Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Discovery of non-sulfonamide carbonic anhydrase IX inhibitors through structure-based virtual screening.

Carbonic anhydrase IX (CA IX) is a subtype of the human carbonic anhydrase (hCA) family and exhibits high expression in various solid tumors, rendering it a promising target for tumor therapy. Currently, marketed carbonic anhydrase inhibitors (CAIs) are primarily composed of sulfonamides derivatives, which may have impeded their potential for further expansion. Therefore, we have developed a structure-based virtual screening approach to explore novel CAIs exhibiting distinctive structures and anti-tumor potential in the FDA database. In vitro experiments demonstrated that 3-pyridinemethanol (0.42 µM), procodazole (8.35 µM) and pamidronic acid (8.51 µM) exhibited inhibitory effects on CA IX activity. The binding stability and interaction mode between the CA IX and the hit compounds are further investigated through molecular dynamics simulations and binding free energy calculations. Furthermore, the ADME/Tox prediction results indicated that these compounds exhibited favorable pharmacological properties and minimal toxic side effects. Our study successfully applied computational strategies to discover three non-sulfonamide inhibitors of carbonic anhydrase IX (CA IX) that demonstrate inhibitory activity in vitro. These findings have significant implications for the development of CA IX inhibitors and anti-tumor drugs, contributing to their progress in the field.

The relationship between carbonic anhydrase IX (CAIX) and patient survival in breast cancer: systematic review and meta-analysis.

PURPOSE: Hypoxia is a characteristic of many solid tumours and an adverse prognostic factor for cancer therapy. Hypoxia results in upregulation of carbonic anhydrase IX (CAIX) expression, a pH-regulating enzyme. Many human tissue studies have examined the prognostic value of CAIX expression in breast cancer but have yielded inconsistent results. Therefore, a systematic review and meta-analysis was undertaken to assess the prognostic value of CAIX expression for breast cancer patients. METHODS: The electronic databases were systematically searched to identify relevant papers. The clinical outcomes included disease-free survival (DFS), recurrence-free survival (RFS) and overall survival (OS) in breast cancer patients. Review Manager version 5.4 was employed to analysis data from 23 eligible studies (containing 8390 patients). RESULTS: High CAIX expression was associated with poorer RFS [HR = 1.42, 95% CI (1.32-1.51), p < 0.00001], DFS [HR = 1.64, 95% CI (1.34-2.00), p < 0.00001], and OS [HR = 1.48, 95% CI (1.22-1.80), p < 0.0001]. Heterogeneity was observed across the studies. There was an effect of the CAIX antibody employed, scoring methods, and tumour localisation on CAIX expression. CONCLUSION: CAIX overexpression was significantly associated with poorer RFS, DFS, and OS in breast cancer patients. However, further work in high quantity tissue cohorts is required to define the optimal methodological approach.

Development of potent nanosized carbonic anhydrase inhibitor for targeted therapy of hypoxic solid tumors.

Overexpression of two carbonic anhydrase (CA) isoforms, CA IX and XII, in several hypoxic solid tumors provides an extracellular hypoxic microenvironment, interferes with extra- and intracellular pH regulation, thus favoring hypoxic tumor cell survival, proliferation and metastasis. In the current study, a selective inhibitor for human CA isoforms IX and XII (isatin-bearing sulfonamide, WEG-104), was incorporated into nanosized spherical niosomes at high encapsulation efficiency to allow for an enhanced and sustained antitumor activity. In vivo, administration of WEG-104 that is either free (10 mg/kg) or loaded into niosomes (5 mg/kg) into a mice model of Ehrlich ascites solid tumor resulted in comparable efficacy in terms of reduction of tumor weight and volume. Administration of WEG-104-loaded niosomes (10 mg/kg) exhibited superior antitumor activity compared to the free drug, evidenced by reduced tumor weight and volume, marked reduction in the activity of CA IX and XII, and suppression of HIF-1α and MMP-2. Moreover, prominent increase of caspase 3 and pronounced decrease in VEGF immune expression were observed in the treated animals. Hence, loading of molecularly designed compounds that targets CAs in hypoxic solid tumors into nanosized delivery systems provided an auspicious strategy for limiting solid tumor progression and malignancy.

Dual Antigen T Cell Engagers Targeting CA9 as an Effective Immunotherapeutic Modality for Targeting CA9 in Solid Tumors.

Glioblastomas (GBM), the most common malignant primary adult brain tumors, are uniformly lethal and are in need of improved therapeutic modalities. GBM contain extensive regions of hypoxia and are enriched in therapy resistant brain tumor-initiating cells (BTICs). Carbonic anhydrase 9 (CA9) is a hypoxia-induced cell surface enzyme that plays an important role in maintenance of stem cell survival and therapeutic resistance. Here we demonstrate that CA9 is highly expressed in patient-derived BTICs. CA9+ GBM BTICs showed increased self-renewal and proliferative capacity. To target CA9, we developed dual antigen T cell engagers (DATEs) that were exquisitely specific for CA9-positive patient-derived clear cell Renal Cell Carcinoma (ccRCC) and GBM cells. Combined treatment of either ccRCC or GBM cells with the CA9 DATE and T cells resulted in T cell activation, increased release of pro-inflammatory cytokines and enhanced cytotoxicity in a CA9-dependent manner. Treatment of ccRCC and GBM patient-derived xenografts markedly reduced tumor burden and extended survival. These data suggest that the CA9 DATE could provide a novel therapeutic strategy for patients with solid tumors expressing CA9 to overcome treatment resistance. .

Carbonic Anhydrase IV Selective Inhibitors Counteract the Development of Colitis-Associated Visceral Pain in Rats.

Persistent pain affecting patients with inflammatory bowel diseases (IBDs) is still very difficult to treat. Carbonic anhydrase (CA) represents an intriguing pharmacological target considering the anti-hyperalgesic efficacy displayed by CA inhibitors in both inflammatory and neuropathic pain models. The aim of this work was to evaluate the effect of inhibiting CA IV, particularly when expressed in the gut, on visceral pain associated with colitis induced by 2,4-di-nitrobenzene sulfonic acid (DNBS) in rats. Visceral sensitivity was assessed by measuring animals' abdominal responses to colorectal distension. Repeated treatment with the selective CA IV inhibitors AB-118 and NIK-67 effectively counteracted the development of visceral pain induced by DNBS. In addition to pain relief, AB-118 showed a protective effect against colon damage. By contrast, the anti-hyperalgesic activity of NIK-67 was independent of colon healing, suggesting a direct protective effect of NIK-67 on visceral sensitivity. The enzymatic activity and the expression of CA IV resulted significantly increased after DNBS injection. NIK-67 normalised CA IV activity in DNBS animals, while AB-118 was partially effective. None of these compounds influenced CA IV expression through the colon. Although further investigations are needed to study the underlying mechanisms, CA IV inhibitors are promising candidates in the search for therapies to relieve visceral pain in IBDs.

Design and Comparative Evaluation of the Anticonvulsant Profile, Carbonic-Anhydrate Inhibition and Teratogenicity of Novel Carbamate Derivatives of Branched Aliphatic Carboxylic Acids with 4-Aminobenzensulfonamide.

Epilepsy is one of the most common neurological diseases, with between 34 and 76 per 100,000 people developing epilepsy annually. Epilepsy therapy for the past 100+ years is based on the use of antiepileptic drugs (AEDs). Despite the availability of more than twenty old and new AEDs, approximately 30% of patients with epilepsy are not seizure-free with the existing medications. In addition, the clinical use of the existing AEDs is restricted by their side-effects, including the teratogenicity associated with valproic acid that restricts its use in women of child-bearing age. Thus, there is an unmet clinical need to develop new, effective AEDs. In the present study, a novel class of carbamates incorporating phenethyl or branched aliphatic chains with 6-9 carbons in their side-chain, and 4-benzenesulfonamide-carbamate moieties were synthesized and evaluated for their anticonvulsant activity, teratogenicity and carbonic anhydrase (CA) inhibition. Three of the ten newly synthesized carbamates showed anticonvulsant activity in the maximal-electroshock (MES) and 6 Hz tests in rodents. In mice, 3-methyl-2-propylpentyl(4-sulfamoylphenyl)carbamate(1), 3-methyl-pentan-2-yl-(4-sulfamoylphenyl)carbamate (9) and 3-methylpentyl, (4-sulfamoylphenyl)carbamate (10) had ED50 values of 136, 31 and 14 mg/kg (MES) and 74, 53, and 80 mg/kg (6 Hz), respectively. Compound (10) had rat-MES-ED50 = 13 mg/kg and ED50 of 59 mg/kg at the mouse-corneal-kindling test. These potent carbamates (1,9,10) induced neural tube defects only at doses markedly exceeding their anticonvuslnat-ED50 values. None of these compounds were potent inhibitors of CA IV, but inhibited CA isoforms I, II and VII. The anticonvulsant properties of these compounds and particularly compound 10 make them potential candidates for further evaluation and development as new AEDs.

The novel CA IX inhibition antibody chKM4927 shows anti-tumor efficacy in vivo.

Carbonic anhydrase IX (CA IX) is an attractive target for cancer therapy. Many anti-CA IX antibodies have been reported but few have been shown to possess inhibition activity. Furthermore, effective use of CA IX-inhibition antibodies for cancer immunotherapy has not been well-validated since data are mainly limited to in vitro assays. In this study, we established that chKM4927, an anti-CA IX chimeric antibody, recognizes CA IX and has CA IX-specific inhibition activity. ChKM4927 also retains antibody-dependent cellular cytotoxicity (ADCC) activity against CA IX-expressing cancer cells. Compared to controls, chKM4927 treatment (10 mg/kg) showed anti-tumor activity in the VMRC-RCW xenograft model in vivo. ChKM4927-attenuated ADCC activity showed equally effective anti-tumor activity. These results suggest that the CA IX-inhibition antibody chKM4927 has an anti-tumor effect in the VMRC-RCW xenograft model via an ADCC-independent mechanism.

Carbonic anhydrase expression in kidney and renal cancer: implications for diagnosis and treatment.

Four different carbonic anhydrases are expressed in the human nephron, the functional unit of the kidney. These are specifically expressed in different nephron segments, emphasizing the critical role carbonic anhydrases play in maintaining the homeostasis of this crucial organ.Whereas the localization of carbonic anhydrases in the kidney has been long established, interest in carbonic anhydrases has increased dramatically for renal cancer, in particular for the clear cell variant of renal cell carcinoma (ccRCC) because carbonic anhydrase IX is specifically expressed in ccRCC. Therefore carbonic anhydrase IX is being studied as potential diagnostic and therapeutic target, despite carbonic anhydrase IX expression in non-renal tissues.

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase: a novel biotechnology-based blood substitute that transports both oxygen and carbon dioxide and also acts as an antioxidant.

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (PolyHb-SOD-CAT-CA) is a therapeutic antioxidant that also transports both oxygen and carbon dioxide. This is formed by crosslinking Hb with SOD, CAT, and CA using glutaraldehyde. Crosslinking stroma free Hb from red blood cell (rbc) reduces CA activity to 55%. Addition of more CA resulted in a preparation with the same CA activity as RBC. PolyHb in the complex acts as a buffer to prevent large pH changes as carbon dioxide is converted to carbonic acid. We then prepare and optimize a novel PolyHb-SOD-CAT-CA, a therapeutic antioxidant that also transports both oxygen and carbon dioxide.

Carbonic anhydrase inhibitors.

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread enzymes in all organisms, catalyzing CO2 hydration to bicarbonate and protons. Their inhibition is exploited clinically for decades for various classes of diuretics and systemically acting antiglaucoma agents. In the last years novel applications of CA inhibitors (CAIs) emerged, such as topically acting antiglaucoma, anticonvulsants, antiobesity, antipain, and antitumor agents/diagnostic tools. Such CAIs target diverse isozymes of the 13 catalytically active alpha-CA isoforms present in mammals. CAs belonging to the alpha-, beta-, gamma-, delta-, and zeta-families are found in many organisms all over the phylogenetic tree, and their inhibition was studied ultimately for some pathogenic protozoa (Plasmodium falciparum), fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae), and bacteria (Helicobacter pylori, Mycobacterium tuberculosis, and Brucella suis). Novel interesting chemotypes, in addition to the sulfonamide and sulfamate CAIs, such as coumarins, phenols, and fullerenes, were also reported recently, together with their mechanism of inhibition. This class of enzyme inhibitors shows promise for designing interesting pharmacological agents and understanding in detail protein-drug interactions at molecular level.

Carbonic anhydrases--an overview.

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes all over the phylogenetic tree, with at least 4 distinct gene families encoding for them. At least 16 different alpha-CA isoforms were isolated in mammals, where these enzymes play crucial physiological roles. Representatives of the beta-delta-CA family are highly abundant in plants, diatoms, eubacteria and archaea. These enzymes are efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate, but at least the alpha-CAs possess a high versatility, being able to catalyze different other hydrolytic processes The catalytic mechanism of the alpha-CAs is understood in detail: the active site consists of a Zn(II) ion co-ordinated by three histidine residues and a water molecule/hydroxide ion. The latter is the active species, acting as a potent nucleophile. For beta- and gamma-CAs, the zinc hydroxide mechanism is valid too, although at least some beta-class enzymes do not have water directly coordinated to the metal ion. CAs are inhibited by two classes of compounds: the metal complexing anions and the sulfonamides and their isosteres (sulfamates, sulfamides etc.) possessing the general formula RXSO(2)NH(2) (R = aryl; hetaryl; perhaloalkyl; X = nothing, O or NH). At least 25 clinically used drugs/agents in clinical development show applications as diuretics and antiglaucoma drugs, anticonvulsants, with some compounds being developed as anticancer agents/diagnostic tools for tumors, antiobesity agents, and antimicrobials/antifungals (inhibitors targeting CAs from pathogenic organisms such as Helicobacter pylori, Mycobacterium tuberculosis, Plasmodium falciparum, Candida albicans, etc). Several important physiological and physio-pathological functions are played by CA isozymes present in organisms all over the phylogenetic tree, related to respiration and transport of CO(2)/bicarbonate between metabolizing tissues and the lungs, pH and CO(2) homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the gluconeogenesis and ureagenesis among others (in animals), CO(2) fixation (in plants and algae), etc. The presence of these ubiquitous enzymes in so many tissues and in so different isoforms, represents an attractive goal for the design of inhibitors or activators with biomedical applications.

Evaluation of renal cell carcinoma vaccines targeting carbonic anhydrase IX using heat shock protein 110.

Carbonic anhydrase IX (CA9) is a renal cell carcinoma (RCC)-specific tumor protein that is targeted using heat shock protein 110 (hsp110). The chaperoning ability of hsp110 can be utilized to form a complex with CA9 (hsp110 + CA9) in vitro, which can be administered as a highly concentrated tumor vaccine. In a tumor prevention model, hsp110 + CA9 prevented the growth of RENCA tumors in BALB/c mice, and produced IFN-gamma response measured using ELISPOT and an antibody response measured using ELISA. To test a second vaccine strategy, hsp110 complexed to a previously described CA9 peptide prevented tumor growth and produced a very weak IFN-gamma response, but no antibody response. A plasmid vector containing grp170, a member of the hsp110 family, linked to CA9 did not produce an antitumor response and produced no IFN-gamma response or antibodies. In a model of metastatic RCC, RENCA cells were injected intradermally prior to vaccination. Hsp110 + CA9 decreased tumor growth compared to control vaccinations. These studies suggest that recombinant hsp110 complexed to CA9 should be evaluated for treatment of RCC.

A study on the prevention and treatment of myopia with nacre on chicks.

The contents of mineral elements and amino acids in the hydrolysate of the traditional Chinese mineral medicine nacre have been determined. It has long since been proved by the practice of doctors of traditional Chinese medicine that pearl can be used to treat eye diseases. Based on such an understanding, a study is made of the influence of the said medicine on the incidence of myopia. First a form-sense-deprived model (FDM) for chicks is developed and the effect of the said medicine on the elongation of axis oculi is determined with an Abbe's comparator and an A-mode ultrasound instrument. The activity of superoxide dismutase (SOD), nitric oxide synthetase (NOS), and the content of nitric oxide (NO) in the retino-pigmental epithelium choroid homogenate are also analysed. The role of the said traditional Chinese mineral medicine in preventing and treating myopia is explained with respect to the above findings. The results obtained will provide a basis for using nacre, a traditional Chinese mineral medicine, to prevent and treat myopia.