Characterization and modulation of anti-αßTCR antibodies and their respective binding sites at the ßTCR chain to enrich engineered T cells.

T cell engineering strategies offer cures to patients and have entered clinical practice with chimeric antibody-based receptors; αßT cell receptor (αßTCR)-based strategies are, however, lagging behind. To allow a more rapid and successful translation to successful concepts also using αßTCRs for engineering, incorporating a method for the purification of genetically modified T cells, as well as engineered T cell deletion after transfer into patients, could be beneficial. This would allow increased efficacy, reduced potential side effects, and improved safety of newly to-be-tested lead structures. By characterizing the antigen-binding interface of a good manufacturing process (GMP)-grade anti-αßTCR antibody, usually used for depletion of αßT cells from stem cell transplantation products, we developed a strategy that allows for the purification of untouched αßTCR-engineered immune cells by changing 2 amino acids only in the TCRß chain constant domain of introduced TCR chains. Alternatively, we engineered an antibody that targets an extended mutated interface of 9 amino acids in the TCRß chain constant domain and provides the opportunity to further develop depletion strategies of engineered immune cells.

GMP-Grade Manufacturing of T Cells Engineered to Express a Defined γδTCR.

γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αßTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αßTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.

The FOAM study: is Hysterosalpingo foam sonography (HyFoSy) a cost-effective alternative for hysterosalpingography (HSG) in assessing tubal patency in subfertile women? Study protocol for a randomized controlled trial.

BACKGROUND: Tubal pathology is a causative factor in 20% of subfertile couples. Traditionally, tubal testing during fertility work-up is performed by hysterosalpingography (HSG). Hysterosalpingo-foam sonography (HyFoSy) is a new technique that is thought to have comparable accuracy as HSG, while it is less expensive and more patient friendly. HyFoSy would be an acceptable alternative for HSG, provided it has similar effectiveness in terms of patient outcomes. METHODS/DESIGN: We aim to compare the effectiveness and costs of management guided by HyFoSy or by HSG. Consenting women will undergo tubal testing by both HyFoSy and HSG in a randomized order during fertility work-up. The study group will consist of 1163 subfertile women between 18 and 41 years old who are scheduled for tubal patency testing during their fertility work-up. Women with anovulatory cycles not responding to ovulation induction, endometriosis, severe male subfertility or a known contrast (iodine) allergy will be excluded. We anticipate that 7 % (N = 82) of the participants will have discordant test results for HyFoSy and HSG. These participants will be randomly allocated to either a management strategy based on HyFoSy or a management strategy based on HSG, resulting in either a diagnostic laparoscopy with chromopertubation or a strategy that assumes tubal patency (intrauterine insemination or expectant management). The primary outcome is ongoing pregnancy leading to live birth within 12 months after randomization. Secondary outcomes are patient pain scores, time to pregnancy, clinical pregnancy, miscarriage rate, multiple pregnancy rate, preterm birth rate and number of additional treatments. Costs will be estimated by counting resource use and calculating unit prices. DISCUSSION: This trial will compare the effectiveness and costs of HyFoSy versus HSG in assessing tubal patency in subfertile women. TRIAL REGISTRATION: Dutch Trial Register (NTR 4746, http://www.trialregister.nl ). Date of registration: 19 August 2014.

The next step toward GMP-grade production of engineered immune cells.

Removing less potent T cell subsets as well as poorly- or non-engineered cells can optimize effectiveness of engineered T cell therapy against cancer. We have recently described a novel, GMP-ready method for the purification of engineered immune cells that might further boost the clinical success of cancer immunotherapy.

FUNCTIONAL LIMITATIONS AND ADIPOKINES IN HIV-INFECTED OLDER ADULTS.

BACKGROUND: There is a significant increase in the number of HIV-infected older adults (HOA). This population may experience functional decline at a much younger age. Little is known about the relationship between functional limitations and systemic adipokines in HOA. OBJECTIVE: Our study aimed to evaluate the relationship between functional limitations and systemic adipokine levels in HOA population. DESIGN: Cross-sectional. SETTING: Academic hospital-based infectious disease clinic. PARTICIPANTS: The study investigated community-dwelling HIV-infected adults >50 years old and compared this group with age, gender and BMI comparable healthy controls. MEASUREMENTS: We measured functional status, body composition and plasma concentrations of adipokines. RESULTS: Fifty-four HOA were studied (mean: age 57 years, BMI 29 kg/m2, CD4 604, duration of HIV 17 years) and compared with thirty-two age, gender and BMI comparable healthy controls. The HOA group showed significantly higher functional limitations compared to the age, gender and BMI comparable controls (p<0.05). Levels of adipokines were significantly different between the two groups (p<0.05). Multiple regression analyses indicated that adiponectin and visfatin were significantly correlated with several physical function measures after controlling for age, sex, and metabolic comorbidities. Adiponectin was negatively correlated with functional limitations, and this relationship was stronger in the control group compared to the HOA group. Conversely, visfatin was positively correlated with functional limitations only in the HOA group. CONCLUSION: HOA have significant functional limitations and alteration in adipokine levels compared to controls. Adiponectin and visfatin were associated with functional limitations. Visfatin was a correlate of physical function only in the HOA group. Prospective longitudinal studies could provide further insight on the role of adipokines in HIV-related functional decline.

Orchestrating an immune response against cancer with engineered immune cells expressing αßTCRs, CARs, and innate immune receptors: an immunological and regulatory challenge.

Over half a century ago, the first allogeneic stem cell transplantation (allo-SCT) initiated cellular immunotherapy. For several decades, little progress was made, and toxicity of allo-SCT remained a major challenge. However, recent breakthroughs have opened new avenues to further develop this modality and to provide less toxic and equally efficient interventions for patients suffering from hematological or solid malignancies. Current novel cellular immune interventions include ex vivo expansion and adoptive transfer of tumor-infiltrating immune cells or administration of drugs which antagonize tolerizing mechanisms. Alternatively, transfer of immune cells engineered to express defined T cell receptors (TCRs) and chimeric antigen receptors (CARs) has shown its potential. A valuable addition to 'engineered' adaptive immunity has emerged recently through the improved understanding of how innate immune cells can attack cancer cells without substantial side effects. This has enabled the development of transplantation platforms with limited side effects allowing early immune interventions as well as the design of engineered immune cells expressing innate immune receptors. Here, we focus on innate immune interventions and their orchestration with TCR- and CAR-engineered immune cells. In addition, we discuss how the exploitation of the full potential of cellular immune interventions is influenced by regulatory frameworks. Finally, we highlight and discuss substantial differences in the current landscape of clinical trials in Europe as compared to the USA. The aim is to stimulate international efforts to support regulatory authorities and funding agencies, especially in Europe, to create an environment that will endorse the development of engineered immune cells for the benefit of patients.

Evaluation of strategies regarding management of imminent preterm delivery before 32 weeks of gestation: a regional cohort study among 1375 women in the Netherlands.

OBJECTIVE: To evaluate the management of imminent preterm delivery with respect to prescription of antenatal corticosteroids (ACS) and referral to a tertiary center. STUDY DESIGN: A retrospective cohort study existing of 1 perinatal center and 9 referring hospitals. All women who received their first dose of ACS in 1 of the 10 hospitals between 24+0 and 32+0 weeks of gestation and/or delivered before 32 weeks of gestation from 2005 until 2010. Patients were identified using the electronic database of hospital pharmacies. Main outcome measures were time interval from administration to delivery for different indications and number of women who were not referred in time to a tertiary center. RESULTS: In total, 1375 women received ACS. Main indications were suspected preterm labor (44.7%), preterm prelabor rupture of membranes (15.9%), maternal indication (12.8%), fetal indication (9.2%) and vaginal blood loss (8.4%). Overall, 467 (34.0%) women delivered ≤7 days after ACS administration; 8.7% of women with vaginal blood loss and 54.5% of women with maternal indication. Among the 931 women who received ACS in the secondary hospitals, 452 (48.5%) women were referred to a tertiary hospital and 89 (6.5%) women delivered in a secondary hospital with a gestational age of less than 32 weeks. CONCLUSION: One-third of all women receiving ACS delivered within 7 days and half of the women who received ACS in a secondary hospital were referred to a tertiary center. There seems to be room for improvement regarding the timing of ACS administration and subsequently referral to a tertiary center.

The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity.

BACKGROUND: HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ. METHODS: The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. In vivo studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions. RESULTS: The gefitinib and RAD001 combination inhibited cell growth in vitro in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated in vitro with cell line dependent increases in cytotoxicity and cytostasis while treatment in vivo promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (in vitro and in vivo) and EGFR signaling in vivo relative to the single drugs. CONCLUSIONS: The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.

The incidence of herpes zoster in patients with Hodgkin's disease. An analysis of prognostic factors.

The incidence of zoster in 717 patients with Hodgkin's disease was determined by a retrospective chart review. All patients had been treated and followed in one of six cancer centers. Prognostic factors that might predict the subsequent incidence of zoster were examined by univariate and multivariate analytic techniques. Intensity of treatment was a key factor in the incidence of zoster. Thirty-six months after initiation of treatment, patients receiving chemotherapy-radiation-chemotherapy had twice the attack rate (27.3%) of those receiving radiation alone (11.5%). The pediatric age group had a significantly higher attack rate (26.6%) than did adults (18.7%). Stage, histology, and laparotomy did not influence the incidence of zoster.

The prognostic value of calcitonin immunostaining in medullary carcinoma of the thyroid.

This study was undertaken to evaluate the prognostic value of calcitonin (CT) immunostaining in medullary carcinoma of the thyroid (MCT). Primary tumors and metastases from 44 patients with MCT were stained for CT using the immunoperoxidase method. According to the number of cells stained in the primary tumors, the patients were subdivided into 3 groups. Group A included 9 patients with CT-poor tumors (less than 25% of cells stained), group C consisted of 21 patients with CT-rich tumors (greater than 75% of cells stained), and group B included 14 patients with intermediate tumors (25-75% of cells stained). Group A and B patients presented with more advanced disease and had a higher rate of recurrence than group C patients, but the difference was not statistically significant. The number of cells stained correlated well with survival, which was significantly longer for group C than groups B (P = 0.044) and A (P = 0.001). Group B patients survived longer than did those of group A (P = 0.036). The 5-yr survival rates were 52.7%, 93%, and 100% for groups A, B, and C, respectively. Eighty-three percent of patients with multiple endocrine neoplasia IIa had CT-rich tumors, whereas 78.3% of those with sporadic disease had CT-poor ones (P less than 0.001). CT-rich metastases were compatible with prolonged survival even if they were affecting vital organs, whereas CT-poor metastases were virulent and carried a poor prognosis. Therefore, CT immunostaining is recommended for all patients with MCT, as it can predict the course of the disease. It also can be used as a staging procedure and may help the clinician in making a decision regarding the therapeutic approach.

Medullary carcinoma of the thyroid. A study of the clinical features and prognostic factors in 161 patients.

The natural history and prognostic factors of medullary carcinoma of the thyroid (MCT) were studied in 161 patients seen at the University of Texas M. D. Anderson Hospital and Tumor Institute at Houston between 1944 and 1983. One hundred twenty-five patients (77.6%) had the sporadic variety of MCT, 31 patients (19.3%) had multiple endocrine neoplasm (MEN) type IIa and 5 patients (3.1%) had MEN-IIb. The disease occurred equally in both sexes (M:F ratio 1:1.05). Thyroid nodules were the most common presenting feature especially in patients with the sporadic disease and MEN-IIb. Fifteen patients with MEN-IIa had occult MCT; the diagnosis was made through screening of family members with calcitonin measurement before and after stimulation with calcium or pentagastrin. Sixteen patients with MEN-II had pheochromocytoma and 7 had hyperparathyroidism. Total thyroidectomy was the most commonly performed operation. The lowest incidence of recurrence occurred in patients who underwent total thyroidectomy and modified neck dissection. Radioactive 131I was used as adjunct to surgery in 19 patients but it did not improve the survival or lower the incidence of recurrence. Patients who received postoperative radiotherapy had significantly lower adjusted survival rates than those treated by surgery alone, but we tended to irradiate patients with more advanced disease. Chemotherapy was administered to 11 patients with disseminated metastases but the response was poor. The 5- and 10-year adjusted survival rates of all the patients with MCT were 78.2% and 61.4%, respectively. Patients with MEN-IIa had much better rates than patients with sporadic disease (p = 0.0005), who were 7.74 times more likely to die of MCT. The stage of the disease at presentation was a major prognostic factor. Patients with stages III or IV disease were 7.31 times more likely to die of MCT than those with stages I or II. There was no significant difference in survival between patients with stages I and II or III and IV. The presence of cervical lymph node metastases did not affect the survival adversely. Direct extension with involvement of tissue was a bad prognostic sign. Patients younger than 40 years old at the time of diagnosis of MCT had a significantly better adjusted survival rate than those who were older. Women had a better prognosis than men, who were 1.89 times more likely to die of MCT. Diarrhea was a bad prognostic sign. However, it occurred more frequently in patients with advanced stages of the disease and larger tumor mass.(ABSTRACT TRUNCATED AT 400 WORDS)

Radioactive iodine in the treatment of medullary carcinoma of the thyroid.

To determine the value of adjunct 131I therapy in medullary carcinoma of the thyroid (MCT), two groups of patients with histologically proved MCT were studied. Group A consisted of 15 patients (6 men and 9 women) treated by surgery, followed by an ablative dose of 131I, and group B included 84 patients (39 men and 45 women) treated by surgery alone. Patients in group A were followed for 1.5-14 yr (median, 53 months), and those in group B were followed for 1-27 hr (median, 75 months). Seven patients (46.6%) from group A and 36 patients (42.9%) from group B developed recurrence or metastasis. The 5- and 10-yr survival rates were 87.5% and 75% for group A and 89% and 74% for group B; the difference was not significant (P greater than 0.05). The changes in serum calcitonin levels in 4 patients of group A were not different from those in patients who did not receive 131I. We conclude that 131I has no value as an adjunct to surgery in the management of MCT.