Antibody-mediated Cell Cytotoxicity Research Articles

Abstract LB056: IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in solid tumors

Abstract Many solid tumors, such as colorectal cancer (CRC) and lung cancer, are dependent on epidermal growth factor receptor (EGFR) pathway for tumor growth. Antibodies that inhibit EGFR signaling, such as Cetuximab, show clinical efficacy but are associated with on-target toxicities that potentially limit the drug efficacy. To enhance the EGFR signaling inhibition and reduce on-target toxicities, we performed bioinformatics analysis for EGFR/TAA co-expression. We found that B7-H3, a member of the B7 family of immune checkpoint proteins, is broadly expressed in many solid tumors and their expression correlates with EGFR expression in multiple cancers, including lung cancer, head and neck cancer, pancreatic cancer and esophageal cancer. Using Innovent’s proprietary Innobody platform, we developed a bispecific antibody, IBI334, against B7-H3 and EGFR. IBI334 has an EGFR arm for signal blockage and is coupled with a fine-tuned B7-H3 arm with optimal affinity and binding domain. With the aid of B7-H3, IBI334 showed enhanced EGFR signal inhibition than Zalutumumab (EGFR mAb) and Amivantamab (c-met/EGFR bsAb). In addition, IBI334 is afucosylated to enhance its antibody-mediated cell cytotoxicity (ADCC) effects as compared to wild type antibody. IBI334 showed significantly better in vivo efficacy than Amivantamab in NCI-H358 (bronchioalveolar carcinoma with KRAS-G12C mutation), NCI-H292 (lymph node metastasis of a pulmonary mucoepidermoid carcinoma), SK-MES1 (lung cancer) and SK-MES1-LTC (lung cancer with EGFR L858R, T790M and C797S mutations) xenograft models and comparable efficacy in NCI-H322-EGFRexon20ins (lung cancer) model. Cetuximab, an EGFR monoclonal antibody, had high on-target toxicity with 50% early mortalities in cynomolgus monkeys at 120/75 mg/kg/week. With the careful design of B7-H3/EGFR bispecific antibody, IBI334 ameliorated the on-target toxicities and showed only slight hardening of the skin at the injection site at 120 mg/kg/week. As a result, IBI334 has a large therapeutic window of >200 folds. In conclusion, IBI334 is a potent B7-H3/EGFR bispecific antibody with broad application in many EGFR-driven solid tumors. The large therapeutic window enables safe and effective cancer treatment at high exposure levels. Citation Format: Jian Guan, Shuaixiang Zhou, Weiwei Wu, Tianyu Zhu, Lei Cao, Ming Wu, Ninghuan Li, Fenggen Fu, Zhengguan Liao, Shuming Lin, Zeyu Liu, Mengjia Zhu, Nana Luo, Ying Zhang, Tiong Sun Chia, Bingliang Chen, Kaijie He. IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB056.

881 Enhanced antibody-mediated phagocytosis and antibody-mediated cell cytotoxicity using tetravalent, bispecific innate cell engagers (ICE®) in 3D spheroids

BackgroundThe redirected optimized cell platform (ROCK®) enables the generation of customizable innate cell engagers (ICE®) of varying valency, affinity, and pharmacokinetic profiles. Preclinical and clinical studies have demonstrated the advantage and unique features of this first-in-class ICE® antibodies across a multitude of cancers and its differentiation to monoclonal antibodies. ICE® are tetravalent, bispecific antibodies that bivalently bind to a unique epitope on CD16A, which is selectively expressed on natural killer (NK) cells and macrophages, while the other domains target a tumor antigen. In addition to promoting antibody-dependent cellular cytotoxicity (ADCC) of NK cells, ICE® can also promote tumor targeting of macrophages eventually inducing antibody-dependent cellular phagocytosis (ADCP).MethodsADCP and ADCC assays were performed using monocyte-differentiated macrophages and NK cells derived from healthy donor PBMCs. Target tumor lines and patient-derived xenograft line-derived spheroids were labelled and co-cultured with macrophages or NK cells. Live-cell imaging (IncuCyte®) was used to measure ADCP and ADCC events.ResultsWe show that ICE® molecules can enhance ADCP of tumor cells mediated by various functional/phenotypic subsets of macrophages derived from in vitro differentiation of human monocytes. ICE®-induced ADCP of tumor target cells was seen across different macrophage subtypes (M1 and M2). We further investigated the expression of immune-suppressive checkpoint programmed death-ligand 1 (PD-L1) on macrophages upon ICE® treatment that could be a key anti-tumor molecule within the suppressive tumor microenvironment. Based on patient-derived xenograft line-derived spheroids (3D) generated from primary tumor samples of patients suffering from various malignancies, we could demonstrate robust ADCC and ADCP mediated by NK cells and macrophages, respectively.ConclusionsICE® molecules are able to mount robust NK cell- and macrophage-mediated anti-tumoral innate immune responses. This combined immune activity has the potential to not only fight tumor cells directly but also to initiate a full immune response comprised of innate and adaptive components of the immune system.

EXTH-43. GENERATION OF A B-CELL-BASED VACCINE FOR THE TREATMENT OF GLIOBLASTOMA

Abstract Immunotherapy has revolutionized the treatment of many tumors. However, most glioblastoma (GBM) patients have not, so far, benefited from such successes. With the goal of exploring ways to boost anti-GBM immunity, we developed a B-cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism and IFNg stimulation. BVaxmigrate to key secondary lymphoid organs and are proficient at antigen cross-presentation, which promotes both the survival and functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. This treatment elicited immunologic memory that prevented the growth of new tumors upon subsequent re-injection in cured mice. GBM patient-derived BVax were successful in activating autologous CD8+ T cells; these T cells showed a strong ability to kill autologous glioma cells. In addition to the role in activating CD8+ T cells, BVax produce tumor-specific antibodies able to control tumor growth via antibody-mediated cell cytotoxicity. In conclusion, BVax tackles GBM immunosurveillance escape by using both cellular (CD8+ T-cell activation) and humoral (anti-tumor antibody production) immunity. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic.

P-D8 New insights on the human anti-HIV-1 Env antibody-mediated cell cytotoxicity (ADCC) against HIV-1 virus: Allosteric regulation of FcRs binding upon antigen engagement

HIV-1 vaccine field is rapidly evolving and a deeper knowledge of the mechanisms of defense against HIV-1 infection is needed. In this regard, several studies highlighted the relevance of the antibody-mediated cell cytotoxicity (ADCC) in the context of HIV-1 infection, linking Fc-effector functions to protection against HIV-1 acquisition. The current model for ADCC activation is based on the concept that antibodies, bound to their respective antigens on the surface of HIV-1 sensitized cells, form aggregates which engage the FcRs, activating the effector cells. Our aim is to understand the molecular basis of ADCC and to identify the critical factors that lead to the triggering of the cytotoxicity against HIV-1 virus. Utilizing multiple approaches, such as ELISA, FCS (Fluorescence Correlation Spectroscopy), H/DX MS (Hydrogen/Deuterium Exchange Mass Spectroscopy) and crystallography, we studied the very first step of ADCC activation: the monovalent binding of viral antigen. Here, we demonstrate an allosteric regulation in anti-HIV gp120 Cluster A mAbs resulting from immune complex (IC) formation with a monomeric gp120-CD4 chimera antigen. We established that IC formation dramatically increases the efficiency of Ab interaction to low affinity FcRs compared to free IgG, impacting, in turn, the activation of the cytotoxicity against HIV-1 positive targets. In conclusion, we believe that monomeric antigen-antibody IC formation might be the very first step of HIV-1-specific ADCC triggering that likely precedes the IgG aggregation required for Fc receptors binding and in turn, effector cell activation. This might be a mechanism that enables the fine tuning of Fc-effector functions in vaccine regimens or HIV-1 passive treatments.

In Vitro Neutralization Is Not Predictive of Prophylactic Efficacy of Broadly Neutralizing Monoclonal Antibodies CR6261 and CR9114 against Lethal H2 Influenza Virus Challenge in Mice.

Influenza viruses of the H1N1, H2N2, and H3N2 subtypes have caused previous pandemics. H2 influenza viruses represent a pandemic threat due to continued circulation in wild birds and limited immunity in the human population. In the event of a pandemic, antiviral agents are the mainstay for treatment, but broadly neutralizing antibodies (bNAbs) may be a viable alternative for short-term prophylaxis or treatment. The hemagglutinin stem binding bNAbs CR6261 and CR9114 have been shown to protect mice from severe disease following challenge with H1N1 and H5N1 and with H1N1, H3N2, and influenza B viruses, respectively. Early studies with CR6261 and CR9114 showed weak in vitro activity against human H2 influenza viruses, but the in vivo efficacy against H2 viruses is unknown. Therefore, we evaluated these antibodies against human- and animal-origin H2 viruses A/Ann Arbor/6/1960 (H2N2) (AA60) and A/swine/MO/4296424/06 (H2N3) (Sw06). In vitro, CR6261 neutralized both H2 viruses, while CR9114 only neutralized Sw06. To evaluate prophylactic efficacy, mice were given CR6261 or CR9114 and intranasally challenged 24 h later with lethal doses of AA60 or Sw06. Both antibodies reduced mortality, weight loss, airway inflammation, and pulmonary viral load. Using engineered bNAb variants, antibody-mediated cell cytotoxicity reporter assays, and Fcγ receptor-deficient (Fcer1g-/-) mice, we show that the in vivo efficacy of CR9114 against AA60 is mediated by Fcγ receptor-dependent mechanisms. Collectively, these findings demonstrate the in vivo efficacy of CR6261 and CR9114 against H2 viruses and emphasize the need for in vivo evaluation of bNAbs.IMPORTANCE bNAbs represent a strategy to prevent or treat infection by a wide range of influenza viruses. The evaluation of these antibodies against H2 viruses is important because H2 viruses caused a pandemic in 1957 and could cross into humans again. We demonstrate that CR6261 and CR9114 are effective against infection with H2 viruses of both human and animal origin in mice, despite the finding that CR9114 did not display in vitro neutralizing activity against the human H2 virus. These findings emphasize the importance of in vivo evaluation and testing of bNAbs.

Abstract B179: Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers

Abstract Different components of the tumor microenvironment hamper the immune response against cancer cells and are a critical impediment for the success of cancer immunotherapy. Among these, the accumulation of regulatory T cells (Treg) and the reduction of the effector T cells (Teff)/Treg ratio favors tumor progression and is associated with worse prognosis in several human cancers. Treg elimination in the tumor microenvironment can be achieved through antibodies that target molecules expressed preferentially in Treg and that induce antibody-mediated cell cytotoxicity (ADCC). CTLA-4 is highly expressed on tumor-infiltrating Treg and therapy with anti-CTLA-4 has been effective for cancer treatment in murine models and in humans. In mice, we and others have shown that anti-CTLA-4 depletes Treg in the tumor but not in peripheral lymphoid organs. This site specificity in mice is given by the expression of the Fcγ-receptor IV (FcγRIV) in myeloid cells present in the tumor microenvironment but not in the periphery. However, the role of FcγRs in the effectiveness of anti-CTLA-4 therapy or therapies with other immunomodulatory antibodies in humans is not known. We therefore investigated the density of targets of clinically relevant immunomodulatory antibodies in different subpopulations of tumor-infiltrating immune cells as well as the expression of FcγR subtypes in murine tumor models. Because of the inter-species differences in the repertoire and the cellular distribution of FcγRs, we also studied this in humanized-FcγR (huFcγR) mice, in which there is no expression of murine FcγRs but of their human homologues. We compared this with samples of human melanoma and observed surprisingly similar expression profiles of both FcγRs and targets of immunomodulatory antibodies, including CTLA-4. We then asked whether anti-CTLA-4 would also lead to selective Treg depletion in the tumor microenvironment in huFcγR mice. Using an antibody targeting mCTLA-4 with a human IgG1 isotype, we observed that was the case. Furthermore, mutation of the Fc region of the antibody to enhance its affinity to activating huFcγRs resulted in more effective selective Treg depletion in the tumor. These findings are not only relevant for CTLA-4 but also for other targets expressed preferentially on Treg instead of Teff and must be taken into account when designing immunomodulatory antibodies for clinical use. Citation Format: Frederick Arce Vargas, Andrew J. Furness, Marta H. Lesko, Martin Pule, Jeffrey V. Ravetch, Karl S. Peggs, Sergio A. Quezada. Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B179.

Abstract 2473: KIR3DL2 is a novel target for antibody-therapy of T cell lymphomas

Abstract Intro: The killer immunoglobulin-like receptor, three domains, long cytoplasmic tail 2 (KIR3DL2) is a transmembrane glycoprotein expressed by natural killer (NK) cells and subsets of T cells. On NK cells, KIRD3L2 modulates NK cell activation by transducing inhibitory signals upon ligation by human leukocyte antigen (HLA) class I molecules. Interestingly, KIR3DL2 is also expressed on several subtypes of T cell lymphomas/leukemias, such as Sézary Syndrome and transformed Mycosis Fungoides. This expression pattern provides a promising target for antibody-based therapy. Recently, antibody-based therapies have been shown to synergize with co-stimulatory immunomodulation via agonistic immunotherapeutic antibodies, such as anti-CD137. Methods: We evaluated the efficacy of an anti-KIR3DL2 monoclonal antibody, IPH4102 (Innate Pharma S.A.), in inducing effector cell cytotoxicity of KIR3DL2+ tumors. In vitro experiments utilized primary samples and three KIR3DL2+ cell lines: HUT78, a Sézary cell line with constitutive KIR3DL2 expression; a RAJI-KIR3DL2 high-expression sub clone with ∼28,000 KIR3DL2 antigen sites per cell; and a RAJI-KIR3DL2 low-expression sub clone with ∼ 6,000 antigens sites per cell. All antibody-mediated cell cytotoxicity (ADCC) experiments used 7-AAD and Annexin-V to identify apoptotic cells. Results: In a CD107a /IFN-γ assay, NK cells co-cultured with IPH4102-coated HUT78 cells demonstrated increased activation profiles relative to NK cells co-cultured with untreated HUT78 cells. Activated NK cells displayed elevated expression of CD137, OX40, and CD69 and produced more CD107a and IFN-γ. In allo-ADCC assays using HUT78 cells at varying target to effector ratios, IPH4102 treatment induced more target apoptosis than treatment with positive control anti-CD52 alemtuzumab. In additional allo-ADCC assays, RAJI-KIR3DL2 high-expression tumors demonstrated more apoptosis than RAJI-KIR3DL2 low-expression tumors. The IPH4102-mediated cytotoxicity could be enhanced by the concurrent administration of an agonistic anti-CD137 monoclonal antibody. In ADCC assays of primary Sézary cells with autologous NK cells, we confirmed the upregulation of KIR3DL2 and observed enhanced NK cytotoxicity following tumor treatment with IPH4102. Again, the combination of anti-KIR3DL2 and anti-CD137 therapy proved synergistic leading to increased tumor eradication relative to either therapy alone. Conclusion: Our findings support a role for anti-KIR3DL2 monoclonal antibodies in the treatment of KIR3DL2+ tumors. IPH4102 effectively increased ADCC of KIR3DL2+ tumors by NK cells. The addition of agonistic anti-CD137 antibodies will further augment the anti-tumor efficacy of this therapeutic strategy. Citation Format: Cariad Chester, Sean Lim, Anne Marie-Cardine, Naren Rajasekaran, Hélène Sicard, Youn Kim, Holbrook Kohrt. KIR3DL2 is a novel target for antibody-therapy of T cell lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2473. doi:10.1158/1538-7445.AM2015-2473

Fcγ-receptor tag team boosts anti-tumor immunity

Anti-tumor monoclonal antibodies eliminate tumor cells through different mechanisms including antibody-mediated cell cytotoxicity and generation of long-term anti-tumor T cell responses. In a recent publication, DiLillo and Ravetch demonstrate how this vaccinal effect is mediated by engagement of Fcγ receptors expressed on antigen-presenting cells.

Targeting B cells in severe thrombotic thrombocytopenic purpura–a road to cure?*

Targeting B cells in severe thrombotic thrombocytopenic purpura–a road to cure?*

Ofatumumab for newly diagnosed and relapsed/refractory chronic lymphocytic leukemia

Monoclonal antibodies have become an increasingly utilized treatment option for many hematological malignancies, including chronic lymphocytic leukemia (CLL). Ofatumumab is a second-generation fully human anti-CD20 monoclonal antibody that binds to the small extracellular loop of CD20, thereby producing complement-dependent cell lysis and antibody-mediated cell cytotoxicity in cells expressing CD20. Ofatumumab has shown efficacy in the treatment for relapsed or refractory CLL. This success has resulted in the recent US FDA approval of ofatumumab for the treatment of CLL in patients who are refractory to fludarabine and alemtuzumab-based regimens. Major side effects of ofatumumab include infusion reactions, neutropenia and increased risk for infection. This article provides an overview of the current data supporting the use of ofatumumab for CLL and projects the future role of ofatumumab as monotherapy and combination therapy.

Immunohistochemical analysis on the expressions of maturation associated antigens, Fcγ receptors and CD14 in normal and diseased human liver macrophages

The functional heterogeneity of human liver macrophages was analyzed by utilizing monoclonal antibodies (MoAb) that recognize antigens specific for the monocyte-macrophage lineage (Mo/Mac). Most liver macrophages were positive for CD68, while there were few mature macrophages detected by 25-F9. Most liver macrophages in the normal liver were negative for CD14, a receptor for lipopolysaccharide (LPS) and LPS binding protein (LBP) complexes. Liver macrophages in liver diseases were activated to express CD14 at varying degrees and were involved in the clearance of LPS-LBP complexes. Fcγ RI, a receptor for monomeric IgG that is involved in antibody-mediated cell cytotoxicity (ADCC), was negative in the normal liver, but was expressed in liver macrophages at inflammatory sites such as piecemeal and focal necrosis in diseased livers. These results suggest that liver macrophages are heterogeneous in maturation and function.

Immunohistochemical phenotyping of liver macrophages in normal and diseased human liver

The phenotypical heterogeneity of human liver macrophages was analyzed with monoclonal antibodies that recognize antigens specific for the monocyte-macrophage lineage. Most liver macrophages in normal and diseased liver were positive for CD68, whereas fewer matured macrophages were detected by 25-F9. Comparative staining of mirror sections revealed some to be doubly positive and others to be singly CD68 positive. Quantitative analysis confirmed the difference, suggesting heterogeneity of maturation in liver macrophages. Most liver macrophages in the normal liver were negative for CD14, a receptor for lipopolysaccharide and lipopolysaccharide-binding protein complexes. Liver macrophages in liver diseases were activated to express CD14 at varying degrees and were involved in the clearance of lipopolysaccharide-lipopolysaccharide-binding protein complexes. Fc gamma RI, a receptor for monomeric IgG that is involved in antibody-mediated cell cytotoxicity, was negative in the normal liver, but was expressed in liver macrophages at inflammatory sites (e.g., in piecemeal and focal necrosis) in diseased livers. Fc gamma RII was expressed in most liver macrophages, as well as in sinusoidal endothelial cells; Fc gamma RIII was expressed in a smaller number of liver macrophages. Expression of Fc gamma RII and Fc gamma RIII was increased in chronic active hepatitis. These results suggest that liver macrophages are heterogeneous in maturation and function and that they are activated in liver diseases as shown by the novel expression of CD14 and Fc gamma RI. The restricted expression of Fc gamma RI indicates that Fc gamma RI-positive macrophages, in cooperation with cytotoxic T lymphocytes, may play an important role in liver cell injury through antibody-mediated cell cytotoxicity.

Immunohistochemical phenotyping of liver macrophages in normal and diseased human liver

The phenotypical heterogeneity of human liver macrophages was analyzed with monoclonal antibodies that recognize antigens specific for the monocytemacrophage lineage. Most liver macrophages in normal and diseased liver were positive for CD68, whereas fewer matured macrophages were detected by 25–F9. Comparative staining of mirror sections revealed some to be doubly positive and others to be singly CD68 positive. Quantitative analysis confirmed the difference, suggesting heterogeneity of maturation in liver macrophages. Most liver macrophages in the normal liver were negative for CD14, a receptor for lipopolysaccharide and lipopolysaccharide-binding protein complexes. Liver macrophages in liver diseases were activated to express CD14 at varying degrees and were involved in the clearance of lipopolysaccharide-lipopolysaccharide-binding protein complexes. FcγRI, a receptor for monomeric IgG that is involved in antibody-mediated cell cytotoxicity, was negative in the normal liver, but was expressed in liver macrophages at inflammatory sites (e.g., in piecemeal and focal necrosis) in diseased livers. FcγRII was expressed in most liver macrophages, as well as in sinusoidal endothelial cells; FcγRIII was expressed in a smaller number of liver macrophages. Expression of FcγRIII and FcγRIII was increased in chronic active hepatitis. These results suggest that liver macrophages are heterogeneous in maturation and function and that they are activated in liver diseases as shown by the novel expression of CD14 and FcγRI. The restricted expression of FcγRI indicates that FcγRI-positive macrophages, in cooperation with cytotoxic T lymphocytes, may play an important role in liver cell injury through antibody-mediated cell cytotoxicity. (Hepatology 1994;20:317–325.)

Effects of mitogens on ADCC activity and Fc receptor bearing cells.

Murine spleen cells, when activated by various mitogens (Con A, PHA, LPS, BCG) or Con-A conditioned medium show altered behavior as effector cells of antibody-mediated cell cytotoxicity (ADCC) which does not correlate with the expression of Fc-receptors (FcR) at the effector cell surface.

The effects of Muramyldipeptide (MDP) in cell-mediated immunity

N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) was tested in cell-mediated systems. A preparation of MDP, which yielded comparable activity to Freund's complete adjuvant in a humoral response against bovine serum albumin, was used to examine the degree of correlation between in vitro and in vivo models of cell-mediated immunity: (a) The proliferative T cell response in vitro was found to be most strongly enhanced by MDP at low antigen concentrations. The stimulation indices (SIs), however, were only enhanced at very low antigen concentrations because of a mitogenic effect of MDP in the absence of any added antigen. In vivo the proliferative response was measured in a graft-versus-host reaction where MDP caused a nonspecific (systemic) proliferation. In a host-versus-graft situation, however, MDP significantly enhanced the local proliferative response, besides causing an increased systemic ‘background’ proliferation. (b) The cytotoxic T cell response in vitro was enhanced with suboptimal and optimal antigen concentrations; with supraoptimal antigen concentrations a strong decrease in lytic activity was observed. In vivo, MDP enhanced the cytotoxic activity of peritoneal exudate cells in the same allogeneic system (H-2b anti H-2a) as the one used in vitro. This enhanced activity did not, however, enhance adoptive protection in the immunoincompetent host. (c) Cytotoxic T memory function was unaffected by MDP, both in an in vitro system using subcellular material to elicit the cytotoxic response and in vivo, when an adoptive transfer system was used to assay T memory cells for their protective capacity against tumor in the immunoincompetent host. (d) Antibody-mediated cell cytotoxicity was slightly suppressed when MDP was present in vitro; in vivo-pretreated spleen cells exhibited enhanced activity, but only at low antibody concentrations where a macrophage activity was superimposed on the K cell activity. (e) Macrophages could be activated both in vitro and in vivo to kill tumor cells effectively.

Antibody-Mediated Cell Cytotoxicity in a Defined System: Regulation by Antigen, Antibody, and Complement

The use of a serum-free environment and target cells carrying defined amounts of radiolabeled antigen allowed a quantitative study of the role of antigen, antibody, and complement on antibody-mediated cell cytotoxicity (AbMC). For lysis to occure, a minimum number of antigen molecules must be present on the target cell. 51Cr release from target cells with lower antigen density requires larger concentration of effector cells and antibodies. Target cell-bound complement, itself unable to mediate cytotoxicity, reduces the number of IgG molecules required for lysis. The antibody and complement, however, have to be bound to the same target cell. Bystander complement-coated erythrocytes, present in the same reaction mixture with IgG-coated targets, are not lysed. Blocking of AbMC is effected only by antigen, either soluble or in immune complexes prepared in antigen excess. Antigen competes at the level of the target cell. Blocking at the level of the effector cell, by use of immune complexes prepared at equivalence or in antibody excess, is difficult to achieve. The large number of cells with Fc receptors contained in mouse spleens may explain this finding. Arming of effector cells by passive binding of immune complexes is poorly effective as a means of obtaining lysis of the target cells. In all situations, the outcome of the reaction is determined by the presence of free antibody-combining sites, alone, or in immune complexes, that are able to combine with the target cell membrane antigen. The requirements for lysis are rather stringent.