Antitumor Cytotoxic Activity Research Articles

Immunogenomics of Colorectal Cancer Response to Checkpoint Blockade: Analysis of the KEYNOTE 177 Trial and Validation Cohorts

Background & AimsColorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy.MethodsWe selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME.ResultsIn hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages.ConclusionsOur study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

Bifunctional TGF-β trap/IL-15 protein complex elicits potent NK cell and CD8+ T cell immunity against solid tumors

Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ Tcells invitro and invivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-β neutralizing activity invitro and sequesters plasma TGF-β invivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ Tcells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.

Natural Killer Cell Interactions With Myeloid Derived Suppressor Cells in the Tumor Microenvironment and Implications for Cancer Immunotherapy.

The tumor microenvironment (TME) is a complex and heterogeneous environment composed of cancer cells, tumor stroma, a mixture of tissue-resident and infiltrating immune cells, secreted factors, and extracellular matrix proteins. Natural killer (NK) cells play a vital role in fighting tumors, but chronic stimulation and immunosuppression in the TME lead to NK cell exhaustion and limited antitumor functions. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive activity that gradually accumulate in tumor tissues. MDSCs interact with innate and adaptive immune cells and play a crucial role in negatively regulating the immune response to tumors. This review discusses MDSC-mediated NK cell regulation within the TME, focusing on critical cellular and molecular interactions. We review current strategies that target MDSC-mediated immunosuppression to enhance NK cell cytotoxic antitumor activity. We also speculate on how NK cell-based antitumor immunotherapy could be improved.

Green Synthesis and Biomedical Applications of ZnO Nanoparticles: Role of PEGylated-ZnO Nanoparticles as Doxorubicin Drug Carrier against MDA-MB-231(TNBC) Cells Line

The present study aimed to develop the synthesis of zinc oxide nanoparticles (ZnO-NPs) using the green method, with Aloe barbadensis leaf extract as a stabilizing and capping agent. In vitro antitumor cytotoxic activity, as well as the surface-functionalization of ZnO-NPs and their drug loading capacity against doxorubicin (DOX) and gemcitabine (GEM) drugs, were also studied. Morphological and structural properties of the produced ZnO-NPs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersion X-ray diffraction (EDX), UV-Vis spectrophotometry, Fourier-transform infrared analysis (FTIR), and X-ray diffraction (XRD). The prepared ZnO-NPs had a hexagonal shape and average particle size of 20–40 nm, with an absorption peak at 325 nm. The weight and atomic percentages of zinc (50.58% and 28.13%) and oxygen (26.71% and 60.71%) were also determined by EDAX (energy dispersive x-ray analysis) compositional analysis. The appearance of the FTIR peak at 3420 m–1 confirmed the synthesis of ZnO-NPs. The drug loading efficiency (LE) and loading capacity (LC) of unstabilized and PEGylated ZnO-NPs were determined by doxorubicin (DOX) and gemcitabine (GEM) drugs. DOX had superior LE 65% (650 mg/g) and higher LC 32% (320 mg/g) than GEM LE 30.5% (30 mg/g) and LC 16.25% (162 mg/g) on ZnO-NPs. Similar observation was observed in the case of PEG-ZnO-NPs, where DOX had enhanced LE 68% (680 mg/g) and LC 35% (350) mg/g in contrast to GEM, which had LE and LC values of 35% (350 mg/g) and 19% (190 mg/g), respectively. Therefore, DOX was chosen to encapsulate nanoparticles, along with the untreated nanoparticles, to check their in vitro antiproliferative potential against the triple-negative breast cancer (TNBC) cell line (MDA-MB-231) through the MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) assay. This drug delivery strategy implies that the PEGylated biogenically synthesized ZnO-NPs occupy an important position in chemotherapeutic drug loading efficiency and can improve the therapeutic techniques of triple breast cancer.

Targeting Antibody Checkpoint Fcgriib Using Monoclonal Antibody BI-1206 to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a rare but aggressive B-cell non-Hodgkin's lymphoma that represents 6% of all lymphomas in the United States. Recent therapies including anti-CD20 antibody rituximab, BTK inhibitors, and BCL-2 inhibitors alone or in combination have shown great anti-MCL efficacy. However, primary and acquired resistance to one or multiple therapies commonly occurs, resulting in poor clinical outcome. Therefore, resistance to such therapies is currently an unmet clinical challenge in MCL patients. Therapeutic strategies to overcome this resistance holds promise to significantly improve survival of refractory/relapsed MCL patients. Recent studies showed Fc gamma receptors (FcγRs) play important roles in enhancing the efficacy of antibody-based immunotherapy. In particular, FcgRIIB (CD32B), an inhibitory member of the FcγR family, is implicated in the immune cell desensitization and tumor cell resistance through the internalization of therapeutic antibodies such as rituximab. Based on our flow cytometry analysis, we demonstrated that FcgRIIB is highly expressed on the cell surface of MCL cell lines (n=10) and primary MCL patient samples (n=22). This indicates that FcgRIIB may play an important role in MCL malignancy and identifies FcgRIIB is a potential therapeutic target for the treatment of MCL. To address this, we tested the in vivo efficacy of BI-1206, a fully humanized monoclonal antibody targeting FcgRIIB, alone, or in combination with clinically approved or investigational drugs including rituximab, ibrutinib and venetoclax. In the first in vivo cohort, BI-1206, as a single agent, dramatically inhibited the tumor growth of ibrutinib-venetoclax dual-resistant PDX tumor models, suggesting that targeting FcgRIIB by BI-1206 alone has high anti-MCL activity in vivo. Next, we assessed whether BI-1206 can boost anti-MCL activity of antibody-based therapy such as rituximab in combination with ibrutinib or venetoclax using additional mice cohorts of cell line-derived xenograft and patient-derived xenograft models. BI-1206 significantly enhanced the in vivo efficacy of ibrutinib plus rituximab, and venetoclax plus rituximab, on tumor growth inhibition, including the JeKo-1 derived xenograft models, previously proven to be partially resistant to ibrutinib and venetoclax in vivo. This tumor-sensitizaton effect was further confirmed in the ibrutinib and venetoclax dual-resistant PDX models of MCL where BI-1206 was combined with venetoclax and rituximab. More detailed mechanistic studies are currently ongoing to reveal the mechanism of action of BI-1206-based combinations or as single therapy with the possibility that BI-1206 itself may have a cytotoxic anti-tumor direct activity in MCL. In conclusion, BI-1206 as single agent showed potent efficacy in overcoming ibrutnib-venetoclax dual resistance. Moveover, BI-1206 enhanced the in vivo efficacy of ibrutinib plus rituximab and venetoclax plus rituximab and overcomes resistance to these treatments resulting in enhanced anti-tumor effects. Disclosures Karlsson: BioInvent International AB: Current Employment. Mårtensson:BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Kovacek:BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Teige:BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Frendéus:BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Wang:Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; Dava Oncology: Honoraria; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Acerta Pharma: Research Funding; VelosBio: Research Funding; MoreHealth: Consultancy; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Lu Daopei Medical Group: Honoraria.

A RARE TRIGGER OF ASTHMA EXACERBATION: THE SCOTCH MARIGOLD

SESSION TITLE: Medical Student/Resident Allergy and Airway Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Calendula officinalis, the common marigold, is a plant which has been ascribed a range of applications in traditional medicine, in both topical and oral forms, for the treatment of pain, superficial infections, and as a wound salve. We present a case of severe respiratory failure as a consequence of an asthma exacerbation in response to exposure to this agent. CASE PRESENTATION: A 26-year-old female with a history of mild intermittent asthma presented to the emergency department (ED) with acute respiratory distress. The patient reported mild productive cough over the preceding week. Earlier in the day, the patient had consumed the extract of Calendula officinalis, an herbal supplement, for the first time in an effort to alleviate chest soreness. About 30 minutes following ingestion, she developed progressive dyspnea and confusion. At presentation, the patient exhibited disorientation with visual hallucinations. She was hypoxic, with associated tachycardia (147 beats per minute), tachypnea (26 breaths per minute), and hypertension (193/100 mm Hg). She had diffuse expiratory wheezing. Arterial blood gas (ABG) on supplemental oxygen via nasal cannula demonstrated severe respiratory acidosis (pH: 6.9, pCO2: 123, pO2: 136), and she had leukocytosis (27.8 /µl). Chest radiography was unremarkable. She was intubated, and mechanical ventilation was initiated. Epinephrine, methylprednisolone, diphenhydramine, and albuterol were administered, given suspicion for an allergic etiology. A diagnosis of status asthmaticus was made, and she was started on methylprednisolone, inhaled bronchodilators, and empiric antibiotics. Her clinical course was complicated by laryngeal edema which made extubation challenging, but she improved and was successfully extubated. A discussion regarding the risks of unpredictable adverse effects of herbal supplements followed, and the patient was discharged with instructions to avoid similar supplements in the future. DISCUSSION: Calendula is a genus of plants which have been proposed to possess anti-inflammatory, anti-fungal, antipyretic, antispasmodic, anticancer, and other pharmacologic properties, prompting its use as oral tinctures, creams, and lotions in traditional medicine. C. officinalis has been demonstrated to have immunomodulatory properties, such as lymphocyte proliferation and granulocyte stimulation. Considerable evidence has illustrated the role of lymphocytes in modulating airway inflammation, and therefore may have played a role in the causation of the asthma exacerbation in this patient. There is scant documentation of any significant adverse reactions to this plant; to our knowledge, this is the first documented report of an asthma exacerbation triggered by Calendula. CONCLUSIONS: Healthcare providers should advise patients to exercise caution when using Calendula or other herbal remedies with unclear adverse reaction profiles. Reference #1: Arora D, Rani A, Sharma A. A review on phytochemistry and ethnopharmacological aspects of genus Calendula. Pharmacogn Rev. 2013;7(14):179–187. doi:10.4103/0973-7847.120520 Reference #2: Jiménez-Medina E, Garcia-Lora A, Paco L, Algarra I, Collado A, Garrido F. A new extract of the plant Calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation. BMC Cancer. 2006;6:119. Published 2006 May 5. doi:10.1186/1471-2407-6-119 Reference #3: Cruceriu D, Balacescu O, Rakosy E. Calendula officinalis: Potential Roles in Cancer Treatment and Palliative Care. Integr Cancer Ther. 2018;17(4):1068–1078. doi:10.1177/1534735418803766 DISCLOSURES: No relevant relationships by Dan Kazmierski, source=Web Response No relevant relationships by Pius Ochieng, source=Web Response No relevant relationships by NISHANT SHARMA, source=Web Response No relevant relationships by Yichen Wang, source=Web Response

Exosomes derived from Vδ2-T cells control Epstein-Barr virus-associated tumors and induce T cell antitumor immunity.

Treatment of life-threatening Epstein-Barr virus (EBV)-associated tumors remains a great challenge, especially for patients with relapsed or refractory disease. Here, we found that exosomes derived from phosphoantigen-expanded Vδ2-T cells (Vδ2-T-Exos) contained death-inducing ligands (FasL and TRAIL), an activating receptor for natural killer (NK) cells (NKG2D), immunostimulatory ligands (CD80 and CD86), and antigen-presenting molecules (MHC class I and II). Vδ2-T-Exos targeted and efficiently killed EBV-associated tumor cells through FasL and TRAIL pathways and promoted EBV antigen-specific CD4 and CD8 T cell expansion. Administration of Vδ2-T-Exos effectively controlled EBV-associated tumors in Rag2-/-γc-/- and humanized mice. Because expanding Vδ2-T cells and preparing autologous Vδ2-T-Exos from cancer patients ex vivo in large scale is challenging, we explored the antitumor activity of allogeneic Vδ2-T-Exos in humanized mouse cancer models. Here, we found that allogeneic Vδ2-T-Exos had more effective antitumor activity than autologous Vδ2-T-Exos in humanized mice; the allogeneic Vδ2-T-Exos increased the infiltration of T cells into tumor tissues and induced more robust CD4 and CD8 T cell-mediated antitumor immunity. Compared with exosomes derived from NK cells (NK-Exos) with direct cytotoxic antitumor activity or dendritic cells (DC-Exos) that induced T cell antitumor responses, Vδ2-T-Exos directly killed tumor cells and induced T cell-mediated antitumor response, thus resulting in more effective control of EBV-associated tumors. This study provided proof of concept for the strategy of using Vδ2-T-Exos, especially allogeneic Vδ2-T-Exos, to treat EBV-associated tumors.

Overview of Global Trends in Classification, Methods of Preparation and Application of Bacteriocins.

This paper summarizes information about the division of bacteriocins into classes (Gram-negative bacteria, Gram-positive bacteria, and archaea). Methods for producing bacteriocins have been studied. It is known that bacteriocins, most successfully used today are products of secondary metabolism of lactic acid bacteria. It is established that the main method of bacteriocin research is PCR analysis, which makes it possible to quickly and easily identify the presence of bacteriocin encoding genes. The mechanism of cytotoxic action of bacteriocins has been studied. It is proved that the study of cytotoxic (antitumor) activity in laboratory conditions will lead to the clinical use of bacteriocins for cancer treatment in the near future. It is established that the incorporation of bacteriocins into nanoparticles and targeted delivery to areas of infection may soon become an effective treatment method. The delivery of bacteriocins in a concentrated form, such as encapsulated in nanoparticles, will increase their effectiveness and minimize potential toxic side effects. The analysis of publications on this topic confirmed that diverse research on bacteriocins is relevant.

Abstract 542: T-cell engager bispecific formats of an AML patient-derived antibody targeting a unique sialylated CD43 epitope induce kill of melanoma cells in vitro and in vivo

Abstract Introduction CD43s, a unique sialylated form of CD43 expressed by myeloid malignancies, is a novel target and AT1413, an antibody targeting CD43s was shown to have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was isolated from a high-risk AML patient who successfully cleared the leukemia after allogeneic stem cell transplantation. Because CD43 is also expressed in non-hematopoietic cells we studied whether CD43s is also present and can also be targeted on non-hematopoietic tumors. Materials and methods AT1413 binding on a panel of tumor cell lines was analyzed by flow cytometry. AT1413 was constructed into a bispecific T-cell engaging format (AT1413 bTCE) by linking the full-length AT1413 IgG to two single chain variable fragments against CD3ϵ with a combination of site-specific enzymatic and chemical coupling. A monovalent T-cell engager was produced as heterotrimer consisting of one AT1413 monovalent chain and one anti-CD3 scFV fused to AT1413 monovalent chain. Point mutations in the IgG heavy chain were introduced to prevent interactions between AT1413 T-cell engagers and Fc-receptors. The cytotoxicity-inducing activities were established using PBMCs as effector and tumor cells as target cells using standard cytotoxic assays in vitro and in vivo in a mouse model carrying human immune cells. Results We observed that AT1413 binds to non-hematopoietic tumor cells, such as melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody dependent cellular cytotoxicity against melanoma cell lines and primary melanoma samples. However, AT1413 was unable to affect growth of melanoma cells in vivo. To increase the efficacy of AT1413 it was formatted as a bispecific T-cell engaging antibody (TCE): one binding bivalently (bTCE) to CD43s and monovalently to CD3ϵ and the other monovalently (KiH) to both CD43s and CD3ϵ. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with different potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth. Conclusion AT1413 recognizes a sialylated epitope on CD43 shared by melanoma, AML and MDS cells. Two different bispecific TCE forms of AT1413 induce strong anti-tumor cytotoxic activities in vitro and in vivo. These data indicate a broad therapeutic potential of AT1413. Citation Format: Greta de Jong, Lina Bartels, Martijn Kedde, Els Verdegaal, Marijn A. Gillissen, Sophie E. Levie, Madalina G. Cercel, Susan E. van Hal-van Veen, Christien Fatmawati, Dorien van de Berg, Etsuko Yasuda, Yvonne Claassen, Arjen Q. Bakker, Remko Schotte, Julien Villaudy, Koen Wagner, Hergen Spits, Mette D. Hazenberg, Pauline M. van Helden. T-cell engager bispecific formats of an AML patient-derived antibody targeting a unique sialylated CD43 epitope induce kill of melanoma cells in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 542.

Abstract 4235: A combination of CAR-NK and CAR-T cells results in rapid and persistent anti-tumor efficacy while reducing CAR-T cell mediated cytokine release and T-cell proliferation

Abstract CAR-NK cells show rapid and potent anti-tumor cytotoxic activity even at very low effector-to-target ratios, however, they do not typically expand upon encounter with antigen and lack long term persistence commonly associated with CAR-T cells. Conversely, CAR-T cells generally exhibit persistent killing activity, even though they are activated somewhat more slowly. In addition, the explosive expansion of CAR-T cells is frequently associated with a variety of toxicities (e.g. CRS, CRES, etc.). To address these shortcomings, Nkarta has developed a novel platform to combine both CAR-NK and CAR-T cells as a novel cancer therapeutic. To evaluate the overall outcomes of combining CAR-NK and CAR-T cells as an anti-tumor therapy, we have developed two novel in vitro assays: (a) IncuCyte-based matrixed cytotoxicity assay, which allows direct assessment of tumor killing kinetically with different E:T and NK:T ratios; (b) IncuCyte-based tumor re-challenge assay, which provides a convenient and quick assessment of anti-tumor persistence. For proof-of-concept studies, a CD19 directed CAR (CAR19) was retrovirally engineered into NK and T cells. Our newly developed assays revealed that the combination of CAR-NK and CAR-T cells clearly outperformed CAR19-NK or CAR19-T cells alone in anti-tumor efficacy and persistence. Additionally, the combination also showed an improved cytokine profile. To further investigate the cellular mechanism(s) underlying the beneficial effects of combining CAR-NK and CAR-T cells, we performed FACS-based phenotyping and a Violet Cell Trace based proliferation assay. When challenged with tumor cells, the presence of CAR19-NK cells reduced the proliferation of CAR19-T cells. Surprisingly, the presence of CAR19-T cells could trigger the proliferation of CAR19-NK cells. Finally, we evaluated the NK plus T platform in vivo with a Nalm6 xenograft model. Treatment with CAR19-NK cells combined with a low dose of CAR19-T cells (5e5 cells/animal) delayed tumor progression. Combined treatment with a medium dose of CAR19-T cells (2.5e6 cells/animal) largely prevented tumor relapse, and consequently enhanced the overall survival of the animals. In summary, our in vitro and in vivo studies strongly demonstrate that the NK plus T platform can harness the advantages of both CAR-NK and CAR-T cells, allowing for both rapid and persistent killing while potentially minimizing the toxicities associated with CAR-T cells. Citation Format: Guangnan Li, Xiumin Wu, Ivan H. Chan, James B. Trager. A combination of CAR-NK and CAR-T cells results in rapid and persistent anti-tumor efficacy while reducing CAR-T cell mediated cytokine release and T-cell proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4235.

Dyeing Performance of Disperse Dyes on Polyester Fabrics Using Eco-Friendly Carrier and Their Antioxidant and Anticancer Activities.

Both non eco- and eco-friendly carriers were utilized for accelerating the dyeing rate of polyethylene terephthalate fabrics (PET) dyed with disperse dyes at 100 °C. Fastness properties of the dyed fabrics showed good and excellent results. Finally, the prepared disperse dyes 1 and 2 showed potent anti-tumor cytotoxic activity in vitro using MCF-7 cells (human breast cancer cell line), HepG-2 cells (human Hepatocellular carcinoma), HCT-116 (colon carcinoma), A-549 cells (Lung carcinoma cell line), and anti-oxidant activities.

The biosynthesis of camptothecin derivatives by Camptotheca acuminata seedlings

10-hydroxycamptothecin and 9-methoxycamptothecin, naturally occurring camptothecin derivatives, are reportedly present in Camptotheca acuminata with a powerful cytotoxic effect and strong antitumor activity. In this paper, we studied the derivatization reaction of camptothecin catalyzed by C. acuminata seedlings. HPLC traced the reaction between exogenous camptothecin and C. acuminata seedlings. The results showed that the exogenous camptothecin was converted into 10-hydroxycamptothecin and 9-methoxycamptothecin by the tender roots and stems of C. acuminata seedlings, which would be a new method for the synthesis of two camptothecin derivatives.

Immunomodulatory Activity of Tyrosine Kinase Inhibitors to Elicit Cytotoxicity Against Cancer and Viral Infection.

Tyrosine kinase inhibitors (TKIs) of aberrant tyrosine kinase (TK) activity have been widely used to treat chronic myeloid leukemia (CML) for decades in clinic. An area of growing interest is the reported ability of TKIs to induce immunomodulatory effects with anti-tumor and anti-viral activity, which appears to be mediated by directly or indirectly acting on immune cells. In selected cases of patients with CML, TKI treatment may be interrupted and a non-drug remission may be observed. In these patients, an immune mechanism of increased anti-tumor cytotoxic activity induced by chronic administration of TKIs has been suggested. TKIs increase some populations of natural killer (NK), NK-LGL, and T-LGLs cells especially in dasatinib treated CML patients infected with cytomegalovirus (CMV). In addition, dasatinib increases responses against CMV and is able to inhibit HIV replication in vitro. Recent studies suggest that subclinical reactivation of CMV could drive expansion of specific subsets of NK- and T-cells with both anti-tumoral and anti-viral function. Therefore, the underlying mechanisms implicated in the expansion of this increased anti-tumor and anti-viral cytotoxic activity induced by TKIs could be a new therapeutic approach to take into account against cancer and viral infections such as HIV-1 infection. The present review will briefly summarize the immunomodulatory effects of TKIs on T cells, NKs, and B cells. Therapeutic implications for modulating immunity against cancer and viral infections and critical open questions are also discussed.

Recent Achievements in Medicinal and Supramolecular Chemistry of Betulinic Acid and Its Derivatives ‡.

The subject of this review article refers to the recent achievements in the investigation of pharmacological activity and supramolecular characteristics of betulinic acid and its diverse derivatives, with special focus on their cytotoxic effect, antitumor activity, and antiviral effect, and mostly covers a period 2015–2018. Literature sources published earlier are referred to in required coherences or from historical points of view. Relationships between pharmacological activity and supramolecular characteristics are included if such investigation has been done in the original literature sources. A wide practical applicability of betulinic acid and its derivatives demonstrated in the literature sources is also included in this review article. Several literature sources also focused on in silico calculation of physicochemical and ADME parameters of the developed compounds, and on a comparison between the experimental and calculated data.

Alginate Enhances Memory Properties of Antitumor CD8+ T Cells by Promoting Cellular Antioxidation.

Alginate, an FDA-approved natural biomaterial usually used as a therapeutic adjuvant, drug carrier, and biological scaffold, reportedly assists the immune system to activate cytotoxic T cells in antitumor assays. In this study, we investigated the direct effect of alginate on cytotoxic T cell function. By incubating sorted cytotoxic CD8+ T cells with alginate, we found that this material facilitated the antitumor cytotoxic activities of T cells. Alginate incubation significantly promoted memory properties of CD8+ T cells and elevated the proportion of CD62L+CD44+ central memory T cell (TCM), a less differentiated T cell subset with high immune activity. Mechanistically, alginate reduced reactive oxide species in CD8+ T cells by increasing intracellular glutathione generation, which was critical for conferring T cells with memory properties. Further, we found that guluronic acid, a constituent component (G unit) of alginate, is responsible for inducing glutathione and promoting TCM. Collectively, we reported new biological activities of alginate on scavenging reactive oxide species and regulating the function of cytotoxic T cells, which suggests that alginate and guluronic acid may be used for improving cytotoxic T cell functions in immunotherapy.

Abstract CT167: A Phase Ib study to assess safety, tolerability, pharmacokinetics, and preliminary antitumor activity of sitravatinib in combination with tislelizumab in patients with advanced solid tumors

Abstract Background: Sitravatinib is an investigative, orally bioavailable, spectrum-selective receptor tyrosine kinase (RTK) inhibitor with potential antitumor activity that has been shown to potently inhibit split kinase receptors (eg, VEGFR2, KIT) and TAM receptors (eg, AXL, MER). Inhibition of RTKs by sitravatinib may also modulate effects on the tumor microenvironment to overcome resistance to checkpoint inhibitors, including enhanced M1/suppressed M2 macrophage cytokine response, abrogated negative regulation of antitumor NK cell activity, and depleted regulatory T- and myeloid-derived suppressor cells with enhanced antitumor cytotoxic T-cell activity. Tislelizumab is an investigational, humanized IgG4 monoclonal antibody that has been shown to have high affinity and binding specificity for programmed cell death receptor-1 (PD-1). Combining an agent that has both immune modulatory and antitumor properties with an immunotherapeutic PD-1 checkpoint inhibitor could enhance the antitumor efficacy observed with either agent alone. The primary objective of this trial is to examine the safety and tolerability of sitravatinib combined with tislelizumab in patients with advanced solid tumors. Methods: Adult patients with histologically or cytologically confirmed, locally advanced or metastatic, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), or ovarian cancer (OC) are enrolling in the open-label, multicenter, nonrandomized, phase 1b clinical trial. Entry criteria include Eastern Cooperative Oncology Group Performance Status ≤ 1 and adequate end-organ function. All patients will receive sitravatinib 120 mg PO QD in combination with tislelizumab 200 mg IV Q3W until progressive disease, unacceptable toxicity, death, withdrawal of consent, or study termination by the sponsor. Approximately 100 patients are expected to be enrolled in 5 cohorts (n=20 patients per cohort): Cohort A: anti-PD-(L)1 antibody-refractory/-resistant, metastatic, nonsquamous NSCLC; Cohort B: anti-PD-1(L)1 antibody-naive, metastatic, nonsquamous NSCLC; Cohort C: anti-PD-(L)1 antibody-refractory/-resistant, metastatic, clear cell RCC; Cohort D (China only): metastatic or advanced RCC without prior therapy; Cohort E: anti-PD-(L)1 antibody-naive, recurrent, platinum-resistant, epithelial OC. The primary endpoint is to assess safety and tolerability of the combination by monitoring adverse events (AEs) and serious AEs. Secondary endpoints include overall response rate, duration of response, disease control rate, progression-free survival assessed by RECIST version 1.1, and plasma concentrations and derived pharmacokinetic parameters of single- and repeated-dose sitravatinib. Seven patients have enrolled as of 3 January 2019 at the sites in Australia and China. Clinical trial registry number: NCT03666143 Citation Format: Jeffrey Goh, Ben Gao, Ben Markman, Hui Gan, Mark Voskoboynik, Yi-Long Wu, Jun Guo, Qing Zhou, Jun Zhao, Cheng Chen, Jingjun Qiu, Yingying Xu, Liu Yang, Michael Millward. A Phase Ib study to assess safety, tolerability, pharmacokinetics, and preliminary antitumor activity of sitravatinib in combination with tislelizumab in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT167.

Abstract A156: PDA tumor cell death as following combination anti-PD-1 blockade and CXCR4 blockade is a direct effect of CD8+ T-cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer death in the United States. Despite the fact that the human PDA microenvironment contains a mixture of infiltrating immune cells, including activated effector CD8+ T-cells, attempts to apply immune checkpoint inhibitor therapy have not yet been successful in achieving a clinical response (1). Combining blockade of the immune checkpoint receptor PD-1 with blockade of the chemokine receptor CXCR4 in a mouse model of PDA produced T-cell-mediated tumor cell killing (2). We therefore hypothesized that, in a human PDA slice culture model system that maintains the intact tumor microenvironment (3), combination of CXCR4 blockade with PD-1 blockade would lead to anti-tumor T-cell activation. Methods: Cores (6 mm) were taken from freshly resected, sterile human PDA specimens and cut into 250 µm thick slices using a vibratome. The slices were treated with either IgG antibody isotype control, AMD3100 (a CXCR4 blocking small molecule drug) plus antibody isotype control, anti-PD-1 antibody, or a combination of anti-PD-1 antibody and AMD3100 for two days. The slices were then stained with fluorescently labelled antibodies for CD8 and EpCAM, as well as with SR-FLICA, a reagent that produces a fluorescent signal when bound to activated Caspase 3 and 7 enzymes. Live slices were maintained in a humidified, temperature-controlled CO2 chamber for time-lapse confocal imaging for 1 hour. Similarly, untreated slices were imaged for 90 minutes prior to and following combined anti-PD-1 and AMD3100 drug treatment. Each slice was imaged at 3 different positions to observe CD8+ T-cells, EpCAM+ tumor cells, and SR-FLICA+ apoptotic cells throughout the slice. Each cell type was counted at each position over the entire time course imaged. Results: Compared to antibody isotype control or monotherapy, slices treated with combined PD-1 and CXCR4 blockade contained a greater fraction of EpCAM+ tumor cells that were undergoing apoptosis, indicated by SR-FLICA positivity. This treatment also increased the proportion of EpCAM+ tumor cells with a CD8+ T-cell within 20 µm, and EpCAM+ cells had increased evidence of apoptosis when a CD8+ cell was nearby. When individual slices were imaged before and after combined PD-1 and CXCR4 blockade, a greater proportion of EpCAM+ cells were found to have a CD8+ cell nearby after treatment compared to before treatment, and the tumor cells with a CD8+ cell nearby were more like to be undergoing apoptosis compared to before treatment. Conclusion: Direct visualization of live pancreatic cancer slice cultures demonstrates that combined PD-1 and CXCR4 blockade enhances both the migration of CD8+ T-cells towards epithelial tumor cells and their anti-tumor cytotoxic activity.

γδ T Cells and Tumor Microenvironment: From Immunosurveillance to Tumor Evasion.

γδ T cells possess cytotoxic antitumor activity mediated by production of proinflammatory cytokines, direct cytotoxic activity, and regulation of the biological functions of other cell types. Hence, these features have prompted the development of therapeutic strategies in which γδ T cells agonists or ex vivo-expanded γδ T cells are administered to tumor patients. Several studies have shown that γδ T cells are an important component of tumor-infiltrating lymphocytes in patients affected by different types of cancer and a recent analysis of ~18,000 transcriptomes from 39 human tumors identified tumor-infiltrating γδ T cells as the most significant favorable cancer-wide prognostic signature. However, the complex and intricate interactions between tumor cells, tumor microenvironment (TME), and tumor-infiltrating immune cells results in a balance between tumor-promoting and tumor-controlling effects, and γδ T cells functions are often diverted or impaired by immunosuppressive signals originating from the TME. This review focuses on the dangerous liason between γδ T cells and tumoral microenvironment and raises the possibility that strategies capable to reduce the immunosuppressive environment and increase the cytotoxic ability of γδ T cells may be the key factor to improve their utilization in tumor immunotherapy.

Myeloid-Derived Suppressor Cells Specifically Suppress IFN-γ Production and Antitumor Cytotoxic Activity of Vδ2 T Cells.

γδ T cells represent less than 5% of circulating T cells; they exert a potent cytotoxic function against tumor or infected cells and secrete cytokines like conventional αβ T cells. As αβ T cells γδ T cells reside in the typical T cell compartments (the lymph nodes and spleen), but are more widely distributed in tissues throughout the body. For these reasons, some investigators are exploring the possibility of immunotherapies aimed to expand and activate Vδ2 T cells, or using them as Chimeric Antigen Receptor carriers. However, the role of immunosuppressive microenvironment on Vδ2 T cells during infections and cancers has not been completely elucidated. In particular, the effects of myeloid-derived suppressor cells (MDSC), largely expanded in such pathologies, were not explored. In the present work, we demonstrated that MDSC may inhibit IFN-γ production and degranulation of phosphoantigen-activated Vδ2 T cells. Moreover, the Vδ2 T cells cytotoxic activity against the Burkitt lymphoma cell line Daudi and Jurkat cell line were impaired by MDSC. The Arginase I seems to be involved in the impairment of Vδ2 T cell function induced by both tumor cells and MDSC. These data open a key issue in the context of Vδ2-targeted immunoteraphy, suggesting the need of combined strategies aimed to boost Vδ2 T cells circumventing tumor- and MDSC-induced Vδ2 T cells suppression.

Tribody [(HER2)2xCD16] Is More Effective Than Trastuzumab in Enhancing γδ T Cell and Natural Killer Cell Cytotoxicity Against HER2-Expressing Cancer Cells.

An enhanced expression of human epidermal growth factor receptor 2 (HER2, ErbB2) often occurs in an advanced stage of breast, ovarian, gastric or esophageal cancer, and pancreatic ductal adenocarcinoma (PDAC). Commonly, HER2 expression is associated with poor clinical outcome or chemoresistance in ovarian and breast cancer patients. Treatment with humanized anti-HER2 monoclonal antibodies, such as trastuzumab or pertuzumab, has improved the outcome of patients with HER2-positive metastatic gastric or breast cancer, but not all patients benefit. In this study, the bispecific antibody [(HER2)2xCD16] in the tribody format was employed to re-direct CD16-expressing γδ T lymphocytes as well as natural killer (NK) cells to the tumor-associated cell surface antigen HER2 to enhance their cytotoxic anti-tumor activity. Tribody [(HER2)2xCD16] comprises two HER2-specific single chain fragment variable fused to a fragment antigen binding directed to the CD16 (FcγRIII) antigen expressed on γδ T cells and NK cells. Our results revealed the superiority of tribody [(HER2)2xCD16] compared to trastuzumab in triggering γδ T cell and NK cell-mediated lysis of HER2-expressing tumor cells, such as PDAC, breast cancer, and autologous primary ovarian tumors. The increased efficacy of [(HER2)2xCD16] can be explained by an enhanced degranulation of immune cells. Although CD16 expression was decreased on γδ T cells in several PDAC patients and the number of tumor-infiltrating NK cells and γδ T cells was impaired in ovarian cancer patients, [(HER2)2xCD16] selectively enhanced cytotoxicity of cells from these patients. Here, unique anti-tumor properties of tribody [(HER2)2xCD16] are identified which beyond addressing HER2 overexpressing solid tumors may allow to treat with similar immunoconstructs combined with the adoptive transfer of γδ T cells and NK cells refractory hematological malignancies. A major advantage of γδ T cells and NK cells in the transplant situation of refractory hematological malignancies is given by their HLA-independent killing and a reduced graft-versus-host disease.