Immunotherapy of human glioma xenografts with unlabeled, 131I-, or 125I-labeled monoclonal antibody 425 to epidermal growth factor receptor.

Monoclonal antibody (mAb) 425 (IgG2a) binds to the external domain of the epidermal growth factor receptor. This determinant is highly expressed by human glioma tissues but rarely by normal brain tissues, and is absent on peripheral blood lymphocytes and bone marrow cells. The mAb exerts variable cytotoxic effects against cultured human glioma cells in conjunction with human and murine effector cells. Inhibition of growth of s.c. glioma xenografts in nude mice by the mAb may be mediated by murine macrophages or may be related to the capacity of the mAb to antagonize growth stimulation of glioma cells by epidermal growth factor. In approaches to radioimmunotherapy of human glioma with mAb 425, the 125I-labeled mAb 425 exhibited more significant antitumor effects than the 131I-labeled mAb both in vitro and in vivo in xenotransplanted nude mice. These differences may be due to enhanced nuclear damage caused by 125I-labeled versus 131I-labeled fragments following their internalization into the glioma cells. Our studies provide the rationale for immunotherapy of glioma patients with either unlabeled or 125I-labeled anti-epidermal growth factor receptor mAb 425.

Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors.

Tumor angiogenesis is mediated by tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] play an important role in vascular permeability and tumor angiogenesis. Previously, we reported on the development of anti-Flk-1 and antikinase insert domain-containing receptor monoclonal antibodies (mAbs) that potently inhibit VEGF binding and receptor signaling. Here, we report the effect of anti-Flk-1 mAb (DC101) on angiogenesis and tumor growth. Angiogenesis in vivo was examined using a growth factor supplemented (basic fibroblast growth factor + VEGF) Matrigel plug and an alginate-encapsulated tumor cell (Lewis lung) assay in C57BL/6 mice. Systemic administration of DC101 every 3 days markedly reduced neovascularization of Matrigel plugs and tumor-containing alginate beads in a dose-dependent fashion. Histological analysis of Matrigel plugs showed reduced numbers of endothelial cells and vessel structures. Several mouse tumors and human tumor xenografts in athymic mice were used to examine the effect of anti-Flk-1 mAb treatment on tumor angiogenesis and growth. Anti-Flk-1 mAb treatment significantly suppressed the growth of primary murine Lewis lung, 4T1 mammary, and B16 melanoma tumors and growth of Lewis lung metastases. DC101 also completely inhibited the growth of established epidermoid, glioblastoma, pancreatic, and renal human tumor xenografts. Histological examination of anti-Flk-1 mAb-treated tumors showed evidence of decreased microvessel density, tumor cell apoptosis, decreased tumor cell proliferation, and extensive tumor necrosis. These findings support the conclusion that anti-Flk-1 mAb treatment inhibits tumor growth by suppression of tumor-induced neovascularization and demonstrate the potential for therapeutic application of anti-VEGF receptor antibody in the treatment of angiogenesis-dependent tumors.

Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model.

The epidermal growth factor receptor (EGFR) is a protein tyrosine kinase expressed on many types of tumor cells, including breast, ovarian, bladder, head and neck, and prostatic carcinoma. There seems to be an association between up-regulation of the EGFR and poor clinical prognosis for a number of human cancers. The 225 antibody is a highly specific murine monoclonal antibody that binds specifically to the human EGFR with an affinity equal to its ligand, competes with the ligand for binding, and blocks activation of the receptor tyrosine kinase. In addition, 225 has been shown to inhibit the growth of human tumor xenografts in athymic nude mice. The 225 antibody has recently been chimerized with human IgG1 in its constant region to increase its clinical utility by decreasing the potential for generation of human antimouse antibodies in recipients. This report compares the biological effects of 225 and its chimeric counterpart (designated C225) against established A431 tumor xenografts in nude mice. The results of these experiments indicated that C225 was more effective than 225 in inhibiting tumor growth in this model. In addition, many of the animals treated with C225 were tumor free at the end of each treatment protocol. It was determined that the dissociation constant of C225 was about 5-fold lower than 225. This suggested that the increased capacity of C225 to compete with ligand for binding to the EGFR was responsible for its enhanced in vivo antitumor effect.

Mouse-human chimeric anti-epidermal growth factor receptor antibody C225 inhibits the growth of human renal cell carcinoma xenografts in nude mice.

The epidermal growth factor (EGF) receptor and its ligand transforming growth factor-alpha (TGF-alpha) are overexpressed in human renal cell carcinoma (RCC). The chimeric anti-EGF receptor monoclonal antibody C225 was used to determine the effects of blocking the EGF receptor on RCC growth both in vitro and in vivo. A panel of RCC cell lines all tested positive at various levels for EGF receptor cell surface expression. C225 inhibited DNA synthesis of cultured A498, Caki-1, SK-RC-4, SK-RC-29, and SW839 cells in a dose-dependent manner, ranging from 20 to 45% inhibition compared with untreated controls. C225 also inhibited exogenous ligand-stimulated tyrosine phosphorylation of EGF receptor on RCC cells. The antitumor effects of C225 on RCC tumor growth were evaluated in ascites, s.c., and orthotopic RCC xenograft models. Mice treated with C225 in a Caki-1 ascites xenograft model showed a significant increase in survival (P = 0.002). All control mice died with ascites tumors by week 9, whereas >70% of C225-treated mice survived beyond 12 weeks. C225 also inhibited the growth of s.c. SK-RC-29 tumors in a dose-dependent manner. Mice treated with C225 (1 mg/dose) displayed a significant decrease in tumor volume compared with mice treated with control antibody (P < 0.05) or vehicle alone (P < 0.01). Lastly, C225 inhibited the growth and metastasis of RCC tumors growing orthotopically in the renal subcapsule of nude mice. Histological examination of RCC tumors from mice treated with C225 showed a substantial decrease in proliferating cell nuclear antigen staining and an increase in tumor cell apoptosis. These data suggest that C225 affects growth of RCC tumors by inhibiting EGF receptor-dependent proliferation and demonstrate the potential for therapeutic application of C225 in the treatment of human renal cancer.

Monoclonal anti-idiotypic antibodies that mimic the epitope on gp120 defined by anti-HIV-1 monoclonal antibody 0.5 beta.

OBJECTIVE: To develop effective, specific and safe anti-idiotypic antibody (Ab2) vaccines against HIV-1. DESIGN: Murine monoclonal Ab2 were generated against anti-HIV-1 antibody 0.5 beta (Ab1), which binds to gp120, neutralizes HIV-1 and inhibits virus-induced syncytia formation. METHODS: Mice were immunized with Ab1, and Ab2 were produced from immunized mice by the hybridoma technique. The Ab2 were characterized in vitro, injected into rabbits, and the anti-anti-idiotypes (Ab3) induced in the rabbits were analyzed for binding and antiviral reactivities by enzyme-linked immunosorbent assay, p24gag release and syncytia formation assays. RESULTS: Seven Ab2 bound to the antigen-combining site of Ab1, one of which (UD7) induced Ab3 in rabbits that were Ab1-like in their binding reactivities to PB1 (recombinant gp120 fragment) or peptides of gp120, and shared idiotypes with the Ab1. Crude Ab3-containing sera specifically and effectively neutralized the virus. CONCLUSION: Monoclonal Ab2 UD7 has potential as a vaccine against HIV-1.

Neuropsychiatric disorders, myoclonus, and dystonia in calcification of basal ganglia pathways.

Two cases of basal ganglia calcification involving the globus pallidus are presented. Both patients had cognitive dysfunction, temporal lobe-like symptoms (including amnestic state, perceptual distortions, or complex visual hallucinations), and myoclonus. Patient 1 manifested depression, auditory hallucinations, anxiety, paranoia, and postural tremor; patient 2 manifested multifocal dystonia with dystonic tremor. These cases supplement other reports of psychotic features and dementia associated with pallidal pathology. Additionally, the phenomena encountered in these cases are considered in light of recent advances in our understanding of basal ganglia functional pathways. These cases afford a potential pathophysiological window to the possible role of the globus pallidus in these neuropsychiatric conditions. In concert with other recent findings, these cases suggest specific pathway involvement in hallucinations, paranoia, depression, myoclonus, and dystonia. Further research will indicate if these pathways play a role in schizophrenia, mood disorders, and anxiety disorders.

Anti-idiotypic antibodies against anti-CD4 antibodies MT151 and OKT4A.

Anti-idiotypic antibodies (Ab2) binding to the antigen-combining site of other antibodies may functionally and even structurally mimic antigen. Ab2 to antibodies directed against the lymphocyte CD4 receptor for human immunodeficiency virus type 1 (HIV-1) may mimic the receptor and therefore inhibit viral infectivity. We have produced Ab2 against monoclonal anti-CD4 receptor antibodies (Ab1). The Ab1 strongly inhibit HIV-1 binding to the receptor. Six monoclonal rat Ab2 and two polyclonal rabbit Ab2 were produced against the Ab1 MT151 and nine monoclonal Ab2 against the Ab1 OKT4A. These Ab2 bound only to Ab1 and not to a panel of nine unrelated murine monoclonal antibodies (MAbs). The Ab2 completely inhibited the binding of the homologous Ab1 to CD4-positive target cells, and recombinant soluble CD4 inhibited binding of Ab2 to Ab1. Thus, the Ab2 seemed to mimic the Ab1-binding site of the CD4 receptor, although the results of inhibition assays did not exclude steric hindrance of antibody-combining sites. However, none of the 17 Ab2 bound to gp120 of HIV-1 envelope or inhibited syncytia formation between cells infected and uninfected with HIV-1. These results suggest that the Ab2 do not mimic the HIV-1 binding site of the CD4 receptor. They further suggest that the Ab1 may not bind within the virus-binding site of the CD4 receptor.

Polyclonal antiidiotypic antibodies mimicking gp120 of HIV-1.

Rabbit antiidiotypic antibodies (Ab2) were produced against anti-HIV-1 antibody 0.5 beta (Ab1), which binds to gp120 of HIV-1 and shows virus-neutralizing activity. The Ab2 bound specifically to the Ab1 and their binding to Ab2 was inhibited by a recombinant fragment of gp120 (PB1) or a peptide (residues 301-324 of gp120), both expressing the Ab1-defined epitope. The Ab2 induced in rats antiantiidiotypic antibodies (Ab3) that were Ab1-like in their binding reactivities to PB1, native gp120 or peptide and shared idiotopes with the Ab1. However, the Ab2 did not induce virus-neutralizing Ab3, probably a reflection of the low avidity of the Ab3 as compared to the Ab1.

Anti-tumor and cell cycle responses in KB cells treated with a chimeric anti-EGFR monoclonal antibody in combination with cisplatin.

Overexpression of the epidermal growth factor receptor (EGFR) has been found to correlate with a poor prognosis for many cancers. The EGFR appears to play an important role in regulating cell growth during tumorigenesis and blockade of the EGFR/ligand interaction may be a means of inducing cell cytotoxicity, terminal differentiation, or apoptosis. In this report, we show that the growth of well-established xenografts of the human epidermoid carcinoma cell line KB could be significantly inhibited by the combination of cisplatin plus C225, a chimeric anti-EGFR monoclonal antibody, whereas the individual treatments had no effect on tumor growth. Substantive changes in the protein expression levels of the EGFR as well as several important cell cycle regulatory proteins were found in cells treated with the combination. In addition, cisplatin plus C225 inhibited the overall phosphorylation patterns including EGFR activation. These in vitro data suggest a mechanism of action for the in vivo therapeutic effects of C225 plus cisplatin.

Effects of electroconvulsive shock on food and water intake in the rat.

Forty-two male rats were habituated to laboratory conditions for four weeks under constant light conditions. Food and water intakes were recorded daily. Following the habituation period half of the animals were given electroconvulsive shock (ECS) and half Sham-ECS (SECS). ECS treatments produced significant decrements in both food and eater intakes which returned to baseline levels after three days.

Apoptosis mediates the selective toxicity of caffeic acid phenethyl ester (CAPE) toward oncogene-transformed rat embryo fibroblast cells.

The active component of the folk medicine propolis, caffeic acid phenethyl ester (CAPE), displays selective toxicity toward cloned rat embryo fibroblast (CREF) cells transformed by a spectrum of diverse acting oncogenes. Identification of the mode of action of CAPE should provide useful information for possible applications of this compound for cancer therapy. The present study uses a series of oncogene transformed, oncogene-reverted and CAPE-resistant oncogene transformed CREF cells to investigate the mechanism underlying the increased sensitivity of transformed cells to CAPE. A direct relationship exists between the cytotoxic effects of CAPE and the induction of DNA fragmentation and apoptosis. DNA degradation into nucleosomal fragments and apoptotic shifts in DNA cell cycle profiles occur in CAPE-treated CREF cells transformed by wild-type 5 adenovirus (Ad5), a mutant Ad5 (H5hr1), the wild-type Ad5 E1A transforming gene, v-src, Ha-ras and the human papilloma virus type 18 transforming genes (HPV-18). In contrast, untransformed CREF cells, human fibroblast expression library-induced morphological revertants of Ad5- and v-src-transformed CREF cells, and Krev-1 expressing revertant Ha-ras-transformed CREF cells are resistant to CAPE-induced toxicity and apoptosis. Similarly, mutant Ad5-transformed CREF cells selected by step-wise growth in increasing concentrations of CAPE are resistant to growth inhibition and apoptosis induced by CAPE. These findings indicate that expression of the transformed phenotype by rodent cells evokes sensitivity to CAPE induced toxicity through apoptosis. The acquisition of CAPE sensitivity in rodent cells is independent of the mode of action of the oncogenic agent. CAPE may prove useful as an antiproliferative agent in cancer cells transformed by mechanistically diverse acting oncogenes.

Factors in the modification by isolation of electroconvulsive shock-produced retrograde amnesia in the rat.

It has been demonstrated that placing rats in a sensory-restricted environment during the electronconvulsive shock- (ECS) retention interval following 1-trial appetitive learning will prevent or eliminate ECS-produced retrograde amnesia. The first study replicated this finding with a 1-trial aversive learning task, indicating that this effect is not task specific. The second study attempted to determine whether illumination or the restricted environment was the crucial factor in this penomenon. Rats placed in the light during the ECS-retention interval, whether restricted or in the colony, demonstrated retrograde amnesia after ECS. However, rats left in the dark during this interval demonstrated little, if any, retention deficit particularly if this condition was combined with sensory isolation. These studies further indicate that manipulation of the general environment of the animal after ECS can alter ECS-produced retention losses.

Epidermal growth factor receptor blockade by antibody IMC-C225 inhibits growth of a human pancreatic carcinoma xenograft in nude mice.

BACKGROUND: Pancreatic carcinoma is associated with a poor prognosis, and treatment options for patients with this disease are limited. The epidermal growth factor (EGF) receptor and its ligands are overexpressed in human pancreatic carcinoma and may contribute to the pathophysiology of these tumors. METHODS: The anti-EGF receptor monoclonal antibody IMC-C225 was used to determine the effects of EGF receptor blockade on the growth of human pancreatic carcinoma BxPC-3 cells in vitro. Athymic mice bearing established (200 mm(3)) subcutaneous BxPC-3 xenografts were treated with IMC-C225 (17 or 33 mg/kg every 3 days) alone or in combination with 5-fluorouracil (17 mg/kg twice weekly). RESULTS: IMC-C225 inhibited exogenous ligand-stimulated tyrosine phosphorylation of the EGF receptor on BxPC-3 tumor cells. Treatment of BxPC-3 cells with IMC-C225 inhibited DNA synthesis (23.8%) and colony formation in soft agar (45.6%). IMC-C225 treatment significantly suppressed the growth of BxPC-3 tumors compared with treatment with vehicle alone (P = 0.003). Combination therapy with IMC-C225 and the chemotherapeutic agent 5-fluorouracil enhanced the antitumor effects compared with either agent alone and resulted in regression of pancreatic tumors in several animals. Histologic examination of pancreatic tumors from mice treated with IMC-C225 showed extensive tumor necrosis that coincided with a substantial decrease in tumor cell proliferation and an increase in tumor cell apoptosis. CONCLUSIONS: These data suggest that IMC-C225 affects the growth of pancreatic tumors by inhibiting EGF receptor-dependent proliferation and survival, and demonstrates the potential for therapeutic application of IMC-C225 antibody in the treatment of human pancreatic carcinoma.

The biologic effects of C225, a chimeric monoclonal antibody to the EGFR, on human prostate carcinoma.

For prostate cancer, a correlation exists between overexpression of the epidermal growth factor receptor (EGFR) and poor clinical prognosis. In addition, late-stage metastatic disease is characterized by a change from a paracrine to an autocrine mode of expression for TGF-alpha, the ligand for the EGFR. These observations suggest that activation of the EGFR may be important for the growth of prostatic carcinoma in situ, and blockade of the receptor-ligand interaction may offer a means of therapeutic intervention for this disease. We describe the biologic effects of a chimeric anti-EGFR monoclonal antibody, C225, on several human prostate tumor cell lines in culture and the tumor inhibitory properties of the antibody for the treatment of human prostate carcinoma xenografts in nude mice. In vitro analysis of the EGFR from androgen-responsive and independent prostatic carcinoma cell lines revealed that C225 blocked EGF-induced receptor activation and induced internalization of the receptor. In vivo, a treatment regimen of C225 alone or antibody plus doxorubicin significantly inhibited tumor progression of well-established DU145 and PC-3 xenografts in nude mice. These results suggest that C225 may have utility for the treatment of human prostate carcinoma in a clinical setting.

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine.

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoaffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector.

Monoclonal anti-idiotypic antibody functionally mimics the human gastrointestinal carcinoma epitope GA733.

Anti-idiotypic antibodies (Ab2) that bind to the antigen-combining region of anti-tumor antibodies (Ab1) may functionally, and even structurally, mimic tumor antigen. We have previously demonstrated that polyclonal goat Ab2 directed against anti-human gastrointestinal carcinoma Ab1 GA733 induces anti-anti-idiotypic antibodies (Ab3) in animals that are Ab1-like in their binding specificity and idiotope expression. To obtain more defined Ab2 vaccines with potentially increased specificity and efficacy, a monoclonal Ab2 (FG1) was produced against Ab1 GA733 in rats. The monoclonal Ab2 FG1, similar to the polyclonal Ab2 described previously, induced Ab3 in rabbits that were Ab1-like in their idiotope expression and binding specificity to tumor cells and antigen. Antigen-specific Ab3 induced by Ab2 FG1 were easily detected in unprocessed rabbit sera, whereas the demonstration of such Ab3 after polyclonal Ab2 immunization required purification of the Ab3 from the rabbit sera. In addition, Ab2 FG1 induced antigen-specific humoral and cellular immunity in mice. Murine Ab3 bound specifically to antigen-positive tumor cells. Ab2-immunized mice showed antigen-specific delayed-type hypersensitivity (DTH) reaction, and cultured splenocytes from the immune mice demonstrated specific proliferation and cytokine (interferon-gamma and interleukin-4) secretion upon stimulation with GA733 antigen. However, immune mice were not protected against a challenge with syngeneic GA733 antigen-expressing colon carcinoma cells.