Ralph A. Reisfeld

Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels

A single intravascular injection of a cyclic peptide or monoclonal antibody antagonist of integrin alpha v beta 3 disrupts ongoing angiogenesis on the chick chorioallantoic membrane (CAM). This leads to the rapid regression of histologically distinct human tumors transplanted onto the CAM. Induction of angiogenesis by a tumor or cytokine promotes vascular cell entry into the cell cycle and expression of integrin alpha v beta 3. After angiogenesis is initiated, antagonists of this integrin induce apoptosis of the proliferative angiogenic vascular cells, leaving preexisting quiescent blood vessels unaffected. We demonstrate therefore that ligation of integrin alpha v beta 3 is required for the survival and maturation of newly forming blood vessels, an event essential for the proliferation of tumors.

A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis

Abstract Protein zones may be stained by Coomassie blue without any destaining of the background and with a sensitivity at least as high as that of amido black. A vastly increased sensitivity may be achieved with Coomassie blue at the price of background destaining.

Anti-GD2 Antibody with GM-CSF, Interleukin-2, and Isotretinoin for Neuroblastoma

BACKGROUND Preclinical and preliminary clinical data indicate that ch14.18, a monoclonal antibody against the tumor-associated disialoganglioside GD2, has activity against neuroblastoma and that such activity is enhanced when ch14.18 is combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2. We conducted a study to determine whether adding ch14.18, GM-CSF, and interleukin-2 to standard isotretinoin therapy after intensive multimodal therapy would improve outcomes in high-risk neuroblastoma. METHODS Patients with high-risk neuroblastoma who had a response to induction therapy and stem-cell transplantation were randomly assigned, in a 1:1 ratio, to receive standard therapy (six cycles of isotretinoin) or immunotherapy (six cycles of isotretinoin and five concomitant cycles of ch14.18 in combination with alternating GM-CSF and interleukin-2). Event-free survival and overall survival were compared between the immunotherapy group and the standard-therapy group, on an intention-to-treat basis. RESULTS A total of 226 eligible patients were randomly assigned to a treatment group. In the immunotherapy group, a total of 52% of patients had pain of grade 3, 4, or 5, and 23% and 25% of patients had capillary leak syndrome and hypersensitivity reactions, respectively. With 61% of the number of expected events observed, the study met the criteria for early stopping owing to efficacy. The median duration of follow-up was 2.1 years. Immunotherapy was superior to standard therapy with regard to rates of event-free survival (66±5% vs. 46±5% at 2 years, P=0.01) and overall survival (86±4% vs. 75±5% at 2 years, P=0.02 without adjustment for interim analyses). CONCLUSIONS Immunotherapy with ch14.18, GM-CSF, and interleukin-2 was associated with a significantly improved outcome as compared with standard therapy in patients with high-risk neuroblastoma. (Funded by the National Institutes of Health and the Food and Drug Administration; ClinicalTrials.gov number, NCT00026312.)

Antibody targeted drugs as cancer therapeutics

Treatment of cancer is a double-edged sword: it should be as aggressive as possible to completely destroy the tumour, but it is precisely this aggressiveness which often causes severe side effects — a reason why some promising therapeutics can not be applied systemically. In addition, therapeutics such as cytokines that physiologically function in a para- or autocrine fashion require a locally enhanced level to exert their effect appropriately. An elegant way to accumulate therapeutic agents at the tumour site is their conjugation/fusion to tumour-specific antibodies. Here, we discuss recent preclinical and clinical data for antibody–drug conjugates and fusion proteins with a special focus on drug components that exert their antitumour effects through normal biological processes.

Targeting tumor-associated macrophages as a novel strategy against breast cancer

Tumor-associated macrophages (TAMs) are associated with tumor progression and metastasis. Here, we demonstrate for the first time that legumain, a member of the asparaginyl endopeptidase family functioning as a stress protein, overexpressed by TAMs, provides an ideal target molecule. In fact, a legumain-based DNA vaccine served as a tool to prove this point, as it induced a robust CD8+ T cell response against TAMs, which dramatically reduced their density in tumor tissues and resulted in a marked decrease in proangiogenic factors released by TAMs such as TGF-beta, TNF-alpha, MMP-9, and VEGF. This, in turn, led to a suppression of both tumor angiogenesis and tumor growth and metastasis. Importantly, the success of this strategy was demonstrated in murine models of metastatic breast, colon, and non-small cell lung cancers, where 75% of vaccinated mice survived lethal tumor cell challenges and 62% were completely free of metastases. In conclusion, decreasing the number of TAMs in the tumor stroma effectively altered the tumor microenvironment involved in tumor angiogenesis and progression to markedly suppress tumor growth and metastasis. Gaining better insights into the mechanisms required for an effective intervention in tumor growth and metastasis may ultimately lead to new therapeutic targets and better anticancer strategies.

Enhancement of sheep red blood cell human lymphocyte rosette formation by the sulfhydryl compound 2-amino ethylisothiouronium bromide.

Abstract In vitro treatment of sheep red blood cells with the sulfhydryl compound 2-aminoethylisothiouronium bromide (AET) under suitable experimental conditions greatly enhances their ability to form rosettes with human lymphoid cells. The effect of AET depends on the concentration, pH of the solution and time of incubation. AET treated sheep red blood cells lose their ability to form rosettes after varying time periods depending on the temperature of storage and on the concentration of the AET solution. Treatment of human peripheral lymphocytes with AET abolishes their capability to form rosettes with both normal and AET treated sheep red blood cells. Lymphocytes from some patients with chronic lymphocytic leukemia react differently with AET-treated sheep red blood cells than with normal sheep red blood cells.

A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth

Tumor cells are elusive targets for immunotherapy due to their heterogeneity and genetic instability. Here we describe a novel, oral DNA vaccine that targets stable, proliferating endothelial cells in the tumor vasculature rather than tumor cells. Targeting occurs through upregulated vascular-endothelial growth factor receptor 2 (FLK-1) of proliferating endothelial cells in the tumor vasculature. This vaccine effectively protected mice from lethal challenges with melanoma, colon carcinoma and lung carcinoma cells and reduced growth of established metastases in a therapeutic setting. CTL-mediated killing of endothelial cells indicated breaking of peripheral immune tolerance against this self antigen, resulting in markedly reduced dissemination of spontaneous and experimental pulmonary metastases. Angiogenesis in the tumor vasculature was suppressed without impairment of fertility, neuromuscular performance or hematopoiesis, albeit with a slight delay in wound healing. Our strategy circumvents problems in targeting of genetically unstable tumor cells. This approach may provide a new strategy for the rational design of cancer therapies.

Targeting tumor-associated fibroblasts improves cancer chemotherapy by increasing intratumoral drug uptake

Tumor-associated fibroblasts are key regulators of tumorigenesis. In contrast to tumor cells, which are genetically unstable and mutate frequently, the presence of genetically more stable fibroblasts in the tumor-stromal compartment makes them an optimal target for cancer immunotherapy. These cells are also the primary source of collagen type I, which contributes to decreased chemotherapeutic drug uptake in tumors and plays a significant role in regulating tumor sensitivity to a variety of chemotherapies. To specifically kill tumor-associated fibroblasts, we constructed an oral DNA vaccine targeting fibroblast activation protein (FAP), which is specifically overexpressed by fibroblasts in the tumor stroma. Through CD8+ T cell-mediated killing of tumor-associated fibroblasts, our vaccine successfully suppressed primary tumor cell growth and metastasis of multidrug-resistant murine colon and breast carcinoma. Furthermore, tumor tissue of FAP-vaccinated mice revealed markedly decreased collagen type I expression and up to 70% greater uptake of chemotherapeutic drugs. Most importantly, pFap-vaccinated mice treated with chemotherapy showed a 3-fold prolongation in lifespan and marked suppression of tumor growth, with 50% of the animals completely rejecting a tumor cell challenge. This strategy opens a new venue for the combination of immuno- and chemotherapies.

T cell homeostatic proliferation elicits effective antitumor autoimmunity

Development of tumor immunotherapies focuses on inducing autoimmune responses against tumor-associated self-antigens primarily encoded by normal, unmutated genes. We hypothesized that such responses could be elicited by T cell homeostatic proliferation in the periphery, involving expansion of T cells recognizing self-MHC/peptide ligands. Herein, we demonstrate that sublethally irradiated lymphopenic mice transfused with autologous or syngeneic T cells showed tumor growth inhibition when challenged with melanoma or colon carcinoma cells. Importantly, the antitumor response depended on homeostatic expansion of a polyclonal T cell population within lymph nodes. This response was effective even for established tumors, was characterized by CD8(+) T cell-mediated tumor-specific cytotoxicity and IFN-gamma production, and was associated with long-term memory. The results indicate that concomitant induction of the physiologic processes of homeostatic T cell proliferation and tumor antigen presentation in lymph nodes triggers a beneficial antitumor autoimmune response.

Electrophoretic Heterogeneity of Polypeptide Chains of Specific Antibodies

Heavy and light polypeptide chains isolated from different specific antibodies to haptens and from γG-immunoglobulin of normal rabbits have been resolved into distinct, multiple components by disc electrophoresis in polyacrylamide gels in the presence of urea. In spite of the resolution of these chains into multiple bands, different specific antibodies and normal rabbit γG-immunoglobulin were indistinguishable from each other by this method.