The role of coagulation factor V in breast cancer

In addition to the well-known link between cancer and risk of thrombotic diseases, it is known that increased coagulation activity is associated with tumor progression. Studying the underlying molecular mechanisms could lead to a better understanding of the association between cancer and hemostasis, and possibly lead to a more individualized treatment for patients suffering cancer or cancer-related thrombosis. The role of coagulation factor V (FV) in cancer has not been studied extensively, but FV is known to express both procoagulant and anticoagulant properties contributing to a balance in the coagulation system. Also, several F5 SNPs are reported to be associated with risk of breast cancer. The aim of this thesis was to gain a better understanding of how coagulation FV relates to breast cancer, using both a clinical breast cancer material and in vitro breast cancer cells. The clinical significance of FV was studied in a merged breast cancer dataset (n=1881, from GOBO) and in patients from the OsloII study (FV plasma protein levels from n=366 and FV tumor mRNA levels from n=152). A FV overexpression cell model was used to study FV wild type and the FV gene variants rs6025 (FV Leiden), rs6028 and a novel mutation (A2184T) in the MDA-MB-231 breast cancer cell line, and their effects on gene expression, protein secretion as well as functional effects on cell growth, cell death and inflammatory markers. Increased FV mRNA expression was observed in patients with more aggressive tumor subtypes; ER-negative, HER2-enriched, basal-like, and high-grade tumors. Interestingly, within these patient subgroups, high FV mRNA expression was associated with increased survival rates. F5 was found associated, directly or indirectly, to cell proliferation, differentiation and immune response. Through the in vitro studies we found that FV wt was highly overexpressed, showed reduced cell growth, increased apoptosis but decreased necrosis and elevated levels of pro-inflammatory cytokines. The FV variants showed varying differences to FV wt, with increase in cell growth, unchanged cell death and increase in inflammatory markers. Deviating, FV rs6028 showed a more elevated increase in cell growth than the other FV variants, and FV A2184T highly increased levels of cell death and pro-inflammatory markers. In conclusion, the results of this thesis indicate that F5 acts as a suppressor gene, showing increased survival in patients with increased levels. F5 may be a good candidate gene for new treatment procedures for patients suffering breast cancer or cancer-related thrombosis. Genetic variation in F5 does affect the suppressor effect, mostly due to reduced levels of FV. Nevertheless, a link to altered biological function caused by the A2184T was assumed.I tillegg til den velkjente sammenhengen mellom kreft og trombotiske sykdommer, er økt koagulasjonsaktivitet kjent for å være forbundet med tumorprogresjon. Å studere de underliggende molekylære mekanismene kan føre til en bedre forståelse av sammenhengen mellom kreft og hemostase, og muligens føre til en mer individualisert behandling for pasienter som lider av kreft eller kreft-relatert trombose. Rollen til koagulasjonsfaktor V (FV) i kreft er ikke nøye studert, men FV er kjent for å uttrykke både prokoagulante og antikoagulante egenskaper som bidrar til en balanse i koagulasjonssystemet. Dessuten er det rapportert en assosiasjon mellom flere F5 SNPs og risiko for brystkreft. Målet med denne avhandlingen var å få en bedre forståelse av hvordan FV er relatert til brystkreft, studert ved hjelp av både et klinisk brystkreft materiale og in vitro brystkreft celler. Den kliniske betydningen av FV ble undersøkt i et sammenslått brystkreft datasett (n = 1881, fra GOBO) og i pasienter fra OsloII studien (FV plasma protein nivåer fra n = 366 og FV tumor mRNA nivåer fra n = 152). En FV overekspresjonscellemodell ble benyttet for å studere FV villtype og FV genvariantene rs6025 (FV Leiden), rs6028 og en ikke tidligere rapportert mutasjon (A2184T) i brystkreftcellelinjen MDA-MB-231, og deres effekt på genekspresjon, proteinsekresjon, samt funksjonelle virkninger på cellevekst, celledød og inflammatoriske markører. Økt FV mRNA ekspresjon ble observert hos pasienter med tumorer med aggressive karakteristika; ER-negative tumorer, HER2-enriched tumorer, basal-lignende tumorer, og tumorer av høy grad. Blant pasienter med disse karakteristika ble høyt FV mRNA uttrykk forbundet med økt overlevelse. F5 ble funnet assosiert direkte eller indirekte, til celleproliferasjon, celledifferensiering og immunrespons. Gjennom in vitro studiene fant vi at FV wt viste høyt FV overutrykk, redusert cellevekst, økt apoptose men redusert nekrose, og forhøyede nivåer av proinflammatoriske cytokiner. FV variantene viste varierende forskjeller for FV wt, med økning i cellevekst, uforandret celledød og økning i inflammatoriske markører. FV rs6028 avvikte med økt cellevekst i forhold til de andre FV variantene, og FV A2184T avvikte med økte nivåer av celledød og pro-inflammatoriske markører i forhold til de andre FV variantene. For å konkludere tyder resultatene i denne avhandlingen på at F5 har tumor-suppressor-gen-egenskaper sett av økt overlevelse hos pasienter med økte FV nivåer. F5 kan være et godt kandidat gen for nye behandlingsmetoder for pasienter som lider av brystkreft eller kreft-relatert trombose. Genetisk variasjon i F5 påvirker suppressor-gen-effekten, hovedsakelig på grunn av reduserte FV nivåer. I tillegg ble det antatt en link til endret biologisk funksjon forårsaket av A2184T.M-BIOTE

Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions

Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria

Abstract Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions

Titers of antibodies against ancestral SARS-CoV-2 correlate with levels of neutralizing antibodies to multiple variants

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 serum samples. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.publishedVersio