Importance of antibody isotypes in antitumor immunity by monocytes and complement using human-immune tumor models.

Monoclonal antibodies (mAbs) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a preclinical human immune - three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer cultures, of human CD20+ B-cell lymphomas. We demonstrate that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, and when combined with apoptosis-inducing RTX-IgG2 isotype the therapeutic effect can be substantially enhanced. The results show further that the treatment outcome by RTX isotypes is influenced by tumor morphology and expression of the complement inhibitor CD59. Hence, the human immune-3D tumor model is a clinical relevant and attractive ex vivo system to predict mAbs for best efficacy in cancer immunotherapy.

Molecular engineering to improve antibodies' anti-lymphoma activity.

The human/mouse chimeric CD20 antibody rituximab has significantly improved the survival of lymphoma patients. However, translational research into pharmacology and effector mechanisms of rituximab has identified several limitations of this prototypic antibody. For example, humanized or fully human next-generation antibodies demonstrated reduced immunogenicity, which may translate into improved applicability in certain patient populations. Furthermore, novel technologies of antibody engineering offer the potential to tailor antibody effector functions. Here, glyco- or protein engineering of antibodies' Fc region has demonstrated promising activity in preclinical models. However, these novel molecules are still in early phases of clinical development, and data from on-going and future studies will determine whether promising preclinical results will indeed translate into improved drugs for the treatment of lymphoma patients.

Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing.

Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human κ and anti-His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs.

More is not necessarily better: prozone-like effects in passive immunization with IgG.

Despite a century of study, the relationship between Ag-specific Ig concentration and protection remains poorly understood for the majority of pathogens. In certain conditions, administration of high Ab doses before challenge with an infectious agent can be less effective than smaller Ab doses, a phenomenon which is consistent with a prozone-like effect. In this study, the relationship between IgG1, IgG2a, IgG2b, and IgG3 dose, infective inocula, and protection was investigated in a mouse model of Cryptococcus neoformans infection. The activity of each IgG subclass ranged from protective to disease-enhancing depending on both the Ab dose and infective inocula used. Enhanced dissemination to the brain was observed in mice given a high IgG2a dose and a relatively low inoculum. Ab administration had immunomodulatory effects, with cytokine expression in lung, brain, and spleen varying as a function of the infective inoculum Ab dose and IgG subclass. In vitro studies did not predict or explain the mechanism of in vivo prozone-like effects, because all isotypes were opsonic and elicited NO release from macrophages. IgG2a was most efficient in inducing a macrophage oxidative burst. These results reveal that an individual Ab can be protective, nonprotective, or disease-enhancing depending on its concentration relative to a challenge inoculum. Our findings have implications for the potential contribution of Ab responses to defense against microbial diseases because Ab-mediated immunity may be protective, nonprotective, or even deleterious to the host.

Functional in vivo characterization of human monoclonal anti-D in NOD-scid mice.

The prophylaxis of the hemolytic disease of the newborn requires significant amounts of plasma-derived polyclonal human anti-D. Because of procurement problems, there is a growing interest in replacing plasma-derived anti-D by in vitro-produced human monoclonal anti-D. Hundreds of monoclonal anti-D have been prepared, but the selection of the most potent for in vivo use is difficult because it cannot be predicted by in vitro characterization. This study evaluated the possibility of using nonobese diabetic/severe combined immunodeficient (NOD-scid) mice for the in vivo evaluation of human monoclonal anti-D. Human red blood cells (RBCs) were found to circulate normally in the blood of NOD-scid mice previously injected with a physiologic amount of human immunoglobulin G (10 mg). The addition of a small amount of anti-D (1 to 5 microg) resulted in the clearance of Rh D(+) RBCs within 4 hours. The comparative testing of 8 monoclonal anti-Ds showed a wide range of potency (15% to 87%) relative to plasma-derived polyclonal anti-D. There was no strong correlation between the in vivo potency index and the immunoglobulin G isotype, affinity, or fine specificity of the antibodies. These results show the usefulness of NOD-scid mice for the initial in vivo screening of human monoclonal anti-D before testing the most active antibodies in clinical trials done in human volunteers.

Engineered antibodies with increased activity to recruit complement.

This manuscript describes two sites in a human IgG1 that, when mutated individually or in combination, result in a dramatic increase in C1q binding and complement-dependent cytotoxicity activity. These two residues, K326 and E333, are located at the extreme ends of the C1q binding epicenter in the C(H)2 domain of a human IgG. A mutation to tryptophan at K326 debilitates Ab-dependent cell-mediated cytotoxicity activity. In addition, substitutions of the residues E333 with serine and of K326 with tryptophan in a human IgG2 confer biological activity in the complement-dependent cytotoxicity assay in which the wild-type IgG2 is inactive. This study reveals that the residues K326 and E333 play a significant role in the control of the biological activity of an IgG molecule and can rescue the activity of an inactive IgG isotype.

Peptides derived from IgE heavy chain constant region induce profound IgE isotype-specific tolerance.

(BALB/c x SJL)F1 mice, perinatally injected with peptide-N-glyconase F-treated, deglycosylated IgE heavy chain or recombinant IgE heavy chain (CH epsilon 2-CH epsilon 4), were profoundly inhibited in antigen-specific IgE production. There exist minimally two tolerogenic IgE peptides, residing in the CH epsilon 2 and CH epsilon 4 domains. Peptide I, generated by V8 protease, comprises 39 amino acids within CH epsilon 2, beginning at amino acid 103. Peptide E begins at amino acid 312 of the CH epsilon 4 domain and extends through the CH epsilon 4 domain. The total lack of antigen-specific IgE responses in IgE peptide-treated mice was not due to overproduction of interferon-gamma, nor lack of interleukin (IL)-4, as predicted by the Th2/IL-4 paradigm for IgE production. IgE-tolerant mice exhibited comparable levels of circulating anti-IgE antibodies to those of PBS-treated control mice. IgG obtained from sera of both sources failed to inhibit IgE responses in vitro. Moreover, IgE responses of spleen cells from IgE peptides-treated mice were restored by CD4+ T cells from PBS-treated control mice. We hypothesize that regulation of antigen-specific IgE responses is mediated by CD4+ T cells which normally recognize IgE peptides on IgE precursor B cells, and can be rendered tolerant by perinatal IgE peptide treatment.

Target-specific activation of mast cells by immunoglobulin E reactive with a renal cell carcinoma-associated antigen.

Immunoglobulin E (IgE) that specifically binds to antigens present on carcinoma cells may represent a useful tool to combat carcinomas. Induction of an inflammatory response at the tumor site by tumor-specific IgE may result in reduced tumor growth and tumor regression. Local mast cells may be activated to release TNF-alpha and other mediators that attract inflammatory cells, such as eosinophils and macrophages, to the tumor site. It may even be expected that eosinophils perform IgE-mediated lysis of tumor cells. The G250 IgE binds an antigen present on renal cell carcinoma. Mast cells were assayed for activation by G250 IgE in vitro in the presence of G250-positive tumor cells, by determination of the release of TNF-alpha and histamine. In parallel, G250 IgG1, IgG2a, and IgG2b, bound to tumor cells, were tested for their ability to activate mast cells. Tumor-specific IgE was capable of activating mast cells in the presence of tumor cells. This activation was specific and required the presence of the antigen on the tumor cell surface and recognition of the tumor cell by the IgE. G250 IgE mediated mast cell activation when present in the medium as well as being preloaded on either tumor cells or mast cells. Preincubation of mast cells with irrelevant IgE did not block specific G250 IgE-mediated mast cell activation. Upon activation, mast cells released histamine and TNF-alpha, as was detected in cytotoxicity assays with TNF-alpha-sensitive target cells (WEHI). G250 IgG2a also induced efficient mast cell activation, comparable to the effect of G250 IgE. Mast cells can be triggered to release mediators such as TNF-alpha by IgE in the presence of tumor cells expressing specific antigen. Whether mast cell activation contributes to antitumor effects observed during antibody-based immunotherapy of tumors deserves further investigation.

Regulation of complement activity by immunoglobulin. I. Effect of immunoglobulin isotype on C4 uptake on antibody-sensitized sheep erythrocytes and solid phase immune complexes.

Intravenous Ig, composed principally of IgG, prevents complement attack by inhibiting C3 and C4 uptake onto target cells and tissues. Using two different models, Ab-sensitized SRBC and BSA-anti BSA solid phase immune complexes, we have examined the complement inhibitory capacity of three Ig classes (IgG, IgM, IgA) focussing on inhibition of C4 uptake. It was found that on both a weight and molar basis, monomeric serum IgA and IgM were far more active than IgG (weight efficiency ratios were 1.0, 20.8, and 236.3, and molar efficiency ratios 1.0, 24.0, and 1382.9 for polyclonal IgG, IgA1, and IgM, respectively). Monoclonal IgM were less active than polyclonal IgM (50% inhibition was achieved in SRBC model by 0.022, 0.30, 1.6, and 1.6 mg/ml of polyclonal IgM and monoclonal IgM from patients Lew, Will, and Pri). Secretory IgA was less active than serum IgA1 and similar in inhibitory activity to IgG (weight and molar efficiency ratios 1.5 and 0.6 compared with IgG). All tested preparations were less active in the solid phase immune complex model than in the sensitized cell model. A mixture of Igs of different isotypes was somewhat more active than any isotype alone. These results suggest that polyclonal serum IgA and IgM can also be considered for active therapy in diseases accompanied by the activation of classical complement pathway.

Molecular analysis of the modulatory factors of the response to HBsAg in mice as an approach to HBV vaccine enhancement.

Mice were injected with immune complexes containing the recombinant hepatitis B surface antigen (HBsAg) vaccine (S + preS2) bound to different monoclonal antibodies (mAbs), in order to determine whether an enhancement of the response to a human vaccine could be obtained and observed. Enhancement and indifference were observed, as well as a decrease in immunogenicity. No relationship could be established between any effect and affinity or isotype of the bound mAbs. The preS2 region was rendered more immunogenic when an IgG2a mAb was bound to the S region of the HBsAg. The response to the S region was not modulated, whereas immunogenicity of the preS2 colinear region was decreased by antibody shielding. The mAb which was the most efficient as an enhancer of the antibody response also increased binding of the complexed immunogen to antigen presenting cells. The binding of a human mAb to the sole S region, but not to the preS2 region, should be tested as a potentiating agent of the anti-preS2 human immune response.

Immune complexes increase nitric oxide production by interferon-gamma- stimulated murine macrophage-like J774.16 cells.

Murine macrophage-like J774.16 cells were tested for changes in nitric oxide production upon incubation with immune complexes. Cryptococcus neoformans capsular polysaccharide and polysaccharide-specific monoclonal antibodies were added to J774.16 cells in the presence and absence of recombinant murine interferon-gamma (IFN-gamma). The effect of immune complexes on nitrite synthesis was both concentration dependent and isotype dependent. In the presence of IFN-gamma, immune complexes of IgG1, IgG2, IgG2b, or IgG3 isotype increased nitrite levels, whereas complexes of IgM isotype did not. Immune complexes did not alter nitrite production by unstimulated macrophages. Antibody alone, antigen alone, and antigen with irrelevant IgG1 antibody did not augment nitrite formation, either in the presence or absence of IFN-gamma, indicating a requirement for Fc gamma R cross-linking. These results suggest that IgG isotypes may offer additional protection against pathogens by enhancing macrophage nitric oxide production.

An analysis, using monoclonal antibodies, of the role of interferon-gamma in ovine immune responses.

A mAb (IFN-9) which neutralizes biologically active ovine and bovine IFN-gamma was used to deplete levels of the cytokine in vivo in sheep and examine the consequences for immune responses to adjuvanted antigen and skin reactivity to Bacillus-Calmette-Guerin (BCG). Groups of sheep were immunized with ovalbumin in the adjuvants, Quil A or dextran sulphate (MW 500,000; DXS), both of which elicit production of IFN-gamma. MAb anti-IFN-gamma or an isotype control mAb (anti-carbonic anhydrase positive particles) were inoculated i.v. during primary and/or secondary responses. Reactions monitored in efferent prefemoral lymph indicated that anti-IFN-gamma effectively depleted levels of IFN-gamma in lymph but had no effects on the magnitude and kinetics of lymphocyte and lymphoblast traffic, or total or isotypic titres of specific Ig. When incubated in vitro with ovalbumin, antigen-reactive cells from anti-IFN-gamma treated sheep did not produce IFN-gamma, suggesting an on-going modification to cytokine production. In contrast, skin reactions to purified-protein derivative in sheep immunized with BCG were reduced by < 40% by anti-IFN-gamma. The results indicate that IFN-gamma production may not be obligatory for delayed-type hypersensitivity reactions or for the adjuvant action of Quil A or DXS, and that specific mAb can alter the profile of cytokines produced by antigen-reactive cells in sheep.

Effect of anti-IL-5 and IL-5 on airway hyperreactivity and eosinophils in guinea pigs.

Chronic ovalbumin challenge of sensitized guinea pigs induces bronchoalveolar lavage (BAL) eosinophilia, neutrophilia, and tracheal hyperreactivity. In the present study, the influence of monoclonal antibody to murine interleukin-5 (anti-IL-5) on these phenomena is examined. In ovalbumin-sensitized guinea pigs treated with isotype-matched control antibody and challenged daily with ovalbumin for 8 days, the number of BAL eosinophils and neutrophils is increased significantly six- and fivefold, respectively, compared with saline-challenged animals. The maximal contractions of tracheal rings to histamine and arecoline in ovalbumin-challenged animals are enhanced significantly to 155% compared with saline-challenged animals. In sensitized guinea pigs treated with anti-IL-5, the BAL eosinophil number is markedly inhibited compared with control antibody treatment in both saline- and ovalbumin-challenged animals. In contrast, the number of neutrophils is not affected by anti-IL-5 treatment. In guinea pigs treated with anti-IL-5, the development of hyperreactivity to histamine and arecoline after ovalbumin challenge is completely inhibited. The contractions to histamine and arecoline of tracheal rings isolated from guinea pigs treated with recombinant murine IL-5 for 3 or 7 days are enhanced significantly to approximately 140% compared with controls. Treatment with IL-5 for 7 days tends to increase the number of eosinophils in BAL fluid. It can be concluded that IL-5 is involved in airway eosinophilia and in the development of hyperreactivity in this animal model, but other cytokines may contribute. Development of IL-5 synthesis inhibitors and/or receptor antagonists could provide another therapeutic class of anti-asthma drugs.

Studies on monoclonal anti-isotypic and anti-idiotypic antibodies against leukemia and myeloma: V. The effects of monoclonal antibodies and interferon on the levels of cyclic nucleotides in leukemic cell lines.

After the leukemic cell lines were treated with monoclonal antibodies (McAbs) and interferon (IFN-alpha), the changes of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in the corresponding leukemic cell lines were measured by radioimmunoassay. The results showed that when the ratio of antigen to antibody was 80 to 1, the cAMP levels in the leukemic cell lines were obviously higher than those in the controls while the cGMP levels were obviously lower after being treated with the corresponding McAbs for 16-24 h (P < 0.001). The average level of intracellular cAMP was remarkably increased and that of cGMP underwent no significant changes in the leukemic cell lines after treatment with IFN-alpha.