Novel Humanized Monoclonal Antibodies for Targeting Hypoxic Human Tumors via Two Distinct Extracellular Domains of Carbonic Anhydrase IX

2021 ◽  
Author(s):  
Miriam Zatovicova ◽  
Ivana Kajanova ◽  
Monika Barathova ◽  
Martina Takacova ◽  
Martina Labudova ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Cancer Progression ◽  
Standard Therapy ◽  
Catalytic Domain ◽  
Conformational Epitope ◽  
Carbonic Anhydrase Ix ◽  
Acidic Environment ◽  
Primary Tumors ◽  
Ca Ix

Abstract Background Hypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy. Methods Parental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma we obtained 25 clones. Each clone was tested for ADCC and CDC activity, affinity, extracellular pH measurement, multicellular aggregation analysis and real-time monitoring of invasion with xCELLigence system. ResultsBoth CA9hu-1 and CA9hu-2 are IgG1 antibodies and they were both examined in vivo. Here we describe anti-CAIX antibodies that can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME. CA9hu-1 is directed at the conformational epitope of the catalytic domain, while CA9hu-2 targets the sequential epitope of the proteo-glycan domain. They are both able to induce an immune response, have high affinity, as well as ADCC and CDC activity. While the first one internalizes after binding to the antigen, the second one is able to reduce metastases formation. More importantly, they have both proved the ability to block the acidification of the extracellular environment. ConclusionCA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.

scholarly journals A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells

2019 ◽  
Vol 476 (10) ◽  
pp. 1497-1513 ◽  
Author(s):  
Mam Y. Mboge ◽  
Zhijuan Chen ◽  
Daniel Khokhar ◽  
Alyssa Wolff ◽  
Lingbao Ai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Expression Patterns ◽  
Extracellular Ph ◽  
Carbonic Anhydrase Ix ◽  
Monocarboxylate Transporter ◽  
Proton Efflux ◽  
Ca Ix

AbstractThe most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+ shuttle system in CA IX to facilitate proton efflux through MCT4.

scholarly journals Dual-functional Liposomes with Carbonic Anhydrase IX Antibody and BR2 Peptide Modification Effectively Improve Intracellular Delivery of Cantharidin to Treat Orthotopic Hepatocellular Carcinoma Mice

Molecules ◽  
2019 ◽  
Vol 24 (18) ◽  
pp. 3332 ◽  
Author(s):  
Zhang ◽  
Lin ◽  
Chan ◽  
Liu ◽  
Lu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Carbonic Anhydrase ◽  
Therapeutic Potential ◽  
Intracellular Delivery ◽  
Carbonic Anhydrase Ix ◽  
Ca Ix ◽  
Dual Functional ◽  
Peptide Modification

Liposomal nanotechnology has a great potential to overcome the current major problems of chemotherapy. However, the lack of penetrability and targetability retards the successful delivery of liposomal carriers. Previously, we showed that BR2 peptide modification endowed cantharidin-loaded liposomes with intracellular penetration that enhanced the drug cytotoxic effects. Here, we aimed to improve the targeting delivery of drugs into cancer cells via highly expressed carbonic anhydrase IX (CA IX) receptors by modifying our previous catharidin-loaded BR2-liposomes with anti-CA IX antibody. A higher cellular uptake of dual-functional liposomes (DF-Lp) than other treatments was observed. Induction of CA IX over-expressing resulted in a higher cellular binding of DF-Lp; subsequently, blocking with excess antibodies resulted in a decreased cancer-cell association, indicating a specific targeting property of our liposomes towards CA IX expressed cells. After 3h tracking, most of the liposomes were located around the nucleus which confirmed the involvement of targeting intracellular delivery. Cantharidin loaded DF-Lp exhibited enhanced cytotoxicity in vitro and was most effective in controlling tumor growth in vivo in an orthotopic hepatocellular carcinoma model compared to other groups. Collectively, our results presented the advantage of the BR2 peptide and CA IX antibody combination to elevate the therapeutic potential of cantharidin loaded DF-liposomes.

scholarly journals Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX

2020 ◽  
Vol 21 (15) ◽  
pp. 5277
Author(s):  
Katarina Koruza ◽  
A. Briana Murray ◽  
Brian P. Mahon ◽  
Jesse B. Hopkins ◽  
Wolfgang Knecht ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Carbonic Anhydrase ◽  
Small Angle ◽  
Catalytic Domain ◽  
Carbonic Anhydrase Ix ◽  
Biophysical Characterization ◽  
X Ray ◽  
Ca Ix ◽  
X Ray Scattering ◽  
Ray Scattering

Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.

scholarly journals Type 1 Sodium Calcium Exchanger Forms a Complex with Carbonic Anhydrase IX and Via Reverse Mode Activity Contributes to pH Control in Hypoxic Tumors

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1139 ◽  
Author(s):  
Veronika Liskova ◽  
Sona Hudecova ◽  
Lubomira Lencesova ◽  
Filippo Iuliano ◽  
Marta Sirova ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Ph Regulation ◽  
Ph Control ◽  
Carbonic Anhydrase Ix ◽  
Proximity Ligation Assay ◽  
Reverse Mode ◽  
Ca Ix

Hypoxia and acidosis are among the key microenvironmental factors that contribute to cancer progression. We have explored a possibility that the type 1Na+/Ca2+ exchanger (NCX1) is involved in pH control in hypoxic tumors. We focused on changes in intracellular pH, co-localization of NCX1, carbonic anhydrase IX (CA IX), and sodium proton exchanger type 1 (NHE1) by proximity ligation assay, immunoprecipitation, spheroid formation assay and migration of cells due to treatment with KB-R7943, a selective inhibitor of the reverse-mode NCX1. In cancer cells exposed to hypoxia, reverse-mode NCX1 forms a membrane complex primarily with CA IX and also with NHE1. NCX1/CA IX/NHE1 assembly operates as a metabolon with a potent ability to extrude protons to the extracellular space and thereby facilitate acidosis. KB-R7943 prevents formation of this metabolon and reduces cell migration. Thus, we have shown that in hypoxic cancer cells, NCX1 operates in a reverse mode and participates in pH regulation in hypoxic tumors via cooperation with CAIX and NHE1.

scholarly journals From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography

Crystals ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 434 ◽  
Author(s):  
Katarina Koruza ◽  
Bénédicte Lafumat ◽  
Maria Nyblom ◽  
Wolfgang Knecht ◽  
Zoë Fisher
Keyword(s):  
Carbonic Anhydrase ◽  
Membrane Protein ◽  
Single Crystals ◽  
Catalytic Domain ◽  
Sparse Matrix ◽  
Carbonic Anhydrase Ix ◽  
Ca Ix ◽  
Human Carbonic Anhydrase ◽  
Neutron Protein Crystallography

Human carbonic anhydrase IX (CA IX) is a multi-domain membrane protein that is therefore difficult to express or crystalize. To prepare crystals that are suitable for neutron studies, we are using only the catalytic domain of CA IX with six surface mutations, named surface variant (SV). The crystallization of CA IX SV, and also partly deuterated CA IX SV, was enabled by the use of microseed matrix screening (MMS). Only three drops with crystals were obtained after initial sparse matrix screening, and these were used as seeds in subsequent crystallization trials. Application of MMS, commercial screens, and refinement resulted in consistent crystallization and diffraction-quality crystals. The crystallization protocols and strategies that resulted in consistent crystallization are presented. These results demonstrate not only the use of MMS in the growth of large single crystals for neutron studies with defined conditions, but also that MMS enabled re-screening to find new conditions and consistent crystallization success.

scholarly journals Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

2019 ◽  
Vol 75 (10) ◽  
pp. 895-903 ◽  
Author(s):  
K. Koruza ◽  
B. Lafumat ◽  
M. Nyblom ◽  
B. P. Mahon ◽  
W. Knecht ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Crystal Form ◽  
Carbonic Anhydrase Ix ◽  
Structural Comparison ◽  
X Ray ◽  
Ca Ix ◽  
Human Carbonic Anhydrase

Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.

scholarly journals A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells

2018 ◽  
Author(s):  
Mam Y. Mboge ◽  
Zhijuan Chen ◽  
Daniel Khokhar ◽  
Alyssa Wolff ◽  
Lingbao Ai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Carbonic Anhydrase ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Expression Patterns ◽  
Extracellular Ph ◽  
Carbonic Anhydrase Ix ◽  
Monocarboxylate Transporter ◽  
Proton Efflux ◽  
Ca Ix

ABSTRACTThe most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX cooperates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple negative breast cancer (TNBC), appears to coordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+shuttle system in CA IX to facilitate proton efflux through MCT4.

scholarly journals Distinct Expression Patterns of Carbonic Anhydrase IX in Clear Cell, Microcystic, and Angiomatous Meningiomas

2019 ◽  
Vol 78 (12) ◽  
pp. 1081-1088
Author(s):  
Rati Chkheidze ◽  
Patrick J Cimino ◽  
Kimmo J Hatanpaa ◽  
Charles L White ◽  
Manuel Ferreira ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Clear Cell ◽  
Expression Patterns ◽  
Carbonic Anhydrase Ix ◽  
World Health ◽  
Loss Of Function ◽  
Ca Ix ◽  
Hypoxia Marker ◽  
Other World ◽  
Health Organization

Abstract Clear cell, microcytic, and angiomatous meningiomas are 3 vasculature-rich variants with overlapping morphological features but different prognostic and treatment implications. Distinction between them is not always straightforward. We compared the expression patterns of the hypoxia marker carbonic anhydrase IX (CA-IX) in meningiomas with predominant clear cell (n = 15), microcystic (n = 9), or angiomatous (n = 11) morphologies, as well as 117 cases of other World Health Organization recognized histological meningioma variants. Immunostaining for SMARCE1 protein, whose loss-of-function has been associated with clear cell meningiomas, was performed on all clear cell meningiomas, and selected variants of meningiomas as controls. All clear cell meningiomas showed absence of CA-IX expression and loss of nuclear SMARCE1 expression. All microcystic and angiomatous meningiomas showed diffuse CA-IX immunoreactivity and retained nuclear SMARCE1 expression. In other meningioma variants, CA-IX was expressed in a hypoxia-restricted pattern and was highly associated with atypical features such as necrosis, small cell change, and focal clear cell change. In conclusion, CA-IX may serve as a useful diagnostic marker in differentiating clear cell, microcystic, and angiomatous meningiomas.

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