Studies on Ligand Directed Enzyme Prodrug Therapy and Production of Long Acting Protein Therapeutics for Targeted Cancer Treatment

Targeting cancer therapy is one of the powerful tools that has been implemented for cancer treatment. One of the targeted cancer strategies is known as Antibody Directed Enzyme Prodrug Therapy (ADEPT). Three main components in this strategy, enzyme, cancer-specific antibody and a prodrug. The enzyme that has been used so far and reached clinical trials is carboxypeptidase G2 (CPG2) and the antibody chosen depends on the type of cancer to be treated. To overcome the need to conjugate a cancer-specific antibody to CPG2, our work showed that a small known peptide motif "Aspargine-Glycine-Arginine (NGR)" in place of an antibody fused to CPG2 can direct the enzyme to aminopeptidase N (APN ) that is highly expressed tumor. The CPG2 then converts the prodrug into the drug near the cancerous tissue. We called this strategy Ligand-targeted enzyme prodrug therapy (LDEPT). We have successfully demonstrated the ability of our new conjugates to bind specifically to cancer cells and CPG2 to produce the cytotoxic drug to kill the tumor cells. The ADEPT or LDEPT have disadvantages such as immunogenicity over CPG2 that undermine the efficacy of the therapy. We have therefore started the tasks to produce long acting CPG2 new variants. First and for the first time, we produced two CPG2 conjugates using life extenders PEGylation and gene fusion of HSA Our produced CPG2 variants (PEG-CPG2 and HSA-CPG2) showed a longer half-life under physiological conditions in addition to lower ex-vivo immunotoxicity . Second, we produced a long acting peptide-CPG2 that can specifically bind to tumor and has a long half-life, these are single and double fusion of PEG-CPG2-CNGRC. The resulting conjugates; single (CNGRC-CPG2) and double (CNGRC-CPG2-CNGRC) have been found to specifically target and bind APN, which is known to be highly expressed on the surface of tumor cells. In addition, the conjugates generated showed higher stability and significantly reduced ex vivo immunotoxicity. CPG2 in both conjugates has the ability to convert prodrug into a cytotoxic drug to kill the cancer cells. The work presented in this thesis paves the way for further in vivo and clinical studies to improve two of the important cancer targeting strategies, ADEPT and LDEPT

Calreticulin regulates Src kinase in osteogenic differentiation from embryonic stem cells

Calreticulin, the major Ca2+ buffer of the endoplasmic reticulum plays an important role in the choice of fate by embryonic stem cells. Using the embryoid body method of organogenesis, we showed impaired osteogenesis in crt−/− cells vis-à-vis calreticulin-containing osteogenic WT cells. In the non-osteogenic crt−/− cells, c-Src- a non-receptor tyrosine kinase- was activated and its inhibition rescued osteogenesis. Most importantly, we demonstrated that calreticulin-containing cells had lower c-Src kinase activity, and this was accomplished via the Ca2+-homeostatic function of calreticulin. Specifically, lowering cytosolic [Ca2+] in calreticulin-containing osteogenic WT cells with BAPTA-AM, activated c-Src and impaired osteogenic differentiation. Conversely, increasing cytosolic [Ca2+] in crt−/− cells with ionomycin deactivated c-Src kinase and restored osteogenesis. The immediate effector of calreticulin, the Ser/Thr phosphatase calcineurin, was less active in crt−/− cells, however, its activity was rescued upon inhibition of c-Src activity by small molecule inhibitors. Finally, we showed that higher activity of calcineurin correlated with increased level of nuclear Runx2, a transcription factor that is the master regulator of osteogenesis. Collectively, our work has identified a novel pathway involving calreticulin regulated Ca2+ signalling via c-Src in osteogenic differentiation of embryonic stem cells

Production of active long lasting CNGRC-CPG2 fusion protein using PEGylation to be used in Ligand Directed Cancer Therapy

Aminopeptidase N (APN) is one of the important enzymes highly expressed in metastatic cancers, thus employed as a marker to target tumor cells. CPG2-CNGRC fusion protein is produced to target high APN expressing cancer cells, which with the prodrug results in high toxic effect. Since PEGylation of CPG2 has shown an improved favorable in vitro stability and immunotoxicity, we performed a site-directed PEGylation (thiol group directed) of the CPG2-CNGRC fusion protein and examined the effect of PEGylation on the resulting fusion protein's therapeutic efficacy. CPG2 kinetic activity was substantially enhanced following PEGylation of the single fusion protein (PEG CPG2-CNGRC). The binding affinity of the produced PEGylated fusion proteins to their cellular marker (APN) was notably reduced in case of the double fusion protein compared with non-PEGylated ones. Moreover, the cytotoxic effect of methotrexate and ZD2767P (prodrug) in association of the PEGylated fusion proteins was investigated and found that the cytotoxic effect of prodrug with PEGylated single fusion protein was improved significantly (low cell survival). Similar finding was found following MTX treatment where lower binding and kinetic activity of the PEGylated double fusion proteins resulted in higher MTX toxic effect (lower cell survival) in comparison with the non-PEGylated double fusion protein. Thus, although PEGylation is known for its usually favorable effect on the protein/drug pharmacodynamics, our results indicated that with our different fusion proteins (single and double fusion proteins) PEGylation improved their properties differintially

In vitro studies on CNGRC-CPG2 fusion proteins for ligand-directed enzyme prodrug therapy for targeted cancer therapy

The sequence asparagine-glycine arginine (NGR), flanked by Cysteine (Cys) residues so as to form a disulfide-bridge (CNGRC), has previously been found to target and bind specifically to aminopeptidase N (APN), which is highly expressed on the surface of tumor cells. The goal of this study was to develop and evaluate the potential of fusion proteins carrying the CNGRC sequence linked to the enzyme carboxypeptidase G2 (CPG2) for targeted cancer therapy. We refer to this strategy as ligand-directed enzyme prodrug therapy (LDEPT). We constructed two forms of the CNGRC-CPG2 fusions, containing one or two copies of the cyclic NGR motif and designated CNGRC-CPG2 (X-CPG2) and CNGRC-CPG2-CNGRC (X-CPG2-X), respectively. binding assays of the purified constructs showed that both X-CPG2 and X-CPG2-X bound with high affinity to cancer cells expressing high levels of APN, compared to their binding to cells expressing low levels of APN. Further studies of the constructs to assess the therapeutic potential of LDEPT were carried out using cells expressing high and low levels of APN. Using methotrexate, it was demonstrated that cancer cell survival was significantly higher in the presence of the fusion proteins, due to the hydrolysis of this cytotoxic drug by CPG2. Conversely, when the prodrug ZD2767P was used, cancer cell killing was higher in the presence of the fused CPG2 constructs than in their absence, which is consistent with CPG2-mediated release of the cytotoxic drug from the prodrug. Furthermore, the doubly-fused CPG2 construct (X-CPG2-X) was significantly more effective than the singly-fused construct (X-CPG2)

Effect of sera from elite athletes on cytokine secretion and insulin signaling in preadipocytes and skeletal muscle cells

Introduction: The immunomodulatory effect of physical activity can impact insulin signaling differentially in adipose tissues and skeletal muscle cells, depending on sport intensity. In this study, the effect of serum from elite athletes with varying endurance levels and playing different power sports on cytokine secretion and insulin signaling in preadipocyte and skeletal muscle cell lines was investigated. Methods: Preadipocytes (3T3-L1) and skeletal muscle cells (C2C12) were cultured in media containing pooled sera from elite athletes who play high-endurance (HE), high-power (HP), or low-endurance/low-power (LE/LP) sports for 72 h. Secreted cytokines (IL-6 and TNF-alpha) were assessed in the supernatant, and insulin signaling phosphoproteins levels were measured in lysates following treatment using cells multiplex immunoassays. Results: Sera from LE/LP and HP induced TNF-α secretion in C2C12, while serum from HE reduced IL-6 secretion compared to non-athlete serum control. All elite athlete sera groups caused decreased insulin sensitivity in 3T3-L1 cells, whereas in C2C12 cells, only HE athlete serum reduced insulin signaling, while LE/LP and HP caused increased insulin sensitivity. Conclusion: Sera from elite athletes of different sport disciplines can affect the inflammatory status and insulin signaling of preadipocytes and myoblasts differently, with risk of developing insulin resistance. Furthermore, investigation of the functional relevance of these effects on exercise physiology and pathophysiology is warranted.This research was sponsored by the award no. QUCG-BRC-21/22-1 (ME and NR), IRCC-2022-467 (ME and MS) from Qatar University. The statements made herein are solely the responsibility of the authors

Assessment of Serum Cytokines and Oxidative Stress Markers in Elite Athletes Reveals Unique Profiles Associated With Different Sport Disciplines

© Copyright © 2020 Sohail, Al-Mansoori, Al-Jaber, Georgakopoulos, Donati, Botrè, Sellami and Elrayess. Objectives: Circulating cytokines and oxidative stress markers vary in response to different exercise regimens. This study aims to compare the immune-inflammatory and oxidative stress profiles of elite athletes from different sport disciplines as potential biomarkers of muscle damage, and cardiovascular demand. Methods: Serum samples from 88 consented elite male athletes from different sports disciplines (aquatics, n = 11, athletics, n = 22, cycling, n = 19, football, n = 28 and weightlifting, n = 8) collected at the anti-doping lab in Italy were screened for 38 cytokines and oxidative stress markers. Comparisons were made between different level of power, cardiovascular demand (CD) and endurance, as well as among the sport types. Results: The anti-inflammatory interleukin (IL)-10 was higher (p = 0.04) in moderate power compared with the high power group. Conversely, superoxide dismutase (SOD; p = 0.001) and malondialdehyde (MDA; p = 0.007) levels were greater in the higher power groups compared with the lower power counterpart. Among athletes who belong to different CD ranks, IL-1β and monocyte chemoattractant protein-1(MCP1) levels were higher (p = 0.03) in the low CD-rank group compared with high CD counterpart, whereas, SOD levels were higher (p = 0.001) in high and moderate CD-rank groups compared to low counterpart. For endurance groups, IL-10 and macrophage inflammatory protein (MIP)-1beta were increased (p = 0.03) in low/moderate endurance compared with the high endurance group. Finally, MIP1-beta, SOD and catalase varied significantly among the sports groups. Conclusion: Specific markers of inflammation and oxidative stress are associated with different sports disciplines and could be utilized as potential biomarkers of athletes’ health, performance, and recovery from injury.We would like to thank Qatar National Research Fund (QNRF) for funding this project

Age and sport intensity-dependent changes in cytokines and telomere length in elite athletes

Background: Exercise-associated immune response plays a crucial role in the aging process. The aim of this study is to investigate the effect of sport intensity on cytokine levels, oxidative stress markers and telomere length in aging elite athletes. Methods: In this study, 80 blood samples from consenting elite athletes were collected for anti-doping analysis at an anti-doping laboratory in Italy (FMSI). Participants were divided into three groups according to their sport intensity: low-intensity skills and power sports (LI, n = 18); moderate-intensity mixed soccer players (MI, n = 31); and high-intensity endurance sports (HI, n = 31). Participants were also divided into two age groups: less than 25 (n = 45) and above 25 years old (n = 35). Serum levels of 10 pro and anti-inflammatory cytokines and two antioxidant enzymes were compared in age and sport intensity groups and telomere lengths were measured in their respective blood samples. Results: Tumor necrosis factor-alpha (TNF-α) was the only cytokine showing significantly higher concentration in older athletes, regardless of sport intensity. Interleukin (IL)-10 increased significantly in HI regardless of age group, whereas IL-6 concentration was higher in the older HI athletes. IL-8 showed a significant interaction with sport intensity in different age groups. Overall, significant positive correlations among levels of IL-6, IL-10, IL-8 and TNF-α were identified. The antioxidant catalase activity was positively correlated with levels of TNF-α. Telomere length increased significantly with sport intensity, especially in the younger group. Conclusion: HI had longer telomeres and higher levels of pro-and anti-inflammatory cytokines, suggesting less aging in HI compared to low and moderate counterparts in association with heightened immune response. Investigation of the functional significance of these associations on the health and performance of elite athletes is warranted

In Vitro and In Vivo Validation of GATA-3 Suppression for Induction of Adipogenesis and Improving Insulin Sensitivity

Impaired adipogenesis is associated with the development of insulin resistance and an increased risk of type 2 diabetes (T2D). GATA Binding Protein 3 (GATA3) is implicated in impaired adipogenesis and the onset of insulin resistance. Therefore, we hypothesize that inhibition of GATA3 could promote adipogenesis, restore healthy fat distribution, and enhance insulin signaling. Primary human preadipocytes were treated with GATA3 inhibitor (DNAzyme hgd40). Cell proliferation, adipogenic capacity, gene expression, and insulin signaling were measured following well-established protocols. BALB/c mice were treated with DNAzyme hgd40 over a period of 2 weeks. Liposomes loaded with DNAzyme hgd40, pioglitazone (positive), or vehicle (negative) controls were administered subcutaneously every 2 days at the right thigh. At the end of the study, adipose tissues were collected and weighed from the site of injection, the opposite side, and the omental depot. Antioxidant enzyme (superoxide dismutase and catalase) activities were assessed in animals’ sera, and gene expression was measured using well-established protocols. In vitro GATA3 inhibition induced the adipogenesis of primary human preadipocytes and enhanced insulin signaling through the reduced expression of p70S6K. In vivo GATA3 inhibition promoted adipogenesis at the site of injection and reduced MCP-1 expression. GATA3 inhibition also reduced omental tissue size and PPARγ expression. These findings suggest that modulating GATA3 expression offers a potential therapeutic benefit by correcting impaired adipogenesis, promoting healthy fat distribution, improving insulin sensitivity, and potentially lowering the risk of T2D.Qatar University H3P grant number QPH3P-BRC-2021-451 (MAE, HN, LM)

GATA-3 as a potential therapeutic target for insulin resistance and type 2 diabetes mellitus.

Impaired adipogenesis plays an important role in the development of obesity-associated insulin resistance and type 2 diabetes as it leads to ectopic fat deposition. The anti-adipogenic transcription factor GATA-3 was identified as one of the potential molecular targets responsible for impairment of adipogenesis. The expression of GATA-3 is higher in insulin resistant obese individuals compared to BMI-matched insulin sensitive counterparts. Adipose tissue inflammation is a crucial mediator of this process. Hyperglycemia mediates the activation of immune system, partially through upregulation of GATA-3, causing exacerbation of the inflammatory state associated with obesity. This review discusses evidence supporting the inhibition of GATA-3 as a useful therapeutic strategy in obesity-associated insulin resistance and type 2 diabetes, through up-regulation adipogenesis and amelioration of the immune response.This research was sponsored by QNRF; Grant no. UREP26-038-3-015 (MAE). Qatar National Library (QNL) has funded the publication of this article

Bio-vehicles of cytotoxic drugs for delivery to tumor specific targets for cancer precision therapy

Abnormal structural and molecular changes in malignant tissues were thoroughly investigated and utilized to target tumor cells, hence rescuing normal healthy tissues and lowering the unwanted side effects as non-specific cytotoxicity. Various ligands for cancer cell specific markers have been uncovered and inspected for directional delivery of the anti-cancer drug to the tumor site, in addition to diagnostic applications. Over the past few decades research related to the ligand targeted therapy (LTT) increased tremendously aiming to treat various pathologies, mainly cancers with well exclusive markers. Malignant tumors are known to induce elevated levels of a variety of proteins and peptides known as cancer “markers” as certain antigens (e.g., Prostate specific membrane antigen “PSMA”, carcinoembryonic antigen “CEA”), receptors (folate receptor, somatostatin receptor), integrins (Integrin αvβ3) and cluster of differentiation molecules (CD13). The choice of an appropriate marker to be targeted and the design of effective ligand-drug conjugate all has to be carefully selected to generate the required therapeutic effect. Moreover, since some tumors express aberrantly high levels of more than one marker, some approaches investigated targeting cancer cells with more than one ligand (dual or multi targeting). We aim in this review to report an update on the cancer-specific receptors and the vehicles to deliver cytotoxic drugs, including recent advancements on nano delivery systems and their implementation in targeted cancer therapy. We will discuss the advantages and limitations facing this approach and possible solutions to mitigate these obstacles. To achieve the said aim a literature search in electronic data bases (PubMed and others) using keywords “Cancer specific receptors, cancer specific antibody, tumor specific peptide carriers, cancer overexpressed proteins, gold nanotechnology and gold nanoparticles in cancer treatment” was carried out.Qatar National Research Fun