Lymphokine-activated Killer Cell Activity Research Articles

NK-92 serial killers - characterization of potential limitations to cytotoxic capacity.

Abstract The NK cell line NK-92 and transgenic variants are receiving attention as adoptive transfer immunotherapies to treat a range of malignancies. However, since NK-92s are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients’ immune systems. In the present study, we investigated NK-92 serial killing against various tumor cells as well as the effects of gamma-irradiation and ligation of the death receptor Fas (CD95) on NK-92 cells and the susceptibility of NK-92s to attack by primary NK cells. To evaluate serial killing, we used a 51Cr-release assay with very low effector to target (E:T) Raji, Daudi or K562 tumor cell ratios at 2, 4, 6, and 8 hour time points. NK-92s that were maintained in high concentrations of recombinant IL-2 were able to kill as many as 14 Raji cells per NK-92 cell in 8 hours at an E:T of 1:32. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but lost >50% activity 24 hours after just 7 Gy irradiation. Despite strong expression of CD95, NK-92s maintained viability after 24 hours of Fas-ligation with an anti-CD95 IgM as compared to cells treated with an isotype control antibody. While NK-92s survived 16 hours of exposure to unstimulated primary NK cells, they were killed when exposed to IL-2 activated primary LAK cells. In conclusion, we recommend that adoptive transfer of irradiated NK-92 cells into patients be made as quickly as possible post-irradiation of the NK-92s. Genetic modification of NK-92 cell lines to secrete IL-2 as an autocrine growth factor may need reconsideration because in vivo activation of endogenous LAK cells by IL-2 could reduce the number of NK-92 cells within tumors and thus decrease therapeutic efficacy.

Abstract 3968: OT-101/Chemotherapy - A novel mechanism of action (MOA) in pancreatic cancer immunization therapy

Abstract Background: OT-101 is a phosphorothioate antisense oligodeoxynucleotide targeting the human TGF-β2 mRNA. OT-101 breaks down immune tolerance and activates immunity against the tumor. The Subsequent Chemotherapy (SC) releases tumor antigens and boosts the immunity response along with further epitope expansion. The phase 1/2 clinical data for OT-101/SC is presented here along with supporting preclinical MOA studies. Methods: Total of 37 pts, 2nd line and beyond received OT-101 with option to go on SC (OT-101/SC) or Best Supportive Care (BSC) (OT-101/BSC). Overall survival (OS) was compared using log-rank statistics. Stratification by treatment line, schedule, metastasis location, disease control (DC), and baseline CA19-9, was performed. For cytokine analysis, plasma levels of 31 cyto-/chemokine were measured over 8 time points during 3 cycles of intravenous OT-101 therapy and a subset of 12 patients. Results: In vitro cell kill assay and in vivo xenograft study were performed with human PBMC and OT-101. OT-101 reduced TGF-β2 secretion and increased LAK cell-activity against all tumor lines by 400% and 364% in comparison to the untreated control and the Lipofectin control, respectively. Addition of active rh-TGF-β2 protein suppressed the cytotoxic activity of the immune cells in a dose dependent manner. Preclinical- LS174T xenograft was treated with PBMC or PBMC + OT-101. OT-101 significantly enhanced the activity of PBMC against the xenograft. Median OS (mOS) of the 18 pts receiving OT-101/SC was 9.4mos vs. the 19 pts on OT-101/BSC (2.8mos, p=0.0004). Pts with only liver mets had a mOS of 9.5mos while those with liver mets and others only had a mOS of 4.7mos (p=0.0077). Among the former, one patient had complete response beyond 77.3mos and another had stable disease with OS of 40.3mos. OS was higher for liver mets. only group with OT-101/SC - 12.4mos versus 1.9mos, p=0.0006. There were 16 of 37 pts with DC, with mOS of 9.7mos vs. 3.0mos (p<0.0001). OS was higher for DC group with OT-101/SC - 11.8mos vs. 5.0mos, p=0.0021. Patients exhibited spike in IL-8 level which returned to basal level at treatment stop. R2 relating the IL-8 spike and OS were 0.8522 and 0.9895 and p values were 0.0011 and 0.0053 for pts treated subsequently with SC and BSC, respectively. The two lines intersect at the origin with higher response for those with OT-101/SC, suggesting exaggerated response expected for antigen boost during the SC phase. Conclusions: Escalating Intratumoral heterogeneity (IH) resulting in xenogenization, which is countered by overexpression of TGF-β. Here we report on the use of OT-101/SC to break immune tolerance to pancreatic cancer (PC) for the cure. The MOA for OT-101/SC is consistent with the reactivation of immunity during TGF-β suppression and subsequent boosting/expansion of immunity during SC. Contrary to traditional tumor vaccine- this is universally applicable to all patients. Citation Format: David Nam, Larn Hwang, Vuong Trieu. OT-101/Chemotherapy - A novel mechanism of action (MOA) in pancreatic cancer immunization therapy [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 3968.

A Tumor Cell-Selective Inhibitor of Mitogen-Activated Protein Kinase Phosphatases Sensitizes Breast Cancer Cells to Lymphokine-Activated Killer Cell Activity.

Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is worthy of further exploration.

117. Development of Novel Cancer Therapy Combination of Ad-SOCS Gene Therapy and LAK Cell Immunotherapy for the Treatment of Prostate Cancer

Background Suppressor of cytokine signaling 3 (SOCS3) is a promising molecule for cancer gene therapy. SOCS3 suppresses tumor growth through inhibition of mutiple signaling pathways including Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase (ERK), and focal adhesion kinase (FAK). The constitutive activation of JAK/STAT3 pathway is known to be involved in oncogenesis of various types of cancers including prostate cancer. We investigated the anti-tumor activity of Ad-SOCS, which is the recombinant adenovirus vectors carrying the SOCS3 gene, against prostate cancer cell line in vitro. Furthermore, to improve the antitumor effect against prostate cancer, we combined LAK (Lymphokine activated killer) cell immunotherapy with Ad-SOCS gene therapy. Materials and Methods LNCaP, human prostate cancer cell line was used in this study. The cytotoxicity of Ad-SOCS against LNCaP was measured by XTT assay. LAK cells were generated from PBMCs isolated from fresh blood by Ficoll-paque (GE Healthcare, Munich, Germany) density gradient centrifugation. PBMC were incubated in medium with interleukin (IL)-2 and anti CD3 antibody (OKT3) to generate LAK cells. The cytotoxicity of LAK cells against LNCaP cells infected with Ad-SOCS was investigated by the in vitro cytotoxicity assay. Expression level of SOCS3, STAT3 and ULBPs were measured by real-time PCR. Results Ad-SOCS showed the significantly higher cell growth inhibitory effect compared to Ad-LacZ and PBS controls in LNCaP cells. In real-time PCR, Ad-SOCS significantly increased the level of SOCS3 mRNA expression. Sequentially, the expression of STAT3 mRNA was decreased by Ad-SOCS infection. Upregulation of SOCS3 mRNA and downregulation of STAT3 mRNA were not observed in both Ad-LacZ and PBS infected LNCaP cells. In cytotoxicity assay, LAK cells exhibited the significantly higher cytotoxicity against LNCaP cells infected with Ad-SOCS at all effector: target cell ratios, 1:1,5:1, 10:1, compared with that of non-infected or Ad-LacZ infected cells. The mRNA expression levels of ULBP 1-5, which are NKG2D ligands to stimulate LAK cell activities, were measured and the mRNA expressions of ULBP 1, 3 and 4, in LNCaP infected with Ad-SOCS were significantly increased compared to the treatments. Conclusion In the present study, we demonstrated that Ad-SOCS could inhibit the cell growth of LNCaP cells and increased the sensitivity of LAK cell in vitro. These findings suggested that the Ad-SOCS and LAK cell combination therapy warranted the further development of novel therapeutic approach for advanced prostate cancer.

Follicular lymphoma: in vitro effects of combining lymphokine-activated killer (LAK) cell-induced cytotoxicity and rituximab- and obinutuzumab-dependent cellular cytotoxicity (ADCC) activity.

Follicular lymphoma (FL) is a disease of paradoxes-incurable but with a long natural history. We hypothesized that a combination of lymphokine-activated killer (LAK) cells and monoclonal antibodies might provide a robust synergistic treatment and tested this hypothesis in a phase II clinical trial (NCT01329354). In this trial, in addition to R-CHOP, we alternated the administration of only rituximab with rituximab and autologous LAK cells that were expanded ex vivo. Our objective was to determine the in vitro capability of LAK cells generated from FL patients to produce cytotoxicity against tumor cell lines and to determine rituximab- and obinutuzumab-induced cytotoxicity via antibody-dependent cellular cytotoxicity (ADCC) activity. We analyzed the LAK cell-induced cytotoxicity and rituximab (R)- and obinutuzumab (GA101)-induced ADCC activity. We show that LAK cells generated from FL patients induce cytotoxicity against tumor cell lines. R and GA101 enhance cytolysis through ADCC activity of LAK cells. Impaired LAK cell cytotoxicity and ADCC activity were detected in 50 % of patients. Percentage of NK cells in LAK infusions were correlated with the R- and GA101-induced ADCC. Our results indicate that the combination of R or GA101 and LAK cells should be an option as frontline maintenance therapy in patients with FL.

Immunotherapy of breast cancer by single delivery with rAAV2-mediated interleukin-15 expression.

Recombinant adenovirus-associated vector serotype 2 (rAAV2) is one of the most promising gene transfer vectors due to its advantage of causing non-pathogenic infection, low immunogenicity, and long-term gene expression in human clinical trials. Human interleukin 15 (hIL15) has been implicated in modulation of antitumor activity of lymphokine-activated killer (LAK) cells, including T cells and NK cells. In this study, the rAAV2-hIL15 vector was produced and subjected for treatment with xenograft JC breast cancer model. Results showed that tumor onset was significantly delayed, the tumor growth was suppressed, and the lifespan of tumor-bearing mice were prolonged by rAAV2-hIL15. In addition, rAAV2-hIL15 was able to produce a substantial expression of IL15 protein that ultimately activated the cytotoxic activity of LAK cells. Furthermore, prominent apoptosis was observed in tumor lesions following injection of rAAV2-hIL15. Taken together, our results suggested that rAAV2-hIL15 appears as a new potential therapeutic tool for breast cancer immunotherapy.

Impact of IL-2 and IL-2R SNPs on proliferation and tumor- killing activity of lymphokine-activated killer cells from healthy chinese blood donors.

One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity through in vitro techniques using peripheral blood collected from patients. However, cancer patients generally have poor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilities and less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimal clinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells. Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on single nucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRM- PCR. LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequency and tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killing assays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors had significantly greater proliferation, tumor-killing activity, and IFN-γ production than LAK cells from TT donors (P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing were significantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL- 2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneic LAK cell immunotherapy.

Gemcitabine sensitizes lung cancer cells to Fas/FasL system‐mediated killing

Gemcitabine is a chemotherapy agent commonly used in the treatment of non-small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine-induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real-time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL-expressing cells was evaluated by flow cytometry, and caspase-8 and caspase-3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine-activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti-Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane-bound FasL (mFasL) and of mFasL-positive apoptotic H292 cells, as well as caspase-8 and caspase-3 cleavage. Moreover, gemcitabine increased CH11-induced caspase-8 and caspase-3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine-treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up-regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas-dependent apoptosis mediated by caspase-8 and caspase-3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing.

Evaluation of host quality of life and immune function in breast cancer patients treated with combination of adjuvant chemotherapy and oral administration of Lentinula edodes mycelia extract

PurposeAnthracycline-based chemotherapies for breast cancer are well known to have adverse effects and can also negatively affect host immune function. There is therefore a necessity for an adjuvant that maintains the quality of life (QOL) and immune function of cancer patients receiving anthracycline-based chemotherapies.Patients and methodsThe present study investigated the effectiveness of the concomitant use of Lentinula edodes mycelia extract (LEM), an oral immunomodulator, with FEC75 (5-fluorouracil + epirubicin + cyclophosphamide) therapy on host QOL and immune function in breast cancer patients with nodal metastases. Ten breast cancer patients with nodal metastases receiving surgery were enrolled in this study. Treatment with 5-fluorouracil (500 mg/m2), epirubicin (75 mg/m2), and cyclophosphamide (500 mg/m2) was performed every 21 days for two courses, and LEM (1800 mg/day by mouth) was administered during the second course.ResultsIn the first course, hematological toxicity was observed and host QOL and immune function were exacerbated. In the second course, however, the number of white blood cells and lymphocytes did not decrease and host QOL was maintained. Furthermore, the cytotoxic activities of natural killer (NK) and lymphokine-activated killer cells and the proportion of activated NK and NK T-cells in lymphocytes were maintained in the second course.ConclusionIt has been suggested that the concomitant use of LEM with FEC75 therapy can maintain host QOL and immune function, and offer important implications for an application of LEM as a useful oral adjuvant to anthracycline-based chemotherapies.

Long-term expression of rAAV2-hIL15 enhances immunoglobulin production and lymphokine-activated killer cell-mediated human glioblastoma cell death

Glioblastoma is the most aggressive primary brain tumor and its prognosis remains poor despite different treatment modalities including surgery, radiotherapy and chemotherapy. Therefore, more useful treatments for glioblastoma patients are required. Human interleukin 15 (hIL15) is an immunomodulator that has antitumor activities. hIL15 combined with gene therapy method is also currently under cosideration as a treatment option. Since recombinant adeno-associated virus serotype 2 (rAAV2) with low immunogenicity and long-term gene expression in human clinical trials has been demonstrated, rAAV2 encoding hIL15 (rAAV2-hIL15) were used to inhibit human glioblastoma growth in the present study. rAAV2-hIL15, which is able to express IL15 protein with bioactivity, was successfully produced and purified. Data of this study demonstrated that the long-term expression of rAAV2-hIL15 enhances immunoglobulin (Ig) production and the cytotoxic activity of lymphokine-activated killer (LAK) cells. In addition, results of the present study showed that rAAV2-hIL15 delays tumor growth on a xenografted human glioblastoma mice model. Taken together, these results indicated that rAAV2-hIL15 constitutes a powerful and potent gene immunotherapy method for human glioblastoma treatment.

Immunomodulatory properties of surfactant preparations

Surfactant replacement significantly decreased acute pulmonary morbidity and mortality among preterm neonates with respiratory distress syndrome. Besides improving lung function and oxygenation, surfactant is also a key modulator of pulmonary innate and acquired immunity regulating lung inflammatory processes. In this review, we describe the immunomodulatory features of surfactant preparations. Various surfactant preparations decrease the proinflammatory cytokine and chemokine release, the oxidative burst activity, and the nitric oxide production in lung inflammatory cells such as alveolar neutrophils, monocytes and macrophages; they also affect lymphocyte proliferative response and immunoglobulin production, as well as natural killer and lymphokine-activated killer cell activity. In addition, surfactant preparations are involved in airway remodeling, as they decrease lung fibroblast proliferation capacity and the release of mediators involved in remodeling. Moreover, they increase cell transepithelial resistance and VEGF synthesis in lung epithelial cells. A number of different signaling pathways and molecules are involved in these processes.Because the inhibition of local immune response may decrease lung injury, surfactant therapeutic efficacy may be related not only to its biophysical characteristics but, at least in part, to its anti-inflammatory features and its effects on remodeling processes. However, further studies are required to identify which surfactant preparation ensures the highest anti-inflammatory activity, thereby potentially decreasing the inflammatory process underlying respiratory distress syndrome. In perspective, detailed characterization of these anti-inflammatory effects could help to improve the next generation of surfactant preparations.

Immunotherapy with LAK Cells and Anti-CD20 Monoclonal Antibodies for Follicular Lymphoma: Enhanced Antibody-Dependent Cell Cytotoxicity of LAK Cells in Association with GA101 Rather Than Rituximab

AbstractAbstract 4881 BackgroundThe current gold standard treatment for follicular lymphoma (FL) consists in the association of rituximab, that is an anti-CD20 monoclonal antibody (mAb), and one among many possible chemotherapy regimens, followed by maintenance with the same mAb. However, LF is still considered incurable. Hence, it is necessary to keep investigating new strategies aiming at improving the overall clinical results.As an anti-tumor mechanism, antibody-dependent cellular cytotoxicity (ADCC) depends on both the immune system effector arm and the antibody structure. The culture of peripheral blood lymphocytes with IL-2 activates killer cell subpopulations (LAK cells), mainly consisting of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), both with significant cytotoxic capacity. The combination of LAK cells with a mAb could achieve a synergistic effect by enhancing ADCC and potentially achieving clinical efficacy. As structural modifications in mABs may alter their biological activity, new anti-CD20 with structural changes in the Fc portion of the immunoglobulin have been synthesized in order to obtain a greater therapeutic effect. GA101 is a new anti-CD20 mAB that binds with higher affinity to the CD20 type II epitope, increasing the ADCC effect as compared to to rituximab. Currently, no data support the notion of a possible synergistic effect of GA101 and LAK cells. AimThe objective of this work was to assess the cytotoxic capacity of LAK cells generated from the peripheral blood of patients with FL. In addition, we aimed at ascertaining whether the combination of rituximab and LAK could improve the functional activity of LAK alone. Finally, we conducted a comparative study of two anti-CD20 antibodies structurally different, namely rituximab and GA101, used in combination with LAK cells. Material and methodsLAK cells were expanded in vitro from 39 peripheral blood samples of patients with FL. Cytotoxicity studies were performed using chromium release assays in which LAK cells targeted K562 (as NK target), Daudi (as LAK cells target) and CRL-1596 (CD20 positive) cells. Overall, mononuclear cells were isolated and basal cytotoxicity was measured in 39 peripheral blood samples from patients with FL. The ability of two anti-CD20 antibody (rituximab and GA101) to enhance LAK cell activity was evaluated against the CRL-1596 cell line. As a control, an irrelevant antibody (cetuximab) was used. To perform these studies, target cells were incubated with effector cells and antibody at 10 mg/ml concentration. ResultsResults are expressed as mean percentages. Basal cytotoxicity against K562, Daudi and CRL-1596 cells was 19.4%, 11.9% and 1.5% respectively. After culture with IL-2, the cytotoxicity activity against the same cell lines was reassessed and in all cases a statistically significant increase was observed (p<0,05). In particular, cytotoxicity against K562, Daudi and CRL-1596 was 39.5%, 39.6% and 21.1%, respectively.Furthermore, we studied the rituximab effect on the cytotoxic capacity against CRL-1596. The observed cytotoxicity of LAK cells with rituximab was 39.9% vs 23.6% for LAK cells alone (p<0,001). In parallel, the GA101 effect on the cytotoxic capacity against CRL-1596 was also evaluated. The cytotoxicity of LAK cells with GA101 was 52.3% vs 23.6% for LAK cells alone (p<0,001). Vice versa, no difference was observed in the cytotoxic activity of LAK cells against CRL1596 cell line when the effector cells were incubated with the irrelevant antibody cetuximab (26.0% vs 23.6%).Finally, when comparing the ability of both anti-CD20 antibodies to enhance ADCC, we observed a significant difference in favor of GA101 (GA101: 52.3%, rituximab: 39.9%; p<0,001). ConclusionsLAK cells generated from peripheral blood lymphocytes by culture with IL-2 in patients with LF show a higher cytotoxic activity than naive lymphocytes. The observed cytotoxic capacity of these LAK cells against a CD20 positive cell line (CRL-1596) is enhanced by the addition of anti-CD20 mAbs. Interestingly, GA101 was consistently more effective than rituximab in enhancing the cytotoxic capacity of LAK cells. Disclosures:No relevant conflicts of interest to declare.

Outpatient Intravenous Interleukin-2 with Famotidine Has Activity in Metastatic Melanoma

Daily short intravenous interleukin-2 (IL-2) infusions have been developed to decrease toxicity while maintaining the anticancer activity of this agent against melanoma. Such IL-2 schedules have previously been shown to promote lymphokine-activated killer cell (LAK) activity. Famotidine may increase LAK activity by increasing IL-2 internalization by the IL-2 receptor on lymphocytes. Twenty-one patients with metastatic melanoma were treated with IL-2 18 million IU/m² intravenously (i.v.) over 15-30 minutes and famotidine 20 mg i.v. daily for 3 days for 6 consecutive weeks on an outpatient basis. Cycles were repeated every 8 weeks. 13 males/8 females, median age, 51 (range: 26-79), and median Eastern Cooperative Oncology Group performance status, 1; common metastatic sites: lymph nodes (16), lungs (14), subcutaneous (8), liver (7), and bone (7). Prior systemic therapy: chemotherapy (7); IL-2 (7); and interferon (5). Most common toxicities were myalgia/arthralgia, rigors, nausea/emesis, and mild elevation of liver function tests. No patients required hospitalization for toxicity of therapy. One patient (5%) has had a complete response (ongoing at 29+ months), while 4 other patients (19%) had partial responses (total response rate: 24%; 95% confidence interval: 9%-48%). Responses occurred in lung, spleen, bones, lymph nodes, and subcutaneous sites. Median response duration=20+ months. Outpatient intravenous IL-2 and famotidine has activity in melanoma.

Immunotherapy for SV40 T/t antigen-induced breast cancer by recombinant adeno-associated virus serotype 2 carrying interleukin-15 in mice

Human interleukin-15 (hIL15) exerts anticancer effects through the activities of lymphokine-activated killer (LAK) cells. However, its short half-life hinders its clinical application. Recombinant adeno-associated virus serotype2 (rAAV2) is used for hIL15 gene transfer vectors, because of its low immunogenicity and long-term gene expression in human clinical trials. SV40T/t antigens are related with many human epithelial cancers and are generally found in human breast cancer. In order to demonstrate the anticancer effects of hIL15 on SV40T/t antigen-induced breast cancer, rAAV2-hIL15 was constructed and an SV40T/t antigen-induced transgenic mouse breast cancer model was established. Our study showed that rAAV2-hIL15 could express the hIL15 protein with anticancer bioactivity. In addition, rAAV2-hIL15 could activate the cytotoxic activity of LAK cells invivo. Furthermore, the rAAV2-hIL15 successfully delayed cancer growth and eventually led to cancer cell death in SV40T/t antigen-induced breast cancer transgenic mice. In summary, our study indicates that rAAV2-hIL15 may be applied for cancer immunotherapy of SV40T/t antigen-induced breast cancer.

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma.

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44(lo)) or elevated (CD44(hi)) expression of CD44 are generated and that the CD44(hi) cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

Granzyme B-mRNA expression by equine lymphokine activated killer (LAK) cells is associated with the induction of apoptosis in target cells

Lymphokine-activated killer (LAK) cells are a subset of cytotoxic cells capable of lysing freshly isolated tumor cells. While LAK activity is typically measured using the 51Cr-release assay, here we used a non-radioactive flow cytometric method to demonstrate equine LAK activity. Equine peripheral blood mononuclear cells (PBMC) were stimulated in vitro with recombinant human interleukin 2 (hIL-2) to generate LAK cells. An equine tumor cell line, EqT8888, labeled with carboxyfluorescein succinimidyl ester (CFSE) was used as target cells. Following incubation of the targets with different concentrations of LAK cells, Annexin V was added to identify the early apoptotic cells. With increasing effector to target cell ratios, EqT8888 apoptosis was increased. We also measured interferon-gamma, granzyme B and perforin mRNA expression in the LAK cell cultures as possible surrogate markers for cytotoxic cell activity and found granzyme B mRNA expression correlated best with LAK activity. Also, we found that the reduced LAK activity of young horses was associated with decreased granzyme B mRNA expression. Our results indicate that fluorescence-based detection of LAK cell activity provides a suitable non-radioactive alternative to 51Cr-release assays and mRNA expression of granzyme B can be used as surrogate marker for these cytotoxic cells.

Abstract 779: Role of CD44 in LAK cell-mediated killing of melanoma

Abstract Lymphokine-activated killer cells play an important role in various immunologically-based treatments of cancer, including in the use of IL-2 therapy for the treatment of malignant melanoma. In previous studies, we demonstrated that the expression of CD44 on splenocytes is significantly elevated following exposure to IL-2. Therefore, in the current study we examined the potential significance of CD44 expression on lymphokine activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells and NKT cells. Treatment of melanoma bearing CD44WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma bearing CD44KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2 treated CD44KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2 treated CD44WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred IL-2-activated splenocytes from CD44KO mice were significantly less effective at controlling tumor growth in vivo. Next the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44lo) or elevated (CD44hi) expression of CD44 are generated and that the CD44hi cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 779. doi:10.1158/1538-7445.AM2011-779

Anticancer effect of polysaccharide isolated from Polyporus sp. M05.

Objective To study effect and mechanism of fungus polysaccharide PSM-a of Polyporus sp. M05 on S180 bearing mice. Methods MTT method was used to detect the inhibiting role of PSM-a on S180 cells proliferation in vitro. S180 mice model was established, and was administered by gavage. Tumor volume was detected, and the ratio of tumor to mile weight and inhibiting tumor rate. The activity of NK and LAK cells on the target cells was analyzed by MTT colorimetric assay; HE stain was used to detect the necrosis of tumor cells. Restilts PSM-a could inhibit S180 cells grouth in vitro. PSM-a could decrease the tumor weight and increase the ratio of tumor volume and mice weight; Tumor inhibiting rate reached 80% and above when treated with 250μg/ml PSM-a. PSM-a could increase the activity of NK and LAK cells, and necrosis happened. Conclusion PSM-a could significantly inhibit the growth of S180 cells, and the mechanism bnay be related with the increased killing activity of immunne cells to tumor cells. Key words: Polyporaceae; polysaccharides; Neoplasms

High-Dose Intensity Pulse Interleukin-2 with Famotidine in Metastatic Kidney Cancer

Lymphokine-activated killer cell (LAK) activity against tumor cell lines may be induced by intravenous (i.v.) interleukin-2 (IL-2). Daily short infusions (pulses) have been developed to decrease toxicity while maintaining the anticancer activity of this agent against kidney cancer. The anthihistamine, famotidine, may increase IL-2 uptake by the IL-2 receptor on lymphocytes. We have treated 12 patients with metastatic kidney cancer, using pulse IL-2 (18 million IU/M(2) i.v.) over 15-30 minutes, preceded by famotidine (20 mg I.V. daily for 5 days) on an oncology inpatient unit. Cycles were repeated every 3 weeks until disease progression. Patient characteristics were as follows: 9 males with a median age of 66 years (range, 48-74), and median Eastern Cooperative Oncology Group performance status of 1; common metastatic sites included in the lungs 9 and lymph nodes 3. Median number of cycles received was 2 (range, 1-5). The most common toxicities were fever, rigors, and hypomagnesemia. Two (2) patients had partial responses (17% response rate). Responses occurred in the liver (11.5 months) and lung, pleura, and lymph nodes (3 months). Pulse IL-2 with famotidine shows activity in kidney cancer.

Significant Impairment in Immune Recovery After Cancer Treatment

Although immunosuppression from cancer adjuvant therapy has been documented, how these suppressed immune responses recover to baseline values after completion of cancer adjuvant therapy has not been studied systematically. The objective of this study was to examine the probability of immune recovery after cancer adjuvant therapy and the potential impact of cancer adjuvant therapy type and cancer stage on immune recovery in patients with newly diagnosed breast cancer. In a repeated-measures design, immune responses were measured four times in 80 patients with early-stage breast cancer: before and at 2, 6, and 12 months from the beginning of cancer adjuvant therapy. Natural killer cell activity, lymphokine-activated killer cell activity, lymphocyte proliferation, CD subsets (CD4, CD8, and CD56), and cytokines (interferon-gamma, interleukin [IL]-2, IL-4, IL-6, and IL-1alpha) were selected for their relevance to breast cancer. Immune recovery was defined by the level of immune response reaching to and above baseline levels. Data were analyzed using a multivariate generalized linear mixed-model approach. Delayed immune recovery to pretreatment baseline levels continued to the 12-month time point in all parameters. The percentages of immune recovery ranged from 6% to 76% of the patients, varying among immune parameters. Overall, immune recovery was poorer for interferon-gamma, IL-2, IL-4, lymphocyte proliferation, and natural killer cell activity than was for CD subsets and IL-6. The type of cancer adjuvant therapy, not cancer stage, showed selective influence on immune recovery. Chemotherapy or chemotherapy and radiotherapy combination significantly delayed IL-2 recovery, whereas radiotherapy significantly delayed IL-4 recovery. Immune recovery after breast cancer adjuvant therapy is delayed significantly for an extended time period in numerous immune parameters. The type of cancer adjuvant therapy has selective influence on immune recovery. Future investigations are warranted to elucidate the time course of immune recovery, clinical significance of poor immune recovery, and factors influencing immune recovery to develop potential interventions.