Bispecific molecules directed to the Fc receptor for IgA (Fc alpha RI, CD89) and tumor antigens efficiently promote cell-mediated cytotoxicity of tumor targets in whole blood

The FcR for IgA (Fc alpha RI, CD89) is primarily expressed on cytotoxic immune effector cells. By chemically cross-linking F(ab') fragments of the FcR for IgA (Fc alpha RI)-specific mAb (A77) with tumor Ag-specific mAb (anti-HER2/neu and anti-epidermal growth factor receptor), we have developed bispecific molecules (BSM) that simultaneously bind to respective tumor Ags and Fc alpha RI-expressing effector cells in whole blood. These BSM mediated up to 55% of specific lysis of appropriate tumor Ag-expressing target cells (from a variety of tumors) with purified polymorphonuclear leukocytes, monocytes, or whole blood effector cells without preactivation with exogenous cytokines. To our knowledge, this is the first demonstration of Ab-dependent cell-mediated cytotoxic activity via Fc alpha RI in whole blood. Also, monocyte-derived macrophages mediated phagocytosis of HER2/neu-expressing tumor cells (>95% tumor cell loss). These BSM-mediated cytotoxic activities were completely inhibited by F(ab')2 of A77, demonstrating the specific role of Fc alpha RI as a trigger molecule. Furthermore, the binding of these BSM to monocytes or polymorphonuclear leukocytes in whole blood did not induce modulation of Fc alpha RI in the absence of the target Ag. Therefore, immune effector cells may be "armed" with Fc alpha RI-directed BSM in whole blood. These Fc alpha RI-directed BSM may offer new treatment options for various malignancies and other disease conditions.     

Structure and function of human IgA Fc receptors (Fc alpha R)

Receptors for the Fc region of IgA are expressed by many human cell types, especially phagocytes located in mucosal areas, where IgA is the prevalent antibody isotype. Binding of IgA-opsonized particles (e.g., bacteria, viruses) to Fc alpha R may trigger a plethora of cell-mediated immune effector functions designed to rid the body of the foreign invader. The IgA receptor present on myeloid cells such as neutrophils, eosinophils, and monocytes (Fc alpha RI or CD89) is a transmembrane glycoprotein that binds both IgA isotypes with similar affinity. Genetic characterization showed Fc alpha RI to be a more distantly related member of the Ig receptor gene family. Recently, Fc alpha RI was found to associate with the FcR gamma-chain signaling molecule through a unique charge-based mechanism. Fc alpha RI is, thus, connected to the intracellular machinery via the ITAM signaling motifs located within the cytoplasmic tail of FcR gamma-chain. Evidence exists in support of receptors for IgA (distinct from Fc alpha RI) on human T and B cells. IgA Fc receptors may, therefore, play a role in both the induction and control of an efficient (mucosal) immune response.     

Rat IgG subclasses mediating binding and phagocytosis of target cells by homologous macrophages

Attachment and ingestion of 51Cr-labelled TNP-SRBC sensitized by rat IgG1, IgG2a or IgG2b-type antibodies by homologous, elicited peritoneal macrophages were studied. IgG1 was found to be the most efficient isotype in mediating these functions. The antibody doses required for a significant attachment were found to differ with the isotype of Ab, while doses needed for a significant phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) varied between 400-700 Ab/SRBC with all the isotypes studied. Both binding and phagocytosis were also influenced by the degree of hapten conjugation when target cells were sensitized by IgG1. Inhibition of these functions by soluble immune complexes and monomeric immunoglobulins suggests the involvement of two Fc gamma R in binding of the three isotypes. Based on the present work and on previous results we conclude that IgG2a interacts with a receptor binding complexed IgG only (Fc gamma RII), IgG2b binds to a different receptor which appears to bind monomeric ligand as well (Fc gamma RI), while IgG1 seems to interact with both types of receptor. We propose that phagocytosis can be mediated by both Fc gamma RI and Fc gamma RII.     

Effects of r-metHuG-CSF on polymorphonuclear leucocyte kinetics and function in patients on continuous ambulatory peritoneal dialysis

End-stage renal failure (ESRF) patients undergoing continuous ambulatory peritoneal dialysis (CAPD) are immunocompromised and exhibit abnormal circulating polymorphonuclear leucocyte (PMN) function, including reduced phagocytosis and intracellular killing. Six uraemic patients on CAPD were each given 300 microg granulocyte colony stimulating factor (G-CSF) every day for 5 d and PMN function tests were performed daily. By day 5 of the study CD11b expression was significantly decreased in response to N-formylmethionylleucylphenylalanine (fMLP) and opsonized Staphylococcus epidermidis stimulation, and expression of L-selectin (CD62L) was significantly decreased in response to opsonized Staphylococcus epidermidis stimulation. Further, superoxide anion production and Fc gammaRI (CD64) expression were found to be significantly increased and Fc gammaRII (CD16) expression was lowered. Circulating white cell and PMN counts were significantly elevated in response to treatment. Administration of G-CSF did not appear to have corrected the abnormalities in phagocytosis and intracellular killing. This study suggests that G-CSF does no harm to ESRF patients and influences uraemic PMN function in a manner that is comparable to its effects on PMN in non-uraemic subjects.     

Colorectal stapled anastomoses. Experiences and results

The mechanism whereby passive Rh (D) immunoglobulins suppress the fetomaternal alloimmunization is still unclear. New in vitro tests are needed to better characterize the functional properties of polyclonal anti-Ds. The DAF assay was developed to monitor the antibody-dependent cell-mediated cytotoxicity (ADCC) and the phagocytosis of anti-Rh (D)-sensitized RBCs by effector cells. The principle of this test is based on the oxydization of the 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of free hemoglobin. The reaction is proportional to the hemoglobin concentration. This test was performed to determine and emphasize the efficacy of different polyclonal anti-D immunoglobulin preparations to mediate lysis and phagocytosis of sensitized RBCs by human peripheral mononuclear cells. The functional properties of different human RhD monoclonal antibodies were also analyzed and compared. The test was found to be convenient to perform and allowed the avoidance of radioactive labelling of RBCs for ADCC studies. It is mainly useful for the direct quantitation of phagocytosis.     

Increased natural killer cell activity correlates with low or negative expression of the HER-2/neu oncogene in patients with breast cancer

BACKGROUND: Increased expression of the HER-2/neu oncogene in breast cancer correlates with decreased estrogen receptor concentration and seems to be an important prognostic factor. The authors investigated whether there is a correlation between HER-2/neu expression and immunologic parameters representing tumor defense in patients with breast cancer. METHOD: A Western blot analysis was used to investigate HER-2/neu expression, whereas a chromium-release assay using the K562 cell line as target was used to measure natural killer (NK) cell activity. RESULTS: In patients with breast cancer, NK cell activity was significantly higher compared with patients with benign tumors (P = 0.006) or healthy control subjects (P = 0.002). Moreover, 23.3% of patients with breast cancer showed an overexpression of HER-2/neu protein. Within this group of patients, NK cell activity was significantly lower (45.6 +/- 16.1%) compared with the group with no HER-2/neu overexpression (57.3 +/- 11.0%). NK cell activity did not increase in patients with HER-2/neu overexpression. Thus, there was a statistically significant correlation of cytolytic effector cell function with HER-2/neu expression of the tumor (P = 0.003), and HER-2/neu overexpression correlated with a negative estrogen receptor status (P = 0.005). CONCLUSION: These data add further evidence to previous observations from the authors' laboratory that certain tumor characteristics may be associated with reactions of the host with breast cancer.     

High-titer HER-2/neu protein-specific antibody can be detected in patients with early-stage breast cancer

PURPOSE: To evaluate HER-2/neu-specific antibody immunity in patients with breast cancer, to determine the rate of occurrence of serum antibodies to HER-2/neu in patients with breast cancer, and to relate the presence of specific immunity to overexpression of HER-2/neu protein in primary tumor. METHODS: The antibody response to HER-2/neu protein was analyzed in 107 newly diagnosed breast cancer patients. Sera was analyzed for the presence of HER-2/neu-specific antibodies with a capture enzyme-linked immunosorbent assay (ELISA) and verified by Western blot. Sera from 200 volunteer blood donors was used as a control population. RESULTS: The presence of antibodies to HER-2/neu correlated with the presence of breast cancer. HER-2/neu antibodies at titers of > or = 1:100 were detected in 12 of 107 (11%) breast cancer patients versus none of 200 (0%) normal controls (P < .01). The presence of antibodies to HER-2/neu also correlated to overexpression of HER-2/neu protein in the patient's primary tumor. Nine of 44 (20%) patients with HER-2/neu-positive tumors had HER-2/neu-specific antibodies, whereas three of 63 (5%) patients with HER-2/neu-negative tumors had antibodies (P = .03). The antibody responses could be substantial. Titers of greater than 1:5,000 were detected in five of 107 (5%). CONCLUSION: The presence of HER-2/neu antibodies in breast cancer patients and the correlation with HER-2/neu-positive cancer implies that immunity to HER-2/neu develops as a result of exposure of patients to HER-2/neu protein expressed by their own cancer. These findings should stimulate further studies to develop the detection of immunity to oncogenic proteins as tumor markers, as well as the development and testing of vaccine strategies to induce and augment immunity to HER-2/neu for the treatment of breast cancer or prevention of recurrent disease.     

Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (Fc gamma RIII) expressed by human natural killer (NK) cells

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.     

Immunologic injury in measles virus infection. III. Presence and characterization of human cytotoxic lymphocytes

Human peripheral blood leukocytes (PBL) from patients with chronic measles virus infection (SSPE) or from immune, adult humans (convalescent from acute childhood measles virus infection) are cytotoxic for target cells infected with measles virus as measured by a 51Cr assay. Specific release of 51Cr by immune PBL occurred both with and without human antibodies added to measles virus-infected cultures. Maximal killing in the absence of added antibodies to measles virus was usually detected only after 15 to 18 hr of incubation and with a high PBL to target ratio (100:1). When antibody to measles virus was added, PBL-mediated killing of virus-infected cells was not blocked. Instead, killing was enhanced and maximal lysis occurred with fewer PBL and a shorter incubation time. This cytotoxic reaction was inhibited in a dose-response manner upon the addition of Fab fragments of IgG containing antibodies to measles virus. On the average 4 to 5 x 105 antibody molecules bound per infected target cell before initiation of antibody-enhanced PBL killing. Depletion of either glass-adhering or E-rosette-forming cells did not reduce PBL killing of measles virus-infected target cells in either system. In contrast, removal of non-E rosette or of EAC rosette-forming population of PBL almost completely abrogated cytotoxicity. When Fc-bearing cells were removed, killing of virus-infected target cells was concomitantly reduced. Lysis of measles virus-infected target cells did not require histocompatibility between the PBL and the target cell. Further, immunospecific lymphocyte killing was not enhanced by such a histocompatibility fit. These experiments indicate first, that the effector PBL involved in lysis of measles virus-infected targets are not T cells but are probably K cells and, second, that PBL obtained from patients with SSPE are competent in killing measles virus-infected targets. Moreover, sera from SSPE patients did not contain a factor(s) that blocked PBL-mediated cytotoxicity.     

Antibody-dependent cell-mediated cytotoxicity in rheumatoid arthritis. Effect of rheumatoid serum fractions on normal lymphocytes

Reduced antibody-dependent cell-mediated cytotoxicity (ADCC) was demonstrated in lymphocytes of patients with rheumatoid arthritis (RA). Rheumatoid factor (RF) positive sera inhibited ADCC of normal lymphocytes by reacting both with effector and target cells (sensitized chicken red blood cells). These sera were fractionated by specific adsorption or gradient ultracentrifugation, and isolated RF or RF negative fractions were tested for their ability to inhibit ADCC by reacting with normal human lymphocytes or target cells. RF was ineffective on normal lymphocytes but it strongly inhibited the reaction by interaction with target cells. IgG RF negative fractions of certain sera were inhibitory by direct interaction with effector cells.     

Resistance of HER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared to HER2/neu-nonexpressing lines. HER2/neu+ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in "cold" target inhibition experiments. HER2/neu+ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and beta 1 integrins was comparable to that of HER2/neu- targets. In contrast, the HER2/neu+ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis of HER2/neu- carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression on HER2/neu+ cells contributes to their resistance. The modest binding and lysis of HER2/neu+ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 on HER2/neu+ targets. The following also supported deficiencies in post-binding events when HER2/neu+ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin. HER2/neu+ cell lines were still resistant to LAK cytolysis, and (b) HER2/neu+ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance of HER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.     

Human NK cell and ADCC reactivity against xenogeneic porcine target cells including fetal porcine islet cells

In vitro studies of human NK cell-mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity. Maximum reactivity was reached following 3-6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA-induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo- and xeno-stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL-2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD16+ or CD56+ lymphoblasts. Natural xenoreactive human anti-porcine antibodies are mainly of IgM and IgG2 subclasses, but antibodies in xenoimmunised patients reactive against porcine lymphocytes and fetal porcine islet cells were also of IgG1 and IgG3 subclasses. The same subclass distribution was found among antibodies specific for gal(alpha)1,3 gal epitopes as shown by tests performed with alpha1,3 galactosyltransferase-transfected Raji cells (human Burkitt lymphoma cells). Natural antibodies did not mediate ADCC, whereas gal(alpha)1,3 gal-specific antibodies in sera from xenoimmunised patients did. Fetal porcine islet cells were sensitive to human NK cell-mediated cytotoxicity and to ADCC mediated by xenoimmune sera.     

Human IgG3 can adopt the disulfide bond pattern characteristic for IgG1 without resembling it in complement mediated cell lysis

In this paper we describe the construction of mouse-human IgG3 mutant antibodies resembling IgG1 in their disulfide bond pattern between the heavy and light chain (H-L) and between the two heavy chains (H-H). The effector functions of these mutant antibodies were compared to normal IgG3 and IgG1. Changing only the disulfide bond pattern between the heavy and light chains did not alter the ability to induce complement mediated cell lysis (CML), regardless of the amount of corresponding antigen that had been introduced to the surface of the target cells. However, alteration of the disulfide bond pattern between the two heavy chains had a large effect on CML due to shortening of the hinge from 62 to 15 amino acids. No difference between the mutants and normal antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC) was observed. This suggests that IgG3 can adopt the H-L disulfide bond pattern of IgG1 without obtaining the CML activity characteristic for IgG1.     

Fc receptors are not required for antibody-mediated protection against lethal malaria challenge in a mouse model

The mechanisms by which Abs mediate protection during blood-stage malaria infections is controversial, with some evidence pointing to the direct effect of Abs on parasite invasion and growth, while other studies suggest that Abs act in cooperation with monocytes to achieve parasite inhibition. To determine whether the effector phase of protection in vivo to the rodent parasite Plasmodium yoelii yoelii requires Fc receptor bearing cells, we passively transferred immune sera into FcR gamma-chain knockout mice. Inflammatory macrophages from these knockout mice were unable to mediate phagocytosis or Ab-dependent cell-mediated cytotoxicity (ADCC) through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Passive transfer of either P. y. yoelii hyperimmune sera or anti-GST-PYC2 sera directed to the major merozoite surface protein (MSP-1) of this parasite enabled both BALB/cByJ mice and FcR gamma-chain-deficient mice to resist lethal P. y. yoelii 17XL (Py17XL) challenge. mAb302, a protective IgG3 Ab, also passively protected both strains of mice. Most of these samples contain Ab isotypes that would not be able to protect mice if their protective effects required Ab-dependent cell-mediated cytotoxicity. These results establish that, in this infection, protection is directly mediated by Abs and does not require the participation of Fc receptors.     

Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity

Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in Fc gamma IIR, Fc gamma IIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CDIIb (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.     

Torsional properties of stainless steel and nickel-titanium endodontic files

American cutaneous leishmaniasis (ACL) presents a spectrum of clinical and immunological manifestations. Since the nature of the cellular response appears to play a fundamental role in determining the characteristics of the immunoglobulin isotype of specific antibody responses, we have compared the relative levels of specific antibodies of the four IgG isotypes against Leishmania in sera from patients with different clinical manifestations of ACL. Using a specific antibody capture assay, significant levels of antibodies of the IgG1, 2 and 3 isotypes were detected in localized cutaneous leishmaniasis (LCL); the average level of IgG4 antibodies was low and they were not detected in 10/20 sera. Sera from muco-cutaneous leishmaniasis (MCL) gave a comparatively strong IgG1 response. Sera from diffuse cutaneous leishmaniasis (DCL), the rare form characterized by antigen-specific anergy of cell-mediated immunity, showed highly significant levels of IgG4 antibodies compared to antibody levels of this isotype in the other groups; IgG1 and IgG2 levels were also elevated. Based on other studies of the relationship between the IgG isotype response and cell-mediated immunity, these results confirm a Th1-like CD4+ T cell response in LCL and MCL and a significant Th2-like response in DCL.     

Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells

Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.     

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay for specific IgG antibody produced in vitro

IgG antibody production by immune spleen cells in vitro was assayed by antibody-dependent cell-mediated cytolysis (ADCC). The use of this technique as a sensitive and reproducible alternative to indirect haemolytic plaque assays is examined.     

Structural bases of Fc gamma R functions

This review describes structures which determine the biological activities triggered by Fc gamma R and account for the cell-mediated functions of IgG antibodies in physiology and pathology. The binding specificity and affinity of Fc gamma R depend primarily on IgG-binding structures, in their immunoglobulin-like extracellular domains. Binding is however also influenced by subunits that associate to multichain Fc gamma R. Effector and regulatory intracytoplasmic sequences that are unique to molecules of the Fc gamma RIIB family determine the internalization properties of these receptors. Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) are intracytoplasmic effector sequences shared by Fc gamma R and other receptors involved in the recognition of antigen, which trigger cell activation and internalization. Immunoreceptor Tyrosine-based Inhibition Motifs (ITIMs) are intracytoplasmic sequences, shared by Fc gamma RIIB and a growing number of negative coreceptors which negatively regulate cell activation via ITAM-bearing receptors. Altogether, these structures enable IgG antibodies to exert a variety of finely tuned biological effects during the immune response.