Tumor-killing Effect Research Articles

Intratumoural MDSC recruitment by chemotherapeutic agent, 5-FU offsets the anti-tumor activity of immune-checkpoint inhibitor in HCC

Abstract Background Current hepatocellular carcinoma (HCC) immunotherapy trials only yield modest outcome, suggesting that possibly strong intrinsic immunosuppressive network needed to be tackled. Conventional chemotherapies are found to exert immunomodulatory effect on the tumor microenvironment (TME). Our study investigated anti-tumor activity of 5-FU in combination with anti-PD-L1, using pre-clinical HCC mouse model, and evaluated the immunomodulation in TME after drug treatments. Methods For the orthotopic HCC in vivo experiment, 6-8-week-old male C57BL/6 mice were injected intrahepatically with 5x10^5 RIL-175 luc+ cells. In order to assess its immunomodulatory effect on HCC TME rather than on direct tumor-killing effect, 5-FU was administrated (i.p.) 3 times per week at a relative low dose—20mg/kg. Whilst, anti-PD-L1(10mg/kg) was delivered (i.p.) every 5 days. Tumor growth was monitored via in vivo imaging. Tumor, liver, spleen and blood was collected afterwards, followed by immune profiling analysis via flow cytometry. Results In terms of in vivo imaging results, mice with single anti-PD-L1 treatment showed significant response in tumor growth amongst other groups. Meanwhile 5-FU monotherapy and combined treatment group showed no difference with vehicle group. We found an increased amounts of T lymphocytes and NK cells in the tumor site after anti-PD-L1 single treatment, suggesting that these immune active cells contributed to the anti-tumor effect. Tumor-infiltrating myeloid cells, particularly P-MDSC, elevated upon 5-FU treatment in both single and combined group, suggesting that they may play a role in mediating immunosuppression. Conclusion This study illustrated that anti-PD-L1 monotherapy enriched tumor-infiltrating T lymphocytes and NK cells, possibly accounting for the deferred tumor growth. However, the accumulation of myeloid cells in 5-FU treated mice hinders the anti-tumor activity of anti-PD-L1 from this orthotopic HCC mouse model. Therefore, low dose of 5-FU may initiate immunosuppressive mechanism to alter the effectiveness of anti-PD-L1 in HCC. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

First-in-Human CD4 CAR Clinical Trial on Peripheral T-Cell Lymphoma

Background CD19 CAR T cell therapy has achieved success in treating acute lymphoblastic leukemia. However, the treatment for Sezary syndrome, an aggressive form of cutaneous peripheral T cell lymphoma (PTCL) has remained a challenge. Despite patients with Sezary syndrome typically receiving multiple treatments within their disease progression, the prognosis is poor with 5 year survival rate of only 24%. Therefore, it is crucial to establish a novel treatment for PTCLs. CD4 is uniformly expressed on most mature T cell lymphoma, which makes it a promising target for treating PTCLs. Here, we present the efficacy of CD4 CAR T cell in our preclinical study and the success in level 1 dose escalation clinical trial on patients with Sezary syndrome. Methods We engineered a CD4 CAR with scFv (single-chain variable fragment) with CD28 and 4-1BB co-activators fused to CD3zeta and a leader sequence of CD8. The efficacy of CD4 CAR was tested with CD4+ leukemic cell line, primary CD4+ PTCL patient samples and multiple mouse models. An alemtuzumab safety switch has also been established to ensure the elimination of CAR T cells following tumor eradication. Children and adults with PTCLs were enrolled in our phase 1 dose escalation trial to evaluate the safety and efficacy of CD4 CAR T cell antitumor activity. Results Coculture assays results showed that CD4 CAR T cells displayed profound tumor killing effects in leukemia cell lines, primary patient samples and multiple mouse model systems. Our preclinical findings suggest that CD4 CAR T cells is an effective approach in treating PTCLs. Patients enrolled in the phase 1 dose escalation trial have shown remarkable response to CD4 CAR T cells treatment. Noticeably, a 54-yr-old patient diagnosed with Sezary syndrome had achieved complete remission with CD4 CAR T cell therapy. Prior to admission, he had been having symptoms of erythroderma, pruritus and scaling of the skin for over 10 years and had been resistant to multiple lines of chemotherapy. Before the initiation of CAR therapy, patient's body skin has extensive leukemia infiltrate (Fig. 1A) confirmed with skin biopsy (Fig. 1B) with bone marrow and blood comprising 50% leukemic cells (Fig. 1C). Patient received a total dose of 3x10^6 /kg single dose CAR T cells, following which fluconazole and valacyclovir were administrated for infection prophylaxis. Since patient received CD4 CAR T cell infusion, the percentage of CAR T cells (Fig 1. D) in peripheral blood had continue to increase as well as NK cells. (Fig 1. E) On day 13, patient had achieved complete remission with the percentage of leukemia cells in blood decreased to zero (Fig. 1C). On day 28, the appearance of the skin had undergone drastic change from what was before the treatment. Noticeable skin regeneration on both legs of the patients was observed (Fig 1. F). Flow cytometry of bone marrow and peripheral blood confirmed the absence of tumor cells. In addition, Skin biopsy on multiple sites demonstrated absence of leukemia infiltrates post CAR treatment (Fig. 1G). Patient was subsequently discharged with no additional medication needed. Throughout the treatment, patient had developed no infections with Grade II CRS toxicity noted. No other toxicities were observed. Updated results on other patients enrolled in this clinical trial including adverse events will be presented. Conclusion Our first-in-human clinical trial demonstrates promising efficacy of CD4 CAR T cell therapy in treating patients with refractory Sezary syndrome. cCAR is able to eradicate leukemia blasts, exerting a profound tumor killing effect that is superior to traditional chemotherapies. Disclosures Pinz: iCell Gene Therapeutics LLC: Employment. Ma:iCAR Bio Therapeutics Ltd: Employment. Wada:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC: Consultancy, Equity Ownership, Research Funding; iCAR Bio Therapeutics Ltd: Consultancy, Equity Ownership, Research Funding.

Impact of Different Doses of Fludarabine in Fludarabine-Based Conditioning Regimen for Unrelated Bone Marrow Transplantation

Background: The purine analog fludarabine (Flu) is recognized as an agent having strong immunosuppressive effects and plays a central role in reduced-intensity conditioning (RIC) and myeloablative reduced-toxicity conditioning (RTC) regimens because of limited nonhematologic toxicities. Combinations of Flu and busulfan (Bu) (FB group) or melphalan (Mel) (FM group) are the commonly used as RIC and RTC regimens. Flu inhibits lymphocyte proliferation, induces apoptosis of hematologic malignant cells and has a synergistic tumor-killing effect with alkylating agents such as Bu and Mel. Few reports assess the impact of different dose of Flu on the clinical outcome. In this study, to compare the impact of Flu doses in RIC or RTC, we retrospectively analyzed clinical outcomes in patients who received FB or FM conditioning by using nationwide registration data of Japan Society for Hematopoietic Cell Transplantation. Method: Patients aged 16 years of older with acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia and malignant lymphoma, who received FB or FM conditioning regimen, and who underwent bone marrow transplantation from an unrelated donor between 2007 and 2016 were included in this study. We excluded patients who received oral Bu. Also, patients who had renal dysfunction in Hematopoietic Cell Transplantation-Comorbidity Index was excluded because Flu dose needs an adjustment due to renal function. As a result, 2809 patients were included in this study. All transplantation data in the present study were obtained from the Transplant Registry Unified Management Program (TRUMP), which included clinical data of hematopoietic cell transplantation performed in Japan. Result: In the FB group (n=1647), high-dose Flu (180 mg/m2; HFB) and low-dose Flu (150/125 mg/m2; LFB) were used in 1334 and 313 patients, respectively. The probabilities of 3-year OS and PFS in the HFB group were significantly higher than that in the LFB group (49% vs 38%, P<0.001; 44% vs 35%, P=0.007). The cumulative incidences of relapse at 3 years were 30.4% and 36.6% (P=0.058), and NRM at 3 years were 25.1% and 28.1% (P=0.24) in the HFB and LFB groups, respectively. In multivariate analysis for OS and relapse, Flu doses was identified as an independent prognostic factor (HR 0.82, P=0.03; HR 0.8, P=0.043). In the FM group (n=1162), high-dose Flu (180 mg/m2; HFM) and low-dose Flu (150/125 mg/m2; LFM) were used in 118 and 1044 patients, respectively. There was no significant difference in the OS, relapse and NRM rates between HFM and LFM (at 3 years, 51.4% vs 48.5%, P=0.596; 22.4% vs 27.0%, P=0.362; 30.7% vs 30.3%, P=0.783). Conclusion: In this large study, we suggested that high-dose Flu was associated with favorable outcome in the FB group, but not in the FM group. Prospective studies and further investigations are needed to clarify the benefit of high-dose Flu in the setting of FB regimen. Disclosures Ozawa: Kyowa-Hakko Kirin: Honoraria; Astellas Pharma Inc.: Honoraria; Pfizer Japan Inc.: Honoraria; Novartis: Honoraria. Kanda:Daiichi Sankyo Company: Honoraria; Bristol-Meyers Squib: Honoraria; Celgene: Honoraria; MSD: Honoraria; JCR Pharmaceuticals: Honoraria; Takeda: Honoraria; Otsuka: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai: Honoraria; Astellas: Honoraria; Novartis: Honoraria; NextGeM Incorporation: Patents & Royalties: 2019-011392. Atsuta:Kyowa Kirin Co., Ltd: Honoraria; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria.

DRES-07. TMZ-LEV- IFN COCKTAIL REGIMEN SIGNIFICANTLY INHIBITED THE GROWTH OF GLIOMA IN VIVO

Abstract OBJECTIVE Temozolomide (TMZ), is the first line chemotherapeutic drug for glioma. Previous studies have suggested that interferon (IFN) and levetiracetam (LEV) could respectively reverse the resistance of TMZ by down-regulating MGMT expression. This study, we aim to investigate the therapeutic effect of a cocktail chemotherapy regimen combining TMZ, LEV, IFN in vivo. METHODS Glioma cell lines U251 and SKMG-4 (MGMT protein expression positive), U138 and GSC-1(MGMT protein expression negative) were used for producing xenograft tumors. The xenograft tumors were established by subcutaneously injecting 1×106 glioma cells into female BALB/C nude mice and divided into 5 treatment groups: Control, TMZ, TMZ+IFN, TMZ+LEV, TMZ+LEV+IFN. The treatment with TMZ (50 mg/kg, i.p.), IFN (2×105 IU, s.c.), LEV (150 mg/kg, i.p.) once a day for five consecutive days and xenograft tumors were measured every two days. RESULTS We identified that U138, U251, SKMG-4 tumor growth among TMZ, TMZ+IFN, TMZ+LEV, TMZ+LEV+IFN were all significantly inhibited (P< 0.05), compared with the control. As for U251 and SKMG-4, tumor killing effect of all 4 treatment groups were not different (P > 0.05). In the treatment of mice bearing U138 glioma, the tumor weight of TMZ+LEV+IFN (0.2688±0.1169 g) group was the lowest and significantly lower than that of TMZ+LEV (0.6574±0.08174g, P=0.0261), TMZ+IFN(0.6108±0.07317 g, P=0.0381), and TMZ (0.9054±0.07154 g, P=0.0017) group. Glioma stem cells GSC-1 was highly resistant to TMZ, tumor volume of TMZ group was not different from control group (P >0.05). While compared with TMZ (1.993±0.1274 g) group, in TMZ+IFN (1.506±0.1223g, P=0.0203), TMZ+LEV (1.178±0.1807g, P=0.0042), and TMZ+LEV+IFN (1.049±0.2171 g, P=0.0038) groups, GSC-1 tumor growth were significantly inhibited(P< 0.05). CONCLUSION Our data demonstrate that both IFN and LEV can sensitize TMZ effect on glioma in vivo, even for MGMT(+) tumors, and TMZ-LEV-IFN cocktail regimen seems the best. Key words: glioma, TMZ, LEV, IFN

Overexpression of Ras-Related C3 Botulinum Toxin Substrate 2 Radiosensitizes Melanoma Cells In Vitro and In Vivo.

Radioresistance is the major obstacle in the radiotherapy of the malignant melanoma. Thus, it is of importance to increase the radiosensitivity of melanoma cells. In the present study, the radioresistant melanoma cell line OCM-1 with inducible overexpression of Ras-related C3 botulinum toxin substrate 2 was established based on a radiation-inducible early growth response gene (Egr-1) promoter. The effects of Ras-related C3 botulinum toxin substrate 2 overexpression on the radiosensitivity of melanoma cells exposed to either X-rays or carbon ion beams were evaluated in cultured cells as well as xenograft tumor models. In addition, both reactive oxygen species yield and the NADPH oxidase activity were measured in the irradiated melanoma cells. It was found that the radiation-inducible overexpression of Ras-related C3 botulinum toxin substrate 2 sensitized the melanoma cells to both X-rays and carbon ion irradiation by enhancing the NADPH oxidase activity and the subsequent reactive oxygen species production. Besides, the overexpression of Ras-related C3 botulinum toxin substrate 2 enhanced the tumor-killing effect of radiotherapy in xenograft tumors significantly. The results of this study indicate that Ras-related C3 botulinum toxin substrate 2 is promising in increasing the radiosensitivity of melanoma cells, which provides experimental evidence and theoretical basis for clinical radiosensitization of the malignant melanoma.

The analysis of peripheral blood T lymphocyte subsets in patients with colorectal cancer: a single center cross-section study

The distribution of peripheral blood lymphocyte subsets were compared between patients with colorectal cancer and healthy controls. The number of natural killer(NK) cells and CD(8)(+)T cells and the percentage of naive CD(4)(+) T cells were all decreased significantly in patients. On the contrary, the percentages of memory CD(4)(+) T cells, HLA-DR(+) CD(8)(+) T cells and CD(38)(+) CD(8)(+) T cells were significantly increased. It suggests that the tumor killing effect of cytotoxic lymphocytes in peripheral blood is impaired in patients with colorectal cancer, whereas the immune response is over stimulated.

Spontaneously formed porous structure and M1 polarization effect of Fe3O4 nanoparticles for enhanced antitumor therapy

In this study, we developed a novel Fe3O4 nanoparticles-doxorubicin (DOX)-Hyaluronic acid (HA) nanoparticles on the basis of firstly discovered “formed porous structure” in spontaneously assembled Fe3O4 nanoparticles. The Mechanism of Action (MOA) behind this porous DOX-loading cargo was tested and confirmed. A multi-functional Fe3O4-DOX+HA nanoparticle was further constructed by incorporating HA into our system. In vitro and in vivo studies exhibited that Fe3O4-DOX+HA owned enhanced antitumor efficacy with significantly prolonged survival time due to the combination of M1 polarization ability of Fe3O4 nanoparticles, tumor killing effect of DOX and tumor and TAM-targeting effect of HA. All in all, our studies offer a novel strategy to develop a multifunctional antitumor system with a simple preparation method and an enhanced therapeutic outcome.

Effects of mesenchymal stem cells harboring the Interferon-β gene on A549 lung cancer in nude mice

Interferon-β (IFN-β) exhibits a tumor-killing effect; however, injection of IFN-β alone for lung cancer is often accompanied by side effects. This study investigated the possibility of using umbilical cord mesenchymal stem cells (MSCs) as cellular carriers of IFN-β.Isolated umbilical cord MSCs were transfected with a lentivirus packaging IFN-β-overexpression plasmid. A549 cells were subcutaneously injected into nude mice to establish a non-small cell lung cancer (NSCLC) mouse model. A total of 50 mice were randomly assigned to 5 different groups: a control group, IFN-β group, IFN-β-MSCs group, MSCs-lentivirus group, and MSCs group. Next, the IFN-β-MSCs, MSCs-lentivirus, and MSCs were injected into the A549 lung cancer-bearing mice in the IFN-β-MSCs, MSCs-lentivirus and MSCs groups, respectively. Mice in the control and IFN-β groups were injected with solvent or IFN-β solution. The tumors in nude mice in the IFN-β and IFN-β-MSCs groups grew at significantly slower rates than tumors in the control group, and tumors in the MSCs-lentivirus and MSC groups also grew slowly. The rates of tumor cell apoptosis in the IFN-β and IFN-β-MSCs groups were significantly higher than those in the MSCs-lentivirus and MSCs groups. The livers, lungs, and kidneys of nude mice in the IFN-β group displayed hyperemia, exudation, and pathological lesions, while those of nude mice in the IFN-β-MSCs group showed no abnormal changes. Both INF-β-MSCs and INF-β inhibited the growth of subcutaneously implanted lung tumors; however, INF-β-MSCs specifically targeted the tumor cells, and did not produce the damage to internal organs caused by the use of INF-β alone.

MACC1 facilitates the escape of nasopharyngeal carcinoma cells from killing by natural killer cells

This study examined the regulation of phenotype and function of natural killer (NK) cells by nasopharyngeal carcinoma (NPC) cells with metastasis-associated in colon cancer-1 (MACC1) overexpression. Thirty patients with NPC and 20 healthy subjects were included. Peripheral blood (5 mL) was collected, and NK cells were purified using Ficoll and negative separation. NPC HONE1 cells were transfected with pcDNA3.1-MACC1 or negative control plasmids, and culture supernatant was collected for co-culture with NK cells. Flow cytometry was used to determine the expression of activated and inhibitory receptors on the cells. Western blotting was used to determine the expression of proteins. The results showed that the ratio of NK cells in the peripheral blood of NPC patients was elevated, but the activity and tumor-killing effect of NK cells were reduced. MACC1 promoted the escape of HONE1 cells from killing by NK cells. HONE1 with overexpression of MACC1 down-regulated the expression of NKG2D in NK cells, inhibited the proliferation of NK cells and escaped the immune surveillance by NK cells. MACC1 down-regulated the expression of NKG2D in NK cells by up-regulating the expression of TGF-β1 in HONE1 cells, thereby inhibiting the activity of NK cells. MACC1 inhibited the cell cycle of NK cells via TGF-β1 pathway. The study demonstrated that MACC1 elevated the expression of TGF-β1 by NPC HONE1 cells, thereby inhibiting NKG2D expression and G1/S transition in NK cells. MACC1 helped NPC HONE1 cells avoid killing by NK cells.

New Cell-Penetrating Peptide (KRP) with Multiple Physicochemical Properties Endows Doxorubicin with Tumor Targeting and Improves Its Therapeutic Index

Cell-penetrating peptides (CPPs) are considered as promising drug carriers by virtue of their potent cell-penetrating capacity. However, lack of targetability still represents a bottleneck for their systemic administration. Here, we synthesized a lysine-rich CPP named KRP and developed a tumor-targeted drug delivery system (DDS) by linking KRP and doxorubicin (DOX) with stable covalent bonds (thioether bond and amide bond). Through in vitro and in vivo tests, we confirmed that the multiple physicochemical properties of KRP endow KRP-DOX with multiple synergistic functions, including good biocompatibility and biodistribution, selective accumulation in tumor tissues, inclination to remain in tumor tissues and be internalized by tumor cells; stable covalent bonds prevent free DOX release from KRP-DOX in blood stream, shield normal tissues from the toxic effect of DOX, and lead to the majority of DOX delivery into tumor cells by KRP; lysosome escape of KRP-DOX ensures its tumor-killing effect. In addition, the simple chemical composition and modification reduce the risk of immunogenicity and metabolite toxicity. Our study provides a simple, safe, and efficient platform for tumor-targeted DDS.

First-in-Human CLL1-CD33 Compound CAR T Cell Therapy Induces Complete Remission in Patients with Refractory Acute Myeloid Leukemia: Update on Phase 1 Clinical Trial

▪BackgroundCD19-specific chimeric antigen receptor (CAR) T cell therapy has achieved high efficacy in acute lymphoblastic leukemia patients. However, the treatment of acute myeloid leukemia (AML) has remained a particular challenge due to the heterogeneity of AML bearing cells, which renders single antigen targeting CAR T cell therapy ineffective. CLL1 and CD33 are often used as targets for AML CAR T cell therapy. CLL1 is associated with leukemia stem cells and disease relapse, and CD33 is expressed on the bulk AML disease. Previously, we demonstrated the profound anti-tumor activity of CLL1-CD33 compound CAR (cCAR) T cells. Here we present the efficacy of cCAR in preclinical study and update the success in level 1 dose escalation clinical trial on relapsed/refractory AML patients.MethodsWe engineered a cCAR comprising of an anti-CLL1 CAR linked to an anti- CD33 CAR via a self-cleaving P2A peptide and expressing both functional CAR molecules on the surface of a T-cell cell. We tested the anti-leukemic activities of CLL1-CD33 cCAR using multiple AML cell lines, primary human AML samples, human leukemia cell line (REH cells) expressing either CLL1 or CD33, and multiple mouse models. An alemtuzumab safety switch has also been established to ensure the elimination of CAR T cells following tumor eradication. Children and adults with relapsed/refractory AML were enrolled in our phase 1 dose escalation trial with primary objective to evaluate the safety of cCAR and secondary objective to assess the efficacy of cCAR anti-tumor activity.ResultsCo-culture assays results showed that cCAR displayed profound tumor killing effects in AML cell lines, primary patient samples and multiple mouse model systems. Our preclinical findings suggest that cCAR, targeting two discrete AML antigens: CLL1 and CD33, is an effective two-pronged approach in treating bulk AML disease and eradicating leukemia stem cells. Patients enrolled in the phase 1 dose escalation trial have shown remarkable response to cCAR treatment. Noticeably, a 6-yr-old female patient diagnosed with a complex karyotype AML including FLT3-ITD mutation had achieved complete remission. The patient was diagnosed with Fanconi anemia, which had progressed to juvenile myelomonocytic leukemia and eventually transformed into AML. The patient had been resistant to multiple lines of treatments, including 5 cycles of chemotherapy with FLT3 inhibitor prior to receiving cCAR. Before the treatment, patient's leukemia blasts comprised 73% of the peripheral blood mononuclear cells and 81% of the bone marrow. Patient underwent lymphodepletion therapy (Fludarabine and Cyclophosphamide) prior to cCAR infusion. Two split doses, each consisting of 1x106/kg CAR T cells, were infused on day 1 and day 2 respectively. On day 12, while leukemia blast still counting up to 98% of the bone marrow (Fig. 1A), robust CAR T cell expansion was detected in both peripheral blood and bone marrow. On day 19, patient achieved MRD- complete remission with bone marrow aspirates revealing complete ablation of myeloid cells (Fig. 1B). Flow cytometry confirmed the absence of leukemia blasts and showed that CAR T cells comprised 36% of the PBMC and 60% of the bone marrow. The patient later underwent non-myeloablative hematopoietic cell transplantation with less toxicities compared to conventional total body radiation and high dose chemotherapies. Updated results on other patients enrolled in this clinical trial including adverse events will be presented.ConclusionOur first-in-human clinical trial demonstrates promising efficacy of cCAR therapy in treating patients with relapsed/ refractory AML. cCAR is able to eradicate leukemia blasts and leukemia stem cells, exerting a profound tumor killing effect that is superior to single target CAR T cell therapies. cCAR is also shown to induce total myeloid ablation in bone marrow, suggesting that it may act as a safer alternative to avoid the severe toxicities caused by standard bone marrow ablation regimens without sacrificing the anti-tumor efficacy. This strategy will likely benefit patients who are unable to tolerate total body radiation or high dose chemotherapies. In addition to AML, cCAR also has the potential to treat blast crisis developed from myelodysplastic syndrome, chronic myeloid leukemia, and chronic myeloproliferative neoplasm. DisclosuresPinz:iCell Gene Therapeutics LLC: Employment. Ma:iCAR Bio Therapeutics Ltd: Employment. Wada:iCell Gene Therapeutics LLC: Employment. Chen:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC, iCAR Bio Therapeutics Ltd: Consultancy, Equity Ownership, Research Funding.

SMAR1 promotes immune escape of Tri-negative Breast Cancer through a mechanism involving T-bet/PD-1 Axis

This study was aimed at investigating the specific molecular mechanisms involved in the regulation of immune escape of triple-negative breast cancer (TNBC) by SMRI, so as to provide a new clinical treatment target for the disease. Mouse original 4T1 breast cancer cells were inoculated subcutaneously in BALB/C to establish TNBC mouse model. CD8+T cells with immunological effects were selected from mouse thymus glands for primary culture. The CD8+positive T cells were infected with lentivirus interference vectors, and the proliferation of CD8+ T cells were determined by trypan blue staining and flow cytometry. CD8+T cells and 4T1 cells were cultured together so as to determine the cytotoxic effects of SMAR1-downregulated CD8+ T cells on tumor cells and the expression of cytokines (IFN-γ, TNF-α, IL-2, IL-4 and IL-6). The expressions of SMAR1, T-bet and PD-1 were assayed by Western blot. SMAR1-downregulated CD8+T cells were injected into 4T1 tumor-bearing mice through the caudal vein, and the growth of tumor in mice was monitored. Following the infection of CD8+T cells with SMAR1-downregulated lentiviral system, cell apoptosis level was decreased significantly (control vs. sh-SMAR1: 32.23 ± 12.4 % vs. 18.28 ± 8.93 %, p < 0.05). Results from trypan blue staining experiments showed that the proliferation of CD8+ T cells in the SMAR1-downregulated group was significantly increased; SMAR1-downregulated CD8+ T cells promoted the production of IFN-γ, TNF-α, IL-2, IL-4 and IL-6 in 4T1 breast cancer cells (p < 0.05). Western blot showed that SMAR1 down-regulation led to significant upregulation of T-bet, while PD-1 was downregulated, when compared to the control group (p < 0.05). The downregulation of SMAR1 was associated with significant reduction in tumor size in mice (p < 0.05). SMAR1 downregulation enhances the tumor killing effect of CD8+T cells by activating T-bet and down-regulating PD-1.

Propofol Promotes Activity and Tumor-Killing Ability of Natural Killer Cells in Peripheral Blood of Patients with Colon Cancer.

BackgroundWe investigated the effect of propofol on activities and tumor-killing ability of natural killer (NK) cells in patients with colon cancer.Material/MethodsTwenty colon cancer patients and 20 healthy subjects were included. Peripheral blood (5 ml) was collected from all patients and healthy subjects. NK cells in peripheral blood were separated by negative screening using immunomagnetic beads. Flow cytometry was used to determine expression of activated receptors, inhibitory receptors, killing effector molecules, and proliferation-associated markers on NK cell surfaces. After in vitro treatment with propofol for 24 h, expression of activated receptors, inhibitory receptors, killing effector molecules, and proliferation-associated markers on NK cell surfaces was examined again. In addition, the tumor-killing effect of NK cells was studied by co-culture with K562 cells or colon cancer SW620 cells at a ratio of 1: 1.ResultsThe number of NK cells in peripheral blood from colon cancer patients was increased compared with healthy subjects, but activities and proliferation ability of the NK cells were decreased. The tumor-killing effect of NK cells isolated from colon cancer patients was decreased. Of note, propofol promoted activation of NK cells from colon cancer patients. In addition, propofol increased expression of tumor-killing effector molecules by NK cells and the proliferation ability of NK cells. Propofol also enhanced the killing effect of NK cells on colon cancer cells.ConclusionsThe present study demonstrates that propofol promotes the activity and tumor-killing ability of NK cells in peripheral blood of patients with colon cancer.

Construction and in vitro verification of a new humanized anti-CD19 CAR-T cells with high affinity

目的构建人源化抗CD19嵌合抗原受体T细胞（CAR-T），通过体外实验验证其杀伤白血病细胞的能力。方法将人CD19的鼠源抗体（FMC63）进行了人源化改造，获得高亲和力的人源化CD19抗体；构建携带人源化CD19 CAR慢病毒载体，感染T细胞获得人源化CD19 CAR-T细胞（hCART19）；按不同效靶比将效应细胞[hCART19、未转染的T细胞（阴性组）及对照病毒转染的T细胞（对照组）]及靶细胞（CHO-K1-CD19及Raji细胞）混合培养，LDH释放实验及ELISA法检测hCART19杀伤白血病细胞的能力及细胞因子释放水平；白血病小鼠模型检测hCART19的杀瘤效果。结果LDH释放实验证实随着效靶比的不断增加，对靶细胞的杀伤率逐渐增加，当效靶比为10∶1时hCART19组的杀伤率最大，在Raji细胞中为（87.56±1.99）%，明显高于阴性组[（19.31±1.16）%]及对照组[（21.35±1.19）%]（P值均<0.001）。ELISA法检测显示Raji细胞作为靶细胞时，hCART19组IL-2水平[（10.56±0.88）pg/ml]及IFN-γ[（199.02±12.66）pg/ml]较阴性组[IL-2：（3.55±0.26）pg/ml；IFN-γ：（37.63±0.85）pg/ml]及对照组[IL-2：（2.92±0.32）pg/ml；IFN-γ：（52.07±3.33）pg/ml]明显升高（P值均<0.001）。以上实验在CHO-K1-CD19细胞作为靶细胞时也出现了相似的结果。给予白血病小鼠模型尾静脉分别注射hCART19、对照病毒转染的T细胞及未转染的T细胞，结果显示hCART19组小鼠存活时间>40 d，另外两组小鼠在20~30 d全部死亡，差异有统计学意义（χ2=11.73，P=0.008）。结论成功构建了具有抗白血病活性的人源化CD19 CAR-T细胞，为下一步的临床研究奠定了基础。

Molecular biology progress of small cell lung cancer

Small cell lung cancer (SCLC) is typified by early recurrence and metastasis associated with many genetic changes. The drug ROVA-T composed of the DLL3 antibody Rovalpituzumab and the cytoto-xic drug Tesirine achieves the tumor-killing effect by releasing the Tesirine when bound to DLL3. Nfib promotes the SCLC metastasis by altering the structure of the chromosome. The PRAP, EZH2 and Wee1 inhibitors inhibit the DNA damage repair to improve the antitumor activity of chemotherapeutic drugs. Combination of Ipilimumab and Nivolumab can activate the human immune system to exert antitumor effect. Key words: Carcinoma, small cell; Immunotherapy; Targeted therapy

P3.07-011 Investigation of Autologous Tumor-Killing Effect of Effusion-Associated Lymphocytes in Malignant Pleural Effusion of Lung Cancer

Many lung cancer patients developed malignant pleural effusion during the course of the disease. Previous studies revealed that the effusion associated lymphocytes (EAL) were in dysfunctional state which appeared to be immunosuppressed and unable to kill tumor cells. This study will evaluate the combined effects of IL-2, IL-12, αCD3 antibody (ab), and PD-1 ab on antitumor activity of lymphocytes. The underlying mechanism and relevant immune biomarkers will also be investigated.

Abstract 3750: Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity

Abstract Introduction: Adoptive immune therapy has achieved great success in eradicating blood-borne cancers, prominently, the CD19 CAR T cells in B cell leukemia and lymphomas. However, CAR T cell therapy in solid tumors has been limited due to the scarcity of tumor antigens that are deemed safe for targeting. One strategy to overcome scarcity of tumor antigen is by tuning the affinity of CAR to limit T cell reaction with cells overexpressing target antigen while sparing cells with basal level expression. To rigorously test the idea of “affinity tuning”, we built variants of CARs possessing one million-fold difference in affinity spanning low nanomolar to high micromolar affinity to a target antigen, and examined the influence of CAR affinity on the rate of tumor killing and systemic toxicity. Methods: Antigen-binding domain of CAR was built from the inserted or I domain belonging to integrin LFA-1. Affinity of CAR expressed in T cells was confirmed by ICAM-1 binding by flow cytometry. For in vivo study, mice with systemic growth of ICAM-1 positive thyroid tumor were used, where tumors grew in lungs, liver, and bones. Tumor growth and killing were monitored by whole body luminescence imaging. Sera were collected for cytokine analysis. Body weight, cytokine profile, and overall behavior were used to assess the severity of systemic toxicity. Results: CAR T cells with a step-wise, one million-fold variation in affinity to ICAM-1 resulted in a rate of target killing in proportion to the increase in affinity and in ICAM-1 density. Owing to cross-reaction of human LFA-1 with murine ICAM-1, the influence of CAR affinity on efficacy and on-target, off-tumor toxicity was tested in mice bearing ICAM-1 positive human tumors. In vivo tumor elimination by CAR T cells was in contrast to in vitro affinity-dependent rate of target killing, demonstrating that micromolar affinity CAR T cells was superior to nanomolar affinity T cells in both tumor killing and safety aspects. Highest affinity CAR T cells led to uniform death of the host, caused by on-target, off-tumor toxicity, and high level cytokine release. Conclusion: Our study is the first comprehensive report examining the effect of CAR affinity on the rate of tumor killing, efficacy, and toxicity. In contrast to in vitro tumor killing effect, the increase in affinity of CAR beyond certain threshold was deleterious to T cell persistence and associated with more frequent tumor relapse. Our study highlights that CAR T cells approximating natural T cell receptor affinity is more efficacious in eliminating tumors with overexpressed antigens, and is safer by avoiding potential reaction with normal cells with basal expression of the same antigen. Citation Format: Enda Shevlin, Spencer Park, Yogindra Vedvyas, Marjan Zaman, Susan Park, Irene M. Min, Moonsoo M. Jin. Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3750. doi:10.1158/1538-7445.AM2017-3750

Folate-Engineered Microvesicles for Enhanced Target and Synergistic Therapy toward Breast Cancer.

As an ideal nanovector candidate, microvesicles (MVs) have been gradually utilized for packaging kinds of functional molecules for effective tumor diagnosis and therapy; however, the deficiency of their tumor targeting influenced their therapy efficacy. Through a facile phospholipid substitution strategy, MVs-based drug delivery system (DDS) was apparently endowed with high tumor targeting toward breast cancer thanks to the modified folate onto the membrane of MVs, simultaneously possessing a synergistic antitumor effect, and in vivo tumor imaging attributed to the SA-QDs labeling. Tumor killing effect could be improved up to 15 percentages with the help of the improved tumor targeting ability.

Abstract 2085: Development of a mechanism-based pharmacokinetic/pharmacodynamic model to characterize tumor killing effect of an anti-VISTA monoclonal antibody in tumor bearing mice

Abstract The V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a recently discovered co-inhibitory molecule that shares limited sequence homology with the IgV domain of immune regulators of the B7 family. A first-in-class monoclonal antibody (mAb) that targets VISTA is currently in development for evaluation in patients with non-small cell lung cancer and other types of cancer. Data from a preclinical mouse model (in human VISTA knock-in mice) showed that treatment with its surrogate antibody demonstrated tumor growth inhibition, which is believed to be associated with modulation of the myelomonocytic and T cell compartments. A mechanistic pharmacokinetic/ pharmacodynamic (PK/PD) modeling approach was applied that integrated the in vitro and in vivo information (PK, target occupancy (TO) and tumor growth), and incorporated key physiological processes. A one-compartment pharmacokinetic model with Michaelis-Menten elimination kinetics was used to characterize the nonlinear clearance of the surrogate antibody. The growth and killing of tumor cells was described with a cell distribution tumor growth/ killing model. Tumor killing effects can be described well by a linear relationship to VISTA TO. The developed PK/PD model captured the observed data in mice. This work also presents the potential of how mechanistic modeling and simulation can support first-in-human dose selection and dosing regimen optimization for immunomodulatory mAbs for anticancer immunotherapy. Citation Format: Xiling Jiang, Jocelyn Leu, Indrajeet Singh, Linda A. Snyder, Weirong Wang. Development of a mechanism-based pharmacokinetic/pharmacodynamic model to characterize tumor killing effect of an anti-VISTA monoclonal antibody in tumor bearing mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2085.

A recombinant chimeric protein specifically induces mutant KRAS degradation and potently inhibits pancreatic tumor growth.

Pancreatic cancer is one of the most lethal human diseases, with an all-stage 5-year survival rate below 5%. To date, no effective and specific therapy is available for this disease. Mutations in KRAS are frequently reported in pancreatic and many other cancers; thus, KRAS is an attractive therapeutic target. Our objective was to specifically eliminate mutant KRAS and induce cell death of tumors expressing this mutant protein. We thus constructed several chimeric proteins by connecting the C-terminal domains of several adaptor proteins of E3 ubiquitin ligases such as CBL, CHIP, E6AP, and VHL, as well as VIF encoded by human immunodeficiency virus type 1 (HIV-1), to the Ras binding domain (RBD) of Raf. Although all of these chimeric proteins caused the degradation of mutant KRAS and the death of KRAS-mutant-tumor cell lines, the RBD-VIF with a protein transduction domain (PTD), named PTD-RBD-VIF, had the strongest tumor-killing effect. Intraperitoneally administered recombinant PTD-RBD-VIF potently inhibited the growth of xenografted KRAS-mutant pancreatic cancer cells. Our findings indicate that recombinant PTD-RBD-VIF, a chimeric protein with a combined cellular-viral origin, could be further developed for the treatment of various tumors harboring mutant or over-activated KRAS, especially for cases presenting with pancreatic cancer recurrence after surgery.