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Biomedical subjects

T Keler

Publications and source records attributed to T Keler.

At least 19 recordsLinked to original sources
PubMed2003

Anti-HPA-1a-mediated platelet phagocytosis by monocytes in vitro and its inhibition by Fc gamma receptor (FcgammaR) reactive reagents.

The study was undertaken to delineate mechanisms of platelet destruction by phagocytosis during fetal/neonatal alloimmune thrombocytopenia (FAIT/NAIT) because of maternal antibodies against human platelet antigen 1a (HPA-1a). By employing a platelet phagocytosis assay based on the ORPEGEN flow cytometric bacterial phagocytosis test, we measured monocyte ingestion of platelets mediated by anti-HPA-1a antibodies. Moreover, we tested, as potential therapeutic agents, FcgammaR reactive reagents, for their inhibition of this process. Four of six anti-HPA-1a sera tested mediated phagocytosis of HPA-1a-positive platelets in a concentration-dependent manner. Monocyte ingestion of platelets was almost completely inhibited by cytochalasin D. No anti-HPA-1a-mediated phagocytosis was observed with anti-HPA-1a-negative platelets. The humanised anti-FcgammaRI monoclonal antibody H22 at concentrations 1-100 microg/ml, completely inhibited anti-HPA-1a-mediated phagocytosis as did similar concentrations of ivIg. By contrast, a mouse monoclonal anti-FcgammaRII (IV.3, Fab) at 10 microg/ml caused little or no suppression of platelet phagocytosis mediated by two anti-HPA-1 sera. Furthermore, the addition of anti-FcgammaRII (10 microg/ml) to sub-optimal concentrations of H22 did not significantly increase the inhibitory effect of the latter compound. Monomeric IgG (0.1-10 microg/ml) failed to suppress anti-HPA-1 mediated platelet ingestion by the phagocytes, as did anti-FcgammaRIII. To our knowledge this is a rare example of an assay that measures platelet phagocytosis in vitro. The results suggest that FcgammaRI plays a major role in anti-HPA-1a-mediated platelet phagocytosis by monocytes while FcgammaRIIa, is of little or minor importance only. Moreover, the findings indicate the use of H22 as an alternative to interavenous Ig (ivIg) in the management of FAIT/NAIT.

Adult↗
PubMed2001

Receptor modulation by Fc gamma RI-specific fusion proteins is dependent on receptor number and modified by IgG.

The high-affinity IgG receptor, FcgammaRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcgammaRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcgammaRI causes receptor recycling, while Abs that cross-link FcgammaRI cause rapid down-modulation of surface FcgammaRI. Because studies performed in the absence of ligand may not be representative of FcgammaRI modulation in vivo, we investigated the ability of FcgammaRI-cross-linking Abs and non-cross-linking derivatives to modulate FcgammaRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcgammaRI on the human myeloid cell line, U937, and its high FcgammaRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcgammaRI expression and correlated with internalization of both wH22xeGFP and FcgammaRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcgammaRI, was unable to modulate FcgammaRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcgammaRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcgammaRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.

Adjuvants, Immunologic↗
PubMed2001

Bispecific antibody-mediated destruction of Hodgkin's lymphoma cells.

CD30 is a molecule that is overexpressed on the surface of Hodgkin's lymphoma cells. Therefore, CD30 represents a potential candidate for immunotherapy. In this study, we report the in vitro results of two bispecific molecules (BSMs) that target CD30 to trigger molecules expressed on myeloid effector cells. The first BSM is composed of the Fab' fragment of a CD30-specific antibody, Ki-4, chemically linked to the Fab' fragment of the humanized CD64 (FcgammaRI)-specific antibody, H22 (H22xKi-4). In the second BSM, the H22 Fab' is replaced with the Fab' fragment of the CD89 (FcalphaR)-specific, antibody, A77 (A77xKi-4). Both BSMs were able to bind specifically to lymphoma cell lines expressing CD30. In addition, the H22xKi-4 and A77xKi-4 BSMs were shown to bind cells expressing CD64 and CD89, respectively. Both BSMs mediated potent, dose-dependent antibody dependent cell-mediated cytotoxicity (ADCC) of CD30-expressing tumor cell lines when human monocytes were used as effector cells. In addition, freshly prepared polymorphonuclear leukocytes (PMNs) and effector cells in whole blood were able to mediate the ADCC of targets in conjunction with the A77xKi-4 BSM in some, but not all, experiments. Furthermore, we examined the ability of monocyte-derived macrophages (MDMs) to phagocytose CD30-expressing tumor cell lines in conjunction with the BSM. MDM-mediated phagocytosis was significantly enhanced in the presence of both BSMs. These results demonstrate that targeting lymphoma cells via CD30 to the myeloid high affinity Fc receptor for IgG and to the Fc receptor for IgA results in potent in vitro anti-tumor activity.

Animals↗
PubMed2001

Bispecific antibody-targeted phagocytosis of HER-2/neu expressing tumor cells by myeloid cells activated in vivo.

Studies from our laboratory and others have established that both mononuclear phagocytes and neutrophils mediate very efficient cytotoxicity when targeted through Fc receptors using a suitable monoclonal or bispecific antibody (BsAb). Cross-linking an Fc receptor for IgG (FcgammaR) triggers multiple anti-tumor activities including superoxide generation, cytokine and enzyme release, phagocytosis and antibody-dependent cellular cytotoxicity (ADCC). In this report, using unfractionated leukocytes and two color flow cytometric analysis, we describe the phagocytic capacity of peripheral blood polymorphonuclear cells (PMN) and monocytes isolated from patients enrolled in a phase I clinical trial of MDX-H210 given in combination with IFNgamma. MDX-H210 is a BsAb targeting the myeloid trigger molecule FcgammaRI and the HER-2/neu proto-oncogene product overexpressed on a variety of adenocarcinomas. In this trial, cohorts of patients received escalating doses of MDX-H210 3 times per week for 3 weeks. Interferon-gamma (IFNgamma) was given 24 h prior to each BsAb infusion. Our results demonstrate that monocytes from these patients were inherently capable of phagocytosing the HER-2/neu positive SK-BR-3 cell line and that addition of MDX-H210 into the assay significantly enhanced the number of targets phagocytosed. Two days after administration of an immunologically active dose of MDX-H210 (10 mg/m2), monocytes from these patients were able to phagocytose greater amounts of target cell material, indicating that these cells remained armed with functionally sufficient BsAb for at least 48 h. PMN from these patients very efficiently mediated phagocytosis through FcgammaRI after being treated with IFNgamma, but not before. We conclude that phagocytosis is not only an efficient mechanism of myeloid cell-mediated cytotoxicity, but may also be a mechanism by which antigens from phagocytosed cells can enter a professional antigen presenting cell for processing and presentation.

Animals↗
PubMed2000

Targeting weak antigens to CD64 elicits potent humoral responses in human CD64 transgenic mice.

Previous studies have documented that targeting foreign Ags to IgG FcgammaR leads to enhanced Ag-specific responses in vitro and in vivo. However, the ability to overcome immunologic nonresponsiveness by targeting poorly immunogenic Ags to FcgammaR has not been investigated. To address this question in a simple model, we immunized transgenic mice expressing human CD64 (FcgammaRI) and their nontransgenic littermates with Fab' derived from the murine anti-human CD64 mAb m22. The m22 Fab' served as both the targeting molecule and the Ag. We found that only CD64-expressing mice developed anti-Id titers to m22. Furthermore, chemically linked multimers of m22 Fab', which mediated efficient internalization of the human CD64, were significantly more potent than monomeric m22 F(ab')(2) at inducing anti-Id responses. In all cases, the humoral responses were specific for m22 Id and did not react with other murine IgG1 Fab' fragments. Chemical addition of a second murine Fab' (520C9 anti-human HER2/neu) to m22 Fab' multimers demonstrated that IgG1 and IgG2a anti-Id titers could be generated to 520C9 only in the CD64-expressing mice. These results show that targeting to CD64 can overcome immunological nonresponsiveness to a weak immunogen. Therefore, targeting to CD64 may be an effective method to enhance the activity of nonimmunogenic tumor vaccines.

Animals↗
PubMed2000

Differential effect of cytokine treatment on Fc alpha receptor I- and Fc gamma receptor I-mediated tumor cytotoxicity by monocyte-derived macrophages.

Macrophages represent an important effector cell for Ab-mediated tumor therapy. Previous studies have documented that cytokines can influence Fc receptor (FcR) expression and function. In this study we examined the tumoricidal activities of the type I receptors for IgG (Fc gamma RI) and the IgA FcR (Fc alpha RI) on monocyte-derived macrophages (MDM) cultured in the presence of IFN-gamma, M-CSF, or GM-CSF. Bispecific Abs were used to target a Her2/neu breast carcinoma cell line, SKBR-3, to Fc alpha RI or Fc gamma RI on MDM. Although Fc alpha RI and Fc gamma RI share a common signaling pathway contingent on association with the gamma-chain (FcR gamma subunit), a marked difference in their efficiency in mediating tumoricidal functions was seen in response to specific cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient phagocytosis of SKBR-3 cells by Fc alpha RI and Fc gamma RI; however, IFN-gamma-treated MDM phagocytosed tumor cells only with the Fc gamma RI-directed bispecific Abs. Similarly, IFN-gamma-cultured MDM lysed tumor cells more efficiently via Fc gamma RI then by Fc alpha RI as measured in Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated MDM mediated more efficient lysis of tumor cells via Fc alpha RI than Fc gamma RI, while M-CSF-cultured MDM were relatively less efficient in mediating Ab-dependent cellular cytotoxicity through either receptor. With the exception of IFN-gamma-mediated enhancement of Fc gamma RI expression and Fc gamma RI gamma-chain complexes, the regulation of Fc gamma RI- or Fc alpha RI-mediated activity occurred without significant change in either receptor expression or total complexes with gamma subunit. These data suggest that the efficiency of Ab-mediated tumor therapy, which depends on FcR effector cell functions, may be modified by the use of specific cytokines.

Adjuvants, Immunologic↗
PubMed2000

HPA-1a-mediated platelet interaction with monocytes in vitro: involvement of Fcgamma receptor (FcgammaR) classes and inhibition by humanised monoclonal anti-FcgammaRI H22.

To gain insight into mechanisms of platelet destruction and its possible inhibition during fetal/neonatal alloimmune thrombocytopenia (FAIT/NAIT) the binding to monocytes (Mo) of anti-HPA-1a-sensitised platelets, the initial step in IgG-mediated destruction by effector cells, was evaluated in an in vitro assay. Neonatal Mo were compared with adult Mo as effectors in the assay. Moreover, the potential involvement of Fcgamma receptor (FcgammaR) classes during platelet destruction in the disease was tested by using FcgammaR class-specific reagents as inhibitors of the binding reaction. Neonatal Mo were 37% less active than adult Mo in their interaction with anti-HPA-1a-sensitised platelets (p < 0.05). The FcgammaRI-specific reagents human monomeric IgG and humanised anti-FcgammaRI monoclonal H22 caused virtually complete inhibition of platelet binding to Mo. When compared to an intravenous immunoglobulin preparation the inhibitory activity of H22 was 10-100 x greater than that of the latter compound. Monoclonal anti-FcgammaRII IV.3 and anti-FcgammaRIII 3G8 decreased platelet binding by 70% and 64%, respectively, but only the anti-FcgammaRII inhibition was statistically significant (p < 0.001). Finally, anti-HPA-1a-sensitised platelets bound to 131H- but not to 131R- FcgammaRIIa transfected 3T6 mouse fibroblasts (p < 0.01), in an anti-HPA-1a-concentration-dependent manner. The results suggest that FcgammaRI and FcgammaRIIa may be involved in anti-HPA-1a-mediated platelet destruction by mononuclear phagocytes during FAIT/NAIT. Moreover, the much greater potency ofmonoclonal H22 than of intravenous immunoglobulin as an inhibitor of anti-HPA-1a-mediated Mo-platelet interaction, might render it superior to the latter agent in the maternal therapy of the disorder.

Adult↗
PubMed1999

The FcgammaRIa (CD64) ligand binding chain triggers major histocompatibility complex class II antigen presentation independently of its associated FcR gamma-chain.

Within multi-subunit Ig receptors, the FcR gamma-chain immunoreceptor tyrosine-based activation motif (ITAM) plays a crucial role in enabling antigen presentation. This process involves antigen-capture and targeting to specific degradation and major histocompatibility complex (MHC) class II loading compartments. Antigenic epitopes are then presented by MHC class II molecules to specific T cells. The high-affinity receptor for IgG, hFcgammaRIa, is exclusively expressed on myeloid lineage cells and depends on the FcR gamma-chain for surface expression, efficient ligand binding, and most phagocytic effector functions. However, we show in this report, using the IIA1.6 cell model, that hFcgammaRIa can potentiate MHC class II antigen presentation, independently of a functional FcR gamma-chain ITAM. Immunoelectron microscopic analyses documented hFcgammaRIa alpha-chain/rabbit IgG-Ovalbumin complexes to be internalized and to migrate via sorting endosomes to MHC class II-containing late endosomes. Radical deletion of the hFcgammaRIa alpha-chain cytoplasmic tail did not affect internalization of rabbit IgG-Ovalbumin complexes. Importantly, however, this resulted in diversion of receptor-ligand complexes to the recycling pathway and decreased antigen presentation. These results show the hFcgammaRIa cytoplasmic tail to contain autonomous targeting information for intracellular trafficking of receptor-antigen complexes, although deficient in canonical tyrosine- or dileucine-targeting motifs. This is the first documentation of autonomous targeting by a member of the multichain FcR family that may critically impact the immunoregulatory role proposed for hFcgammaRIa (CD64).

Amino Acid Sequence↗
PubMed1999

Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210.

BACKGROUND: MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcgammaRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcgammaRI is expressed on human monocytes, macrophages, and IFN-gamma activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions. MATERIALS AND METHODS: Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit. RESULTS: Both BsAb MDX-210 (via FcgammaRI) and MoAb 520C9 (mouse IgG1, via FcgammaRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF. CONCLUSIONS: BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.

Animals↗
PubMed1999

Development of a trispecific antibody conjugate that directs two distinct tumor-associated antigens to CD64 on myeloid effector cells.

A trispecific F(ab')3 antibody conjugate (TAC) with specificities for the Fc gamma receptor I (Fc gamma RI/CD64), the epidermal growth factor receptor (EGFR) and the HER2/neu antigen has been developed to redirect effector cell-mediated cytotoxicity against cancer cells expressing both or either of the tumor-associated antigens. The TAC was constructed in two steps using the sulfhydryl-specific cross-linker o-phenylenedimaleimide (o-PDM). In step one, a bispecific antibody was prepared by linking the Fab' fragments of mAb m22 (a murine IgG1 specific for Fc gamma RI) and mAb H425 (a humanized IgG1 antibody recognizing EGFR). The conjugation efficiency was about 60%. In the second step, the Fab' fragment of mAb 520C9, a murine IgG1 specific for HER2/neu, was coupled to the bispecific antibody made in step one. About 40% of the bispecific conjugate was derivatized to form the trispecific antibody. The purity of the TAC was more than 90% after gel filtration purification. The TAC was characterized in vitro for its ability to bind specifically to all the three antigens and to kill target cells expressing the tumor antigens. In contrast to bispecific conjugates that can only target cells expressing either of the tumor antigens, the TAC was able to bind both the antigens more efficiently in cell-binding assays and to kill tumor cells expressing EGFR and HER2/neu antigens.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Characterization of a novel bispecific antibody that mediates Fcgamma receptor type I-dependent killing of tumor-associated glycoprotein-72-expressing tumor cells.

A bispecific antibody was made by chemical conjugation of Fab' fragments from humanized antibodies specific for tumor-associated glycoprotein-72 (TAG-72) and high-affinity immunoglobulin receptor, FcgammaA receptor type I (FcgammaRI). The purified anti-TAG-72 x anti-FcgammaRI (HCC49xH22) bispecific antibody had an approximate Mr of 111,000, consistent with a F(ab')2, and bound specifically to KLEB and LS174T tumor cell lines, which express the TAG-72 tumor antigen. Furthermore, HCC49x H22 was shown to simultaneously bind to KLEB cells and a soluble FcgammaRI fusion protein, demonstrating the bifunctional nature of the molecule. Using IFN-gamma-treated monocytes as effector cells, concentrations of the bispecific antibody in the range of 1-10,000 ng/ml mediated specific lysis of TAG-72-positive tumor cells. In contrast, the bispecific antibody did not promote antibody-dependent cellular cytotoxicity of a cell line that was negative for TAG-72 antigen. Importantly, the antibody-dependent cellular cytotoxicity activity of the bispecific antibody was significantly greater than that of the monoclonal antibody HCC49. These in vitro data indicate that the humanized bispecific antibody HCC49xH22 has the appropriate specificity and functional activity for further evaluation as potential immunotherapy for TAG-72-positive malignancies.

Animals↗
PubMed1997

Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcgamma receptor I bispecific antibodies.

Abs are able to induce inflammatory antitumor responses by recruiting IgG Fc receptor (FcgammaR)-bearing cytotoxic effector cells. We recently described the capacity of the high affinity FcgammaRI (CD64) to trigger cytotoxic activity of neutrophils (PMN) during granulocyte CSF (G-CSF) treatment. To take advantage of FcgammaRI as a cytotoxic trigger molecule on PMN, two Ab constructs were prepared. We show that a chimeric human IgG1 Ab (Ch520C9) and an anti-FcgammaRI bispecific Ab (BsAb; 22x520C9), both directed to the proto-oncogene product HER-2/neu, interact with FcgammaRI. In addition, both Ab constructs mediate enhanced lysis of HER-2/neu-expressing tumor cells by G-CSF-primed PMN. However, engagement of FcgammaRI by Ch520C9 was inhibited by human serum IgG, thereby abrogating the enhanced Ch520C9-mediated cytotoxicity. BsAb 22x520C9, which binds FcgammaRI outside the ligand binding domain, effectively recruits the cytotoxic potential of FcgammaRI on G-CSF-primed PMN regardless of the presence of human serum. These results indicate that under physiologic conditions, serum IgG impairs activation of FcgammaRI-mediated cytotoxicity by conventional antitumor Abs. The IgG blockade can be circumvented with anti-FcgammaRI BsAbs. Using human FcgammaRI transgenic mice we demonstrate that BsAb 22x520C9 is able to engage FcgammaRI in vivo. BsAb 22x520C9 injected i.v. was readily detected on circulating PMN of G-CSF-treated transgenic animals. In addition, we showed that PMN remain "armed" with BsAb 22x520C9 during migration to inflammatory sites, and that after isolation such PMN specifically lyse HER-2/neu-expressing tumor cells. These results point to the possibility of targeting anti-FcgammaRI BsAbs to G-CSF-primed PMN in vivo, endowing them with specific anti-tumor activity.

Animals↗
PubMed1997

Bispecific antibody-dependent cellular cytotoxicity of HER2/neu-overexpressing tumor cells by Fc gamma receptor type I-expressing effector cells.

A bispecific antibody, MDX-H210, was developed to target cytotoxic effector cells expressing Fc gamma receptor type I (Fc gammaRI, CD64) to HER2/neu-overexpressing tumor cells. HER2/neu is an appropriate target for immunotherapy due to the high level of expression of this proto-oncogene in a variety of malignancies. The expression of Fc gammaRI is limited primarily to cytotoxic immune cells, including monocytes, macrophages, and cytokine-activated polymorphonuclear (PMN) cells. Therefore, tumor cells bound with MDX-H210 can be selectively recognized by effector cells with cytotoxic potential. MDX-H210 was prepared by chemical conjugation of Fab' fragments derived from the HER2/neu-specific monoclonal antibody, 520C9, and the Fc gammaRI-specific monoclonal antibody, H22. This bispecific molecule demonstrated specific, dose-dependent, and saturable binding to both HER2/neu- and Fc gammaRI-expressing cells. A solid-phase immunoassay that demonstrated simultaneous and specific binding to both antigens was used to confirm the bispecific nature of MDX-H210. Monocytes and PMN cells mediated MDX-H210-dependent lysis of HER2/neu-overexpressing cell lines derived from breast, ovarian, and lung carcinomas. IFN-gamma treatment of monocytes enhanced antibody-dependent cellular cytotoxicity, whereas IFN-gamma and granulocyte colony-stimulating factor were required for PMN cell-mediated tumor cell lysis. In addition, MDX-H210 elicited tumor necrosis factor-alpha secretion from monocytes when cultured in the presence of HER2/neu-positive target cells. These in vitro data suggest that targeting tumor cells to Fc gammaRI with MDX-H210 may be an effective treatment for malignancies that overexpress HER2/neu. The in vivo cytotoxic potential of MDX-H210 may be enhanced by combination therapy with the cytokines granulocyte colony-stimulating factor and IFN-gamma, which up-regulate Fc gammaRI expression on cytotoxic effector cells.

Antibodies, Bispecific↗
PubMed1997

Humanized mAb H22 binds the human high affinity Fc receptor for IgG (FcgammaRI), blocks phagocytosis, and modulates receptor expression.

About 10-15% of patients with immune thrombocytopenic purpura (ITP) cannot be controlled by corticosteroid therapy and splenectomy. For these patients treatment with high-dose IVIgG induces partial or complete responses. The clinical benefits of IVIgG could be due to blockade of Fc receptors for IgG (FcgammaR), because several model systems clearly show that functional FcgammaR are essential for establishment of ITP and related diseases. However, the specific contributions of the three individual classes of FcgammaR remain to be more completely defined. Recently monoclonal antibody (mAb) H22, which recognizes an epitope on FcgammaRI (CD64) outside the ligand binding domain, was humanized by grafting its complementarity determining regions onto human IgG1 constant domains. Because FcgammaRI has a high affinity for human IgG1 antibodies, we predicted mAb H22 would also bind to FcgammaRI through its Fc domain and block FcgammaRI-mediated phagocytosis. These studies demonstrate that mAb H22 blocked phagocytosis of opsonized red blood cells 1000 times more effectively than an irrelevant IgG. Moreover, cross-linking FcgammaRI with mAb H22 rapidly down-modulated FcgammaRI expression on monocytes without affecting other surface antigens. We conclude that because mAb H22 is a humanized mAb that blocks the FcgammaRI ligand binding domain and down-modulates FcgammaRI expression, it is a particularly good candidate for evaluating the role of FcgammaRI in patients with ITP.

Animals↗
PubMed1996

Development of T-cell lines expressing functional HIV-1 envelope glycoproteins for evaluation of immune responses in HIV-infected individuals.

The human T-lymphoid cell line, CEM, was transfected with gp 160 cDNA of human immunodeficiency virus type 1 (HIV-1)pm213. Three clones expressing the envelope glycoproteins (env), designated CEM-213env1, -env4, and -env7, were isolated. These clones expressed high levels of surface gp41 and gp120, as demonstrated by flow cytometry with anti-HIV env monoclonal antibodies. Processing and function of env was shown by induction of syncytia with CD4-expressing HeLa cells and by immunoblot analysis. The env expression resulted in specific down-regulation of surface CD4 levels, supporting the role of HIV env in CD4 modulation. Furthermore, serum samples from nine of nine HIV-1-infected individuals bound specifically to the env-expressing transfectants, substantiating the presence of conserved antigenic determinants. These sera also mediated antibody-dependent cellular cytotoxicity (ADCC) of the env-expressing cell lines. The env-expressing cell lines provide a relevant, safe, and practical model for qualitative and quantitative analysis of humoral and cellular immune responses and their role in HIV-1 pathogenesis and therapy.

Blotting, Western↗