P38 mitogen activated protein kinase inhibitor improves platelet in vitro parameters and in vivo survival in a SCID mouse model of transfusion for platelets stored at cold or temperature cycled conditions for 14 days

Platelets for transfusion are stored at room temperature (20–24°C) up to 7 days but decline in biochemical and morphological parameters during storage and can support bacterial proliferation. This decline is reduced with p38MAPK inhibitor, VX-702. Storage of platelets in the cold (4–6°C) can reduce bacterial proliferation but platelets get activated and have reduced circulation when transfused. Thermocycling (cold storage with brief periodic warm ups) reduces some of the effects of cold storage. We evaluated in vitro properties and in vivo circulation in SCID mouse model of human platelet transfusion of platelets stored in cold or thermocycled for 14 days with and without VX-702. Apheresis platelet units (N = 15) were each aliquoted into five storage bags and stored under different conditions: room temperature; cold temperature; thermocycled temperature; cold temperature with VX-702; thermocycled temperature with VX-702. Platelet in vitro parameters were evaluated at 1, 7 and 14 days. On day 14, platelets were infused into SCID mice to assess their retention in circulation by flow cytometry. VX-702 reduced negative platelet parameters associated with cold and thermocycled storage such as an increase in expression of activation markers CD62, CD63 and of phosphatidylserine (marker of apoptosis measured by Annexin binding) and lowered the rise in lactate (marker of increase in anaerobic metabolism). However, VX-702 did not inhibit agonist-induced platelet aggregation indicating that it does not interfere with platelet hemostatic function. In vivo, VX-702 improved initial recovery and area under the curve in circulation of human platelets infused into a mouse model that has been previously validated against a human platelet infusion clinical trial. In conclusion, inhibition of p38MAPK during 14-days platelet storage in cold or thermocycling conditions improved in vitro platelet parameters and platelet circulation in the mouse model indicating that VX-702 may improve cell physiology and clinical performance of human platelets stored in cold conditions.

Novel Bispecific Antibody for Synovial-Specific Target Delivery of Anti-TNF Therapy in Rheumatoid Arthritis

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, via the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.

KDM4C contributes to cytarabine resistance in acute myeloid leukemia via regulating the miR-328-3p/CCND2 axis through MALAT1

Aims: Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm, in which relapse due to drug resistance is the main cause for treatment failure and the disease progression. In this study, we aimed to investigate the molecular mechanism of KDM4C-dependent MALAT1/miR-328-3p/CCND2 axis in cytarabine (Ara-C) resistance in the context of AML. Methods: Bioinformatics analysis was performed to predict the targeting relationships among KDM4C, MALAT1, miR-328-3p, and CCND2 in AML, which were validated with chromatin immunoprecipitation and dual-luciferase reporter assay. Methylation-specific polymerase chain reaction was conducted to detect the methylation of MALAT1 promoter. After conducting gain- and loss-of-function assays, we investigated the effect of KDM4C on cell Ara-C resistance. A NOD/SCID mouse model was established to further investigate the roles of KDM4C/MALAT1/miR-328-3p/CCND2 in Ara-C resistant AML cells. Results: KDM4C expression was upregulated in AML. KDM4C upregulation promoted the demethylation in the promoter region of MALAT1 to increase its expression, MALAT1 targeted and inhibited miR-328-3p expression, enhancing the Ara-C resistance of HL-60/A. miR-328-3p targeted and suppressed the expression of CCND2 in HL-60/A to inhibit the Ara-C resistance. Mechanistically, KDM4C regulated miR-328-3p/CCND2 through MALAT1, resulting in Ara-C resistance in AML. Findings in an in vivo xenograft NOD/SCID mouse model further confirmed the contribution of KDM4C/MALAT1/miR-328-3p/CCND2 in the Ara-C resistant AML. Conclusion: Our study demonstrated that KDM4C may up-regulate MALAT1 expression, which decreases the expression of miR-328-3p. The downregulation of miR-328-3p increased the level of CCND2, which induced the Ara-C resistance in AML.

EC313-a tissue selective SPRM reduces the growth and proliferation of uterine fibroids in a human uterine fibroid tissue xenograft model

Uterine fibroids (UFs) are associated with irregular or excessive uterine bleeding, pelvic pain or pressure, or infertility. Ovarian steroid hormones support the growth and maintenance of UFs. Ulipristal acetate (UPA) a selective progesterone receptor (PR) modulator (SPRM) reduce the size of UFs, inhibit ovulation and lead to amenorrhea. Recent liver toxicity concerns with UPA, diminished enthusiasm for its use and reinstate the critical need for a safe, efficacious SPRM to treat UFs. In the current study, we evaluated the efficacy of new SPRM, EC313, for the treatment for UFs using a NOD-SCID mouse model. EC313 treatment resulted in a dose-dependent reduction in the fibroid xenograft weight (p < 0.01). Estradiol (E2) induced proliferation was blocked significantly in EC313-treated xenograft fibroids (p < 0.0001). Uterine weight was reduced by EC313 treatment compared to UPA treatment. ER and PR were reduced in EC313-treated groups compared to controls (p < 0.001) and UPA treatments (p < 0.01). UF specific desmin and collagen were markedly reduced with EC313 treatment. The partial PR agonism and no signs of unopposed estrogenicity makes EC313 a candidate for the long-term treatment for UFs. Docking studies have provided a structure based explanation for the SPRM activity of EC313.

Development of an Immunotherapeutic Monoclonal Antibody Recognizing DKK1-HLA-A2 Complex to Treat Human Hematologic Malignancies

Immunotherapy is a promising option for cancer treatment. Our previous studies demonstrated that DKK1 is widely expressed by various tumor cells including multiple myeloma (MM) and other hematological malignancies but not normal tissues, and DKK1 peptide (such as P20 and P66v, which bind with HLA-A2 molecule) specific cytotoxic T cells specifically kill myeloma and other cancer cells that express DKK1 and HLA-A2, but not HLA-A2+ normal cells, indicating that DKK1+ tumor cells naturally express these peptides, in the context of HLA-A2 molecules, on their surface. To develop cancer therapeutic antibodies, DKK1 peptide P20-HLA-A2 monomer was synthesized and used to immunize mice. Hybridomas secreting monoclonal antibodies (mAbs) recognizing soluble and cell surface-expressed DKKl P20-HLA-A2 complex were obtained and analyzed. The mAbs bind specifically with DKK1-expressing, HLAA2+ cancer cells but not DKK1-expressing, HLA-A2- cancer cells or DKK1- HLA-A2+ normal blood cells. The mAbs exhibited potent in vitro tumoricidal activity on HLA-A2+DKK1+ U266 multiple myeloma cells, HLA-A2+DKK1+ PC-3 prostate cancer cells and T2 cells loaded with DKK1-P20 peptides. Our results also showed that the anti-DKK1-HLA-A2 mAbs effectively lysed cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which were correlated with and dependent on the surface expression of DKK1-HLA-A2 complex on cancer cells. Furthermore, anti-DKK1/HLA-A2 mAbs were also active and therapeutic in vivo. In MM xenografted SCID mouse model, the mAbs were able to eradicate U266 MM cells and more than 60% of mAb-treated mice survived for 3 months, while control mice all died within 2 months. Toxicity and safety assay in MM xenografted A2-SCID mouse model showed that the mAbs had no significant negative effects on different normal tissues. Therefore, these results support clinical development of anti- DKK1-HLA-A2 mAbs as therapeutic agents to treat hematological malignancies and possibly solid tumors that express surface DKK1-HLA-A2. Disclosures No relevant conflicts of interest to declare.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stagePlasmodium falciparumandCryptosporidium parvumin cell-culture studies. Target deconvolution inP. falciparumhas shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of bothPfKRS1 andC. parvumKRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90= 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology betweenPfKRS1 andCpKRS. This series of compounds inhibitCpKRS andC. parvumandCryptosporidium hominisin culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds forPfKRS1 andCpKRS vs. (human)HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

The TLR3 Agonist Poly Inosinic:Cytidylic Acid Significantly Augments the Therapeutic Activity of an Anti-CD7 Immunotoxin for Human T-cell Leukaemia

We have previously shown that antibody-dependent cellular cytotoxicity (ADCC) cooperates with immunotoxin (IT)-mediated killing of human leukaemia cells in an severe combined immunodeficient (SCID) mouse model of human T-cell acute lymphoblastic leukaemia (SCID-HSB-2 mice), but not in an equivalent non-obese diabetic (NOD)/SCID mouse model. In these earlier studies, we reasoned that diminished ADCC due to the functional deficit in natural killer (NK) cell activity in NOD/SCID mice resulted in a failure of effective perforin/granzyme-mediated cytotoxicity necessary for the delivery of the augmentative effect. Poly-inosinic-cytidylic acid [poly (I:C)] is a synthetic dsRNA toll-like receptor 3 (TLR3) agonist that possesses a number of biological properties that includes the in vivo activation of NK cells. We show here that intravenous (i.v.) injection of SCID mice with [poly (I:C)] results in characteristic time-related changes in serum interleukin 2 (IL-2), IL-12, and interferon γ (INFγ) cytokine levels that are consistent with TLR3 driven activation of SCID mouse NK cells. Concomitantly, there are changes in the expression levels of CD2, CD16/32 (FcγRII/RIII), CD161 (NK1.1), and F4/80 in the bulk splenocyte population. These observed changes correlate with an increase in the in vitro lytic capabilities of putative NK cells from within the splenocyte population of [poly (I:C)] treated SCID mice. We demonstrate that the in vivo activation of NK cells with [poly (I:C)] in SCID mice bearing disseminated human T-cell leukaemia xenografts resulted in a significant improvement in the therapeutic activity exerted by an intact murine monoclonal antibody against human CD7. This was also seen for a saporin-based immunotoxin constructed with the same intact antibody (HB2-SAPORIN), but not with an F(ab’)2 derivative of the same antibody or of an IT constructed with the same F(ab’)2 HB2 antibody derivative. This study further demonstrates the previously reported reinforcing role of ADCC for the therapeutic activity of IT in an SCID mouse model of human T-ALL and the potential to significantly boost this further with [poly (I:C)]. Our study provides the rationale to justify the exploration of the clinical utility of IT based therapeutics in combination with TLR3 agonists, such as [poly (I:C)], for the treatment of haematological, and possibly other, malignancies.

Tetraspanin CD82 affects migration, attachment and invasion of rheumatoid arthritis synovial fibroblasts

Tetraspanins function as membrane adaptors altering cell-cell fusion, antigen presentation, receptor-mediated signal transduction and cell motility via interaction with membrane proteins including other tetraspanins and adhesion molecules such as integrins. CD82 is expressed in several malignant cells and well described as tumour metastasis suppressor. Rheumatoid arthritis (RA) is based on persistent synovial inflammation and joint destruction driven to a large extent by transformed-appearing activated synovial fibroblasts (SF) with an increased migratory potential.ObjectiveCD82 is upregulated in RA synovial fibroblasts (RASF) compared with osteoarthritis (OA) SF as well as within RA compared with OA synovial lining layer (LL) and the role of CD82 in RASF was evaluated.MethodsCD82 and integrin immunofluorescence was performed. Lentiviral CD82 overexpression and siRNA-mediated knockdown was confirmed (realtime-PCR, Western blot, immunocytochemistry). RASF migration (Boyden chamber, scrape assay), attachment towards plastic/Matrigel, RASF-binding to endothelial cells (EC) and CD82 expression during long-term invasion in the SCID-mouse-model were evaluated.ResultsCD82 was induced by proinflammatory stimuli in SF. In RA-synovium, CD82 was expressed in RASF close to blood vessels, LL, sites of cartilage invasion and colocalised with distinct integrins involved in tumour metastasis suppression but also in RA-synovium by RASF. CD82 overexpression led to reduced RASF migration, cell-matrix and RASF-EC adhesion. Reduced CD82 expression (observed in the sublining) increased RASF migration and matrix adhesion whereas RASF-EC-interaction was reduced. In SCID mice, the presence of CD82 on cartilage-invading RASF was confirmed.ConclusionCD82 could contribute to RASF migration to sites of inflammation and tissue damage, where CD82 keeps aggressive RASF on site.