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Antibody-mediated inhibition of MHC class I chain-related protein A shedding for tumor therapy

Liptáková, Veronika (2023)
Antibody-mediated inhibition of MHC class I chain-related protein A shedding for tumor therapy.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00024482
Dissertation, Erstveröffentlichung, Verlagsversion

Kurzbeschreibung (Abstract)

The NKG2D/NKG2D ligand pathway mediates cancer immunosurveillance by cytotoxic lymphocytes. Natural killer group 2 member D (NKG2D) is a homodimeric C-type lectin like receptor expressed on almost all human cytotoxic lymphocytes (NK, CD8+ and γδ T cells) and serves as a primary activating receptor on NK cells. In humans, there are eight NKG2D ligands (MICA/MICB and ULBP1-6), among which MICA/B (MHC class I chain-related sequence A/B) have been best characterized. MICA and MICB are ´sister´ molecules that share around 91 % identity in their coding sequences. The domain structure of MICA/B consists of three extracellular domains, the α1α2 MHC class I-like superdomain and the Ig-like α3 domain. Under normal physiological conditions, MICA/B glycoproteins usually are not expressed in healthy tissues, but MICA/B cell surface expression can be induced by various stress conditions (e.g., virus infection, malignant transformation). MICA/B-NKG2D interaction promotes immune recognition and cytolysis of MICA/B-expressing stressed cells. However, tumor cells often escape from NKG2D-mediated recognition, apparently also by shedding of MICA molecules from the cell surface by various metalloproteases (e.g., ADAM10 and ADAM17). There is a negative correlation of elevated soluble MICA/B (sMICA/B) levels in serum of cancer patients (as a consequence of MICA/B shedding) with overall survival. Moreover, sMICA/B was suggested as a cancer marker. Hence, these properties mark the shedding of MICA/B as an attractive target for cancer studies. Numerous clinical studies attempted to target metalloproteases in order to block their action to treat malignant diseases in patients. Unfortunately, these attempts were unsuccessful due to important biological roles of these proteases in processes. As a result, there is an urgent need to develop other means of cancer treatment that lead to inhibition of MICA shedding from tumors.

The aim of this thesis was to characterize the MICA/B-monoclonal antibody (mAb) BAMO3, which inhibits MICA/B shedding, and test the therapeutic efficacy of BAMO3-mediated inhibition of MICA shedding in aggressive syngeneic mouse tumor models. In addition, particular amino acids in the transmembrane and α3 domain in the MICA molecule and their role in MICA shedding and eventually their role in affecting BAMO3-mediated MICA shedding inhibition were studied. Lastly, therapeutic efficacy of another MICA antibody conjugated to toxin was tested. Previously, the Steinle laboratory generated the mAb BAMO3 specific for MICA and MICB. This thesis demonstrates BAMO3 proteolytic shedding inhibition of MICA/MICB from different tumor cells in a dose-dependent and cell-specific manner in vitro. The shedding inhibition was very efficient as BAMO3 was able to prevent MICA/B shedding at very low concentrations, as low as 0.1 µg/ml, and at 10 µg/ml the MICA/B shedding was almost completely blocked. BAMO3 is widely applicable as it prevented shedding of different MICA and MICB allelic variants expressed by various cancer cell lines. To elucidate the molecular action of BAMO3, the epitope of BAMO3 was mapped to the membrane proximal site of the MICA α3 domain suggesting that BAMO3 acts by steric hindrance of metalloprotease cleavage.

MICA is highly polymorphic and there are over 100 different alleles recognized in the current literature. Several alleles possess two, four or even nine GCT repeats, in the transmembrane region of MICA, respectively. The results of this study suggest that the length of alanine repeats in the transmembrane domain of MICA does not affect MICA shedding inhibition by BAMO3.

Next, a cysteine in position 250 in MICA (positioned in the α3 domain), that does not seem to form an intramolecular disulfide bond with any cysteine partner, was studied. Mutation of this cysteine (Cys250Ala) led to mitigation of shedding intensity of Cys250Ala MICA from B16F10 cells. Moreover, the surface expression of Cys250Ala MICA was increased while ADAM10 metalloprotease surface expression was decreased. Further, co-immunoprecipitation experiments indicated a lower intensity interaction between Cys250Ala MICA and ADAM10.

Finally, anti-tumor effects of anti-MICA antibodies were studied in vivo. Administration of BAMO3 delayed growth of RMA-MICA lymphoma in a syngeneic mouse model in MICA transgenic (MICAgen) mice and thereby improved survival of tumor-bearing mice. Tumor-infiltrating NK cells expressed higher NKG2D levels upon BAMO3 treatment as compared to control-treated mice. Overall, these data demonstrated therapeutic efficacy of BAMO3 treatment in the mouse RMA lymphoma model, and, together with other data from our laboratory, endorse BAMO3 treatment as a potential novel immunotherapeutic approach for treatment of human malignant diseases.

In addition, therapeutic efficacy of a drug-conjugated antibody against MICA (19E9-PBD) was tested in MICAgen mice bearing various aggressive mouse tumors. 19E9-PBD demonstrated a tremendous efficiency in delaying the tumor growth and improving survival of MICAgen mice. A decreased MICA expression on immunosuppressive myeloid-derived suppressor cells (MDSCs) was detected in the tumors. Moreover, the combination therapy of 19E9-PBD together with an anti-PD-1 antibody showed therapeutic efficacy in delaying the growth of MICA-expressing tumors.

In conclusion, these results presented powerful antibody-based agents for tumor elimination of MICA-positive tumors. They may be used to complement and enhance the antineoplastic effects of other anti-cancer therapeutics or immune checkpoint inhibitors that are currently used for patients with various malignancies.

Typ des Eintrags: Dissertation
Erschienen: 2023
Autor(en): Liptáková, Veronika
Art des Eintrags: Erstveröffentlichung
Titel: Antibody-mediated inhibition of MHC class I chain-related protein A shedding for tumor therapy
Sprache: Englisch
Referenten: Kolmar, Prof. Dr. Harald ; Steinle, Prof. Dr. Alexander
Publikationsjahr: 2 November 2023
Ort: Darmstadt
Kollation: 112 Seiten
Datum der mündlichen Prüfung: 10 Juli 2023
DOI: 10.26083/tuprints-00024482
URL / URN: https://tuprints.ulb.tu-darmstadt.de/24482
Kurzbeschreibung (Abstract):

The NKG2D/NKG2D ligand pathway mediates cancer immunosurveillance by cytotoxic lymphocytes. Natural killer group 2 member D (NKG2D) is a homodimeric C-type lectin like receptor expressed on almost all human cytotoxic lymphocytes (NK, CD8+ and γδ T cells) and serves as a primary activating receptor on NK cells. In humans, there are eight NKG2D ligands (MICA/MICB and ULBP1-6), among which MICA/B (MHC class I chain-related sequence A/B) have been best characterized. MICA and MICB are ´sister´ molecules that share around 91 % identity in their coding sequences. The domain structure of MICA/B consists of three extracellular domains, the α1α2 MHC class I-like superdomain and the Ig-like α3 domain. Under normal physiological conditions, MICA/B glycoproteins usually are not expressed in healthy tissues, but MICA/B cell surface expression can be induced by various stress conditions (e.g., virus infection, malignant transformation). MICA/B-NKG2D interaction promotes immune recognition and cytolysis of MICA/B-expressing stressed cells. However, tumor cells often escape from NKG2D-mediated recognition, apparently also by shedding of MICA molecules from the cell surface by various metalloproteases (e.g., ADAM10 and ADAM17). There is a negative correlation of elevated soluble MICA/B (sMICA/B) levels in serum of cancer patients (as a consequence of MICA/B shedding) with overall survival. Moreover, sMICA/B was suggested as a cancer marker. Hence, these properties mark the shedding of MICA/B as an attractive target for cancer studies. Numerous clinical studies attempted to target metalloproteases in order to block their action to treat malignant diseases in patients. Unfortunately, these attempts were unsuccessful due to important biological roles of these proteases in processes. As a result, there is an urgent need to develop other means of cancer treatment that lead to inhibition of MICA shedding from tumors.

The aim of this thesis was to characterize the MICA/B-monoclonal antibody (mAb) BAMO3, which inhibits MICA/B shedding, and test the therapeutic efficacy of BAMO3-mediated inhibition of MICA shedding in aggressive syngeneic mouse tumor models. In addition, particular amino acids in the transmembrane and α3 domain in the MICA molecule and their role in MICA shedding and eventually their role in affecting BAMO3-mediated MICA shedding inhibition were studied. Lastly, therapeutic efficacy of another MICA antibody conjugated to toxin was tested. Previously, the Steinle laboratory generated the mAb BAMO3 specific for MICA and MICB. This thesis demonstrates BAMO3 proteolytic shedding inhibition of MICA/MICB from different tumor cells in a dose-dependent and cell-specific manner in vitro. The shedding inhibition was very efficient as BAMO3 was able to prevent MICA/B shedding at very low concentrations, as low as 0.1 µg/ml, and at 10 µg/ml the MICA/B shedding was almost completely blocked. BAMO3 is widely applicable as it prevented shedding of different MICA and MICB allelic variants expressed by various cancer cell lines. To elucidate the molecular action of BAMO3, the epitope of BAMO3 was mapped to the membrane proximal site of the MICA α3 domain suggesting that BAMO3 acts by steric hindrance of metalloprotease cleavage.

MICA is highly polymorphic and there are over 100 different alleles recognized in the current literature. Several alleles possess two, four or even nine GCT repeats, in the transmembrane region of MICA, respectively. The results of this study suggest that the length of alanine repeats in the transmembrane domain of MICA does not affect MICA shedding inhibition by BAMO3.

Next, a cysteine in position 250 in MICA (positioned in the α3 domain), that does not seem to form an intramolecular disulfide bond with any cysteine partner, was studied. Mutation of this cysteine (Cys250Ala) led to mitigation of shedding intensity of Cys250Ala MICA from B16F10 cells. Moreover, the surface expression of Cys250Ala MICA was increased while ADAM10 metalloprotease surface expression was decreased. Further, co-immunoprecipitation experiments indicated a lower intensity interaction between Cys250Ala MICA and ADAM10.

Finally, anti-tumor effects of anti-MICA antibodies were studied in vivo. Administration of BAMO3 delayed growth of RMA-MICA lymphoma in a syngeneic mouse model in MICA transgenic (MICAgen) mice and thereby improved survival of tumor-bearing mice. Tumor-infiltrating NK cells expressed higher NKG2D levels upon BAMO3 treatment as compared to control-treated mice. Overall, these data demonstrated therapeutic efficacy of BAMO3 treatment in the mouse RMA lymphoma model, and, together with other data from our laboratory, endorse BAMO3 treatment as a potential novel immunotherapeutic approach for treatment of human malignant diseases.

In addition, therapeutic efficacy of a drug-conjugated antibody against MICA (19E9-PBD) was tested in MICAgen mice bearing various aggressive mouse tumors. 19E9-PBD demonstrated a tremendous efficiency in delaying the tumor growth and improving survival of MICAgen mice. A decreased MICA expression on immunosuppressive myeloid-derived suppressor cells (MDSCs) was detected in the tumors. Moreover, the combination therapy of 19E9-PBD together with an anti-PD-1 antibody showed therapeutic efficacy in delaying the growth of MICA-expressing tumors.

In conclusion, these results presented powerful antibody-based agents for tumor elimination of MICA-positive tumors. They may be used to complement and enhance the antineoplastic effects of other anti-cancer therapeutics or immune checkpoint inhibitors that are currently used for patients with various malignancies.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Die NKG2D/NKG2D-Liganden Achse ermöglicht die immunologische Kontrolle von entarteten Zellen durch zytotoxische Lymphozyten. NKG2D gehört zur Familie der C-Typ ähnlichen Rezeptoren und wird als Homodimer auf zytotoxischen Lymphozyten (NK, CD8+ und γδ T Zellen) exprimiert, wo es als aktivierender Rezeptor fungiert. Bislang sind acht NKG2D Liganden (NKG2DL) im Menschen beschrieben. Neben MICA und MICB Molekülen, welche eine zentrale Rolle in dieser Arbeit einnehmen, sind die NKG2DL ULBP1-6 zu erwähnen. MICA und MICB weisen drei extrazelluläre Domänen (α1-α3) auf und 91% Sequenzidentität. Unter normalen physiologischen Bedingungen ist keine Expression von MICA/B auf gesunden Zellen nachweisbar. Zahlreiche Stressoren wie z.B. eine Virusinfektion, oder eine maligne Transformation vermögen jedoch die Oberflächenexpression von MICA/B zu induzieren, sodass es zu einer Immunantwort (Zytolyse) durch NKG2D+ Zellen kommen kann. Entartete Zellen können jedoch einer NKG2D-vermittelten Immunantwort entgehen, indem MICA/B Moleküle proteolytisch durch Metalloproteasen (ADAM10/17) von der Zelloberfläche abgetrennt werden („shedding“). Studien zufolge korreliert das Überleben von Krebspatienten negativ mit der Menge an löslichen MICA/B im Serum, weshalb MICA/B als Biomarker in Frage kommen. In zahlreichen klinischen Studien versuchte man Metalloproteasen zu blockieren. Bisher waren diese therapeutischen Ansätze jedoch aufgrund erheblicher Nebenwirkungen erfolglos, da Metalloproteasen auch in wichtige biologische Prozesse involviert sind. Es ist deshalb von großem Interesse eine Therapie zu entwickeln die MICA shedding auf anderem Wege gezielt inhibiert.

Ziel dieser Arbeit war die Charakterisierung des MICA/B-spezifischen monoklonalen Antikörpers BAMO3 hinsichtlich dessen Verwendung als Inhibitor von MICA shedding in vitro und in vivo.

Diese Doktorarbeit zeigt den inhibitorischen Einfluss von BAMO3 auf das proteolytische „Shedding“ von MICA/B in Tumorzellen in vitro. Bereits eine geringe Konzentration [0.1 µg/ml] an BAMO3 vermag das shedding verschiedener MICA/B Allele in unterschiedlichen Tumorzelllinien zu reduzieren. Mit Hilfe von Alaninmutagenese wurde das Epitop von BAMO3 an einer membranproximalen Stelle der MICA α3 Domäne kartiert. In unmittelbarer Nähe liegt die Schnittstelle der Metalloproteasen, weshalb der inhibitorische Effekt von BAMO3 wahrscheinlich auf eine sterische Blockade zurückzuführen ist.

MICA ist polymorph, wobei die Literatur bislang über hundert verschiedene Allele beschreibt. So finden sich auch repetitive GCT Sequenzmotive in der Transmembrandomäne von MICA. Diese Arbeit zeigt, dass die Anzahl der GCT- Repetitionen keinen Einfluss auf die BAMO3 vermittelte Inhibierung des MICA- „Shedding“ hat.

Des weiteren zeigt diese Arbeit, dass die Substitution von Cystein an Position 250 durch Alanin im MICA Molekül (α3 Domäne) zur Verminderung des „Sheddings“ führt. Zugleich nimmt die Oberflächenexpression der Metalloprotease ADAM10 ab. In Folge dessen steigt die Dichte der MICA Moleküle auf der Zelloberfläche. Des Weiteren wurde auch eine verringerte Interaktion zwischen ADAM10 und MICA (C250A) beobachtet.

Abschließend widmet sich diese Arbeit der therapeutischen Verwendung von anti-MICA Antikörpern in Maustumormodellen. Die therapeutische Gabe von BAMO3 vermag nicht nur das Wachstum des MICA+ Lymphoms RMA in einem syngenen MICA-transgenen Mausmodell zu verzögern, sodass das Überleben signifikant verlängert werden konnte. Daneben führte die BAMO3-Behandlung auch zu einer erhöhten NKG2D-Oberflächenexpression auf Tumor-infiltrierenden NK Zellen. Daneben wurde auch wird die Effizienz des Wirkstoff-konjugierten anti-MICA Antikörper 19E9-PBD sowohl in der Monotherapie, als auch in der Kombinationstherapie mit einem anti-PD-1 Antikörper beschrieben. Beide Ansätze zeigen - wie auch die BAMO3 Behandlung – ein verzögertes Tumorwachstum sowie einen positiven Effekt auf das Überleben. Darüber hinaus verringern sie die MICA Expression myeloider Suppressorzellen (MDSC), deren immunsuppressive Eigenschaften gegenüber T-Zellen maßgeblich an der Immunevasion maligner Tumore beteiligt sind. Zusammenfassend demonstriert diese Arbeit die Wirkungsweise und das immunotherapeutische Potential von BAMO3 zur Elimination MICA-positiver Tumore. Eine Kombinationstherapie mit bereits zugelassenen antineoplastischen Krebsmedikamenten könnte synergistisch wirken und wird Bestandteil künftiger Studien sein.

Deutsch
Status: Verlagsversion
URN: urn:nbn:de:tuda-tuprints-244826
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 07 Fachbereich Chemie
07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Biochemie
Hinterlegungsdatum: 02 Nov 2023 14:57
Letzte Änderung: 03 Nov 2023 10:30
PPN:
Referenten: Kolmar, Prof. Dr. Harald ; Steinle, Prof. Dr. Alexander
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 10 Juli 2023
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