Joseph G. Sinkovics

Newcastle disease virus (NDV): brief history of its oncolytic strains

BACKGROUND While genetically engineered viruses are now being tested for the virus therapy of human cancers, some naturally occurring viruses display unmatched oncolytic activity. Newcastle disease virus (NDV) excels as an oncolytic agent. OBJECTIVES As its virulence versus attenuation can be explained on molecular biological bases, it may be possible to develop or select highly oncolytic strains of NDV without adverse toxicity. STUDY DESIGN Questions are posed as to the mechanisms of viral oncolysis, the appropriateness of tests to predict oncolytic activity of a given NDV strain and the best modes of administration for oncolytic effects. Answers are provided based on specific data or on considerations drawn from experience (the authors use NDV oncolysates to immunize against melanoma and kidney carcinoma) or from analogous clinical situations (therapeutic use of mumps or measles viruses). RESULTS AND CONCLUSIONS NDV oncolysates probably suit better for immunotherapy (providing also active tumor-specific immunization) than massive repeated inoculations of NDV strains, especially when the NDV strain used is not proven to be oncolytic by appropriate pre-clinical tests.

New Developments in the Virus Therapy of Cancer: A Historical Review

Since the 1920s, viruses had been used for oncolysis. Natural human viral infections can rarely induce remissions of leukemias or lymphomas. Inoculation of tumor-bearing patients with live viruses very seldom resulted in durable complete remissions. Genetically engineered or tumor-adapted virus strains may perform better. Virally modified tumor cell membrane vaccines can induce in the host rejection strength antitumor immunity. Modern technology and much more work is needed before the optimal procedures for viral oncolysis or active antitumor immunization with virally modified tumor cell vaccines are learned and can be implemented in the clinical practice. Laboratory monitoring of the hosts immunological reactions accompanying failure and success of tumor rejection is essential for the recognition and duplication of the successful and for the avoidance of the unsuccessful interventions.

Natural and genetically engineered viral agents for oncolysis and gene therapy of human cancers

Based on personal acquaintances and experience dating back to the early 1950s, the senior author reviews the history of viral therapy of cancer. He points out the difficulties encountered in the treatment of human cancers, as opposed by the highly successful viral therapy of experimentally maintained tumors in laboratory animals, especially that of ascites carcinomas in mice. A detailed account of viral therapy of human tumors with naturally oncolytic viruses follows, emphasizing the first clinical trials with viral oncolysates. The discrepancy between the high success rates, culminating in cures, in the treatment of tumors of laboratory animals, and the moderate results, such as stabilizations of disease, partial responses, very rare complete remissions, and frequent relapses with virally treated human tumors is recognized. The preclinical laboratory testing against established human tumor cell lines that were maintained in tissue cultures for decades, and against human tumors extricated from their natural habitat and grown in xenografts, may not yield valid results predictive of the viral therapy applied against human tumors growing in their natural environment, the human host. Since the recent discovery of the oncosuppressive efficacy of bacteriophages, the colon could be regarded as the battlefield, where incipient tumor cells and bacteriophages vie for dominance. The inner environment of the colon will be the teaching ground providing new knowledge on the value of the anti-tumor efficacy of phage-induced innate anti-tumor immune reactions. Genetically engineered oncolytic viruses are reviewed next. The molecular biology of viral oncolysis is explained in details. Elaborate efforts are presented to elucidate how gene product proteins of oncolytic viruses switch off the oncogenic cascades of cancer cells. The facts strongly support the conclusion that viral therapy of human cancers will remain in the front lines of modern cancer therapeutics. It may be a combination of naturally oncolytic viruses and wild-type viruses rendered oncolytic and harmless by genetic engineering, that will induce complete remissions of human tumors. It may be necessary to co-administer certain chemotherapeutic agents, advanced cancer vaccines, or even immune lymphocytes, and targeted therapeuticals, to ascertain, that remissions induced by the viral agents will remain complete and durable; will co-operate with anti-tumor host immune reactions, and eventually will result in cures of advanced metastatic human cancers.

Horizontal Gene Transfers with or without Cell Fusions in All Categories of the Living Matter

This article reviews the history of widespread exchanges of genetic segments initiated over 3 billion years ago, to be part of their life style, by sphero-protoplastic cells, the ancestors of archaea, prokaryota, and eukaryota. These primordial cells shared a hostile anaerobic and overheated environment and competed for survival. “Coexist with, or subdue and conquer, expropriate its most useful possessions, or symbiose with it, your competitor” remain cellular life’s basic rules. This author emphasizes the role of viruses, both in mediating cell fusions, such as the formation of the first eukaryotic cell(s) from a united crenarchaeon and prokaryota, and the transfer of host cell genes integrated into viral (phages) genomes. After rising above the Darwinian threshold, rigid rules of speciation and vertical inheritance in the three domains of life were established, but horizontal gene transfers with or without cell fusions were never abolished. The author proves with extensive, yet highly selective documentation, that not only unicellular microorganisms, but the most complex multicellular entities of the highest ranks resort to, and practice, cell fusions, and donate and accept horizontally (laterally) transferred genes. Cell fusions and horizontally exchanged genetic materials remain the fundamental attributes and inherent characteristics of the living matter, whether occurring accidentally or sought after intentionally. These events occur to cells stagnating for some 3 milliard years at a lower yet amazingly sophisticated level of evolution, and to cells achieving the highest degree of differentiation, and thus functioning in dependence on the support of a most advanced multicellular host, like those of the human brain. No living cell is completely exempt from gene drains or gene insertions.

Adult human sarcomas. I. Basic science

When connective tissue undergoes malignant transformation, glioblastomas and sarcomas arise. However, the ancient biochemical mechanisms, which are now operational in sarcomas distorted by mutations and gene fusions in misaligned chromosomes, were originally acquired by those cells that emerged during the Cambrian explosion. Preserved throughout evolution up to the genus Homo, these mechanisms dictate the apoptosis- and senescence-resistant immortality of malignant cells. A ‘retroviral paradox’ distinguishes human sarcomas from those of the animal world. In contrast to the retrovirally induced sarcomatous transformation of animal (avian, murine, feline and simian) cells, human sarcomas have so far failed to yield a causative retroviral isolate. However, the proto-oncogenes/oncogenes transduced from their host cells by retroviruses of animals are the same that are active in human sarcomas. Since the encoded oncoproteins arise after birth, they are recognized frequently by the immune system of the host. Immune lymphocytes that kill autologous sarcoma cells in vitro commonly fail to do so in vivo. Sarcoma vaccines generate immune T- and natural killer cell reactions; even when vaccinated patients do not show a clinical response, their tumors become more sensitive to chemotherapy. The aim of this review is to lay a solid molecular biological foundation for the conclusion that targeting the sarcoma oncogenes will result in regression of the disease.

Adult human sarcomas. II. Medical oncology.

Human sarcoma cells can be killed by radio- and chemotherapy, but tumor cells acquiring resistance frequently kill the patient. A keen understanding of the intracellular course of oncogenic cascades leads to the discovery of small molecular inhibitors of the involved phosphorylated kinases. Targeted therapy complements chemotherapy. Oncogene silencing is feasible by small interfering RNA. The restoration of some of the mutated or deleted tumor-suppressor genes (p53, Rb, PTEN, hSNF, INK/ARF and WT) by demethylation or reacetylation of their histones has been accomplished. Genetically engineered or naturally oncolytic viruses selectively lyse tumors and leave healthy tissues intact. Adeno- or retroviral vectors deliver genes of immunological costimulators, tumor antigens, chemo- or cytokines and/or tumor-suppressor proteins into tumor (sarcoma) cells. Suicide gene delivery results in apoptosis induction. Genes of enzymes that target prodrugs as their substrates render tumor cells highly susceptible to chemotherapy, with the prodrug to be targeted intracellularly. It will be combinations of sophisticated surgical removal of the nonencapsulated and locally invasive primary sarcomas, advanced forms of radiotherapy to the involved sites and immunotherapy with sarcoma vaccines that will cure primary sarcomas. Adoptive immunotherapy with immune lymphocytes will be operational in metastatic disease only when populations of regulatory T cells are controlled. Targeted therapy with small molecular inhibitors of oncogene cascades, the driving forces of sarcoma cells, alteration of the tumor stroma from a supportive to a tumor-hostile environment, reactivation or replacement of wild-type tumor-suppressor genes, and radio–chemotherapy (with much reduced toxicity) will eventually accomplish the cure of metastatic sarcomas.

A Reappraisal of Cytotoxic Lymphocytes in Human Tumor Immunology

Extensive work done at the Section of Clinical Tumor Virology and Immunology, Department of Medicine, The University of Texas M. D. Anderson Hospital, from 1968 to 1978 on the cytotoxicity of lymphocytes to cultured human tumor cells is reviewed. The early results and concepts are now reinterpreted in the light of newer knowledge generated worldwide in the past few years. This work is now leading to adoptive immunotherapy of human tumors by reinfusion of activated autologous tumor killer (ATK) lymphocytes expanded by co-administration of T cell growth factor (interleukin 2).

Clinical Recognition and Treatment of Endotoxinemia

In health we co-exist with our bowel flora without harm from lipopoly-saccharide endotoxins that the gram-negative constituents of this flora produce and release. The lower intestinal tract of patients entering hospitals becomes rapidly colonized by the nosocomial gram-negative flora dominant in that environment. Green salads, faucets, the vase holding cut flowers at the bedside serve as common sources of colonization without tissue invasion. Breaking through natural barriers (indwelling arterial and venous lines, genitourinary and other catheters, mucosal ulcerations of the oral cavity and gastrointestinal tract), leukopenia (including granulocyto- and lymphocytopenia and depletion of monocytes-macrophages) allow for invasion of tissues and blood stream by gut flora containing also the colonizing nosocomial bacteria (Pseudomonas sp., Serratia sp.). Absorption of endotoxin from the compromised gastrointestinal or genitourinary tracts may occur without actual bacterial invasion of the blood stream. Gram-negative bacteria invading soft tissues and blood produce and release endotoxins. These endotoxins exert diverse and most profound biological effects often cascading relentlessly toward irreversible shock and death.

Adoptive immunotherapy for human cancers: Flagmen signal first “open road” then “roadblocks.” A narrative synopsis

There was overwhelming evidence documented in vitro in the early 1970s for lymphocyte-mediated cytotoxicity to autologous cancer cells. Cancer-bearing patients circulated small compact lymphocytes in their blood that promptly killed their tumor cells in vitro. These lymphocytes were identified later as CD8+ immune T cells. Tumor cells were killed by cytoplasmic lysis with perforins and granzymes, or by nuclear clumping by Fas ligand and related ligands. With the discovery of T cell growth factor (interleukin-2), the road for lymphocyte therapy of human cancers appeared wide open. Then emerged the “large granular lymphocytes”. These cells occurred not only in patients with cancer, but in healthy cancer-free individuals. The author of this article served as “negative (healthy) control” in the cytotoxicity assays in the late 1960s and early 1970s. Some project site visitors of the National Cancer Institute could not comprehend that “immune reactions could exist without pre-immunization” and referred to the phenomenon as an “in vitro artifact” (worse than that: they canceled grant support for its study). It was years later, that first in mice and then in human patients the “large granular lymphocytes” were recognized as natural killer cells. Then emerged the “suppressor/regulatory T cells” (TREG). This lymphocyte population is responsible for curtailing autoimmune reactions against “self ”. Tumor cells masquerading as “self ” are protected by TREG against cytotoxicity executed by immune T cells, and even by NK cells. Adoptive immune lymphocyte therapy of human cancer will be effectively resolved when technology develops for the neutralization of the TREG population.

Notable Phenomena of the 1960s Recapitulated. I. Graft-Versus-Leukemia Reaction and Natural Hybridoma Formation in Mice

Joseph G Sinkovics. St. Joseph’s Hospital Cancer Institute, University of South Florida, Tampa, FL. In 1962–3 at MD Anderson Hospital viral leukemia-susceptible Balb/c mice were rendered to be chronically runted with allogeneic disease due to cellular chimerism by injections of lymphoid cells from leukemia virus-resistant C57Bl mice. In these Balb/c mice leukemia viruses replicated to very low titers and induced no or low incidence of leukemia (AACR 1963; TX Rep Biol Med 1965; 7th Internat Symposium: Autologous Marrow and Blood Transplantation, Arlington, TX 1994). Later 20% of Balb/c mice were cured of virally induced leukemias by lethal irradiation followed by inoculations of Balb/c lymphoand hematopoietic cells deriving from donors that were actively immunized with a photodynamically inactivated leukemia virus vaccine (CR 1965; MD Anderson 20th Symposium: Carcinogenesis: A Broad Critique, WW 1967). A virally induced but cell-passaged murine lymphoma (#620) presented with the starry sky histological picture (J Inf Dis 1968; 1969), induced celland antibody-mediated immune reactions in its host and it could be rejected. Immune modulators either accelerated or decelerated its course (Science 1967; J Retic Soc 1970). The diploid lymphoma cells expressed budding retroviral particles and could fuse with immune plasma cells secreting leukemia virus-neutralizing antibodies. The fusion product tetraploid cells (#818) contained both leukemia virus antigens and IgG2b immunoglobulins; grew in suspension cultures and as lethal ascites tumors, while continuously secreting the specific antibody for over 10 years (1968– 1980) (Lancet 1970; MD Anderson 14th Clinical Conference: LeukemiaLymphoma, Yearbook, Chicago 1970; CR 1970; 1981). Antibody-coated lymphoma cells can be phagocytized by macrophages or dendritic cells (hence the starry sky feature of Burkitt lymphoma as the EBV-positive tumor cells are antibody-coated) (Critic Rev Immun 1991) to present tumor antigens to immune T cells. In human lymphomas two-directional FasL to Fas reactions occur between lymphoma cells and immune T cells (Inter J Oncol 2001). A human lymphoma cell line (T1 or #778) established in 1968 (JM Trujillo and the author) replicated unidentified retroviral particles. Lymphoma cells fused with plasma cells of the patient. During the RAG-induced V(D)J somatic hypermutations silent resident retrotransposons may acquire env sequences and emerge as endogenous retroviruses (AACR 2001; ASCO 2002). When these lymphoid cells generate autoantibody production, they may fuse with the antibody-producer plasma cells. Natural hybridoma formation in human lymphomas may be of decisive prognostic significance (Wainwright: Persp Biol Med 1992). This presentation will be illustrated with genuine original data and microphotographs from the 1960s to 1970s.