Phase I/II study of gefitinib and capecitabine in patients with colorectal cancer

Objective The objectives of this phase I/II study were to determine the maximum tolerated dose (MTD), characterise the principal toxicities in the phase I part and assess the efficacy in the phase II part of gefitinib, an oral selective inhibitor of the epidermal growth factor receptor, in combination with capecitabine in patients with advanced colorectal cancer (CRC). Methods and patients Patients with advanced CRC were treated with gefitinib administered daily for 21 days and capecitabine administered twice daily for 14 days of a 21-day cycle. The dose levels of gefitinib (mg) and capecitabine (mg/m² bid) assessed were 250/1000 and 250/ 1250. An expanded cohort was enrolled at the MTD to better characterise toxicity and efficacy. A total of 32 previously treated patients were accrued. In the phase I part 10 subjects were treated, with one dose-limiting toxicity. Overall 26 patients were treated at the MTD of the combination, which was gefitinib 250 mg/day and capecitabine 1250 mg/m² twice daily. Results The most frequent treatment-related adverse events included asthenia, diarrhoea, nausea, rash and anorexia. The incidence profile was very similar in phases I and II. No objective responses were documented but 53% of the patients achieved stable disease as best response to therapy. Conclusions Capecitabine 1250 mg/m² twice daily 14 of 21 days and gefitinib at 250 mg/day can be safely administered in combination. The combination is relatively well tolerated. There were no objective responses, although an interesting stabilisation rate was documented, in previously treated advanced CRC patients.     

Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)–based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

Chimeric antigen receptor (CAR)–engineered T cells have invigorated the field of cancer immunotherapy with their proven ability to treat CD19⁺ malignancies in the clinic (1–4) and continuing progress in solid tumors (5, 6). The synthetic CAR imparts T cells with the ability to recognize antigen independent of peptide presentation by major histocompatibility complexes. This antigen recognition is most often mediated by single-chain variable fragments derived from monoclonal antibodies. As an alternative, naturally occurring ligands or receptors have been used for CAR antigen recognition (7), including interleukin 13 (IL13) (8–10), a proliferation-inducing ligand (APRIL) (11), NKG2D (12), NKp44 (13), and CD27 (14). By leveraging natural binding interactions, these molecules can mediate CAR antigen recognition with minimal additional engineering (8, 14), are fully human in sequence and thus carry potentially lower immunogenicity than other classes of engineered antigen binding domains, and can potentially target multiple cancer biomarkers (11–13). However, the ability to target multiple receptors can also be disadvantageous when binding partners are not implicated in disease.IL13 is one prominent example of a naturally occurring ligand that has been used for CAR antigen recognition (8–10). IL13 interacts strongly with the high-affinity receptor IL13Rα2 (15), which is a versatile therapeutic target due to its rare expression in normal tissue (16) and overexpression in many human cancers, including glioblastoma (GBM) (17), pancreatic ductal adenocarcinoma (18), melanoma (19), ovarian carcinoma (20), clear cell renal cell carcinoma (21), breast cancer (22), and lung cancer (23). A second IL13 receptor family member, IL13Rα1, interacts with IL13 with lower affinity (15) and is ubiquitously expressed in healthy tissue (16). Additionally, IL4Rα can stabilize the IL13Rα1-IL13 complex (15) to mediate signaling through the JAK/STAT6 pathway (24). This receptor pair is coexpressed in pulmonary and other normal tissues (25). Despite this wide expression of IL13 binding partners in healthy tissue, an IL13 ligand–based CAR has shown safety in humans during clinical trials with locoregional central nervous system delivery in GBM (5, 26), suggesting that toxicity from on-target/off-disease binding is not problematic in this context. However, for the treatment of systemic disease, the wide expression of IL13 binding partners outside of the diseased tissue could act as a sink for IL13-based therapy, resulting in safety concerns and possibly impeding trafficking to the disease site. Previous work in the field has attempted to address this problem by generating CARs derived from IL13 variants containing mutations to direct binding away from IL13Rα1/IL4Rα. Mutations at E12 have yielded improved selectivity for IL13Rα2 over IL13Rα1 (8, 9), albeit with the E12Y mutation still allowing measurable recognition of IL13Rα1 in the context of both recombinant antigen and antigen-expressing cancer cells (9). The addition of both E12K and R109K mutations into an IL13-based CAR also showed attenuated, but not abolished, recognition of IL13Rα1-expressing cancer cells relative to IL13Rα2-expressing cancer cells (10). While these examples are encouraging, additional protein engineering is warranted to develop an IL13Rα2-specific IL13 mutant.Structure-based protein engineering and directed evolution approaches offer opportunities to modify the affinity and specificity of binding interactions (27, 28). In this approach, structural information is used to identify residues that contribute to binding interactions, combinatorial libraries are developed through designed or random mutation at the identified residues, and high-throughput in vitro methods are employed to screen for the desired function. Previous applications of this method in the context of cytokines have led to the development of a panel of IL13 mutants with a 6-log affinity range for IL13Rα1 to study the interplay of binding affinity and signal transduction (29), engineering of an orthogonal interleukin 2 (IL2) cytokine-receptor complex system that does not act with the native cytokine or receptor (30), and the development of transforming growth factor beta (TGFβ)-based inhibitors (31), among other examples.Here, we describe the development of IL13-mutein CARs with improved selectivity for IL13Rα2 relative to IL13Rα1 and study their activity in IL13Rα1-expressing, IL13Rα2-expressing, and IL13Rα1/IL4Rα-coexpressing contexts. Prior knowledge of the structures of the IL13 complexes with IL13Rα2 and IL13Rα1/IL4Rα (15) informed the design of an IL13-mutein library that was screened using yeast surface display for diminished binding to IL13Rα1 (29). Characterization of hits yielded two promising candidates, termed C4 and D7, with markedly improved selectivity for IL13Rα2, as shown by affinity characterization. These IL13 muteins were then built into CAR constructs for functional comparison to CARs derived from IL13 wild-type (WT) and IL13 with the E12Y mutation. In vitro and in vivo functional characterization of C4 and D7 IL13-mutein CAR T cells showed decreased activation, degranulation, cytokine release, and cytolytic activity compared to WT and E12Y CAR T cells in the presence of IL13Rα1-expressing cancer cells. Interestingly, C4, but not D7, showed attenuated cytotoxicity relative to WT against IL13Rα1/IL4Rα-coexpressing cancer cells in vitro and in vivo. Conversely, all of the IL13-mutein CAR T cells exhibited similar cytolytic killing of IL13Rα2 targets in vitro and in vivo. Collectively, this work provides insight into the interplay of binding affinity and selectivity in CAR T cell activity and validates IL13-mutein CARs with improved recognition profiles for targeting IL13Rα2-expressing malignancies. Application of these CARs could expand the therapeutic window for systemic administration of IL13Rα2-targeted therapy for a variety of cancers.     

The Relationship between Serum Adipokines,miR-222-3p,miR-103a-3p and Glucose Regulation in Pregnancy and Two to Three Years Post-Delivery in Women with Gestational Diabetes Mellitus Adhering to Mediterranean Diet Recommendations

The San Carlos Gestational Diabetes Mellitus (GDM) prevention study, a nutritional intervention RCT based on a Mediterranean Diet (MedDiet), has been shown to reduce the incidence of GDM. The objective of this study is to investigate the relationship of leptin, adiponectin, interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), insulin and HOMA-IRand circulating miRNAs (miR-29a-3p, miR-103a-3p, miR-132-3p, miR-222-3p) with the appearance of GDM and with MedDiet-based nutritional intervention, at 24–28 gestational weeks (GW), and in glucose regulation 2–3 years post-delivery (PD). A total of 313 pregnant women, 77 with GDM vs. 236 with normal glucose tolerance (NGT), 141 from the control group (CG, MedDiet restricting the consumption of dietary fat including EVOO and nuts during pregnancy) vs. 172 from the intervention group (IG, MedDiet supplemented with extra virgin olive oil (EVOO) and pistachios during pregnancy) were compared at Visit 1 (8–12 GW), Visit 2 (24–28 GW) and Visit 3 (2–3 years PD). Expression of miRNAs was determined by the Exiqon miRCURY LNA RT-PCR system. Leptin, adiponectin, IL-6 and TNF-α, were measured by Milliplex^® immunoassays on Luminex 200 and insulin by RIA. Women with GDM vs. NTG had significantly higher leptin median (Q1–Q3) levels (14.6 (9.2–19.4) vs. 9.6 (6.0–15.1) ng/mL; p < 0.05) and insulin levels (11.4 (8.6–16.5) vs. 9.4 (7.0–12.8) µUI/mL; p < 0.001) and lower adiponectin (12.9 (9.8–17.2) vs. 17.0 (13.3–22.4) µg/mL; p < 0.001) at Visit 2. These findings persisted in Visit 3, with overexpression of miR-222-3p (1.45 (0.76–2.21) vs. 0.99 (0.21–1.70); p < 0.05)) and higher levels of Il-6 and TNF-α. When the IG is compared with the CG lower levels of insulin, HOMA-IR-IR, IL-6 levels at Visit 2 and 3 and leptin levels only at Visit 2 were observed. An overexpression of miR-222-3p and miR-103a-3p were also observed in IG at Visit 2 and 3. The miR-222-3p and miR103a-3p expression correlated with insulin levels, HOMA-IR, IL-6 and TNF-α at Visit 2 (all p < 0.05). These data support the association of leptin, adiponectin and insulin/HOMA-IR with GDM, as well as the association of insulin/HOMA-IR and IL-6 and miR-222-3p and miR-103a-3p expression with a MedDiet-based nutritional intervention.     

Differential Effects of Three Nutritional Supplements on the Nutrient Intake of Pregnant Women Enrolled in a Conditional Cash Transfer Program in Mexico: A Cluster Randomized Trial

Supplementation in malnourished pregnant women should not displace natural healthy foods. Objective: To estimate the differential effects of three nutritional supplements on macro- and micronutrient intake of pregnant women beneficiaries of the conditional cash transfer program Prospera (CCT-POP). Methods: Prospective cluster randomized trial. Communities were randomly assigned to receive a fortified beverage (Beverage), micronutrient tablets (Tablets), or micronutrient powder (MNP). Pregnant women (at <25 weeks) were recruited. The food frequency questionnaire was applied at 25 and 37 weeks of pregnancy and at one and three months postpartum (mpp). Differential effects of the three supplements on the median change in nutrient intake from baseline to each follow-up stage were estimated. Results: Median change in protein intake from dietary and supplement sources were significantly lower for MNP and Tablets than for Beverages (baseline to 37 w: −7.80 ± 2.90 and −11.54 ± 3.00, respectively; baseline to 1 mpp: −7.34 ± 2.90 for MNP, p < 0.001). Compared to Beverages, median increases were higher for the MNP for vitamins C (31.2 ± 11.7, p < 0.01), E (1.67 ± 0.81, p < 0.05), and B12 (0.83 ± 0.27, p < 0.01) from baseline to 37 wk; from baseline to 1 mpp, there was a higher median increase in B12 (0.55 ± 0.25, p < 0.05) and folate (63.4 ± 24.3, p < 0.01); and from baseline to 3 mpp, a higher median increase in iron (2.38 ± 1.06, p < 0.05) and folate (94.4 ± 38.1, p < 0.05). Conclusions: Intake of micronutrients was higher for MNP and Tablets, likely due to food displacement among Beverage consumers. Although iron bioavailability and absorption inhibitors were not considered for the present analyses, the distribution of Tablets or MNP had several advantages in this context where micronutrient deficiency remains high among pregnant women, but macronutrient intake is generally adequate or even high.     

Evaluation of the multimorbidity network and its relationship with clinical phenotypes in chronic obstructive pulmonary disease: The GALAXIA study

BackgroundChronic obstructive pulmonary disease (COPD) is a complex and heterogeneous condition, in which taking into consideration clinical phenotypes and multimorbidity is relevant to disease management. Network analysis, a procedure designed to study complex systems, allows to represent connections between the distinct features found in COPD.MethodsNetwork analysis was applied to a cohort of patients with COPD in order to explore the degree of connectivity between different diseases, taking into account the presence of two phenotypic traits commonly used to categorize patients in clinical practice: chronic bronchitis (CB⁺/CB⁻) and the history of previous severe exacerbations (Ex⁺/Ex⁻). The strength of association between diseases was quantified using the correlation coefficient Phi (ɸ).ResultsA total of 1726 patients were included, and 91 possible links between 14 diseases were established. Although the four phenotypically defined groups presented a similar underlying comorbidity pattern, with special relevance for cardiovascular diseases and/or risk factors, classifying patients according to the presence or absence of CB implied differences between groups in network density (mean ɸ: 0.098 in the CB⁻ group and 0.050 in the CB⁺ group). In contrast, between‐group differences in network density were small and of questionable significance when classifying patients according to prior exacerbation history (mean ɸ: 0.082 among Ex⁻ subjects and 0.072 in the Ex⁺ group). The degree of connectivity of any given disease with the rest of the network also varied depending on the selected phenotypic trait. The classification of patients according to the CB⁻/CB⁺ groups revealed significant differences between groups in the degree of conectivity between comorbidities. On the other side, grouping the patients according to the Ex⁻/Ex⁺ trait did not disclose differences in connectivity between network nodes (diseases).ConclusionsThe multimorbidity network of a patient with COPD differs according to the underlying clinical characteristics, suggesting that the connections linking comorbidities between them vary for different phenotypes and that the clinical heterogeneity of COPD could influence the expression of latent multimorbidity. Network analysis has the potential to delve into the interactions between COPD clinical traits and comorbidities and is a promising tool to investigate possible specific biological pathways that modulate multimorbidity patterns.     

Assessment of ovarian vascularization in the polycystic ovary by three-dimensional power Doppler ultrasonography

Objective. To assess whether there are differences in ovarian echogenicity and vascularization as assessed by three-dimensional power Doppler angiography (3D-PDA) between women with polycystic ovaries (PCO) and women with normal ovaries (NO).

Methods. Eighty-three women were classified into two groups according to the 2003 Rotterdam consensus criteria. The NO group comprised women (n = 45) with regular menstrual cycles and proven fertility, whereas the PCO group comprised women (n = 38) with oligo-anovulation, clinical and/or biochemical features of hyperandrogenism, and polycystic ovary morphology at two-dimensional ultrasound. All women were evaluated by means of 3D-PDA. The parameters studied in both groups were follicle number per ovary (FNPO), ovarian volume (OV), mean gray value (MG) and three vascular indices: vascularization index (VI), flow index (FI) and vascularization flow index (VFI).

Results. The PCO group showed a higher mean OV as well as FNPO. No differences in MG, VI, FI and VFI were found between the groups.

Conclusions. 3D-PDA indices are not useful for discriminating between normal and polycystic ovaries.