Preclinical studies with Fc(gamma)R bispecific antibodies and granulocyte colony-stimulating factor-primed neutrophils as effector cells against HER-2/neu overexpressing breast cancer.

Immunotherapies directed to the proto-oncogene product HER-2/neu, which is overexpressed on a subset of breast and other carcinomas, currently receive considerable attention. We have investigated cell-mediated effector mechanisms of HER-2/neu antibodies against breast cancer cell lines. Compared to unfractionated control blood, whole blood from patients during granulocyte colony-stimulating factor (G-CSF) treatment exhibits significantly enhanced lysis (P < 0.001) of SK-BR-3 cells in the presence of HER-2/neu antibody 520C9. The extent of tumor cell killing correlated positively (r = 0.74) to polymorphonuclear neutrophil (PMN) blood counts. Fractionation of whole blood into plasma, mononuclear cells, and PMNs showed major killing capacity to reside in the granulocyte fraction. PMNs were efficiently cytolytic with a panel of HER-2/neu antibodies and against various breast cancer cell lines. Experiments with blocking antibodies to Fc(gamma)R documented Fc(gamma)RII (CD32) as the major trigger molecule for monoclonal antibody 502C9-mediated cytotoxicity. Killing via 520C9 was significantly influenced by an allotypic polymorphism of Fc(gamma)RIIa, the CD32 molecule expressed on PMNs. In reverse antibody-dependent cell-mediated cytotoxicity experiments with a panel of HER-2/neu-directed bispecific antibodies, Fc(gamma)RIII (CD16) proved to be an efficient trigger molecule in blood from healthy volunteers. During G-CSF treatment, however, Fc(gamma)RI (CD64)-expressed on monocytes and G-CSF primed, but not on healthy donor PMNs-became the predominant cytotoxic trigger molecule. Thus, G-CSF application increased effector cell numbers for HER-2/neu-directed immunotherapy, and G-CSF primed PMNs proved particularly effective with a [HER-2/neu x Fc(gamma)RI] bispecific antibody. These findings support clinical trials with HER-2/neu-directed antibodies in combination with G-CSF in breast cancer patients overexpressing HER-2/neu.

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.

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.

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.

Practical considerations in operation and scale-up of spin-filter based bioreactors for monoclonal antibody production.

Minimum spin-filter fouling and optimum cell retention at high specific perfusion rates are important for efficient operation of a spin-filter based continuous perfusion bioreactor. We examined the effect of operation conditions and spin-filter configuration on the performance of continuous perfusion bioreactors using a perfusion recycle scheme. This study showed that single cell suspensions foul a spin-filter screen, partially but irreversibly, in the early stages of the bioreactor run. A high perfusion rate and cell density contribute to screen fouling, while an increase in rotational velocity and screen surface area per reactor volume reduce screen fouling. An empirical model was developed to describe the effects of these parameters on the perfusion capacity of a spin-filter. Examination of screen samples by scanning electron microscopy confirmed that partial screen fouling occurs at relatively early stages of fermentation. Once the initial partial screen fouling has occurred, further fouling continues slowly due to cell growth on the screen surface, and this gradually leads to the overflow state. The rate of this gradual fouling phase probably depends upon the number of cells deposited on the screen surface during the initial partial fouling. The usefulness of this model was confirmed by successful scale-up of high-cell-density (> 10 x 10(6)/mL) long-term (> 30 days) continuous perfusion process for commercial scale production of monoclonal antibodies.

Cloning and characterization of the sisomicin-resistance gene from Micromonospora inyoensis.

We cloned DNA fragments of sisomicin-producing Micromonospora inyoensis into Streptomyces plasmid vectors and identified Streptomyces lividans TK24 transformants expressing the M. inyoensis sisomicin-resistance (sisA) gene. The sisA gene was compared to the previously reported Micromonospora purpurea Kan-Gen (kanamycin-gentamicin)-resistance gene. While the restriction endonuclease digestion patterns of the two determinants appear to be divergent, the genes are nonetheless closely related, based on similar patterns and levels of aminoglycoside-resistance and their ability to cross-hybridize under stringent conditions. We have transformed recombinant plasmid pMD5-2, which carries the sisA gene, into our M. purpurea gentamicin-production strain and determined that gentamicin biosynthesis was not improved.

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.

Semicontinuous and continuous production of penicillin-G by Penicillium chrysogenum cells immobilized in kappa-carrageenan beads.

Conidia of Penicillium chrysogenum were immobilized in K-carrageenan beads and then incubated in a growth-supporting medium to yield a penicillin producing immobilized cell mass. These in situ grown immobilized cells were used for the semicontinuous (replacement cultures)and continuous (fluidized bioreactor culture) production of penicillin-G. When periodically replaced into a minimal production medium, immobilized cells exhibited a half-life for penicillin production which was ninefold greater than that exhibited by free cells. The half-life of penicillin production and the yield of penicillin from glucose in such a replacement culture were greatly affected by the frequency of replacement and by the production medium's pH and concentration of glucose, phosphate, and trace metal nutrients. A penicillin-producing continuous flow bioreactor (150 mL), employing immobilized cells, was operated for up to 16 days. The best specific penicillin productivity (1.2 mg/g cells/h)yield from glucose (7.0 mg/g glucose) and half-life of production (15 days) were obtained when the feed medium contained 10 g/L of glucose, the pH was maintained at 7.0, the relative dissolved oxygen concentration was ca. 40%; and the residence time was 20 h.

Media replenishment: a tool for the analysis of high-cell density perfusion systems.

A simple method to analyze media consumption and by-product build-up in high-cell-density perfusion cultures has been developed. This technique makes use of a media replenishment strategy which can be used to decouple the two phenomena. Also, the replenished media method can be used to identify limitation by complex and undefined media components such as bovine serum.

Examination of immobilized fungal cells by phase-contrast and scanning electron microscopy.

Conidia of Penicillium urticae were immobilized in kappa-carrageenan beads and then shaken, in a growth-supporting medium to yield an in situ grown population of mycelia. The physical stability of these beads and the degree of mycelial growth inside the beads were significantly affected by the concentrations of kappa-carrageenan and locust bean gum (LBG) in the bead matrix and by the porous or nonporous nature of the interior. Thus 16-h-old porous and nonporous beads, prepared from 1.25% kappa-carrageenan, 0.5% LBG, and conidia, possessed a very dense mycelial mass at the surface. Only the porous beads possessed a moderately dense mycelial mass at the centre. The conidia at the centre of nonporous beads either failed to germinate or formed very small germ tubes. When washed, 36-h-old porous beads were repeatedly (i.e., 48 h) transferred into nitrogen-free medium, the density of mycelia at the centre increased to equal that at the surface after three transfers or 8 days. Mycelia at the surface exhibited signs of physical damage, while those in the centre did not. The addition of 100 micrograms/mL of cycloheximide to these replacement cultures was reflected by the distortion of interior mycelia.

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.

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.

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.

Role of Src in the modulation of multiple adaptor proteins in FcalphaRI oxidant signaling.

Cross-linking of Fc receptors for IgA, FcalphaR (CD89), on monocytes/macrophages is known to enhance phagocytic activity and generation of oxygen free radicals. We provide evidence here that the FcalphaR signals through the gamma subunit of FcepsilonRI in U937 cells differentiated with interferon gamma (IFNgamma). Our results provide the first evidence that FcalphaR-mediated signals modulate a multimolecular adaptor protein complex containing Grb2, Shc, SHIP, CrkL, Cbl, and SLP-76. Cross-linking of FcalphaRI using anti-FcalphaRI induces the phosphorylation of the gamma subunit as detected by mobility retardation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Stimulation of FcalphaRI induced the tyrosine phosphorylation of Shc and increased the association of Grb2 with Shc and CrkL. Grb2 associates constitutively with Sos, and the latter undergoes mobility shift upon FcalphaRI stimulation. The complex adapter proteins, Cbl and SLP-76, are physically associated in myeloid cells and both proteins undergo tyrosine phosphorylation upon FcalphaR stimulation. These data indicate that the stimulation of FcalphaR results in the modulation of adaptor complexes containing tyrosine-phosphorylated Cbl, Shc, SHIP, Grb2, and Crkl. Experiments performed with the Src kinase inhibitor, PP1, provide the first evidence that Src kinase activation is required for FcalphaRI-induced production of superoxide anions and provide insight into the mechanism for FcalphaR-mediated activation of downstream oxidant signaling in myeloid cells.

Human monocyte-derived dendritic cells produce macrophage colony-stimulating factor: enhancement of c-fms expression by interleukin-10.

Human monocyte-derived dendritic cells (DC) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 express c-fms (CD115), the receptor for macrophage-CSF (M-CSF). Expression of c-fms on monocyte-derived DC has been interpreted as the susceptibility of these cells to M-CSF-induced macrophage development. We show here that homogeneous cultures of CD14 DC constitutively produced large amounts of M-CSF. However, presence of M-CSF neither induced macrophage development nor did it prevent terminal maturation into CD83+ DC. M-CSF production by DC was driven by GM-CSF and inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin. M-CSF synthesis was rapidly induced during the first 24 h of DC culture and then declined during the 5-day culture period. Replating of the cells, which was associated by a transient adherence, always induced a strong up-regulation of M-CSF synthesis. Addition of recombinant IL-10 to DC cultures enhanced c-fms expression and induced macrophage development as measured by the strong up-regulation of CD14 expression as well as by enhanced expression of the Fcgamma receptors I, II, and III (CD64, CD32, CD16). Our data demonstrate that immature monocyte-derived DC produce M-CSF which does not induce macrophage development, despite the surface expression of c-fms on DC. IL-10 appears to induce macrophage development by up-regulating c-fms and, thereby, enhancing the sensitivity of the cells to endogenously produced M-CSF.

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.

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.

A pilot trial of GM-CSF and MDX-H210 in patients with erbB-2-positive advanced malignancies.

MDX-H210 is a chemically, cross-linked, half-humanized bispecific antibody composed of F(ab') fragment from monoclonal antibody (mAb) H22 that binds to the high-affinity receptor Fc gamma RI and F(ab') of mAb 520C9 that recognizes the erbB-2 (HER2/neu) oncoprotein. In a previous trial, the murine bispecific, MDX-210 at a dose of 7 mg/m2, was well tolerated and activated monocytes and macrophages in vivo in doses as low as 0.35 mg/m2. In our multidose trial, granulocyte-macrophage colony-stimulating factor, which increases and activates potential effector cells, was given on days 1-4 at 250 micrograms/m2 s.c. and MDX-H210 was given on day 4 weekly for 4 consecutive weeks. Thirteen patients were treated at dose levels of 1, 3.5, 7, 10, 15, and 20 mg/m2 without dose-limiting toxicity. Fever, chills, and rigors occurred during and up to 2 h postinfusion and correlated with the time to peak levels of tumor necrosis factor-alpha (median 88.2 pg/ml; range 15.6-887 pg/ml) and interleukin-6 (median 371 pg/ml; range 175-2,149 pg/ml). By the fourth consecutive week of treatment the side effects and cytokine levels decreased significantly. Human antibispecific antibody (HABA) levels were increased by 200- to 500-fold above pretreatment levels in 5 of 11 evaluable patients after 3 weeks of treatment. The monocyte and granulocyte population increased on days 4 and 11 (median 44%; range 18-68% and 42%; 19-71%), respectively, for monocytes and (60%; 43-75% and 74%; 54-82%) on days 4 and 11 for granulocytes. There was a significant decrease in the monocyte populations immediately after MDX-H210 administration (median decrease 73%; range 42-94%) and (52%; 12-72%) on days 4 and 11, respectively. Ten patients completed 4 weeks of treatment. One patient had a 48% reduction in an index lesions and six patients had stable disease at the time of evaluation. Three patients progressed before the fourth week. The therapy was generally well tolerated with toxicity, primarily, limited to the days of treatment.

In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody.

Neutrophils isolated from cancer patients treated with granulocyte colony-stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G-CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo.