Evaluation of the resistance to bacterial growth of star-shaped poly(ε-caprolactone)-co-poly(ethylene glycol) grafted onto functionalized carbon nanotubes nanocomposites.

Nanocomposites of functionalized carbon nanotubes (CNTsf) as nanofillers, and a copolymer of star-shaped poly(ε-caprolactone) (stPCL) and poly(ethylene glycol) (PEG) as a polymeric matrix were synthesized, characterized, and their resistance to the growth of Staphylococcus aureus and Pseudomonas aeruginosa was evaluated. CNTsf contain hydroxyl, carboxyl and acyl chloride groups attached to their surface. Nanocomposites were prepared by mixing CNTsf to a solution of stPCL-PEG copolymer. Raman and FT-IR spectroscopies confirm the functionalization of carbon nanotubes (CNTs). Star-shaped PCL-PEG copolymer was characterized by Gel permeation chromatography (GPC), and 1H-NMR and 13C-NMR spectroscopies. X-ray photoelectron spectroscopy (XPS) shows that CNTsf are grafted to the stPCL-PEG copolymer. Crystallization behavior of the nanocomposites depends on the amount of CNTsf used in their preparation, detecting nucleation (nanocomposites prepared with 0.5 wt.% of CNTsf) or anti-nucleation (nanocomposites prepared with 1.0 wt.% of CNTsf) effects. Young's Moduli and thermal stability of nanocomposites were higher, but their resistence to the proliferation of Staphylococcus aureus and Pseudomonas aeruginosa was lower than the observed for their pure polymer matrix.

Designing a Low Cost Electrospinning Device for Practical Learning in a Bioengineering Biomaterials Course / Diseño de un dispositivo de electrohilado de bajo costo para la enseñanza práctica en un curso de Biomateriales en Bioingeniería

Abstract The electrospinning device is used in the biomaterials research field nowadays for fabricating nanofibers that can be used for manufacturing artificial skin and muscular tissue, blood vessels (vascular grafts), orthopedic components (bones, cartilages, and ligaments/tendon), and peripheral or central nervous system components. Electrospun nanofibers act as ideal scaffolds for tissue engineering and drug delivery systems because they can mimic the functions of native extracellular matrices. A low cost electrospinning device was designed and built for undergraduate practical learning in the Biomaterials course in the area of Bioengineering at Universidad Autónoma de Baja California, México. The methodology includes 3D CAD designing, manufacturing of the acrylic cabinet, different collectors and the fabrication of poly (vinyl alcohol) nanofibrous scaffolds, in order to validate the functionality of the electrospinning system. The prototype is an affordable device; its cost is 95% less than the laboratory commercial devices.

Resumen El dispositivo de electrohilado es actualmente empleado en la investigación de biomateriales, utilizado para sintetizar nanofibras que ofrecen un potencial para la manufactura de piel artificial y tejido muscular, vasos sanguíneos (implantes vasculares), componentes ortopédicos (hueso, cartílago y tendones/ligamentos) y componentes del sistema nervioso central y periférico. Las nanofibras producidas por electrohilado pueden ser usadas como andamios ideales para ingeniería de tejidos y liberación controlada de fármacos debido a que mimetizan las funciones de la matriz extracelular. El dispositivo de electrohilado de bajo costo fue diseñado y construido para al aprendizaje practico de estudiantes de licenciatura en la asignatura de Biomateriales de la carrera de Bioingeniería. La metodología incluye diseños CAD 3D, manufactura del gabinete de acrílico, diferentes colectores y fabricación de los andamios de nanofibras de Poli (vinil alcohol) para validar la correcta funcionalidad del sistema de electrohilado. El prototipo es un dispositivo accesible económicamente, su costo es un 95% más barato que los dispositivos de tipo comercial.

Characterization of bone cements prepared with functionalized methacrylates and hydroxyapatite.

Bone cements prepared with methyl methacrylate and either methacrylic acid or diethyl amino ethyl methacrylate as comonomers were characterized by infrared spectroscopy, nuclear magnetic resonance, gel permeation chromatography, dynamic mechanical thermal analysis, and mechanical testing. Selected formulations containing these functionalized methacrylates were filled with hydroxyapatite and studied in terms of their properties in tension, compression and bending, and X-ray diffraction. It was found that residual monomer was not greatly affected by the presence of either acid or basic comonomers in the unfilled bone cements. In contrast, molecular weight, curing times, and glass transition temperature were composition dependent. For samples with acidic comonomer, a faster curing time, higher molecular weight, and higher glass transition temperatures were observed with respect to those with the basic comonomer. X-ray diffraction revealed that the crystalline structure was not affected by the nature of comonomer in the bone cement while scanning electron microscopy showed that hydroxyapatite remained as clusters in the bone cement. The mechanical properties of filled bone cements depended mainly on composition and type of testing. Hydroxyapatite-filled bone cements fullfilled the minimum compressive strength (70 MPa) required for bone cement use. However, the minimum tensile strength (30 MPa) was only fullfilled by cements prepared without comonomer and those containing methacrylic acid. The minimum bending strength requirement (50 MPa) was not satisfied by any of the formulations studied.

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.

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.

Uptake of antigen-antibody complexes by human dendritic cells.

Fc receptors specific for IgG (FcγR) potentiate the immune response by facilitating the interaction between myeloid cells and antibody-coated targets (1-3). Monocyte and neutrophil FcyR engagement can lead to the induction of lytic-type mechanisms associated with innate responses. FcyR triggering can also play a key role in adaptive immune responses. For example, FcyR-directed capture and uptake of antigens (Ag) by dendritic cells (DC) results in processing and presentation to naive Ag-specific T cells, leading to their expansion and maturation into effector T-cell populations. This chapter describes methodology currently in use to explore and manipulate antigen-antibody (Ag-Ab) uptake by FcyR expressed on DC.

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.

Triggering Fc alpha-receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy.

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In (51)Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcalphaRI) and CD20, a broad range of B cell lines were effectively killed. FcalphaRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcalphaRI x CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcgammaRI- or FcgammaRIII-directed BsAbs against CD20. Experiments with blood from human FcalphaRI/FcgammaRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcalphaRI x CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.

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.

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.

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.

FcalphaRI (CD89) as a novel trigger molecule for bispecific antibody therapy.

Promising results from clinical trials with unconjugated antibodies stimulated renewed interest in immune effector mechanisms of monoclonal antibodies (MoAbs). We investigated the potential of IgA as antibody isotype for cell- or complement-mediated tumor cell lysis and assessed the potential of its myeloid Fc receptor, FcalphaRI (CD89), as trigger molecule for bispecific antibody (BsAb)-mediated immunotherapy. Comparing hapten-directed antibodies of human IgA2 with IgG1 or IgG3 isotypes, we found all three to mediate effective killing of sensitized tumor target cells in whole blood assays. Analysis of effector mechanisms showed IgG-mediated lysis to be predominantly complement-dependent, whereas IgA-dependent killing was primarily effector cell-mediated. A comparison of effector cell populations in antibody-dependent cell-mediated cytotoxicity (ADCC) showed neutrophils to be most important for IgA-dependent tumor cell killing, involving FcalphaRI as shown with Fc receptor blocking antibodies. Reverse ADCC experiments against target cells sensitized with Fc receptor antibodies, or assays with FcalphaRI-directed bispecific antibodies confirmed FcalphaRI as effective trigger molecule in polymorphonuclear neutrophil (PMN)-mediated lysis. During granulocyte colony-stimulating factor (G-CSF ) therapy, (FcalphaRI x HER-2/neu) bispecific antibodies induced enhanced killing of HER-2/neu positive SK-BR-3 breast cancer cells in whole blood assays. This enhanced cytotoxicity was paralleled by increased PMN counts, which lead to higher effector to target cell ratios in G-CSF-primed blood. Furthermore, bispecific antibodies, directed to FcalphaRI and Candida albicans, enhanced neutrophils' phagocytosis of fungi. In summary, these results identify IgA as an effective antibody isotype for immunotherapy, working primarily via FcalphaRI on neutrophils. They suggest FcalphaRI-directed bispecific antibodies and G-CSF to be an attractive combination for malignant or infectious diseases.

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.

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.

Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor Fc gamma RI (CD64) expressed on human blood dendritic cells.

The mechanisms responsible for efficient sequestration of Ag by cells of the dendritic cell (DC) lineage remain incompletely characterized. One pathway, internalization of Ag-IgG complexes via CD32 (the type II IgG FcR, Fc gamma RII) enhances Ag presentation 100-fold over noncomplexed Ag. Blood leukocytes differentially express two additional IgG FcR, Fc gamma RI (CD64) and Fc gamma RIII (CD16), which may also participate in leukocyte functions such as phagocytosis, Ab-dependent cellular cytotoxicity (ADCC), release of oxygen intermediates, and enhancement of Ag presentation. A phagocytically active form of CD64 was recently demonstrated on human blood DC, but complete functional potential of CD64 on the DC lineage remains undefined. Therefore, highly purified human blood DC (CD33(2)+, CD14-, CD11c2+, HLA-DR3+, CD64+ (CD83+ after overnight culture)) and monocytes (CD33(2)+, CD14(3)+, CD11c2+, HLA-DR+, CD64(2)+, CD83-) were compared for cytokine modulation and effector functions of CD64. Both DC and monocyte CD64 expression was increased by IFN-gamma and IL-10, but while monocyte CD64 was decreased by IL-4, DC CD64 remained unchanged. FcR-mediated functional differences were also evident between the DC and the monocytes. Monocytes generated robust Fc gamma R-dependent superoxide anion release and ADCC activity, while DC failed to release reactive oxygen intermediates and demonstrated minimal ADCC activity, despite apparently normal expression of the gamma-chain subunit and the signaling molecule Syk. In contrast, DC were more efficient than monocytes with respect to T cell activation when Ag was targeted specifically to CD64. These new findings suggest a previously unappreciated potential for CD64 to shape the immune response by dramatically increasing the efficiency with which DC sequester Ag prior to achieving full T cell stimulatory potential.

Clinical significance of IgG Fc receptors and Fc gamma R-directed immunotherapies.

Fc receptors for IgG (Fc gamma Rs) can trigger the inflammatory, cytotoxic and hypersensitivity functions of immune effector cells. Activation or deactivation of effector cells via Fc gamma Rs can be exploited to develop novel therapies for cancer, infectious diseases and autoimmune disorders. Initial results of clinical trials for several Fc gamma R-directed immunotherapies show the potential promise of this approach.

Cytolytic and cytostatic properties of an anti-human Fc gammaRI (CD64) x epidermal growth factor bispecific fusion protein.

A bispecific fusion protein (H22-EGF) that binds simultaneously to the epidermal growth factor receptor (EGF-R) and to the high affinity receptor for the Fc portion of human IgG, Fc gammaRI (CD64), has been successfully constructed and expressed. For this construction, genomic DNA encoding the Fd fragment of humanized anti-Fc gammaRI mAb, H22, which binds Fc gammaRI at an epitope that is distinct from the Fc binding site, was fused to cDNA encoding human epidermal growth factor (EGF), a natural ligand for EGF-R. The resulting H22Fd-EGF-expressing vector was transfected into a myeloma cell line that was transfected previously with a vector containing DNA encoding the H22 kappa-light chain. SDS-PAGE analysis of purified H22-EGF demonstrated that the fusion protein was secreted predominantly as H22Fab'-EGF monomer (approximately 55 kDa), even though a free Cys residue exists in the hinge region of the H22 Fab' component. Using a novel bispecific flow cytometry-binding assay, we demonstrated that the purified bispecific fusion protein, H22-EGF, was able to bind simultaneously to soluble Fc gammaRI and EGF-R-expressing cells. H22-EGF inhibited the growth of EGF-R-overexpressing tumor cells and mediated dose-dependent cytotoxicity of these cells in the presence of Fc gammaRI-bearing cytotoxic effector cells. These results suggest that this fusion protein may have therapeutic utility for EGF-R-overexpressing malignancies.

Phase I study of carboplatin, cisplatin, and cyclophosphamide without and with lenograstim for the treatment of ovarian cancer.

Cisplatin and carboplatin are both active in ovarian cancer with different toxicity profiles; thus, dose intensification may be possible by combining them. The aim of the present study was to determine the maximum tolerated dose of carboplatin combined with fixed doses of cisplatin and cyclophosphamide without and with support of lenograstim. Cisplatin (60 mg/m(2)), cyclophosphamide (600 mg/m(2)) and carboplatin (starting dose 200 mg/m(2)) were given on day 1 every 3 weeks for 4 cycles. Escalated dose levels for carboplatin were planned by increments of 50 mg/m(2) per level. Lenograstim (L) (150 mu g/m(2)/day subcutaneously) was given in case of grade 4 leukopenia (levels without support) or from day 5 up to leukocyte >10,000/mm(3) after nadir (levels with support). Four levels were studied (200, 250, 250 + lenograstim, 300 + lenograstim) with 7, 7, 8, and 7 patients enrolled, respectively. Unacceptable toxicity was induced in 1 patient at the level I (grade 4 thrombocytopenia), in 4 patients at the level 2 (2 prolonged grade 2 leukopenia, 1 grade 4 leukopenia with concomitant grade 4 thrombocytopenia and 1 grade 4 thrombocytopenia), in 1 patient at the level 2 + L (grade 4 thrombocytopenia) and in 3 patients at the level 3 + L (3 grade 4 thrombocytopenia). Thus, 200 mg/m(2) and 250 mg/m(2) were defined as carboplatin MTDs without and with lenograstim support, respectively. Median total platinum (cisplatin + 1/4 carboplatin) delivered dose-intensities were 33, 32, 38 and 44 mg/m(2)/week at the four levels, respectively. Hematological toxicity was overall mild. In no case was febrile neutropenia recorded. Grade 4 thrombocytopenia was always transient and never symptomatic. Grade 3 vomiting was the only severe non-hematological toxicity reported in 5 patients. Out of 16 patients with measurable disease, 11 objective responses were obtained (5 complete and 6 partial) for an overall response rate of 69% (95% exact CL 41-89%). Recommended dose of carboplatin is 200 mg/m(2) without and 250 mg/m(2) with support of lenograstim when combined with cisplatin 60 mg/m(2) and cyclophosphamide 600 mg/m(2). Dose limiting toxicity is persistent leukopenia without and grade 4 thrombocytopenia with support of lenograstim.

[Prevalence of retinopathy of prematurity in very low birth weight infants]. / Prevalência da retinopatia da prematuridade em recém-nascidos de muito baixo peso.

OBJECTIVE: Evaluate the prevalence of retinopathy of prematurity (ROP) in very low birthweight infants (birthweight<1500 g). METHOD: A prospective examination was conducted on 102 neonates with very low birthweight admitted to the BAM-HC (FMUSP) between 01/01/92 and 12/31/93. The mapping of the retina with scleral depression was first conducted between 3rd and the 8th weeks of life, and it was repeated every 1 to 4 weeks until the vascularization of the retina was complete or the ROP was present. To classify the ROP the International Classification of ROP was used. For the purposes of statistical analysis, the most serious phase of ROP presented by the neonate was considered. RESULTS: In this study retinopathy of prematurity was present in 29.09% of the neonates, in 78.5% of those under 1,000 g of birthweight, and 72.73% of those with less than 30 weeks of gestational age. Among the newborns with ROP, 77.05% were in phase 1, 13.11% in phase 2, and 9.84% in phase 3. Oxygen in mechanical ventilation and "CPAP" were statistically significant factors for the development the ROP. CONCLUSION: The ophthalmologic examination between the 3rd and 4th weeks of life was an important instrument for the detection of RP and should be done in all very low birth weight infants (weight<1,500 g), especially in neonates with less than 1,250 g and/or gestational age under 34 weeks.