Immunomagnetic selection or irradiation eliminates alloreactive cells but also reduces anti-tumor potential of cytokine-induced killer cells: implications for unmanipulated cytokine-induced killer cell infusion

Background aimsCytokine-induced killer (CIK) cells may offer a novel therapeutic approach for patients with malignancies relapsing after allogeneic stem cell transplantation. Although CIK cells display negligible alloreactivity and cause minimal graft versus-host-disease (GVHD), high CIK cell doses required during relapse may pose a risk for severe GVHD, specifically in the mismatched or haploidentical transplantation setting. Manipulation of CIK cells may reduce risk for GVHD without affecting the anti-tumor potential.MethodsIn this pre-clinical study, we provide a detailed functional comparison of conventional and irradiated, CD56-enriched or T-cell receptor α/β-depleted CIK cells.ResultsIn vitro analysis showed retained anti-leukemic and anti-tumor potential after CIK cell manipulation. Even being sequentially infused into immunodeficient mice grafted with malignant cells, cytotoxic effects were fewest after irradiation but were improved by CD56 enrichment and were best with conventional CIK cells. Hence, considering the proliferative capacity of inoculated malignancies and effector cells, a single dose of conventional CIK cells resulted in prolonged disease-free survival and elimination of rhabdomyosarcoma cells, whereas sequential infusions were needed to achieve comparable results in leukemia-bearing mice. However, this mouse model has limitations: highly effective conventional CIK cells demonstrated both limited xenogenic GVHD and low alloreactive potential in vitro.ConclusionsOur study revealed that conventional CIK cells demonstrate no significant alloreactive potential but provide the strongest anti-tumor efficacy compared with manipulated CIK cells. Conventional CIK cells may therefore be tested in high numbers and short-term intervals in patients with impending relapse even after mismatched transplantation.     

The apoptotic and proliferative fate of cytokine-induced killer cells after redirection to tumor cells with bispecific Ab

BACKGROUND: Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with co-expression of CD3 and CD56 and NK activity. They have recently been evaluated in a phase I/II clinical trial against malignant lymphoma. Bispecific Ab (bsAb) redirect CIK cells to tumor targets, thus enhancing their cytotoxicity. While bsAb may improve T-cell mediated anti-tumor activity, little is known about the fate of effector cells upon redirection to tumor targets using a bsAb. METHODS: Using ex vivo-activated CIK cells, Her2/neu expressing breast and ovarian cell lines and a F(ab')2 Her2/neu x CD3 bsAb, we investigated the anti-tumor activity and the proliferative and apoptotic outcome of CIK cells. RESULTS: When redirected to tumor targets with bsAb, there was a significant increase in anti-tumor activity as well as an increase in both CIK cell proliferation and apoptosis. The addition of agonistic Ab against CD28 did not significantly increase proliferation or apoptosis of CIK cells redirected to CD80- and CD86- tumor targets. To attempt to reduce T-cell apoptosis, we incubated CIK cells in the presence of the pan-caspase inhibitor z-VAD-fmk, which led to a partial reduction in T-cell apoptosis without increasing cellular cytotoxicity. DISCUSSION: bsAb are effective in redirecting activated T cells to tumor targets and such redirection leads to both T-cell proliferation and apoptosis that are not altered by co-stimulation through CD28. Effector cell apoptosis can be reduced by using a caspase inhibitor but this does not increase CIK cell cytotoxicity.     

Coupling programmed cell death 1-positive tumor-infiltrating T cells with anti-programmed cell death 1 antibody improves the efficacy of adoptive T-cell therapy

Background aimsAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has shown great success in clinical trials. Programmed cell death 1 (PD-1)-expressing TILs show high specificity to autologous tumor cells. However, limited therapeutic efficiency is observed as a result of the tumor immune microenvironment (TIME).MethodsCoupling PD-1⁺ ex vivo-derived TILs with a monoclonal antibody against anti-PD-1 (aPD-1) reinvigorated the anti-tumor response of TILs against solid tumor without altering their high tumor targeting ability.ResultsUsing a melanoma-bearing mouse model, PD-1⁺ TILs blocked with aPD-1 (PD-1⁺ TILs-aPD-1) exhibited a high capability for tumor targeting as well as improved anti-tumor response in TIME. Tumor growth was substantially delayed in the mice treated with PD-1⁺ TILs-aPD-1.ConclusionsThe strategy utilizing TIL therapy coupled with immune checkpoint antibodies may extend to other therapeutic targets of ACT.     

BL-01, an Fc-bearing,tetravalent CD20 × CD5 bispecific antibody,redirects multiple immune cells to kill tumors in vitro and in vivo

Background aimsThe authors describe here a novel therapeutic strategy combining a bispecific antibody (bsAb) with cytokine-induced killer (CIK) cells.MethodsThe authors have designed, produced and purified a novel tetravalent IgG1-like CD20 × CD5 bsAb called BL-01. The bsAb is composed of a fused heavy chain and two free light chains that pair correctly to the heavy chain sequences thanks to complementary mutations in the monoclonal antibody 2 CH1/CL sequences.ResultsThe authors show that BL-01 can bind specifically to CD20 and CD5 with an affinity of 4–6 nM, demonstrating correct pairing of two light chains to the fused heavy chain. The CD20 × CD5 BL-01 bsAb has a functional human IgG1 Fc and can induce up to 65% complement-dependent cytotoxicity of a CD20⁺ lymphoma cell line in the presence of human complement, similar to anti-CD20 rituximab. The bsAb also induces significant natural killer cell activation and antibody-dependent cytotoxicity of up to 25% as well as up to 65% phagocytosis by human macrophages in the presence of CD20⁺ tumor cells. The BL-01 bsAb binds to CD20 and CD5 simultaneously and can redirect CIK cells in vitro to kill CD20⁺ targets, increasing the cytotoxicity of CIK cells by about 3-fold. The authors finally show that the CD20 × CD5 BL-01 bsAb synergizes with CIK cells in vivo in controlling tumor growth and prolonging survival of nonobese diabetic/severe combined immunodeficiency mice inoculated with a patient-derived, aggressive diffuse large B-cell lymphoma xenograft.ConclusionsThe authors suggest that the efficacy of bsAb in vivo is due to the combined activation of innate immunity by Fc and redirection of CIK cells to kill the tumor target.     

Adjuvant cytokine-induced killer cell immunotherapy improves long-term survival in patients with stage I–II non-small cell lung cancer after curative surgery

Background aimsNon-small cell lung cancer (NSCLC) remains the most common cancer worldwide, with an annual incidence of around 1.3 million. Surgery represents the standard treatment in early-stage NSCLC when feasible. However, because of cancer recurrence, only approximately 53% of patients with stage I and II NSCLC survive 5 years after radical surgery. The authors performed a retrospective study to investigate the impact of cytokine-induced killer (CIK) cell immunotherapy on the long-term survival of patients with stage I–II NSCLC after curative resection.MethodsFifty-seven patients with NSCLC were included in the study, with 41 and 16 in the control and CIK groups, respectively. Clinical characteristics were compared using a t-test and χ² test. Survival analysis of patients with NSCLC was performed using the Kaplan–Meier method. The phenotypes and anti-tumor functions of CIK cells were evaluated by flow cytometry.ResultsPatients in the CIK group exhibited significantly longer overall survival (OS) and better disease-free survival (DFS) than those in the control group. Subgroup analysis indicated that patients with a higher risk of recurrence benefited more from CIK treatment and attained longer OS and DFS compared with those in the control group. No severe adverse events related to CIK treatment occurred. CIK cells contained a higher proportion of CD3⁺CD56⁺ natural killer (NK) T cells and CD3⁺ and CD8⁺ T cells and a lower proportion of CD3^–CD56⁺ NK cells compared with peripheral blood mononuclear cells. CIK cells exhibited potent tumor-killing ability, with longer contact times with tumor cells and a greater number of cells exposed to tumor cells.ConclusionsThe authors’ data suggest that adjuvant CIK cell therapy is a safe and effective therapeutic strategy for improving OS and DFS in patients with stage I–II NSCLC after curative resection.     

IL-12 enhances efficacy and shortens enrichment time in cytokine-induced killer cell immunotherapy

Cytokine-induced killer (CIK) cells are T cell derived ex vivo expanded cells with both NK and T cell properties. They exhibit potent anti-tumor efficacy against various malignancies in preclinical models and have proven safe and effective in clinical studies. We combined CIK cell adoptive immunotherapy with IL-12 cytokine immunotherapy in an immunocompetent preclinical breast cancer model. Combining CIK cells with IL-12 increased anti-tumor efficacy in vivo compared to either therapy alone. Combination led to full tumor remission and long-term protection in 75% of animals. IL-12 treatment sharply increased the anti-tumor efficacy of short-term cultured CIK cells that exhibited no therapeutic effect alone. Bioluminescence imaging based in vitro cytotoxicity and in vivo homing assays revealed that short-term cultured CIK cells exhibit full cytotoxicity in vitro, but display different tumor homing properties than fully expanded CIK cells in vivo. Our data suggest that short-term cultured CIK cells can be “educated” in vivo, producing fully expanded CIK cells upon IL-12 administration with anti-tumor efficacy in a mouse model. Our findings demonstrate the potential to improve current CIK cell-based immunotherapy by increasing efficacy and shortening ex vivo expansion time. This holds promise for a highly efficacious cancer therapy utilizing synergistic effects of cytokine and cellular immunotherapy.     

Epstein-Barr virus–specific cytokine-induced killer cells for treatment of Epstein-Barr virus–related malignant lymphoma

Background

Prolonged immunosuppression or delayed T-cell recovery may favor Epstein-Barr virus (EBV) infection or reactivation after allogeneic hematopoietic stem cell transplantation (HSCT), which can lead to post-transplant lymphoproliferative disease (PTLD) and high-grade malignant B-cell lymphoma. Cytokine-induced killer (CIK) cells with dual specific anti-tumor and virus-specific cellular immunity may be applied in this context.

Can the dual-functional capability of CIK cells be used to improve antitumor effects?

Cytokine-induced killer (CIK) cells, which display both potent anti-tumor ability of T lymphocytes and non-major histocompatibility complex (MHC) restricted killing tumor cells capacity of natural killer (NK) cells are capable of recognizing and lysing a broad array of tumor targets. They have begun to be used in clinical care with good prospects for treatment success. CIK cells are a heterogeneous cell population that contain CD3⁺CD56⁺ cells, CD3⁻CD56⁺ natural killer (NK) cells and CD3⁺CD56⁻ T cells on which much attention has been focused. This review will summarize the connections and differences among CD3⁺CD56⁺CIK cells, CD3⁻CD56⁺ NK cells and CD3⁺CD56⁻ T cells in the following aspects: the main cell surface molecule, killing mechanism, and clinical applications so that treatment with CIK cells can be optimized and further to enhance the antitumor effect.     

Long-term clinical efficacy of cytokine-induced killer cell-based immunotherapy in early-stage esophageal squamous cell carcinoma

Background aimsIn this retrospective clinical study, the authors investigated the impact of cytokine-induced killer (CIK) cell-based immunotherapies on the long-term survival of patients with esophageal squamous cell carcinoma (ESCC).MethodsA total of 87 patients with ESCC who received comprehensive treatment were enrolled in the study. Of these patients, 43 were in the control group and 44 were in the CIK treatment group. Flow cytometry analysis was performed to detect the phenotype and anti-tumor function of CIK cells. Clinical characteristics were compared between these two groups, and the survival estimates of ESCC patients were determined using Kaplan–Meier analysis.ResultsCIK cells contained a high proportion of the main functional fraction (CD3⁺CD56⁺ group) and exhibited a strong killing ability for esophageal cancer cells in vitro. Importantly, overall survival (OS) and progression-free survival (PFS) were significantly higher in the CIK group than in the control group in early-stage ESCC. However, patients with advanced-stage ESCC did not benefit from CIK cell-based therapy in terms of OS and PFS compared with the control group.ConclusionsThese results demonstrate that CIK cells combined with conventional treatments potentially prolong long-term survival of patients and may serve as a combined therapeutic approach for the treatment of early-stage ESCC.     

过继免疫治疗中DC-CIK的研究进展

目前,免疫细胞生物学和免疫分子生物学发展迅猛,由于其具备低毒性和高效率的特性,肿瘤免疫治疗在恶性肿瘤治疗中所起的作用引起了学者们的广泛关注,其中细胞介导的过继免疫治疗为当前研究的热点之一。过继免疫治疗(adoptive cellular immunotherapy,ACI)是目前恶性肿瘤治疗的新方向,它通过向细胞免疫功能低下者回输具有抗肿瘤活性的免疫细胞,直接杀伤或间接杀伤肿瘤细胞,使其获得抗肿瘤免疫力。树突状细胞(Dendritic cell DC)是专职抗原递呈细胞(antigen presenting cell APC)之一,在机体免疫应答的启始、调节、维持中发挥核心作用。细胞因子诱导的杀伤(cytokine induced killer CIK)细胞具有高效的MHC非限制性溶瘤活性,具有极其广泛的杀瘤范围。近年来,国内外大量研究表明,联合培养的DC-CIK抗肿瘤活性提升明显,患者预后生存期延长,效果显著。本文就DC与CIK生物学特点及抗肿瘤作用予以简要综述。     

An NK cell line (NK92-41BB) expressing high levels of granzyme is engineered to express the high affinity chimeric genes CD16/CAR

Natural killer (NK) cells are known to play a role in mediating innate immunity and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) based on the reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, devoid of CD16 and derived from a lymphoma patient, has been well characterized. The adoptive transfer of irradiated NK-92 cells demonstrated safety and showed preliminary evidence of clinical benefit for cancer patients. The molecules 41BB and CD3 are commonly used as stimulators in the CAR structure, and their expression in NK cells can promote the activation of NK cells, leading to the enhanced perforin- and granzyme-mediated lysis of tumor cells. This study showed that genetically modified NK-92 cells combined with antibody-mediated ADCC using rituximab and trastuzumab monoclonal antibodies lysed tumor cells more efficient than the NK-92 cell lines. It also showed that the anti-tumor activity of chimeric stimulator molecules of the CAR-modified CD16 receptor was stronger than that of CD16 (allotype V158). These studies provide a rationale for the use of genetically modified NK-92 cells in combination with IgG1 anti-tumor monoclonal antibodies. We also provide a rationale for the chimeric modified CD16 receptor that can improve the anti-tumor effect of NK92 cells via ADCC.

     

Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

Both adoptive immunotherapy and gene therapy hold a great promise for treatment of malignancies. However, these strategies exhibit limited anti-tumor activity, when they are used alone. In this study, we explore whether combination of cytokine-induced killer (CIK) adoptive immunotherapy with oncolytic adenovirus-mediated transfer of human interleukin-12 (hIL-12) gene induce the enhanced antitumor potency. Our results showed that oncolytic adenovirus carrying hIL-12 (AdCN205-IL12) could produce high levels of hIL-12 in liver cancer cells, as compared with replication-defective adenovirus expressing hIL-12 (Ad-IL12). AdCN205-IL12 could specifically induce cytotoxocity to liver cancer cells. Combination of CIK cells with AdCN205-IL12 could induce higher antitumor activity to liver cancer cells in vitro than that induced by either CIK or AdCN205-IL12 alone, or combination of CIK and control vector AdCN205-GFP. Furthermore, treatment of the established liver tumors with the combined therapy of CIK cells and AdCN205-IL12 resulted in tumor regression and long-term survival. High level expression of hIL-12 in tumor tissues could increase traffic of CIK cells to tumor tissues and enhance their antitumor activities. Our study provides a novel strategy for the therapy of cancer by the combination of CIK adoptive immunotherapy with oncolytic adenovirus-mediated transfer of immune stimulatory molecule hIL-12.     

Enhanced antitumor effects of DC-activated CIKs to chemotherapy treatment in a single cohort of advanced non-small-cell lung cancer patients

Cytokine-induced killer (CIK) cells show cytolytic activity against tumor. The purpose of this study was to evaluate the antitumor effect of dendritic cell (DC)-activated CIK cells in vitro and their clinical efficacy of DC-activated CIK cells in combination with chemotherapy (abbreviated below as chemotherapy plus DC + CIK) in patients with advanced non-small-cell lung cancer (NSCLC). A paired study was performed between 61 patients treated with chemotherapy alone (group 1) and 61 patients treated with chemotherapy plus DC + CIK cells (group 2). In group 2, 36 patients with adenocarcinoma and 18 patients with squamous cell carcinoma were analyzed for the survival rate. Compared to unstimulated CIK cells, DC-activated CIK cells significantly enhanced antitumor activity, increased the ratio of CD3⁺CD56⁺ cells, promoted cell proliferation and lessened cell apoptosis. In the paired study, the 1- and 2-year overall survival rates in group 2 were 57.2 and 27.0 %, which were significantly higher than that of group 1 (37.3 and 10.1 %) (P < 0.05). There was no significant difference in the survival rate between the adenocarcinoma and squamous carcinoma patients in group 2. The present study suggests that DC-activated CIK cell has enhanced antitumor effects and chemotherapy plus DC + CIK cells improved the clinical outcomes of chemotherapy for advanced NSCLC patients.     

Pan-Bcl-2 Inhibitor AT-101 Enhances Tumor Cell Killing by EGFR Targeted T Cells

Pancreatic cancer is a deadly disease and has the worst prognosis among almost all cancers and is in dire need of new and improved therapeutic strategies. Conditioning of tumor cells with chemotherapeutic drug has been shown to enhance the anti-tumor effects of cancer vaccines and adoptive cell therapy. In this study, we investigated the immunomodulatory effects of pan-Bcl-2 inhibitor AT-101 on pancreatic cancer (PC) cell cytotoxicity by activated T cells (ATC). The effects of AT-101 on cytotoxicity, early apoptosis, and Granzyme B (GrzB) and IFN-γ signaling pathways were evaluated during EGFR bispecific antibody armed ATC (aATC)-mediated killing of L3.6pl and MiaPaCa-2 PC cells pre-sensitized with AT-101. We found that pretreatment of tumor cells with AT-101 enhanced susceptibility of L3.6pl and MiaPaCa-2 tumor cells to ATC and aATC-mediated cytotoxicity, which was in part mediated via enhanced release of cytolytic granule GrzB from ATC and aATC. AT-101-sensitized L3.6pl cells showed up-regulation of IFN-γ-mediated induction in the phosphorylation of Ser⁷²⁷-Stat1 (pS⁷²⁷-Stat1), and IFN-γ induced dephosphorylation of phospho-Tyr⁷⁰⁵-Stat3 (pY⁷⁰⁵-Stat3). Priming (conditioning) of PC cells with AT-101 can significantly enhance the anti-tumor activity of EGFRBi armed ATC through increased IFN-γ induced activation of pS⁷²⁷-Stat1 and inhibition of pY⁷⁰⁵-Stat3 phosphorylation, and resulting in increased ratio of pro-apoptotic to anti-apoptotic proteins. Our results verify enhanced cytotoxicity after a novel chemotherapy conditioning strategy against PC that warrants further in vivo and clinical investigations.     

Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells

Cytokine-Induced killer (CIK) cells are raising growing interest in cellular antitumor therapy, as they can be easily expanded with a straightforward and inexpensive protocol, and are safe requiring only GMP-grade cytokines to obtain very high amounts of cytotoxic cells. CIK cells do not need antigen-specific stimuli to be activated and proliferate, as they recognize and destroy tumor cells in an HLA-independent fashion through the engagement of NKG2D. In several preclinical studies and clinical trials, CIK cells showed a reduced alloreactivity compared to conventional T cells, even when challenged across HLA-barriers; only in a few patients, a mild GVHD occurred after treatment with allogeneic CIK cells. Additionally, their antitumor activity can be redirected and further improved with chimeric antigen receptors, clinical-grade monoclonal antibodies or immune checkpoint inhibitors. The evidence obtained from a growing body of literature support CIK cells as a very promising cell population for adoptive immunotherapy. In this review, all these aspects will be addressed with a particular emphasis on the role of the cytokines involved in CIK cell generation, expansion and functionalization.     

CD86 Is an Activation Receptor for NK Cell Cytotoxicity against Tumor Cells

CTLA4Ig has been successfully used in the clinic for suppression of T cell activation. However, patients treated with CTLA4Ig experienced reduced incidence of tumors than predicted, but the underlying mechanism remains unknown. In this paper, we showed that brief administration of CTLA4Ig significantly reduced tumor metastasis and prolonged the survival of host mice bearing B16 melanoma. Depletion of NK cells prior to CTLA4Ig administration eliminated the CTLA4Ig-mediated anti-tumor activity. CTLA4Ig enhanced NK cell cytotoxicity to tumor cells via up-regulation of NK cell effecter molecules CD107a and perforin in vivo. In addition, we demonstrated that, upon activation, NK cells could significantly increase the expression of CD86 both in vitro and in vivo, and ligation of CD86 with CTLA4Ig significantly increased the ability of NK cells to kill tumor cells. Furthermore, a human NK cell line that expressed high level of CD86 was directly activated by CTLA4Ig so that killing of tumor targets was enhanced; this enhanced killing could be inhibited by blocking CD86. Our findings uncover a novel function of CTLA4Ig in tumor immunity and suggest that CD86 on NK cells is an activating receptor and closely involved in the CTLA4Ig-mediated anti-tumor response.     

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon α and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells

 Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon γ (IFNγ), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3⁺CD56⁺ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNα but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis^y antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. Received: 9 December 1999 / Accepted: 18 May 2000     

耗竭性T细胞在肿瘤中的作用

耗竭性T细胞(exhausted T cells)是一群效应功能减弱,持续表达抑制性受体的T细胞,在肿瘤中表现为T细胞功能缺陷状态,主要特征为一系列抑制性受体表达增加及细胞因子分泌减少。耗竭性T细胞主要通过细胞表面的抑制性分子,细胞因子和免疫调节细胞类型改变等参与肿瘤免疫负调控,从而引起肿瘤免疫逃逸。而T细胞耗竭状态并非不可逆转,应用相应单克隆抗体靶向免疫调控点可以有效逆转耗竭性T细胞,恢复机体抗肿瘤免疫反应,提高肿瘤控制率。因此,通过逆转肿瘤患者体内的耗竭性T细胞可能是肿瘤免疫治疗的新途径之一。     

Enhanced Gene Delivery and Expression in Human Hepatocellular Carcinoma Cells by Cationic Immunoliposomes

Abstract

A targeted vector allowing enhanced gene transfer to human hepatocellular carcinoma (HCC¹) cells in vitro was developed using cationic liposomes covalently conjugated with the mAb AF-20. This high affinity antibody recognizes a rapidly internalized 180 kDa cell surface glycoprotein which is abundantly expressed on the surface of human HCC and other cancer cells. Quantitative binding analysis of liposomes with target cells by flow cytometry showed specific association of mAb-targeted liposomes with human HCC cells. Using mAb-targeted cationic liposomes containing 20% DOTAP, in the presence or absence of serum, gene expression in HuH-7 cells was enhanced up to 40-fold as compared to liposomes conjugated with an isotype-matched non-relevant control antibody. Transfection specificity was not observed in a control cell line that does not express the antigen recognized by mAb AF-20. This study demonstrates that cationic liposome formulations can be targeted with monoclonal antibodies (mAbs) to enhance specific in vitro gene delivery and expression in the presence or absence of serum.