Judith Kantor

Diversified prime and boost protocols using recombinant vaccinia virus and recombinant non-replicating avian pox virus to enhance T-cell immunity and antitumor responses.

Recombinant vaccinia viruses containing tumor associated genes represent an attractive vector to induce immune responses to weak immunogens in cancer immunotherapy protocols. The property of intense immunogenicity of vaccinia proteins, however, also serves to limit the number of inoculations of recombinant vaccinia viruses. Host immune responses to the first immunization have been shown to limit the replication of subsequent vaccinations and thus reduce effectiveness of boost inoculations. The use of recombinant avian pox viruses (avipox) such as the canarypox (ALVAC) or fowlpox are potential candidates for immunization protocols in that they can infect mammalian cells and express the inserted transgene, but do not replicate in mammalian cells. We report here the construction and characterization of a canarypox (ALVAC) recombinant expressing the human carcinoembryonic antigen (CEA) gene (designated ALVAC-CEA). Antibody, lymphoproliferative and cytolytic T-cell responses as well as tumor inhibition were shown to be elicited by the ALVAC-CEA recombinant in a murine model. The utilization of a diversified immunization scheme using a recombinant vaccinia virus followed by recombinant avian pox virus was shown to be far superior than the use of either one alone in eliciting CEA-specific T-cell responses. Experiments were conducted to determine if the use of a diversified immunization scheme using a recombinant vaccinia virus (rV-CEA) and ALVAC-CEA would be superior to the use of either one alone in eliciting CEA-specific T-cell responses. When mice were immunized with rV-CEA and then ALVAC-CEA. CEA-specific T-cell responses were at least four times greater, and for superior to those achieved with three immunizations of ALVAC-CEA. Multiple boosts of ALVAC-CEA following rV-CEA immunization further potentiated anti-tumor effects and CEA specific T-cell responses. These studies demonstrate the proof of concept of the advantage of diversified immunization protocols employing both recombinant vaccinia and recombinant avipox vectors.

Anti-tumor immunity elicited by a recombinant vaccinia virus expressing CD70 (CD27L).

CD70, a ligand of the T cell costimulatory receptor CD27, is expressed mainly on activated B cells and has been shown to increase cytotoxic activity and proliferation of preferentially unprimed T cells. Reported herein is the construction of a recombinant vaccinia virus encoding CD70 (designated rV-CD70) and a demonstration of its biological effect on naive T cells in vitro and in vivo. In a whole tumor cell vaccine model, the growth of CD70-negative murine colon adenocarcinoma (MC38) tumor cells infected with rV-CD70 (multiplicity of infection [MOI] of 0.1) and transplanted into syngeneic C57BL/6 mice was inhibited completely while control tumors infected with wild-type vaccinia grew rapidly and killed mice within 3-5 weeks. Tumor-free mice previously immunized with rV-CD70-infected tumors were partially protected against rechallenge with wild-type tumors, demonstrating the induction of systemic anti-tumor immunity. In addition, immunization of C57BL/6 mice with rV-CD70 admixed with vaccinia virus encoding carcinoembryonic antigen (rV-CEA) was superior to treatment with rV-CEA alone in inducing CEA-specific lymphoproliferative T cell responses and reducing growth of murine colon carcinomas transduced with CEA. These studies demonstrate for the first time the potential utility of a recombinant vaccinia virus expressing CD70 to enhance T cell responses and mediate anti-tumor immunity.

Beta thalassemia: Mutations which affect processing of the β-globin mRNA precursor

To define the molecular lesion which causes decreased beta-globin synthesis in beta+ thalessemia, four patients of diverse ethnic origin were studied. Each had a 2--3 fold higher concentration of beta-globin mRNA precursor than that found in control bone marrow cells from patients with sickle cell anemia. Globin RNA metabolism was analyzed in two of these patients. Transcription of the beta-globin gene appeared to be normal, since analysis of nuclear RNA indicated that beta-globin mRNA synthesis exceeded that of alpha in a 2 hr pulse but the cytoplasm contained a relative deficiency of labeled beta-globin mRNA. An abnormal RNA species approximately 650 nucleotides in length, which contained sequences transcribed from both the large intron and coding portions of the beta-globin gene, was found in one patients bone marrow cells. The second patients cells contained a significant amount of a 1320 nucleotide RNA species, not initially evident in normal cells, from which part but not all of the large intervening sequence had been removed. Our data thus indicate that mutations which affect RNA processing cause beta thalessemia.

The use of a cationic liposome formulation (DOTAP) mixed with a recombinant tumor-associated antigen to induce immune responses and protective immunity in mice

The cationic liposome DOTAP is a well-known transfection reagent. It has been manufactured and approved for clinical use, is readily available, and can be easily used as an adjuvant. These characteristics prompted us to investigate the effectiveness of DOTAP as an adjuvant to induce immune responses and protective immunity in mice using baculovirus-derived carcinoembryonic antigen (bV-CEA) as a model antigen. Two routes of administration and a dose-response study of bV-CEA were used in BALB/c mice to define the magnitude of the immune response as well as the most effective route of immunization. The results demonstrate differences in antibody titers, immunoglobulin (Ig)G isotype, and T-cell responses between the intravenous (i.v.) or subcutaneous (s.c.) route of immunization. The titer of the anti-CEA antibodies induced by the s.c. immunization was greater than the response by i.v. immunization. The s.c. route enhanced the IgG2a/2b isotype, whereas i.v. immunization elicited primarily IgGl. T-cell proliferation responses and cytokine production paralleled the humoral response (i.e., production was higher in the s.c. immunized animals). No differences in immunological responses were seen using either 25 or 10 μg of bV-CEA three times. An amount of 25 μg of bV-CEA/DOTAP given by s.c. immunization was sufficient in protecting mice from the transplant of syngeneic tumor cells transduced with the human CEA gene. We conclude that the cationic liposome DOTAP may be a useful immunoadjuvant for active anti-tumor immunotherapy in future clinical trials. This study will help to define the most effective way to use such an adjuvant.

Enhanced immune responses and anti-tumor activity by baculovirus recombinant carcinoembryonic antigen (CEA) in mice primed with the recombinant vaccinia CEA

Carcinoembryonic antigen (CEA), a glycosylated protein of Mr 180, is one of the most widely studied oncofetal antigens. A majority of gastrointestinal cancers as well as breast and non-small-cell lung carcinomas express CEA. CEA thus represents a potential target for immunotherapy of several carcinoma types. A recombinant vaccinia-CEA virus (rV-CEA) was previously shown to induce anti-tumor activity in an experimental murine model after three rV-CEA inoculations. However, because the majority of cancer patients have received a previous smallpox vaccination, a long-lasting immune memory and/or induced anamnestic responses against vaccinia proteins may prevent repetitive boosting with the recombinant vaccinia virus expressing CEA. Therefore, other types of vaccines may be required to boost the anti-CEA immune response; one such schema would be the use of purified CEA as a boost in hosts given one administration of rV-CEA. Commercially available sources of CEA are usually derived from liver metastases extracts and are sometimes contaminated with nonspecific cross-reactive antigen. We have previously generated a recombinant source of full-length human CEA using a baculovirus expression system (bV-CEA). bV-CEA was shown to be glycosylated differently than native CEA, but it contains at least 10 epitopes found on native CEA (nCEA). Moreover, bV-CEA was able to induce a humoral response against CEA present on human colorectal cancer cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Adoptive immunotherapy as an in vivo model to explore antitumor mechanisms induced by a recombinant anticancer vaccine.

We have described previously the construction, generation, and in vivo biologic consequences of a recombinant vaccinia virus containing the human CEA gene (rV-CEA) in an experimental murine colon carcinoma model. Immunization of C57BL/6 mice with rV-CEA led to antigen-specific inhibition of tumor growth in both prophylactic and therapeutic settings. Although such antitumor effects were correlated with the induction of CEA-specific T-cell responses, their exact contribution in the tumor rejection mechanism remained unclear. In this study, we examined the mechanism of action of rV-CEA, with emphasis on definition of the immune cells important for such antitumor effects. To that end, a cellular adoptive transfer model was established in vivo, which allowed specific functional analysis of donor-derived immune cells in naive, sublethally irradiated, tumor-bearing recipients. Splenocytes from rV-CEA-immunized donors expressed strong antitumor activity in such tumor-bearing recipients, whereas nonimmune donor cells did not. Depletion of immune T cells before cellular transfer abolished the antitumor response. Moreover, depletion of CD8+ T cells before transfer resulted in the loss of antitumor activity, despite the presence of CD4+ T cells. In contrast, antitumor activity was demonstrable with CD8-containing, CD4-depleted effectors, although it was not as effective as with both T-cell subpopulations combined. Finally, in beta 2-microglobulin/CD8+ T-cell-deficient mice, rV-CEA immunization exerted only partial antitumor protection, compared with the immune-competent controls. Overall, we demonstrated that (a) antitumor activity induced by rV-CEA was essentially mediated by CD8+ effectors; and (b) the combination of both CD8+ and CD4+ lymphocytes led to maximal antitumor therapeutic effects, suggesting an important helper or immunoregulatory contribution of the CD4+ subset. Thus, adoptive cellular transfer strategies may have implications for both the study of recombinant anticancer vaccines and the development of potential clinical applications for cancer immunotherapy.

Transcription of single base oligonucleotides by ribonucleic acid-directed deoxyribonucleic acid polymerase

The synthesis of DNA products complementary to artificial templates by the enzyme RNA-directed DNA polymerase isolated from avian myeloblastosis virus has been studied. Of the single base polyribonucleotides, poly (rC), poly(rA), and poly(rI) were active while poly (rG) and poly (rU) were almost inactive. The minimum length showing activity for an oligo (rC) template was 9; the minimum primer length of oligo(dG) was 3 or 4. In order to examine the fidelity of transcription, single base oligoribonucleotides of defined length were studied. Using (rC)13 as template and (dG)8as primer, the oligo (dG) product coelectrophoresed with the template. However, using (rA)-20 as template and (dT)10 as primer, a large (10-16s) product was formed. Similarly, using oligo (rI) (2.5S) as template and (dC)10 as primer, a large (greater than 22s) product was formed. No significant activity was obtained with oligo (rU) templates. RNA-directed DNA polymerase transcribes the various oligonucleotides differently: slippage with oligo (rA) and oligo (rI), faithful transcription with oligo (rC), and poor transcription with oligo (rU).

Serological and biochemical characterization of recombinant baculovirus carcinoembryonic antigen.

Carcinoembryonic antigen (CEA), a glycosylated protein of M(r) 180 kDa, is one of the most widely used human tumor markers. A majority of gastrointestinal cancers as well as breast and nonsmall cell lung carcinomas express CEA. We have previously described a recombinant baculovirus BVCEA-140 expressing the full-length human CEA and a variant, BVCEA-16, that encodes only the NH2-terminal domain, as well as a recombinant (BVNCA) expressing the closely related molecule nonspecific cross-reactive antigen (NCA). We have now compared a panel of 24 anti-CEA and anti-NCA monoclonal antibodies (MAbs) for their ability to bind to these recombinant CEA and NCA proteins, as well as with a new 60 kDa subgenomic form designated BVCEA-60. The epitope mapping studies indicate that all the CEA specific MAbs can recognize BVCEA-140. We also compared the sugar composition of BVCEA-140 to native CEA, using a lectin-linked immunoradiometric assay. The results demonstrated that both the native and recombinant baculovirus CEA contain simple high-mannose carbohydrates as well as biantennary and biantennary hybrid complexes. However, native CEA also contains triantennary and tetraantennary complex sugars, while the recombinant CEA molecule does not. Immunogenicity of the recombinant CEA molecules was demonstrated in mice. ELISA and Western blot analyses were used to determine the cross-reactivity of the anti-CEA sera. Mice immunized with BVCEA-140 elicit antibodies that are reactive to native CEA. When the BVCEA-16 was used as an immunogen, the antisera failed to detect native CEA or BVCEA-140. These studies demonstrate that minor sugar differences exist between native and baculovirus-derived CEA. However, epitope mapping with a panel of 24 anti-CEA MAbs (recognizing at least 10 CEA epitopes) stowed virtual immunologic identity between these two molecules. Moreover, BVCEA-140 appears to be a more potent humoral immunogen in mice than native CEA. These purified recombinant proteins can thus serve as standards in CEA serum assays for the possible detection and characterization of cell-mediated immune responses to CEA and as a potential source of immunogen (primary or for boosting) for active specific immunotherapy protocols of human carcinomas.

HETEROGENEITY OF MESSENGER RNA DEFECTS IN THE THALASSEMIA SYNDROMES

The aand p-thalassemia syndromes are characterized by reduced or absent synthesis, respectively, of the aor 8-globin subunits of normal adult hemoglobin (Hb A: a,B,).’ A common feature of these syndromes is reduced or absent messenger RNA coding for the affected g l ~ b i n . ~ ~ Elegant studies, reviewed elsewhere in this Annal, have shown that in some conditions (SF thalassemia, hereditary persistence of fetal hemoglobin, the “classical” Asian forms of a thalassemia, the a thalassemia encountered in Blacks, and a few Indian and Pakistani patients with “classical” high Hb A,+ thalassemia) , absence of mRNA coding for the affected globin is due to partial or complete deletion of the appropriate structural genes. However, the most common forms of /3 thalassemia appear to be associated with reduced mRNA accumulation despite the presence of grossly intact B-globin genes. Moreover the presence of apparently complete gene sequences coding for mRNAs accumulating in reduced amounts can be inferred from studies of globins, globin messenger RNAs, and globin genes of at least some patients with the Mediterranean forms of Hb H disease (a thalassemia), the Hb Lepore syndrome, and the Hb Constant Springs syndrome. Thus, in a substantial portion of the patients with

Cancer vaccine development.

A new era involving the evaluation of recombinant cancer vaccines has begun with the concurrent emergence of insights and technologies in the fields of molecular biology and immunology. These advances include: The identification and cloning of an array of genes associated with the neoplastic process, such as oncogenes, suppressor genes, genes encoding oncofoetal antigens and tissue-lineage determinants. The development of a variety of viral and bacterial vectors to deliver and present gene products. The identification of numerous T-cell costimulatory molecules and an understanding of their mode of action. The cloning and analysis of the modes of action of an array of cytokines and other immunomodulatory molecules. More sophisticated knowledge of the mode(s) of antigen presentation and T-cell activation. One current challenge in cancer therapy is the delineation of strategies toward the rational design and implementation of recombinant vaccines that will be of therapeutic benefit to cancer patients and/or members of groups at high risk for specific neoplasias. Numerous concepts are emerging in this regard. The study of immunologic intervention using laboratory animal models demonstrates that no one approach will prevail for all cancer types or, perhaps, for the various stages of the neoplastic process of a given tumour type. The immunological role(s) of CD8+, CD4+, natural killer and other cell types, as well as the roles of antibodies, must all be taken into consideration. This article reviews some of the strategies currently undergoing evaluation toward the development of recombinant vaccines for several carcinoma types.