7 research outputs found
Bivalent EGFR-Targeting DARPin-MMAE Conjugates
Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5)
Bivalent EGFR-Targeting DARPin-MMAE Conjugates
Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5)
Prostate-specific membrane antigen expression in the vasculature of primary lung carcinomas associates with faster metastatic dissemination to the brain
Glioblastomas and brain metastases (BM) of solid tumours are the most common central nervous system neoplasms associated with very unfavourable prognosis. In this study, we report the association of prostate-specific membrane antigen (PSMA) with various clinical parameters in a large cohort of primary and secondary brain tumours. A tissue microarray containing 371 cases of ascending grades of gliomas pertaining to astrocytic origin and samples of 52 cases of primary lung carcinomas with matching BM with follow-up time accounting to 10.4 years was evaluated for PSMA expression using immunohistochemistry. In addition, PSMA expression was studied in BM arising from melanomas and breast carcinomas. Neovascular expression of PSMA was evident alongside with high expression in the proliferating microvasculature of glioblastomas when compared to the tumour cell expression. This result correlated with the results obtained from the in silico (cancer genome databases) analyses. In gliomas, only the vascular expression of PSMA associated with poor overall survival but not the tumour cell expression. In the matched primary lung cancers and their BM (n = 52), vascular PSMA expression in primary tumours associated with significantly accelerated metastatic dissemination to the brain with a tendency towards poor overall survival. Taken together, we report that the vascular expression of PSMA in the primary and secondary brain tumours globally associates with the malignant progression and poor outcome of the patients.Peer reviewe
Cancer neuroscience : cell surface proteins in neural development and cancer
Organ development is orchestrated by a series of complex molecular programs where the cells of the three germ layers transform into an embryo. A perturbation in cellular or molecular interactions can lead to defective organogenesis or even embryonic lethality. The key feature of embryonic development is the stringent regulation of proliferation, migration, differentiation, and apoptosis to mediate normal cellular functions. Cancer cells hijack these molecular programs underlying embryogenesis to promote tumorigenesis. Neural innervation and expression of neural markers have been detected in various tumors. Moreover, the presence of nerves in the tumor microenvironment has been shown to regulate aberrant tissue function and promote cancer progression.
Cancer neuroscience is a newly emerging field of cancer biology focused on understanding the cancer-nervous system interactions. This thesis brings together both developmental neurobiology and cancer biology to study the functions of two different neuron-associated genes (FOLH1 and IGSF3). Further, transcriptomic profiling was performed in patient-derived glioblastoma (GBM) stem cell lines to identify their genetic signatures and cellular states.
Glutamate carboxypeptidase II (GCPII) also known as prostate-specific membrane antigen (PSMA) is a neuro-glial enzyme encoded by the FOLH1 (folate hydrolase 1) gene. My results show that in gliomas vascular expression of GCPII/PSMA is associated with poor overall survival (I). Interestingly, an association between accelerated metastatic dissemination to the brain and vascular GCPII/PSMA expression was observed in primary lung cancers (I).
Through antibody phage display, our laboratory originally identified Immunoglobulin superfamily member 3 (IGSF3) as a metastasis-associated protein. To determine the physiological function of this gene, Igsf3 knockout mice (KO) were generated using CRISPR/Cas9 mediated genome engineering. A defect in neural crest cell migration and intestinal abnormalities were observed in the KO animals (II). A screening of various cancers using the cancer genome atlas (TCGA) datasets revealed that IGSF3 is expressed by gliomas, melanoma, breast, lung, and colorectal carcinomas and that high expression levels correlate with poor survival (unpublished results).
Detailed transcriptomic profiling revealed the heterogeneity of the glioblastoma stem cell lines and allowed their clustering to different GBM subtypes (III). In addition, a high Cluster of Differentiation 109 (CD109) mRNA levels were observed in the mesenchymal subtype of GBMs (III).
This thesis provides a new understanding of neuron-associated genes during development and tumorigenesis. In the future, the Igsf3 KO mouse model will be useful for studying the neural crest and its derivatives while the neuron-specific expression of IGSF3 allows studies focusing on neuronal involvement in cancer progression.Monimutkaiset molekyyliohjelmat säätelevät elinten kehitystä soluista alkioksi. Alkion kehityksen aikainen solujen lisääntymisen, liikkumisen, erilaistumisen ja ohjelmoidun solukuoleman tarkka säätely varmistaa kudosten normaalin kehityksen ja toiminnot. Häiriöt solujen tai molekyylien vuorovaikutuksessa voivat johtaa elintenkehityksen häiriöihin tai jopa alkion kuolemaan. Syöpäsolut käyttävät hyväkseen alkion kehitystä
sääteleviä molekyyliohjelmia kasvaimen kasvun edistämiseksi. Lisäksi kasvaimissa on havaittu hermotusta ja syöpäsoluissa hermoston merkkimolekyylien ilmentymistä. Hermojen on havaittu kasvaimen mikroympäristössä edistävän mm. syöpäkasvaimen kasvua ja syövän etenemistä.
Syövän neurotiede on uusi syöpäbiologian ala, joka keskittyy syövän ja hermoston vuorovaikutuksen ymmärtämiseen. Tämä väitöskirja yhdistää kehityksen aikaisen neurobiologian ja syöpäbiologian kahden eri geenin (FOLH1 ja IGSF3) avulla. Näiden kahden geenin tutkimisen lisäksi väitöskirjassa profiloitiin glioblastoomaa (GBM) sairastavilta potilailta peräisin olevista kantasolulinjoista geenien ilmentymistä koko
genomin laajuudelta.
FOLH1 (folaattihydrolaasi 1) -geenin koodaama glutamaattikarboksypeptidaasi II (GCPII) on entsyymi, joka tunnetaan myös eturauhasspesifisenä kalvoantigeeninä (PSMA). Tulokseni osoittavat, että verisuonistossa ilmentyvä GCPII/PSMA proteiini assosioituu glioomapotilaiden huonoon elinajanennusteeseen (I). Lisäksi havaitsin, että primääreissä keuhkosyövissä verisuonten pinnalla ilmentyvä GCPII/PSMA assosioituu nopeampaan keuhkosyövän etäpesäkkeiden leviämiseen aivoihin (I).
Vasta-ainefaagiselektiomenetelmää hyödyntäen laboratoriomme tunnisti, että immunoglobuliinisuperperheeseen kuuluvan proteiinin 3 (IGSF3) ilmentyminen assosioituu syövän etäpesäkkeisiin. Selvittääksemme tämän geenin fysiologista
toimintaa loimme muuntogeenisen hiirikannan, jossa Igsf3-geenin toiminta poistettiin CRISPR/Cas9 genomin muokkaustekniikan avulla (II). Poistogeenisillä hiirillä havaittiin häiriöitä sekä hermostopienan (neural crest, NC) solujen liikkumisessa että suoliston
hermotuksessa. Julkista genomikartastoa (The Cancer Genome Atlas, TCGA) hyödyntäen tutkimme geenien ilmentymistä eri syöpätyypeisssä. Tämä osoitti, että IGSF3 ilmenee glioomissa ja melanoomissa sekä rinta-, keuhko- ja kolorektaalisyövissä. Analyysimme osoitti myös, että IGSF3:n korkea ilmenemistaso korreloi huonon
ennusteen kanssa (julkaisematon tulos).
Glioblastooman kantasolulinjojen yksityiskohtainen transkriptioprofilointi paljasti niiden heterogeenisyyden ja mahdollisti niiden luokituksen GBM:n eri alatyyppeihin (III). Lisäksi GBM:n mesenkymaalisessa alatyypissä havaittiin hyvin korkea CD109-geenin ilmentyminen (III).
Yhteenvetona voidaan todeta, että tämä väitöskirja tuotti uutta tietoa kahden hermostossa ilmentyvän geenin toiminnasta alkion kehityksen aikana ja syövissä. Igsf3 KO-hiirimallin avulla voidaan jatkossa tutkia hermostopienan ja sen johdannaisten kehitystä ja toimintaa, kun taas IGSF3:n ilmentyminen hermoissa mahdollistaa syöpäsolujen ja
hermoston välisen vuorovaikutuksen tutkimisen
Bivalent EGFR-Targeting DARPin-MMAE Conjugates
Karsten L, Janson N, Le Joncour V, et al. Bivalent EGFR-Targeting DARPin-MMAE Conjugates. International Journal of Molecular Sciences. 2022;23(5): 2468.Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR designed ankyrin repeat protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5)
Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization
Abstract The immunoglobulin (Ig) superfamily members are involved in cell adhesion and migration, complex multistep processes that play critical roles in embryogenesis, wound healing, tissue formation, and many other processes, but their specific functions during embryonic development remain unclear. Here, we have studied the function of the immunoglobulin superfamily member 3 (IGSF3) by generating an Igsf3 knockout (KO) mouse model with CRISPR/Cas9-mediated genome engineering. By combining RNA and protein detection methodology, we show that during development, IGSF3 localizes to the neural crest and a subset of its derivatives, suggesting a role in normal embryonic and early postnatal development. Indeed, inactivation of Igsf3 impairs the ability of the vagal neural crest cells to migrate and normally innervate the intestine. The small intestine of Igsf3 KO mice shows reduced thickness of the muscularis externa and diminished number of enteric neurons. Also, misalignment of neurons and smooth muscle cells in the developing intestinal villi is detected. Taken together, our results suggest that IGSF3 functions contribute to the formation of the enteric nervous system. Given the essential role of the enteric nervous system in maintaining normal gastrointestinal function, our study adds to the pool of information required for further understanding the mechanisms of gut innervation and etiology behind bowel motility disorders