ObjectivesBladder cancer (BlCa) is the tenth most frequent malignancy worldwide and the costliest to treat and monitor. Muscle‐invasive BlCa (MIBC) has a dismal prognosis, entailing the need for alternative therapies for the standard radical cystectomy. Checkpoint blockade immunotherapy has been approved for high‐grade non‐muscle‐invasive BlCa (HG NMIBC) and metastatic disease, but its effectiveness in localised MIBC remains under scrutiny. Herein, we sought to characterise and compare the immune infiltrate of HG NMIBC and MIBC samples, including ICOS expression, a targetable immune checkpoint associated with regulatory T cell(Tregs)‐mediated immunosuppression.MethodsImmunohistochemistry for CD83, CD20, CD68, CD163, CD3, CD8, CD4, FoxP3/ICOS and PD‐L1 was performed in HG NMIBC and MIBC samples (n = 206), and positive staining was quantified in the peritumoral and/or intratumoral tissue compartments with QuPath imaging software.ResultsCD20+ B cells, CD68+ and CD163+ tumor‐associated macrophages were significantly increased in MIBCs and associated with poor prognosis. In turn, higher infiltration of T cells was associated with prolonged survival, with exception of the CD4+ helper subset. Intratumoral expression of CD3 and CD8 was independent prognostic factors for increased disease‐free survival (DFS) in multivariable analysis. Remarkably, Tregs (FoxP3+/FoxP3+ICOS+) were found differentially distributed between tissue compartments. PD‐L1 immunoexpression independently predicted a shorter DFS and associated with higher infiltration of FoxP3+ICOS+ Tregs.ConclusionsImmune infiltrates of HG NMIBC and MIBC display significant differences that may help selecting patients for immunotherapies. Considering ICOS immunoexpression results, it might constitute a relevant therapeutic target, eventually in combination with anti‐PD‐1/PD‐L1 therapies, for certain BlCa patient subsets.

Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time, explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in two different, well established and clinically relevant rodent models of ACD. In addition to counteracting hepcidin driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared to either monotherapy in a qualitative and quantitative manner. Consequently, combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.

The identification of effective biomarkers for early diagnosis, prognosis, and response to treatments remains a challenge in ovarian cancer (OC) research. Here, we present an unbiased high-throughput approach to profile ascitic fluid autoantibodies in order to obtain a tumor-specific antigen signature in OC.We first reported the reactivity of immunoglobulins (Igs) purified from OC patient ascites towards two different OC cell lines. Using a discovery set of Igs, we selected tumor-specific antigens from a phage display cDNA library. After biopanning, 700 proteins were expressed as fusion protein and used in protein array to enable large-scale immunoscreening with independent sets of cancer and noncancerous control. Finally, the selected antigens were validated by ELISA.The initial screening identified eight antigenic clones: CREB3, MRPL46, EXOSC10, BCOR, HMGN2, HIP1R, OLFM4, and KIAA1755. These antigens were all validated by ELISA in a study involving ascitic Igs from 153 patients (69 with OC, 34 with other cancers and 50 without cancer), with CREB3 showing the highest sensitivity (86.95%) and specificity (98%). Notably, we were able to identify an association between the tumor-associated (TA) antibody response and the response to a first-line tumor treatment (platinum-based chemotherapy). A stronger association was found by combining three antigens (BCOR, CREB3, and MRLP46) as a single antibody signature.Measurement of an ascitic fluid antibody response to multiple TA antigens may aid in the identification of new prognostic signatures in OC patients and shift attention to new potentially relevant targets.

During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection.