Effective Antitumor Activity Research Articles

Synthesis, structures and biological activity of novel complexes with trifluorinated anthranilic acid derivatives

Two cobalt complexes [Co2(L)(phen)(Ac)(PLC)] (1), [Co3(HL)4(CH3O)2(H2O)2] (2) and one zinc complex [Zn2(L)(phen)(Ac)(PLC)] (3), (L = (2-carboxylato-5-(trifluoromethyl)phenyl) (3-hydroxy-4-methoxyphenoxy)amide, phen = 1,10-phenanthroline, Ac = acetate, PLC = 2-amino-4-trifluoromethylbenzoate) have been synthesized and characterized by physicochemical and spectroscopic methods. In addition, X-ray crystallography shows that the Co and Zn atoms in each complex are penta- or six-coordinated, adopting a twisted square pyramid or triangular bi-conical geometry. The antitumor activity of complex against A549 (human lung cancer cells) and Hela (human cervical cancer cells) was screened. Results show that 3 exhibits the most effective antitumor activity (IC50 = 1.369–2.129 μM). Antimicrobial studies also show that 3 exhibits the highest activity.

In vivo anti-tumor activity of tumor-targeting pH-sensitive lipoprotein-mimic nanocarrier with paclitaxel loaded

Paclitaxel( PTX) is used as a broad spectrum anti-tumor medicine. However,serious drawbacks restrict clinical application of PTX. In this study,we prepared tumor-targeting and pH-sensitive lipoprotein-mimic nanocarrier containing paclitaxel( BSALC/DOPE-PTX) to study the effective antitumor activity. The in vivo targeting ability of the nanocarrier in tumor bearing nude mice was evaluated by using a Kodak in vivo imaging system FX PRO. The in vivo anti-tumor activity was evaluated in MDA-MB-231 tumor bearing mice,and representative sections were stained with hematoxylin and eosin( H&E),and examined by light microscopy. The results showed that DiR-loaded FA-BSA-LC/DOPE selectively targeted tumor,and had a relatively long residence in the tumor tissue. According to the in vivo anti-tumor activity study,FA-BSA-LC/DOPE-PTX exhibited an outstanding tumor inhibition effect with a tumor growth inhibition rate of 79.3%,and tumor tissue sections stained by hematoxylin and eosin( HE) showed severe necrosis areas and many dead cells with condensed nuclei in the FA-BSA-LC/DOPE-PTX group. Therefore,FA-BSA-LC/DOPE-PTX is a biocompatible,tumor-targeting and pH-sensitive lipoprotein-mimic nanocarrier,with a very marked anti-tumor activity in tumor-bearing mice in vivo.

Synthesis and evaluation of bi-functional 7-hydroxycoumarin platinum(IV) complexes as antitumor agents.

A series of bi-functional 7-hydroxycoumarin platinum(IV) complexes were synthesized, characterized, and evaluated for antitumor activities. The 7-hydroxycoumarin platinum(IV) complexes display moderate to effective antitumor activities toward the tested cell lines and show much potential in overcoming drug resistance of platinum(II) drugs. In reducing microenvironment, the title compounds could be reduced to platinum(II) complex accompanied with two equivalents of coumarin units. By a unique mechanism, the 7-hydroxycoumarin platinum(IV) complex attacks DNA via the released platinum(II) compound, meanwhile it also inhibits the activities of cyclooxygenase by coumarin fragment. This action mechanism might be of much benefit for reducing tumor-related inflammation in the progress of inhibiting tumor proliferation and overcoming cisplatin resistance. The incorporation of 7-hydroxycoumarin leads to significantly enhanced platinum accumulation in both whole tumor cells and DNA. The HSA interaction investigation reveals that the tested coumarin platinum(IV) compound could effectively combine with HSA via van der Waals force and hydrogen bond.

<p>Glucose-responsive mesoporous silica nanoparticles to generation of hydrogen peroxide for synergistic cancer starvation and chemistry therapy</p>

Background: The combination of novel starving therapy with chemotherapy is one of the most promising strategies to achieve an effective antitumor activity.Methods: Herein, we developed a multifunctional mesoporous silica nanoparticle (MSNs-GOx/PLL/HA) coated with poly (L-lysine) (PLL) and hyaluronic acid (HA) for co-delivery of glucose oxidase (GOx) and anticancer drug paclitaxel (PTX) for cancer treatment for the first time. Compared to single chemotherapy, introduction of GOx would not only selectively trigger the consumption of intracellular glucose, leading to the interruption of energy supply, but also elevat the endogenous H2O2 level, inducing stronger therapeutic effects.Results: The novel drug delivery system possessed desirable particle diameter of 40 nm and exhibited a pH-sensitive drug release behavior. An in vitro cellular uptake study indicated that MSNs-GOx/PLL/HA nanoparticles effectively enhanced the cellular uptake of drug in an apparently CD44 receptor-dependent manner, and delivered more cargo into cytoplasm via endolysosomal escape effect in presence of PLL. The nanoplatform has also demonstrated amplified synergistic therapeutic effects for remarkable tumor inhibition in a xenograft animal tumor model.Conclusion: Consequently, the developed synergistic starving-like/chemotherapy may provide a potential platform for next generation cancer therapy.

Coordination bonding-based Fe3O4@PDA-Zn2+-doxorubicin nanoparticles for tumor chemo-photothermal therapy

Multistimuli-responsive vehicles that deliver drugs in temporal-, spatial- and dosage-controlled manners have developed as a potential platform in tumor therapy. However, how to integrate these functions in one single carrier effectively still presents a lot of challenge. Here, we developed a strategy to construct a photothermal and pH responsive platform based on Zn2+ coordination with doxorubicin (DOX) and polydopamine (PDA) on the surface of magnetic nanoparticles (Fe3O4@PDA-Zn2+-DOX NPs). In this architecture, Fe3O4@PDA nanoparticles triggered photothermal effects for tumor hyperthermia, and Zn2+ coordinated DOX was released in responding to the thermal effect to achieve chemotherapy simultaneously. Moreover, DOX release was further promoted at acidic environment due to the pH sensitivity of Zn2+ coordination. Therefore, our system possesses multiple effects in tumor treatment: photothermal therapy with near infrared lighting (NIR), thermosensitive DOX release for chemotherapy, and pH-responsive DOX release to enhance deeper therapy in tumor acidic microenvironment. Our in vivo results provide the evidence that Fe3O4@PDA-Zn2+-DOX NPs upon laser exposure have effective anti-tumor therapeutic activity, indicating this platform is suitable for enhanced chemo-photothermal synergistic tumor therapy.

Development of a series of 4-hydroxycoumarin platinum(IV) hybrids as antitumor agents: Synthesis, biological evaluation and action mechanism investigation

A series of new 4-hydroxycoumarin platinum(IV) complexes were designed, synthesized and evaluated as antitumor agents. All the title compounds display moderate to effective antitumor activities toward the tested cell lines and two prominent compounds were screened out with activities comparable to cisplatin and oxaliplatin. The mechanism investigation demonstrates that the platinum(IV) compounds could be reduced to bivalence and exert significant genotoxicity to tumor cells. Meanwhile the coumarin moiety endows the title compounds with cyclooxygenase inhibitory competence which might favour the reduction of tumor-related inflammation and further influence tumor proliferation. The coumarin platinum(IV) complex could effectively induce apoptosis of SKOV-3 cells through up-regulating the expression of caspase3 and caspase9. Furthermore, the conversion of platinum(II) drugs to platinum(IV) form via the conjunction with 4-hydroxycoumarin enhances the drug uptake in whole cells and DNA simultaneously. Moreover, the 4-hydroxycoumarin platinum(IV) complex could combine with human serum albumin via van der Waals force and hydrogen bond, which would influence their transport and bioactivities in vivo.

Melatonin synergizes BRAF-targeting agent vemurafenib in melanoma treatment by inhibiting iNOS/hTERT signaling and cancer-stem cell traits

BackgroundAs the selective inhibitor of BRAF kinase, vemurafenib exhibits effective antitumor activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after its initial treatment.MethodsImmunohistochemical staining was performed to detect the expression of iNOS and hTERT, p-p65, Epcam, CD44, PCNA in mice with melanoma xenografts. The proliferation and migration of melanoma cells were detected by MTT, tumorsphere culture, cell cycle, cell apoptosis, AO/EB assay and colony formation, transwell assay and scratch assay in vitro, and tumor growth differences were observed in xenograft nude mice. Changes in the expression of key molecules in the iNOS/hTERT signaling pathways were detected by western blot. Nucleus-cytoplasm separation, and immunofluorescence analyses were conducted to explore the location of p50/p65 in melanoma cell lines. Flow cytometry assay were performed to determine the expression of CD44. Pull down assay and ChIP assay were performed to detect the binding ability of p65 at iNOS and hTERT promoters. Additionally, hTERT promoter-driven luciferase plasmids were transfected in to melanoma cells with indicated treatment to determine luciferase activity of hTERT.ResultsMelatonin significantly and synergistically enhanced vemurafenib-mediated inhibitions of proliferation, colony formation, migration and invasion and promoted vemurafenib-induced apoptosis, cell cycle arresting and stemness weakening in melanoma cells. Further mechanism study revealed that melatonin enhanced the antitumor effect of vemurafenib by abrogating nucleus translocation of NF-κB p50/p65 and their binding at iNOS and hTERT promoters, thereby suppressing the expression of iNOS and hTERT. The elevated anti-tumor capacity of vemurafenib upon co-treatment with melatonin was also evaluated and confirmed in mice with melanoma xenografts.ConclusionsCollectively, our results demonstrate melatonin synergizes the antitumor effect of vemurafenib in human melanoma by inhibiting cell proliferation and cancer-stem cell traits via targeting NF-κB/iNOS/hTERT signaling pathway, and suggest the potential of melatonin in antagonizing the toxicity of vemurafenib and augmenting its sensitivities in melanoma treatment.

<p>Chronic chemotherapy with paclitaxel nanoparticles induced apoptosis in lung cancer in vitro and in vivo</p>

AimPaclitaxel (PTX) is an effective antitumor drug. Previous research demonstrated that paclitaxel nanoparticles (PTX-NPs) exhibited the greatest antitumor effect at 15 hours after light onset (15 HALO), but the mechanism in chronic chemotherapy is still unknown. In our study, we investigated whether PTX-NPs regulated Period2 (Per2) during chronic chemotherapy to induce apoptosis in vivo and in vitro.MethodsTo improve the antitumor effect and reduce organ damage induced by PTX treatment, PTX-NPs were prepared using a film dispersion method. Then, A549 cells were treated with PTX-NPs at 0, 5, 10, 15, and 20 HALO. An annexin/PI V-FITC apoptosis kit was measured for apoptosis, and PI was analyzed for cell cycle. The relative mechanism was detected by RT-PCR and Western blotting. Tumor volume and weight were measured to evaluate the antitumor effect of the PTX-NPs, and H&E staining was performed to assess organ damage.ResultsCell cycle analysis demonstrated that PTX-NPs blocked cell cycle in G2 phase and that the ratio of cell death was significantly increased in A549 cells, while the ratios of cells in G2 phase and of apoptotic cells were highest at 15 HALO. Evaluation of in vivo antitumor activity revealed that PTX-NPs inhibited tumor growth and decreased tumor weight at 15 HALO. RT-PCR and Western blotting demonstrated that PTX-NPs upregulated Per2 mRNA and protein expression, and the highest Per2 expression was observed at 15 HALO in vivo and in vitro. Meanwhile, Bax mRNA and protein expression was upregulated, while Bcl-2 mRNA and protein expression was downregulated after PTX-NPs treatment in vivo. Moreover, H&E staining revealed that PTX-NPs reduced liver damage at 15 HALO.ConclusionPTX-NPs exhibited the most effective antitumor activity and reduced liver damage at 15 HALO through upregulation of Per2 expression to induce apoptosis in vivo and in vitro.

Modeling Sézary Syndrome for Immunophenotyping and Anti-Tumor Effect of Ucart and Long-Acting Interleukin-7 Combination Therapy

Background Sezary syndrome (SS) is a highly-morbid T cell leukemic lymphoma with no widely-effective treatments and few preclinical models. SS T cells typically lose CD7 but maintain ubiquitous high CD2 expression. Thus, we generated CD2- and TRAC-deleted anti-CD2 universal CARTs (UCART2) and multiple SS xenograft models (PDXs) as preclinical UCART2 testing platforms. We further tested a stable homodimeric interleukin-7 molecule, the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7), to potentiate UCART2 killing of an SS xenograft in vivo. Methods To generate SS PDX models, we injected NSG-SGM3 mice with ∼2 × 106 mononuclear cells derived from SS patients. We immunophenotyped SS patient blood and PDX engraftment with two 21-color flow cytometry panels assessing major immune subsets, CTCL, and exhaustion markers. To generate UCART2s, we CRISPR/Cas9 multiplexed edited CD2 and TRAC from CART2. For initial testing, we injected NSG mice with 5 × 105 cells from a human Sezary cell line (HH) on day -4 from UCART2 treatment, followed by NT-I7 (10mg/kg SC) on days +1, +15 and +29. Results SS patient blood showed specific defects in monocyte, monocytic dendritic cell, and natural killer cell differentiation, increased skewing toward granulocytes and non-classical CD16+ monocytes (p 90% vs ∼20%, p UCART2-treated HHCBR-GFP mice showed dramatically reduced tumor burden as compared to control UCART19-treated HHCBR-GFP mice (BLI; 10^7 vs. 10^11 photon flux/s at 3 weeks, p Discussion We describe the generation of physiologically-relevant SS preclinical models and the highly effective anti-tumor activity of UCART2- plus NT-I7-mediated killing of SS cells in vivo using an NSG xenograft model.

Construction of novel procoagulant protein targeting neuropilin-1 on tumour vasculature for tumour embolization therapy

The cellular transmembrane receptor Neuropilin-1(NRP-1) is overexpressed in tumour tissue and endothelial cells of tumour vessels, whereas it has limited expression in normal tissues. This study aimed to design a novel recombinant protein tTF-EG3287, which consisting of the truncated tissue factor (tTF) and the NRP-1 targeting peptide EG3287. The procoagulant protein selectively activates blood coagulation in tumour vessels once bound to the cell surface of the tumour vasculature by a targeting peptide EG3287. In this study, procoagulant activity of the recombinant protein tTF-EG3287 was evaluated by Spectozyme FXa assay. NRP-1 targeting ability was analysed by fluorescence confocal microscopy and flow cytometry. The living imaging system was used to assess the tumour targeting ability of recombinant proteins tTF-EG3287 in vivo. Tumour growth inhibition showed effective antitumor activity in HepG2 tumour-bearing nude mice. Histological study showed obvious thrombosis and thromboembolism in tumour vessels and cell necrosis of tumour tissue, without any clear side effect such as thrombosis in other organs.

Intravesical treatment of chemotherapeutic agents sensitizes bacillus Calmette‑Guerin by the modulation of the tumor immune environment.

Intravesical treatment with bacillus Calmette‑Guerin (BCG) is the most common treatment for preventing progression and recurrence of non‑muscle invasive bladder cancer. Our previous study using the N‑butyl‑N‑(4‑hydroxybutyl) nitrosamine (BBN)‑induced orthotopic bladder cancer model demonstrated that intravesical treatment with mitomycin C (MMC) and adriamycin (ADM) suppressed pro‑tumoral immunity, including the aggregation of tumor‑associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. Previous evidence supports the association of resistance to intravesical treatment of BCG with TAMs and Tregs. In the present study, we investigated the antitumoral efficacy of sequential intravesical treatments with chemotherapeutic agents and BCG in a BBN‑induced orthotopic bladder cancer model. Thirty‑six C57BL/6J mice bearing bladder cancer were randomly divided into six treatment groups as follows: control, BCG, MMC, ADM, MMC‑BCG and ADM‑BCG. Intravesical treatment was performed once a week for six weeks. One week after the completion of intravesical treatment, bladder and blood were harvested. MMC‑BCG and ADM‑BCG were more effective antitumor activities than BCG monotherapy. Bladders were subjected to immunohistochemical analysis and revealed that intravesical BCG treatment combined with MMC/ADM promoted the local recruitment of NK cells to the bladder as effectively as BCG monotherapy and reduced TAMs and Tregs in the bladder. Interleukin (IL)‑17 and granulocyte‑colony stimulating factor (G‑CSF) in serum were analyzed by enzyme‑linked immunosorbent assay and these levels were revealed to be elevated in mice treated with sequential treatments similar to levels following monotherapy with MMC and ADM. Our findings indicated that intravesical sequential treatment could suppress the resistance to BCG through the enhancement of antitumor immunity (induction of NK cells) and inhibition of pro‑tumoral immunity (reduction of TAMs and Tregs). Systemic changes in IL‑17 and G‑CSF may be involved in topical immunomodulation. Further studies including clinical trials may be required to establish an appropriate strategy based on the immunomodulation of the tumor microenvironment.

Biochemical Studies of (5-P-chlorophenyl -2-benzo 5, 6-coumarin-3-yelthylidene aminothiazole) as Antitumor Agent

Heterocyclic compounds have a large spectrum of biological activities including antitumor activity. The present study describes the cytotoxic effect of newly synthesized thiazole derivative (TD2) that can prove effective antitumor activity on both in vivo and in vitro studies.
 Objective: the essential objective of this research is to prove the cytotoxic effect of newly synthesized thiazole derivative (TD2) up on EAC bearing mice and many kinds of human cell lines.
 Materials and Methods: Antitumor activity of TD2 was examined on EAC in Swiss albino Mice at dose of 2.5 mg/kg. TD2 was injected for 10 following days after transplantation of tumor. After one day of last dose and 18 hours of fasting, 7 Mice were sacrificed and the remaining was kept to evaluate ILS %.
 Antitumor activity of TD2 was assessed by inspecting tumor volume, tumor weight, viable cell count and nonviable cell count, hematological, biochemical and antioxidant parameters of mice.
 Results: TD2 demonstrated an inhibitory effect on both cancer cell lines in vitro and Ehrlich ascites cells in vivo.TD2 increased in life span of Ehrlich–bearing mice compared to control. Cell cycle and flow cytometric analysis revealed that TD2 directed Ehrlich cells toward apoptosis by increasing of P53 expression.
 Conclusion: It was concluded that TD2 have a potent antitumor activity against Ehrlich ascites carcinoma in mice beside a cytotoxic effect on MCF-7, PC3, HepG2 and HCT-116.

Development of dual-targeted nano-dandelion based on an oligomeric hyaluronic acid polymer targeting tumor-associated macrophages for combination therapy of non-small cell lung cancer

In this study, the novel carrier materials were screened to structure targeting nano-micelles (named ‘nano-dandelion’) for synchronous delivery of curcumin (Cur) and baicalin (Bai), which could effectively overcome the tumor resistance. Mannose (Man) was found to bind better to CD206 receptors on the surface of tumor-associated macrophages (TAMs), thereby increasing the number of nano-dandelion engulfed by TAMs. Furthermore, oligomeric hyaluronic acid (oHA) was able to target CD44 receptors, resulting in recruitment of a higher number of nano-dandelion to locate and engulf tumor cells. The disulfide bond (S–S) in 3,3′-dithiodipropionic acid (DA) could be broken by the high concentration of glutathione (GSH) in the tumor microenvironment (TME). Based on this, we selected DA to connect hydrophobic fragments (quercetin, Que) and oHA. A reduction-sensitive amphiphilic carrier material, quercetin–dithiodipropionic acid–oligomeric hyaluronic acid–mannose–ferulic acid (Que–S–S–oHA–Man–FA; QHMF) was fabricated and synthesized by 1H NMR. Next, QHMF self-assembled into nano-dandelion, i.e. encapsulated Cur and Bai in water. Critical experimental conditions in the preparation process of nano-dandelion that could affect its final properties were explored. Nano-dandelion with a small particle size (121.0 ± 15 nm) and good normal distribution (PI = 0.129) could easily enter tumor tissue through vascular barrier. In addition, nano-dandelion with a suitable surface potential (–20.33 ± 4.02 mV) could remain stable for a long duration. Furthermore, good cellular penetration and tumor cytotoxicity of nano-dandelion were demonstrated through in vitro cellular studies. Finally, effective antitumor activity and reduced side effects were confirmed through in vivo antitumor experiments in A549 tumor-bearing nude mice.

Natural Killer Cell-Based Immunotherapy for Cancer: Advances and Prospects

Abstract Natural killer (NK) cells are key innate immune cells that provide the first line of defense against viral infection and cancer. Although NK cells can discriminate between “self” and “non-self,” recognize abnormal cells, and eliminate transformed cells and malignancies in real time, tumors develop several strategies to escape from NK cell attack. These strategies include upregulating ligands for the inhibitory receptors of NK cells and producing soluble molecules or immunosuppressive factors. Various types of NK cells are currently being applied in clinical trials, including autologous or allogeneic NK cells, umbilical cord blood (UCB) or induced pluripotent stem cell (iPSC)-derived NK cells, memory-like NK cells, and NK cell line NK-92 cells, for the treatment of different types of tumors. Chimeric antigen receptors (CARs)-NK cells have recently shown great potential due to their redirect specificity and effective antitumor activity. In this review, we summarize the mechanisms of tumor escape from NK cell recognition, the current status and advanced progress of NK cell-based immunotherapy, ways of enhancing the antitumor capacity of NK cells in vivo, and major challenges for clinical practice in this field.

A class of novel tubulin polymerization inhibitors exert effective anti-tumor activity via mitotic catastrophe

In current work, a class of novel 4,5-dihydro-1H-pyrazole-1-carboxylate derivatives (E01-E28) were designed, synthesized and evaluated. Among them, the most potent compound E24 exhibited comparable activity against a panel of cancer cells (GI50 ranging 0.05–0.98 μM) and tubulin polymerization inhibition (IC50 = 1.49 μM) with reference drug CA-4(P) (GI50 ranging 0.019–0.32 μM, IC50 = 2.18 μM). The following assays indicated that compound E24 disturbed the dynamics of tubulin catastrophe and rescue, which triggered G2/M arrest, leading to ROS accumulation, cleavage of PARP and apoptosis. Molecular dynamics simulation validated that compound E24 could tightly bind into tubulin heterodimers with β Lys 254 and β Cys 241 of tubulin in the docking pose. Metabolic stability and pharmacokinetics parameters were also determined. The half time (t1/2) displayed species differences in three microsomes. The plasma elimination half-life (t1/2), peak plasma concentration (Cmax), mean retention time (MRT), the area under the curve (AUC0-∞) and distribution volume (Vz) of E24 after intravenous administration were 0.90 ± 0.22 h, 594.50 ± 97.23 ng/mL, 1.09 ± 0.22 h, 413.67 ± 105.64 ng/mL*h and 5.03 ± 1.82 L/kg, respectively. In HeLa-xenografts, compound E24 exhibited obvious antitumor efficacy via the suppression of tumor growth without weight loss of body or organ. In brief, compound E24 might be a hopeful candidate with excellent properties for oncotherapy as tubulin polymerization inhibitor.

A novel tetravalent bispecific antibody targeting programmed death 1 and tyrosine-protein kinase Met for treatment of gastric cancer.

Background Redirecting T cells to tumor cells using bispecific antibodies (BsAbs) is emerging as a potent cancer therapy. The main concept of this strategy is to cross-link tumor cells and T cells by simultaneously binding to cell surface tumor-associated antigen (TAA) and the CD3ƹ chain. However, immune checkpoint programmed cell death ligand-1 (PD-L1) on tumor cells or other myeloid cells upreglulated remarkablely after the treatment of CD3-binding BsAbs, leads to the generation of suppressed microenvironment for immune evasion and tumor progression. Although this resistance could be partially reversed by anti-PD-L1 treatment, targeting two pathways through one antibody-based molecule may provide a strategic advantage over the combination of BsAbs and immune checkpoint inhibitors. Methods We developed two novel BsAbs PD-1/c-Met DVD-Ig and IgG-scFv both targeting PD-1 to restore the immune effector function of T cells and engaging them to tumor cells via binding to cellular-mesenchymal to epithelial transition factor (c-Met). Binding activities, T cell activation and proliferation were analyzed by flow cytometry. Cell Cytotoxicity and cytokine release were measured using LDH release assay and ELISA, respectively. Anti-tumor response in vivo was evaluated by generate xenograft models in NOD-SCID mice. Results These bispecific antibodies exhibited effective antitumor activity against high- and low- c-Met-expressing gastric cancer cell lines in vitro and mediated strong tumor growth inhibition in human gastric cancer xenograft models. Conclusion The engagement of the PD-1/PD-L1 blockade to c-Met-overexpressing cancer cells is a promising strategy for the treatment of gastric cancer and potentially other malignancies.

Myocarditis in a patient treated with Nivolumab and PROSTVAC: a case report

BackgroundImmune checkpoint inhibitors have revolutionized treatment and improved survival in many cancers. However, since immune-related adverse events (irAEs) are potentially fatal, early recognition and prompt treatment are warranted. One of the rarest but most dramatic irAE is myocarditis, which has significant morbidity and mortality if not recognized and treated early.ObjectiveTo report the first case of myocarditis in a patient with metastatic castration-resistant prostate cancer (mCRPC) treated with a combination of nivolumab, an anti-programmed cell death protein 1 antibody, and PROSTVAC, a vector-based therapeutic prostate cancer vaccine.Case ReportA 79-year-old man with mCRPC metastatic to bone and lymph nodes and a history of atrial fibrillation presented with blurred vision and pain and stiffness in the upper back after 8 weeks on a clinical trial with nivolumab (1 mg/kg) and PROSTVAC, both given every 2 weeks. Eye exam was within normal limits, while musculoskeletal exam revealed tenderness in trapezius muscles and decreased motor strength in arms (III/V) and neck (IV/V). The rest of the physical exam was within normal limits, with the exception of an irregular heart rhythm. Laboratory tests were as follows: creatinine kinase (CK) 3200 U/L (normal: 39–308 U/L), CK-MB 65.7 mcg/L (normal: 0–7.6 mcg/L), troponin I 0.209 ng/mL (normal: 0–0.056 ng/mL). Electrocardiogram (ECG) revealed atrial fibrillation with QT prolongation (QTc 514 msec) and left anterior fascicular block, unchanged from baseline. 2D-echocardiogram showed a left ventricular ejection fraction of 65% with an enlarged left atrium, dilated right ventricle, and increased pulmonary artery pressure (45 mmHg). ProBNP was elevated at 1463 pg/mL and peaked at 3066 pg/mL one day after hydration. With a presumed diagnosis of autoimmune myositis and possible myocarditis, the patient was admitted and started on methylprednisolone 1 mg/kg/day. Cardiac MRI showed elevated native myocardial T1 values consistent with myocarditis (Fig. 1). The patient was discharged on a prednisone taper after normalization of cardiac enzymes on day 4. Treatment with PROSTVAC continued for three more months; nivolumab was discontinued. Six months later, patient is doing well, with no residual cardiac damage.DiscussionCardiovascular irAEs are relatively rare (< 1%) and have a variety of clinical presentations. Myocarditis is potentially life-threatening and can range from subclinical to fulminant. Therefore, clinical suspicion, early detection, and prompt treatment are imperative (1). The initial diagnostic workup should include cardiac enzymes, ECG, and 2D-echocardiogram. The most commonly observed ECG changes are generalized repolarization abnormalities, prolonged QT interval, and conduction abnormalities (2). An elevated troponin I in the absence of overt coronary artery disease is suggestive of myocarditis and should be evaluated further. Myocardial biopsy is the standard diagnostic procedure; however, a cardiac MRI can achieve a diagnosis when biopsy is not feasible (3). Advancements in parametric mapping techniques have allowed the use of native myocardial T1 in the detection of myocarditis, as it has superior diagnostic performance and higher sensitivity than older parameters (3). Our patient had been treated with an immune checkpoint inhibitor and a therapeutic cancer vaccine to induce effective antitumor activity through immunogenic intensification and presented with muscle stiffness and elevated CK. Although he had no new cardiovascular symptoms, cardiac enzymes were tested to rule out myocardial involvement. MRI with gadolinium confirmed the diagnosis of myocarditis. To date, none of the 1360 patients treated with PROSTVAC as a single agent have developed myocarditis, while myocarditis has been rarely reported in patients treated with nivolumab (< 1%) (1). Whether the combination of PROSTVAC and nivolumab presents an additional risk of myocarditis is unclear. To our knowledge, this is the first case of myocarditis in a patient with mCRPC receiving simultaneous treatment with an immune checkpoint inhibitor and a prostate cancer vaccine.Our experience highlights the importance of suspicion and early intervention in patients who present with cardiac abnormalities after receiving cancer immunotherapy. We propose following protocol: baseline troponin, ECG, and 2D-echocardiogram prior to treatment, then repeated troponin at 2, 4, and 12 weeks post-treatment, then monthly. If troponin becomes positive without alternative explanation, myocarditis should be ruled out with cardiac MRI or myocardial biopsy, and patient should be admitted for treatment with high-dose steroids as early intervention may minimize myocardial injury.

Selectively Targeting Checkpoint Kinase By Prexasertib Had Potent Anti-Tumor Activity As Single Agent or Combined with Other Chemotherapeutic Drugs in Diffuse Large B-Cell Lymphoma, Especially Double Hit Lymphoma

Background: Relapsed/Refractory diffuse large B-cell lymphoma (DLBCL) patients had a poor prognosis, especially those relapsing within 12 months of completion therapy. Clinically, there is a need to identify and target pathways associated with acquired treatment resistance. Using rituximab-chemotherapy resistant DLBCL cell lines generated in our laboratory, we demonstrated the acquirement of rituximab resistance is associated with changes in the cell cycle distribution (S phase arrest) and expression of cell cycle regulatory proteins (cyclin B, CDC2, CDK7, Wee1, checkpoint kinase 1 [CHK1] and checkpoint kinase 2 [CHK2]). CHK1 and 2 are serine/threonine kinases that control: 1) G2/M phase transition in response of DNA damage, 2) stabilization of DNA replication fork and 3) coordinate the mitosis. Inhibition of CHKs is an attractive strategy in cancer medicine. CHK1 inhibitors are been studied in phase I clinical trials in solid tumor malignancies (i.e. breast, pancreas or lung cancer). We hypothesized that CHK inhibition by Prexasertib has effective anti-tumor activity in DLBCL pre-clinical models. Methods: We used a panel of DLBCL cell lines including rituximab sensitive (Raji, RL, DHL4, DHL6, TMD8, RIVA, and U2932), rituximab resistant (Raji 4RH and RL 4RH), and double hit DLBCL (DOH2, ROS50 and VAL). In addition, experiments were conducted in primary tumor cells isolated from lymphoma patients. Cells were exposed to prexasertib alone or in combination with chemotherapy agents. Cell viability were determined by Presto Blue assay. IC50 was calculated by Graphpad software and coefficient of synergy was calculated using the CalcuSyn software. Subsequently, cells were expose to prexasertib and changes in cell cycle distribution and apoptosis were determined by propidium iodide staining (Pi), annexing V/PI staining and by Caspase3/7 activity detection assay. Changes in mitochondrial potential were determined by DiOC6 staining. Induction of DNA double strain break was investigated by western blot probed with phosphory-H2A.X antibody. At molecular basis, downstream pathway activities ( Mcl-1, phosphor-GSK3β, phosphory-H3, and wee1) were determined by western blot. Results: Prexasertib exhibited a dose dependent anti-lymphoma activity in all all cell lines tested including DHL cell lines. The IC50 of the cells ranged from 11nM to 2.8uM at 72 hours. Additive/synergistic effects were observed by combining prexasertib and doxorubicin, etoposide, vincristine, gemcitabine and proteasome inhibitors. At the dosage of 10nM and 25nM, prexasertib induced cell cycle arrest at G2/M phase. It also induced low membrane potential, caspse3/7 activation and apoptosis in all DLBCL cell lines. Interestingly, western blot also showed CHK inhibitor at 10nM was able to reduce Mcl-1 and p-GSK 3beta. Conclusion: Our data suggests that targeting checkpoint kinase is a promising therapeutic strategy in the lymphoma, including in double hit lymphoma pre-clinical models. In vivo studies are planned. Our data supports the clinical development of prexasertib in DLBCL. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) DisclosuresNo relevant conflicts of interest to declare.

Two-Year Follow-up of Keynote-087 Study: Pembrolizumab Monotherapy in Relapsed/Refractory Classic Hodgkin Lymphoma

Two-Year Follow-up of Keynote-087 Study: Pembrolizumab Monotherapy in Relapsed/Refractory Classic Hodgkin Lymphoma

Modeling Sézary Syndrome for Immunophenotyping and Anti-Tumor Effect of Ucart and Long-Acting Interleukin-7 Combination Therapy

Background: Sézary syndrome (SS) is a highly-morbid T cell leukemic lymphoma with no widely-effective treatments and few preclinical models. We demonstrated effective T cell lymphoma therapy with allogeneic gene-edited anti-CD7 CARTs (Cooper et al, Leukemia, 2018). However, SS T cells typically lose CD7 but maintain ubiquitous high CD2 expression. Thus, we generated CD2- and TRAC-deleted anti-CD2 universal CARTs (UCART2) and multiple SS xenograft models (PDXs) as preclinical UCART2 testing platforms. We further tested a stable homodimeric interleukin-7 molecule, the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7), to potentiate UCART2 killing of an SS xenograft in vivo.Methods: To generate SS PDX models, we injected NOD scid IL2Rgammanull (NSG) mice expressing SCF, GM-CSF, and IL-3 (NSG-SGM3) with ~2x106 mononuclear cells derived from SS patients. We immunophenotyped SS patient blood and PDX engraftment with two 21-color flow cytometry panels assessing major immune subsets, CTCL, and exhaustion markers (Staser et al, Cytometry A, 2018). To generate UCART2s, we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9, a TRAC-targeted gRNA, and a CD2-targeted gRNA followed by viral transduction with an anti-CD2 scFv 3rd generation CAR. For initial UCART2 testing, we injected NSG mice with 5x105 cells from a human Sézary cell line transduced with click beetle red luciferase (HHCBR-GFP) four days prior to UCART2 treatment. Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART2 infusion.Results: SS patient blood showed specific defects in monocyte, monocytic dendritic cell, and natural killer cell differentiation, increased skewing toward granulocytes and non-classical CD16+ monocytes (p<0.01, SS vs. normal PBMCs), and loss of effector memory CD4 cells (8% vs 34%, p<0.001, SS vs. normal PBMCs). SS cells were CD3+CD4+CD2+CD5+CD8- with variable CD7 loss and PD1 gain. Four of six unique human SS samples injected in NSG-SGM3 mice engrafted within ~6 weeks with no signs of xenogeneic GVHD. Following engraftment, SS cells showed near ubiquitous PD1 expression (>90% vs ~20%, p<0.001, SS vs. normal PBMCs), CD7 loss, and increased CD30 and CD26 expression. Immunohistochemistry further revealed atypical CD3+CD4+CD8-CD7- lymphocytes lining the dermo-epidermal junction. Second generation PDXs showed infiltration of the spleens, blood, and bone marrow with CD2+CD7- human cells and developed alopecia, scaling, and subcutaneous and intraperitoneal masses, with immunophenotyping, sequencing, and UCART treatment studies ongoing.To test UCART2's efficacy in killing SS cells in vivo, we injected NSG mice with HHCBR-GFP+ cells. UCART2-treated HHCBR-GFP mice showed dramatically reduced tumor burden as compared to control UCART19-treated HHCBR-GFP mice (BLI; 10^7 vs. 10^11 photon flux/s at 3 weeks, p<0.0001, UCART2 vs. UCART19). Moreover, UCART2-treated HHCBR-GFP mice survived ~40 days as compared to ~21 days in the UCART19 group. Remarkably, UCART2-treated HHCBR-GFP mice receiving NT-I7 showed virtually no tumor burden (maximum 106 photon flux/s vs. 1010 photon flux/s, UCART2+NT-I7 vs. UCART2 only groups) with 100% of UCART2- and NT-I7-treated HHCBR-GFP mice surviving beyond 49 days (Figure 1).Discussion: We describe the generation of physiologically-relevant SS preclinical models, comprehensive immunophenotyping of patient SS samples, clonal SS PDX outgrowth, and the highly effective anti-tumor activity of UCART2- plus NT-I7-mediated killing of SS cells in vivo using an NSG xenograft model. Ongoing studies involve treating primary SS and CTCL PDX models with UCART2 and NT-I7. These preclinical data validate the use of allogeneic “off-the-shelf” adoptive immunotherapy for the treatment of Sézary syndrome, while demonstrating the dramatic enhancement of CART efficacy using a dose-adjustable, clinic-ready long-acting interleukin-7 agonist given in an adjuvant setting. [Display omitted] DisclosuresPark:NeoImmuneTech: Employment. Lee:NeoImmuneTech: Employment. Musiek:Seattle Genetics: Honoraria; Actelion: Other: Scientific Advisory Committee ; Kyowa Kirin: Honoraria.