Application of disaccharides alone and in combination,for the improvement of stability and particle properties of spray-freeze dried IgG

Purpose: Spray-freeze drying (SFD) is a recently applied method to develop pharmaceutical powders. This study aimed to analyze the competence of Trehalose, Mannitol, Lactose, and Sorbitol instability and aerosolization of Immunoglobulin G (IgG) via SFD.

Methods: Induced soluble aggregates were quantified at 0 and 3?months, and 45?°C using size-exclusion chromatography. Conformation and thermogravimetric assessments were done by Fourier transform infrared spectroscopy and differential scanning calorimetry. Laser light scattering was performed to determine the particle sizes. Aerodynamic features were characterized by twin stage impinger and scanning electron microscopy.

Results: Although sugars/polyols preferably stabilized IgG following the process, storage stabilization was achieved in Trehalose, Trehalose-Lactose, Lactose, and Trehalose-Mannitol-based powders with soluble aggregates <5%. The conformation of antibody was preserved with β sheet content from 66.28% to 76.37%. Particle sizes ranged from 5.23 to 8.12?µm. Mannitol exhibited the best aerodynamic behavior, fine particle fraction (FPF: 70%) but high degree of protein aggregation during storage.

Conclusions: SFD could favorably stabilize antibody using Trehalose and its combination with Lactose and Mannitol, and also, Lactose alone. Sorbitol disturbed IgG powder recovery. Incorporation of other types of excipient is required for efficient respiratory delivery of IgG molecules.     

Inhibition of inducible nitric oxide synthase enhances anti-tumour immune responses in rats immunized with IFN-gamma-secreting glioma cells

Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, l-N(6)-(1-Iminoethyl)-l-lysine (l-NIL), and non-selective, N-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l-NIL and l-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma, and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with l-NIL, but not l-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, l-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.     

Landscape of surfaceome and endocytome in human glioma is divergent and depends on cellular spatial organization

Therapeutic strategies directed at the tumor surfaceome (TS), including checkpoint inhibitor blocking antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cells, provide a new armament to fight cancer. However, a remaining bottleneck is the lack of strategies to comprehensively interrogate patient tumors for potential TS targets. Here, we have developed a platform (tumor surfaceome mapping [TS-MAP]) integrated with a newly curated TS classifier (SURFME) that allows profiling of primary 3D cultures and intact patient glioma tumors with preserved tissue architecture. Moreover, TS-MAP specifically identifies proteins capable of endocytosis as tractable targets for ADCs and other modalities requiring toxic payload internalization. In high-grade gliomas that remain among the most aggressive forms of cancer, we show that cellular spatial organization (2D vs. 3D) fundamentally transforms the surfaceome and endocytome (e.g., integrins, proteoglycans, semaphorins, and cancer stem cell markers) with general implications for target screening approaches, as exemplified by an ADC targeting EGFR. The TS-MAP platform was further applied to profile the surfaceome and endocytome landscape in a cohort of freshly resected gliomas. We found a highly diverse TS repertoire between patient tumors, not directly associated with grade and histology, which highlights the need for individualized approaches. Our data provide additional layers of understanding fundamental to the future development of immunotherapy strategies, as well as procedures for proteomics-based target identification and selection. The TS-MAP platform should be widely applicable in efforts aiming at a better understanding of how to harness the TS for personalized immunotherapy.

Cell-surface proteins have a key role in drug development, and approximately two-thirds of approved human drugs target a cell-surface protein (1). Recently, tumor cell–surface proteins integrated with the plasma membrane (tumor surfaceome [TS]) have attracted considerable attention as targets for immunotherapies in cancer. Immune checkpoint-blocking antibodies (e.g., ipilimumab and nivolumab), antibody drug conjugates (ADCs, e.g., trastuzumab emtansin), radioimmunotherapy (RIT, e.g., ⁹⁰Y ibritumomab tiuxetan), and chimeric antigen receptor T (CAR-T) cells are all directed at the TS and currently revolutionize cancer treatment (2–6). With the impressive development of creative methods for antibody and T cell engineering, a remaining challenge is the lack of strategies to comprehensively map potential TS target antigens for the design of more rational, individualized treatments (7). Although advancements in DNA and RNA sequencing provide high throughput data for prediction algorithms, e.g., personalized peptide vaccine trials (8, 9), the predicted proteome derived from these platforms is not necessarily expressed and available for targeting. Moreover, proteomics-based strategies involve analysis of the bulk from disintegrated tumor tissue, resulting in loss of spatial information and limited coverage of the less abundant and hydrophobic TS proteins (10, 11). Of particular relevance, ADC, RIT, and other intracellular drug delivery strategies rely on TS targets that functionally engage in endocytic internalization (12). Clearly, despite its great targeting potential in cancer immunotherapy, the TS remains an elusive treasure for further discovery.Procedures for unbiased mapping of the TS and target identification should include specific labeling of the TS in freshly resected patient tumors with preserved tissue architecture. Enrichment of TS proteins and reduction of noise from intracellular proteins as well as abundant extracellular matrix collagens and glycoproteins would greatly improve downstream mass spectrometry analysis. Moreover, the approach should allow functional and dynamic profiling of TS internalization in an intact tissue environment. With the aim to address these challenges and to provide insight into the complexity of the TS, we have developed a versatile technology for TS mapping (TS-MAP). As proof of concept, we focused on primary brain tumors that remain among the most aggressive forms of cancer and for which attempts to conquer the most common variant, glioblastoma (GBM) (World Health Organization [WHO] grade IV) have failed so far (13). TS-MAP is compatible with spheroids from primary human stem cell–like GBM cultures, as well as mouse and patient brain tumors, and separately profiles surface resident and internalized TS proteins. Moreover, a TS classifier (SURFME) was curated for filtering and categorization of bona fide membrane proteins exposed to the extracellular space. We find significant differences in the TS between the 2D and 3D spheroid format, which underlines the importance of cellular spatial organization. In strong support of the need of individualized approaches, our findings suggest substantial intertumoral heterogeneity in the relative abundance of TS proteins in a cohort of freshly resected patient gliomas.     

Mutational analysis of enhancer domains responsive to trans-activation by the X gene of human hepatitis B virus

Summary The X gene product of human hepatitis B virus (HBV) trans-activates the HBV enhancer. In order to identify domains responsive to trans-activation by the X gene, we introduced a series of mutations into the HBV enhancer and assayed the enhancer activities in the presence of the X gene product. Our results suggest that the EP domain of the enhancer is essential for trans-activation by the X gene.     

Infradiaphragmatic versus supradiaphragmatic Hodgkin lymphoma: a retrospective review of 1,114 patients

Infradiaphragmatic Hodgkin lymphoma (IDH) accounts for 4-13% of cases of stage I-II Hodgkin lymphoma (HD). It has been associated with distinct pre-treatment characteristics and outcomes when compared with supradiaphragmatic HD (SDH). The comparison of IDH vs SDH can only be made in early and intermediate stages (I-II), such a comparison is not possible for advanced stages (III-IV). This study retrospectively compared two groups of 1013 patients with stage I-II SDH and 101 patients with IDH (10%). These two sub-groups of patients were treated in 1988-1993 in 2 prospective randomized clinical trials in Germany for early and intermediate stages of Hodgkin lymphoma. IDH-patients were older (median 39 vs 31 years; p < 0.001), predominantly male (73% vs 52%; p < 0.001) and more often had involvement of 3 lymph node areas (LNA) (80% vs 55%; p < 0.001). Histology in IDH was more likely to be mixed cellularity (46.5% vs 23.6%, p < 0.001) or lymphocyte predominant (20 vs 10%, p = 0.003) and less likely nodular sclerosis (25% vs 63%, p < 0.001). In early-stage unfavorable disease, IDH was associated with a higher treatment failure rate (unadjusted hazard ratio 2, 95% CI, 1.3-3.4; p = 0.003). After controlling for age, sex, stage, histology, B-symptoms and involvement of 3 LNA, the adjusted hazard ratio was 1.25 (95% CI, 0.65-2.4; p = 0.51) so that IDH was no longer associated with a statistically significant treatment failure rate. Poorer outcomes with IDH as compared to SDH are attributable to its association with known adverse prognostic risk factors, but IDH, in itself, is not an independent adverse prognostic factor for treatment failure or survival.     

Childbearing among young Latino women in the United States.

We analyzed 1979 and 1982 data from the Youth Cohort of the National Longitudinal Surveys (NLS) of Labor Market Experience to compare rates of early childbearing among White, Black, Mexican-origin and Puerto Rican women up to age 21. Latino young women fall in between the extremely low rate of the Whites and the extremely high rate of the Blacks. Mexican and Puerto Rican young women have similar proportions of premarital first births, but the marital first birth rate for young Mexicans is twice that of the Puerto Ricans. The bulk of Mexican first births, like births to Whites, occur within marriage, while Puerto Rican first births are similar to those of Blacks, the majority being out-of-wedlock. These racial/ethnic differences in premarital first birth rates do not change greatly when socioeconomic status, and birthplace of respondents and respondents' parents are controlled.     

The outermost N‐terminal region of tapasin facilitates folding of major histocompatibility complex class I

Tapasin (Tpn) is an ER chaperone that is uniquely dedicated to MHC‐I biosynthesis. It binds MHC‐I molecules, integrates them into peptide‐loading complexes, and exerts quality control of the bound peptides; only when an “optimal peptide” is bound will the MHC‐I be released and exported to the cell surface for presentation to T cells. The exact mechanisms of Tpn quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human Tpn, Tpn_1–87 (representing the 87 N‐terminal and ER‐luminal amino acids of the mature Tpn protein). Using a biochemical peptide–MHC‐I‐binding assay, recombinant Tpn_1–87 was found to specifically facilitate peptide‐dependent folding of HLA‐A*0201. Furthermore, we used Tpn_1–87 to generate a monoclonal antibody, αTpn_1–87/80, specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of αTpn_1–87/80 was located to Tpn_40–44, which maps to a surface‐exposed loop on the Tpn structure. Together, these results demonstrate that the N‐terminal region of Tpn can be recombinantly expressed and adopt a structure, which at least partially resembles that of WT Tpn, and that this region of Tpn features chaperone activity facilitating peptide binding of MHC‐I.