E-cadherin expression on human carcinoma cell affects trastuzumab-mediated antibody-dependent cellular cytotoxicity through killer cell lectin-like receptor G1 on natural killer cells

Trastuzumab is a recombinant antibody drug that is widely used for the treatment of HER2-overexpressing breast carcinoma. Despite encouraging clinical results, many HER2-overexpressing carcinomas are primarily resistant to trastuzumab. We attempted to explain trastuzumab resistance and search for solutions. Since the killer cell lectin-like receptor G1 (KLRG1), an inhibitory receptor expressed on subsets of natural killer (NK) cells recognizes E-cadherin as ligands and may inhibit immune responses by regulating the effector function of NK cells, we used HER2-overexpressing carcinoma cells which were expressing E-cadherin to investigate the role of antibody-dependent cellular cytotoxicity (ADCC) through KLRG1 on NK cells in vitro and vivo. The results indicated that HER2-overexpressing carcinoma cells were killed by trastuzumab-mediated ADCC and the ADCC activity was reflected the degree of E-cadherin expression on carcinoma cells. We found that expression of E-cadherin was shown to be a predictor of response to trastuzumab-based treatment for HER2-overexpressing carcinomas, furthermore, trastuzumab-mediated ADCC was markedly enhanced by KLRG1-negative peripheral blood mononuclear cells (PBMCs(KLRG1(-))).     

Modulation of natural killer cell activity, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by andrographolide in normal and Ehrlich ascites carcinoma-bearing mice

Modulation of immune responses is highly relevant in tumor cell destruction. The present study is focused on the effect of Andrographis paniculata extract (APE) and its isolated compound andrographolide (ANDLE) on cell-mediated immune responses in normal and tumor-bearing control animals. Treatment with APE and ANDLE significantly enhanced natural killer cell activity in normal (APE, 46.82% cell lysis; ANDLE, 40.79% cell lysis) and tumor-bearing animals (APE, 48.66% cell lysis; ANDLE, 42.19% cell lysis) on the fifth day, and it was observed earlier than in tumor-bearing control animals (12.89% cell lysis on day 9). Antibody-dependent cellular cytotoxicity was also increased in APE (45.17% cell lysis on day 11) as well as ANDLE (39.92% cell lysis on day 11)-treated normal and tumor-bearing animals (APE, 47.39% cell lysis; ANDLE, 41.48% cell lysis on day 11) compared to untreated tumor-bearing control animals (maximum of 11.76% cell lysis on day 17). An early enhancement of antibody-dependent complement-mediated cytotoxicity was also observed by the administration of APE and ANDLE in normal as well as tumor-bearing animals. APE and ANDLE administration could significantly enhance the mitogen-induced proliferation of splenocyte, thymocyte, and bone marrow cells. Moreover, treatment of APE and ANDLE significantly elevated the production of interleukin-2 and interferon-gamma in normal and Ehrlich ascites carcinoma-bearing animals.     

Heterodimeric bispecific antibody-derivatives against CD19 and CD16 induce effective antibody-dependent cellular cytotoxicity against B-lymphoid tumor cells

Bispecific scFv antibody-derivatives (bsscFvs) recruiting natural killer (NK) cells for the lysis of malignant cells have therapeutic potential. However, a bsscFv specific for the B-lymphoid tumor antigen CD19 and the trigger molecule CD16 on NK cells had similar affinities for both antigens (42 and 58nM, respectively) and was not optimal for cytotoxicity. Therefore, a bispecific tribody (bsTb) was constructed with two binding sites for CD19 and one for CD16. This bsTb contained a CD19-specific Fab fragment carrying a CD16-specific scFv fused to its light chain and a CD19-specific scFv fused to its heavy chain. The bsTb was compared with a bispecific bibody (bsBb) lacking the CD19-specific scFv. The bsTb had 3-fold greater avidity for CD19 than the bsBb (8 and 24nM, respectively), while both had equal affinity for CD16 (56nM). Both molecules mediated antibody-dependent cellular cytotoxicity (ADCC) of leukemia-derived SEM cells and primary cells from leukemia patients. The bsTb showed half-maximum effective concentrations (EC(50)) of 55pM and promoted equal lysis as the bsBb and the bsscFv at 6- and 12-fold lower concentrations, respectively. Among these three molecules the bsTb showed the most promising in vitro properties which are anticipated to be displayed also in vivo.     

Complement-induced cell death by rituximab depends on CD20 expression level and acts complementary to antibody-dependent cellular cytotoxicity.

PURPOSE: The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20+ follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. EXPERIMENTAL DESIGN: We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximab-mediated CDC, ADCC, and the combination. RESULTS: We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. CONCLUSIONS: These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary. These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20+ tumors.     

Modulation of natural killer and antibody-dependent cellular cytotoxicity by interferon and interleukin-2 in chronic myeloid leukemia patients in remission

Our earlier studies demonstrated that about 55% of chronic myeloid leukemia (CML) patients in remission exhibited impaired natural killer (NK) cytotoxicity (low NK responders) while antibody-dependent cellular cytotoxicity (ADCC) of these patients, on chicken red blood cells as targets, was within normal range. In this paper, we have attempted to modulate the NK cytotoxicity of CML patients in remission with interferon (IFN) and interleukin-2 (IL-2) singly or together. ADCC using K562 target-directed monoclonal antibody (MAb) 4.6E10 was also modulated by treating the effectors with IFN or IL-2. Pretreatment of nonadherent mononuclear cells from peripheral blood (NAPBMNC) with IFN or IL-2 was found to result in 20 and 21% increase in target cell lysis in case of healthy donors, 79 and 98% increase in case of CML normal NK responders, and 164 and 159% increase in case of CML low NK responders. Combined use of IFN and IL-2 potentiated further the lymphocytotoxicity to 25% in healthy donors, 135% in normal NK responder CML patients and 283% in low NK responder CML patients. This treatment resulted in restoration of cytotoxicity of the latter group of patients to a normal level. The augmentation was seen in 80-100% CML patients. Although ADCC with chicken red blood cells as targets was within normal range, ADCC mediated with MAb to K562 cells was significantly lower in CML low NK responders (24.5%) than CML normal NK responders (42.4%) and healthy donors (65.9%).(ABSTRACT TRUNCATED AT 250 WORDS)     

苦参碱抑制KG1a细胞生长及提高自然杀伤细胞对其杀伤敏感性的研究

目的:观察苦参碱(matrine)对人急性髓系白血病细胞株KG1a的生长抑制作用,及苦参碱作用前后自然杀伤(natural killer,NK)细胞对KG1a细胞杀伤敏感性的影响.方法:采用CCK-8法和锥虫蓝染色法检测苦参碱对KG1a细胞的生长抑制作用及细胞存活率,FCM检测苦参碱作用前后KG1a细胞周期的变化及表面NK细胞活化性受体(natural-killer group 2,member D,NKG2D)配体(MICA/B、ULBP1、ULBP2和ULBP3)和人类白细胞抗原(human leukocyte antigen,HLA)Ⅰ类分子的表达,乳酸脱氢酶释放法检测苦参碱作用前后NK细胞对KG1a细胞杀伤敏感性的影响.结果:苦参碱能抑制KG1a细胞的生长,随着药物浓度的升高和作用时间的延长,细胞的生长抑制率逐渐升高、存活率逐渐下降;苦参碱作用后细胞发生G1/S期阻滞;细胞表面NKG2D各配体表达率均明显升高,与作用前相比差异有统计学意义(P<0.05),HLA-Ⅰ类分子表达率无明显变化(P>0.05);当效靶比分别为5︰1、10︰1和20︰1时,苦参碱作用前后NK细胞对KG1a细胞的杀伤敏感性差异均有统计学意义(P<0.05).结论:苦参碱可抑制KG1a细胞的生长并改变细胞周期,上调KG1a细胞表面NKG2D各配体的表达率,增强NK细胞对其的杀伤敏感性.     

Enhanced natural killer cell binding and activation by low-fucose IgG1 antibody results in potent antibody-dependent cellular cytotoxicity induction at lower antigen density.

PURPOSE: Recent studies have revealed that fucose removal from the oligosaccharides of human IgG1 antibodies results in a significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) via improved IgG1 binding to FcgammaRIIIa. In this report, we investigated the relationship between enhanced ADCC and antigen density on target cells using IgG1 antibodies with reduced fucose. EXPERIMENTAL DESIGN: Using EL4 cell-derived transfectants with differential expression levels of exogenous human CC chemokine receptor 4 or human CD20 as target cells, ADCC of fucose variants of chimeric IgG1 antibodies specific for these antigens were measured. We further investigated IgG1 binding to natural killer (NK) cells and NK cell activation during ADCC induction to elucidate the mechanism by which low-fucose IgG1 induces ADCC upon target cells with low antigen expression. RESULTS: Low-fucose IgG1s showed potent ADCC at low antigen densities at which their corresponding high-fucose counterparts could not induce measurable ADCC. The quantitative analysis revealed that fucose depletion could reduce the antigen amount on target cells required for constant degrees of ADCC induction by 10-fold for CC chemokine receptor 4 and 3-fold for CD20. IgG1 binding to NK cells was increased by ligating IgG1 with clustered antigen, especially for low-fucose IgG1. Up-regulation of an activation marker, CD69, on NK cells, particularly the CD56(dim) subset, in the presence of both the antibody and target cells was much greater for the low-fucose antibodies. CONCLUSIONS: Our data showed that fucose removal from IgG1 could reduce the antigen amount required for ADCC induction via efficient recruitment and activation of NK cells.     

Inhibition of effector cell functions in natural killer cell activity (NK) and antibody-dependent cellular cytotoxicity (ADCC) in mice by normal and cancer sera

Effector cells mediating natural killer (NK) cell activity and antibody-dependent cellular cytotoxicity (ADCC) have been receiving considerable interest recently as they may represent an additional cellular defense mechanism against maglinancy. Spleen cells from normal young C3H (MTV+) female mice were found to have relatively high natural killer (NK) activities toward RL 1 and YAC-1 tumor cell lines and high antibody-dependent cell-mediated cytotoxicity (ADCC) activities toward chicken RBC and SB tumor targets. Serum inhibition of NK and ADCC activities was either not demonstrable or only seen at low levels in the young mice. Following injection of a transplantable mammary adenocarcinoma that grew progressively in these mice, both NK and ADCC activities decreased and serummediated inhibition of both NK and ADCC increased as the tumors grew. NK and ADCC activities declined with age in C3H (MTV+) mice, and the serum inhibition of NK and ADCC activities of young mice increased with age. Aging mice, however, which had developed spontaneous mammary adenocarcinoma, although having increased levels of inhibitor against both NK and ADCC activities and decreased NK and ADCC activities of their spleen cells as compared to young normal mice, did not have different levels from those of aging mice which had not developed the mammary tumor. Correlation was poor between the amount of serum inhibitor(s) on NK and ADCC activities. NK versus RL 1 and ADCC for chicken RBC were not correlated, but NK versus YAC-1 and ADCC for the tumor cell line SB appeared to be correlated. Decreasing NK and ADCC and increasing serum inhibitory activity toward these functions appeared to correlate with tumor progression. Sera from transplantable tumor-bearing young C3H mice contained increased amounts of circulating immune complexes, but the amount of circulating immune complexes was only weakly correlated with the serum inhibition of NK activity of spleen cells from young animals by sera from tumor-bearing young mice. These findings indicate that a serum blocking factor(s) that can inhibit natural cell-mediated cytotoxicity or antibody-mediated cytotoxicity in vitro must be considered in analyzing host resistance to tumor growth.     

Trastuzumab activates allogeneic or autologous antibody-dependent cellular cytotoxicity against malignant rhabdoid tumor cells and interleukin-2 augments the cytotoxicity.

PURPOSE: Malignant rhabdoid tumor (MRT) is an early childhood cancer with poor prognosis. Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor-2 (HER-2), has been shown to be effective against breast cancer and other cancers. We investigated the effect of trastuzumab on MRT cell lines. EXPERIMENTAL DESIGN: We examined expression of HER-2 on four MRT cell lines and two tumor tissues by indirect immunofluorescence, flow cytometry, and immunohistochemistry. The effect of trastuzumab against MRT cells was examined by cell growth assay. To observe the antibody-dependent cellular cytotoxicity of effector cells, we examined the cytotoxicity of trastuzumab in combination with allogeneic or autologous human peripheral blood mononuclear cells with and without IL-2 using the chromium release assay. RESULTS: All four MRT cell lines and both MRT tissues expressed HER-2 protein. Trastuzumab alone did not reduce the viability of the MRT cell lines. On the other hand, the cytotoxicity of trastuzumab against each of the MRT cell lines was significantly increased by the presence of allogeneic and autologous peripheral blood mononuclear cells (P < 0.01). There was a strong correlation coefficient (r = 0.825) between HER-2 expression and the cytotoxicity enhanced by trastuzumab. Moreover, trastuzumab in combination with peripheral blood mononuclear cells augmented by interleukin-2 (IL-2) was significantly more cytotoxic than trastuzumab alone or IL-2 alone (P < 0.01). CONCLUSIONS: Our results indicate that (1) trastuzumab can exert antitumor effects on MRT cells by using the antibody-dependent cellular cytotoxicity of effector cells and (2) IL-2 can enhance the cytotoxicity of trastuzumab against MRT cells.     

Differences in resistance of metastatic tumor cells and cells from local tumor growth to cytotoxicity of natural killer cells.

Cells from local tumor growth (L-3LL) were compared to metastatic tumor cells (M-3LL) for their susceptibility to the cytotoxicity of natural killer (NK) cells. M-3LL cells were more resistant in vitro to NK cells from normal spleens than were L-3LL cells. A similar phenomenon of relative resistance of metastatic cells to NK activity was found when L-3LL and M-3LL cells were admixed with normal spleen cells and then inoculated into syngeneic mice. Because hybrid resistance was shown to be based on mechanisms that in principle are similar to mechanisms involved in NK activity, we tested the growth of M-3LL and L-3LL cells in semiallogeneic F1 mice. The in vitro effect of NK cells from semiallogeneic mice on M-3LL and L-3LL cells was tested in parallel. In vitro tests showed that irrespective of the haplotype of the spleen cell donors, L-3LL cells were more susceptible to NK activity than were M-3LL cells. In vivo experiments indicated that whereas M-3LL and L-3LL cells grew similarly in syngeneic recipients, M-3LL cells grew far more in F1 mice than did L-3LL cells. Thus metastatic cells are more resistant to NK activity than are cells of the local tumor growth. This relative resistance may determine, among other factors, the metastatic spread and progression of tumor cells.     

Protective effects of interferon-γ on squamous-cell carcinoma of head and neck targets in antibody-dependent cellular cytotoxicity mediated by human natural killer cells

An in vitro model of antibody-dependent cellular cytotoxicity (ADCC) was established, using squamous-cell carcinoma of the head and neck (SCCHN) targets, human/mouse chimeric monoclonal antibodies (cMAbs) SF-25 and 323/A3 and human peripheral blood mononuclear cells (PBMC). We previously showed that natural killer (NK) cells are the main effector population mediating ADCC in the presence of the cMAbs. ADCC was significantly inhibited by the overnight pre-treatment of SCCHN targets with exogenous interferon-γ (IFN-γ). This inhibition was dose-dependent, reproducible and consistently observed with various SCCHN cell lines. SCCHN cells pre-treated with IFN-γ had a significantly higher expression of intercellular adhesion molecule-1 (ICAM-I) and major histocompatibility complex (MHC) class I antigens compared with untreated target cells. No differences in expression of the SCCHN-associated antigens on these targets or in the formation of NK-SCCHN conjugates were found, using flow cytometry. IFN-γ-pre-treated SCCHN cells were less effective in competing with untreated targets in cold target inhibition assays and in inducing cytokine production from NK cells in co-incubation experiments. Protective effects of IFN-γ on target cell sensitivity to lysis were blocked by pre-treatment of target cells with actinomycin-D or cycloheximide. The susceptibility of the target cells was restored by removal of MHC class I antigens from their surface by acid stripping before ADCC. Our results suggest that the decreased ADCC seen with SCCHN targets pre-treated with IFN-γ is related to post-binding events, possibly altered signaling from targets to effector cells, and requires protein synthesis in the target cells. © 1996 Wiley-Liss, Inc.     

Platelets and fibrinogen facilitate each other in protecting tumor cells from natural killer cytotoxicity

The functions of platelets and fibrinogen in protecting tumor cells from natural killer cytotoxicity have been discussed for more than 20 years. However, their exact roles and relationships in the process are still not clear. In this study, we show that tumor cells prefer to adhere to fibrinogen than to platelets, and fibrinogen can enhance the adhesion of tumor cells to platelets. β₃ integrin plays an important role in the adhesion of B16F10 to platelets enhanced by fibrinogen. In the presence of thrombin, fibrinogen forms dense fibrin(ogen) layers around tumor cells. Tumor cells can induce platelets to aggregate and form thrombin. Platelets, as well as thrombin, can help fibrinogen protect tumor cells from lethal contact with natural killer cells and natural killer cytotoxicity. Hirudin, a specific inhibitor of thrombin, can reverse the effect of platelets on fibrinogen in blocking natural killer cytotoxicity. Our results suggest that fibrinogen helps platelets to adhere to tumor cells, and platelets in turn promote more fibrinogen to aggregate around tumor cells by forming thrombin. They facilitate each other in protecting tumor cells from natural killer cytotoxicity. ( Cancer Sci 2009; 100: 859–865)     

Lapatinib inhibits receptor phosphorylation and cell growth and enhances antibody-dependent cellular cytotoxicity of EGFR- and HER2-overexpressing esophageal cancer cell lines

Lapatinib is a dual tyrosine kinase inhibitor of the EGFR and HER2 tyrosine kinase domains. EGFR is expressed in 33.3% and HER2 in 30.3% of esophageal squamous cell carcinomas (ESCCs). To explore the potential utility of Lapatinib for therapy of ESCC patients, we evaluated the effect of Lapatinib on a panel of ESCC cell lines. EGFR and HER2 expression by the cell lines was established, and the effects of Lapatinib on inhibition of the phosphorylation of HER2, antiproliferative effect, apoptosis-inducing activity and accumulation of HER2 and EGFR on cell surface were evaluated. Additionally, the combined effect of Lapatinib together with Herceptin or Cetuximab on cell-mediated cytotoxicity was evaluated. Lapatinib inhibited HER2 phosphorylation in HER2-overexpressing, HER2 gene amplification positive ESCC cell line. Lapatinib also inhibited cell proliferation, induced apoptosis and caused the surface accumulation of HER2 and EGFR in ESCC cell lines. Addition of Lapatinib increased Herceptin-mediated antibody-dependent cell-mediated cytotoxicity by 15-25% with three ESCC target cell lines. Similarly, Cetuximab-mediated antibody-dependent cell-mediated cytotoxicity also increased by 15-30% in two ESCC cell lines on addition of Lapatinib. Cumulatively, the data indicate that Lapatinib has activity in EGFR- and/or HER2-expressing ESCC cells, and the combination therapy of Lapatinib and Cetuximab/Herceptin is a promising strategy in ESCC.     

Trispecific killer engager 161519 enhances natural killer cell function and provides anti-tumor activity against CD19-positive cancers

Objective:Natural killer (NK) cells have gained considerable attention due to their potential in treating “cold tumors,” and are therefore considered as one of the new strategies for curing cancer, by using worldwide development of their new possibilities and interventions with NK cell-related therapeutic products.Methods:We constructed a trispecific killer engager (TriKE) consisting of anti-CD16, IL-15, and anti-CD19. This TriKE was designed to attract CD19⁺ tumor cells to CD16⁺ NK cells, whereas IL-15 sustained the proliferation, development, and survival of NK cells.Results:Treatment with 161519 TriKE in the presence of CD19⁺ targets upregulated expression of CD69, CD107a, TRAIL, IFN-γ, and TNF-α in NK cells, and significantly improved the proliferation and cytotoxicity of NK cells. NK cells “armed” with 161519 TriKE showed stronger cytolysis against CD19⁺ targets compared with that of “unarmed” NK cells. A preclinical model of B-cell lymphoma in human peripheral blood mononuclear cell-reconstituted xenograft mice showed significant inhibition of tumor growth and prolonged overall survival after treatment with 161519 TriKE, when compared with that in control mice or mice treated with 1619 BiKE. Combined use of IL-2 was a more effective treatment with 1619 BiKE, when compared with that using 161519 TriKE.Conclusions:The newly generated 161519 TriKE enhanced the proliferation, activation, cytokine secretion, and cytotoxicity of NK cells in the presence of CD19⁺ tumor cells. The 161519 TriKE aided inhibition of tumor growth and prolonged the overall survival of murine xenografts, and could be used to treat CD19-positive cancers.     

Toll-like receptor agonists and invariant natural killer T-cells enhance antibody-dependent cell-mediated cytotoxicity (ADCC)

huHMFG-1 (AS1402) is a humanised IgG1 against MUC1, which exerts tumour cell killing through antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Here, we explored the capacity of invariant NKT (iNKT) cells, which are known to activate NK cells, and toll-like receptor (TLR) ligands which activate both iNKT and NK cells, to enhance huHMFG-1-ADCC. Addition of iNKT cells, as well as TLR2/6, 7, 8 and 9 agonists to PBMC improved the efficacy of huHMFG-1. These results suggest that transfer of ex vivo expanded iNKT cells or TLR agonist treatment may improve the efficacy of NK cell-mediated antibody-based tumour immunotherapies.     

Disruption of cell-cell adhesion enhances antibody-dependent cellular cytotoxicity: implications for antibody-based therapeutics of cancer

Resistance to antibody-based anticancer approaches has become of considerable interest because of the rapidly growing clinical use of several different monoclonal antibodies as therapeutic agents, coupled with the recent finding that their efficacy may be attributable in part to their participation in host antibody-dependent cellular cytotoxicity. In this proof-of-concept study, we demonstrate the novel ability of an antiadhesive antibody (SHE78-7), targeted at the potent homophilic cell adhesion molecule E-cadherin, to play a dual role as participant in, and sensitizing agent for, host immune-mediated antitumor activity. SHE78-7 disrupted preformed multicellular aggregates (spheroids) of HT29 colon carcinoma cells both in vitro and in vivo in an ascites tumor xenograft model, but had no direct antitumor effect in vitro. In vivo, however, i.p. injection of SHE78-7 significantly prolonged the survival of nude mice carrying established i.p. HT29 xenografts, most notably when injections were given biweekly. This antitumor effect was dependent on the antiadhesive effect of SHE78-7 and could be effectively recapitulated via treatment with a combination of nondisruptive anti-hMHC-I antibodies, capable of recruiting an F(c)-mediated immune response but ineffective as a monotherapy and antiadhesive F(ab')(2) fragments of SHE78-7. Furthermore, additional therapy experiments using such F(ab')(2) fragments, or mice lacking activating F(c)gammaRIII receptors or inhibitory F(c)gammaRIIB, unequivocally indicated a role for host antibody-dependent cellular cytotoxicity, mediated by F(c)gammaRIII and negatively regulated by F(c)gammaRIIB. Taken together, the results suggest a possible means of improving antibody-based therapies of cancer, namely targeting antigens, selectively expressed or up-regulated by target cancer cells, which mediate cell-cell adhesive functions.