD11c expression in spleens. Conclusion Differences in cytokine response to systemic inflammation is unlikley to modulate the increased MM during pregnancy. Altered T-cell and dendritic cell responses in pregnancy may be responsible for the increase in MM.