CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1) specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4+ helper T-cells (Th17) have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in in vitro experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15–35% of CD4+ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab’)2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an important target for modulating autoimmune diseases.
Background : The ubiquitin-proteasomal degradation pathway plays a critical role in protein degradation and regulates a wide variety of cellular functions. This highly conserved post-translational modification of proteolytic processes is mainly carried out by substrate-specific E3 ligases. The deregulation of E3 ligases contributes to cancer development and their overexpression is often associated with poor prognosis. Objectives : We review the current understanding of E3 ligases, their functional role in cancer pathogenesis, current progress and development of certain ubiquitin E3 ligases as targets for therapeutic intervention. Methods : Preclinical and clinical data for E3 ligase inhibitors available in the public domain are discussed. Conclusions : With the growing understanding of their role in cancer development and progression, E3 ligases have emerged as potential anticancer targets for therapeutic intervention.
Conventionally, the successful targets for the drug development in cancer range from the DNA damage, replication, signal transduction pathways, hormones, cytokines, anti-angiogenic agents, and radio/photo-sensitizers. They dominate the therapeutic arena after the initial debulking surgery. More recently, tubulin, the primary constituent of microtubules (MTs), has made a fairly successful debut in the therapeutic armamentarium. Tubulin binding drugs come in two classes: that depolymerize microtubules and that over-polymerize and bundle them. Microtubule (MT) binding drugs are in some ways superior in nature primarily because of their less debilitating side effects when compared to the generalized DNA metabolism targeting agents, and many new promising patents are being funneled into the drug development pipeline. Nevertheless, many of these relatively new agents still face challenges relating to their delivery methods, bioavailability, toxicities, and the inevitable resistance shared by all chemotherapeutics. Finally, we disclose a new genre of anti-MT drugs, noscapinoids that have just begun climbing the clinical trials ladder. The lead compound, noscapine, is a plant derived, orally available, minimally-toxic (if at all) agent that has shown phenomenal promise in the preclinical experimentation and Phase-I clinical trial. A rational approach based upon the precise molecular model of the tubulin-noscapine complex is bound to inspire novel and better therapeutic analogs in future.
This section discusses the potential of noscapine, a plant-derived alkaloid, as a chemotherapeutic drug for cancer. This alkaloid is from Papaver somniferum. The discovery of the compound as a novel microtubule drug, its structure-activity relationship, mechanism of action, effect on healthy and cancerous cells and its non-toxic profile in animals are described. Discussions on noscapine and drug-resistant cancer cells, pharmacokinetic profile, angiogenesis and cytotoxicity are also given. Other medicinal uses are mentioned. Issues on biotechnological production and future uses are also tackled briefly.
Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age. Although FAP is involved in only 1% of all colon cancer cases, over 80% of sporadic cancers harbor somatic mutations of APC. We show here that bromo-onoscapine (EM011), a rationally-designed synthetic derivative of a natural non-toxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G2/M arrest in wild type mouse embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with siRNA. Furthermore, both β-catenin levels and activity fell to half the original levels with a concomitant reduction of cell proliferation-inducing cyclin D1, c-Myc, and induction of cytostatic protein p21 prior to caspase-3 activation. Additionally, we show a statistically significant reduction in the number of newly emerging intestinal polyps (to 35% compared with untreated mice) as well as the mean size of polyps (to 42% compared with untreated mice) in EM011-treated ApcMin/+ mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of ApcMin/+ mice showed evidence of elevated apoptosis as revealed by immunohistochemistry. We failed to detect any evidence of histopathological and hematological toxicities following EM011 treatment. Taken together, our data are persuasive that a clinical trial of EM011 is possible for the prevention/amelioration of polyposis in FAP patients.
BackgroundGiven the pleiotropic functions of transforming growth factor-beta (TGFβ), current approaches to targeting systemic TGFβ will likely lead to suboptimal clinical activity and/or undesirable effects. Epidermal growth factor receptor (EGFR) is one of the most extensively validated tumor-associated antigens. Bicara Therapeutics has developed a novel bifunctional fusion protein, composed of a monoclonal antibody against EGFR and an extracellular domain of human TGFβ receptor II (TGFβRII). We demonstrate BCA101 has the potential to improve anti-tumor response by leveraging the cooperativity between EGFR and TGFβ signaling pathways while restricting TGFβ neutralization to EGFR-expressing tissues.MethodsFunctional neutralization of TGFβ by BCA101 was demonstrated by several in vitro assays which assessed TGFβ-dependent epithelial to mesenchymal transition (EMT), cell invasion, inducible Treg differentiation, as well as allogeneic immune responses in tumor cell/immune cell coculture assays. In vivo, the anti-tumor efficacy of BCA101 was determined in tumor xenograft mouse models, using either human tumor cell lines or patient-derived tumor cells (PDX), as well as in a humanized mouse model.ResultsIn vitro, we showed BCA101 is capable of simultaneously binding EGFR and TGFβ1 with a significantly higher affinity for EGFR. The incorporation of the TGFβRII ”trap” did not sterically interfere with the ability of BCA101 to bind EGFR, inhibit cell proliferation or mediate antibody-dependent cellular cytotoxicity (ADCC). Relative to cetuximab, BCA101 showed improved ability to reverse EMT and preserve ADCC activity. In tumor cell/immune cell co-culture assays, BCA101 increased production of proinflammatory cytokines associated with T and NK cell activation and suppressed VEGF release. Further, BCA101 inhibited differentiation of inducible Treg and displayed an immuno-potentiating profile in the BioMAP® TME model. In vivo, biodistribution studies showed that BCA101 localized to tumor tissues in xenograft mouse models, with comparable kinetics as cetuximab. TGFβ in tissues was neutralized to about 90% at 10 mg/kg of BCA101 while equimolar doses of TGFβRII receptor inhibited TGFβ in tumors by around 50%, confirming improved tumor localization with BCA101. In PDX models derived from head and neck cancer squamous cell carcinoma patients, BCA101 exerted sustained antitumor effect and delayed tumor growth compared to cetuximab. Finally, BCA101 improved the anti-tumor activity of PD1 blockade therapy in humanized HuNOG-EXL mice bearing PC-3 xenografts (figure 1).Abstract 874 Figure 1BCA101 shows superiority over cetuximab in animal models. (A) & (B). Patient derived xenograft (PDX) models. Patient derived tumors were engrafted into female NOG mice. Once tumor reached about 130 mm3, mice were randomized into control and test groups. Test group mice were treated with either BCA101 (A) or cetuximab (B), thrice a week (i.p), whereas control animals received placebo alone. Mice were treated for 27 days followed by a treatment-free phase until Day 79. Tumor volumes and mice weight were recorded twice a week. (C). BCA101 inhibits FaDu tumor xenograft growth (CDX) in vivo. Nude mice were implanted with FaDu cells on flanks. Once tumor reached about 100 mm3, mice were randomized (n=7) and treated with six doses of test compounds, BCA101 and cetuximab. Tumor volume and mice weight were recorded twice a week. (D). BCA101 and anti-PD1 combination studies in hu-NOG-EXL humanized animal model. PC-3 cells were implanted into flank of Hu-NOG-EXL humanized mice and randomized into control and test groups once tumors reached about 120 mm3. Test group mice were treated with cetuximab or BCA101, intraperitoneally for 6 doses. Anti-PD1 antibody (pembrolizumab) was administered intraperitoneally at a dose of 10 mg/kg with a dosage schedule of every fifth-day for 5 doses (Q5Dx5). Statistical analysis for panel (C) & (D) was performed using repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test. Significance was indicated by * = p value ≤0.05, ** = p value ≤0.01 and *** = p value ≤0.001. Tumor volumes are presented as Mean ± SEM.ConclusionsThese results support the clinical development of BCA101 as a targeted immunotherapy with the potential to induce improved anti-tumor response with a wider therapeutic window, either as a monotherapy or in combination with immune checkpoint blockade therapy.AcknowledgementsWe acknowledge Mazumdar Shaw Center for Translational Research for providing the human tissues used for the PDX studies. We thank Syngene International for conducting the PDX and humanized mice studies at their vivarium. We thank Dr. Sreesha Srinivasa for providing suggestions and feedback at the early stages of this project.ReferencesBedi A, Chang X, Noonan K , Pham V, Bedi R, Fertig EJ, Considine M, Califano JA, Borrello I, Chung CH, Sidransky D, Ravi R. Inhibition of TGF-b enhances the in vivo antitumor efficacy of EGF receptor–targeted therapy. Mol Cancer Ther 2012;11:1–11.Yegodayev KM, Novoplansky O, Golden A, Prasad M, Levin L, Jagadeeshan S, Zorea J, Dimitstein O, Joshua B-Z, Cohen L, Khrameeva E, Elkabets M. TGF-Beta-activated cancer-associated fibroblasts limit cetuximab efficacy in preclinical models of head and neck cancer. Cancers 2020;12:1–17.Ethics ApprovalMice were maintained as per the regulations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. All animal experiments were approved by institutional ethical committee and performed under approved protocols. For PDX model, head and neck cancer patient samples were obtained from Mazumdar Shaw Medical Foundation, Bengaluru, India after appropriate approvals were obtained from institutional ethical committee: NHH/MEC-RC2016-404
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