Frank R. Jones

Treatment of cancer chemotherapy-associated thrombotic thrombocytopenic purpura/hemolytic uremic syndrome by protein A immunoadsorption of plasma

Background. Chemotherapy‐associated thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (C‐TTP/HUS) is a condition involving thrombocytopenia, microangiopathic hemolytic anemia, and progressive renal dysfunction that develops in 2–10% of patients with a history of malignant neoplasms treated with certain chemotherapeutic agents. Pathogenesis of the disease may depend on the following: (1) generation of endothelial lesions in the kidney microvasculature, resulting from drug toxic effects and/or generation of small soluble circulating immune complexes (CIC), and (2) generation of autoantibodies and/or CIC that trigger aggregation and deposition of platelets around the lesions.

Treatment of feline leukemia and reversal of FeLV by ex vivo removal of IgG: A preliminary report

Cats that were spontaneously infected with feline leukemia virus (FeLV) were treated with a combination of low‐dose irradiation and extracorporeal immunosorption using formalin and heat‐fixed S. aureus as a non‐specific immunosorbent to remove plasma IgG and immune complexes. The treatment resulted in reduction of circulating lymphoblasts within two weeks and clinical improvement of three of the five animals. A reversal of the FeLV status is reported in five of five cats. Two of the five cats remain FeLV negative and completely tumor free seven and eight months post‐therapy at the time of writing (July 1979). A third cat returned to an FeLV positive state but remained tumor free for 24 weeks. Another cat responded to the therapy by reduction of lymphoblasts and became FeLV negative but died of a hemorrhage during an immunosorption. The last cats status was FeLV positive, then FeLV negative, and finally FeLV positive again. He died 20 weeks after initiation of therapy. During the treatment there was a weight gain in the three cats responding by tumor regression. The results are discussed in terms of a removal of some type of immunoinhibiting factors such as antigen‐antibody complexes or suppressor molecules.

Novel Adenovirus type 5 vaccine platform induces cellular immunity against HIV-1 Gag, Pol, Nef despite the presence of Ad5 immunity

Recombinant Adenovirus serotype 5 (Ad5) vectors have been used as vaccine platforms in numerous animal and human clinical studies. The immune response induced by Ad5 vaccines can be mitigated due to pre-existing Ad5 immunity. We previously reported the use of a novel Ad5 platform to induce cellular immune responses (CMI) against HIV-1 Gag in Ad5 hyper immune mice. Here, the effectiveness of the Ad5 [E1-, E2b-] vaccine platform was evaluated using a triad mixture of HIV-1 Gag, Pol, and Nef as antigenic transgenes. Broad CMI was induced following vaccination with the HIV-1 expressing vectors in Ad5 naïve and Ad5 immunized mice. A mixture of the three vaccines induced CMI against each transgene product even in the presence of hyper Ad5 immunity. These studies revealed that CMI responses to immunization with Ad5 [E1-, E2b-]-gag, Ad5 [E1-, E2b-]-pol or Ad5 [E1-, E2b-]-nef vectors were transgene specific and did not induce CMI responses against irrelevant antigens such as carcinoembryonic antigen (CEA), herpes simplex virus glycoprotein B (HSV), cytomegalovirus (CMV) or influenza virus antigens. We are evaluating this recombinant triad viral vector as an HIV-1 vaccine in a non-human primate model and the data indicate that the vaccine is worthy of clinical evaluation.

A non-oncogenic HPV 16 E6/E7 vaccine enhances treatment of HPV expressing tumors

Human papillomaviruses (HPVs) are the causative factor for >90% of cervical cancers and 25% of head and neck cancers. The incidence of HPV positive (+) head and neck squamous cell carcinomas has greatly increased in the last 30 years. E6 and E7 are the two key viral oncoproteins that induce and propagate cellular transformation. An immune response generated during cisplatin/radiation therapy improves tumor clearance of HPV(+) cancers. Augmenting this induced response during therapy with an adenoviral HPV16 E6/E7 vaccine improves long-term survival in pre-clinical models. Here, we describe the generation of an HPV16 E6/E7 construct, which contains mutations that render E6/E7 non-oncogenic, while preserving antigenicity. These mutations do not allow E6/E7 to degrade p53, pRb, PTPN13, or activate telomerase. Non-oncogenic E6/E7 (E6Δ/E7Δ) expressed as a stable integrant, or in the [E1-, E2b-] adenovirus, lacks the ability to transform human cells while retaining the ability to induce an HPV-specific immune response. Moreover, E6Δ/E7Δ plus chemotherapy/radiation statistically enhances clearance of established HPV(+) cancer in vivo.

Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b-]-CEA

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.

Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine.

Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases.

An Ad5[E1-, E2b-]-HER2/neu vector induces immune responses and inhibits HER2/neu expressing tumor progression in Ad5 immune mice

Immunotherapy is a promising approach for the treatment of cancers. Modified adenovirus 5 (Ad5) vectors have been used as a platform to deliver genes encoding tumor associated antigens (TAA). A major obstacle to Ad5 vector immunotherapy has been the induction of vector immunity following administration or the presence of pre-existing Ad5 immunity, which results in vector mitigation. It has been reported by us that the Ad5[E1-, E2b-] platform with unique deletions in the E1, E2b and E3 regions can induce potent cell mediated immunity (CMI) against delivered transgene products in the presence of pre-existing Ad5 immunity. Here we report the use of an Ad5[E1-, E2b-] vector platform expressing the TAA HER2/neu as a breast cancer immunotherapeutic agent. Ad5[E1-, E2b-]-HER2/neu induced potent CMI against HER2/neu in Ad5 naïve and Ad5 immune mice. Humoral responses were also induced and antibodies could lyse HER2/neu expressing tumor cells in the presence of complement in vitro. Ad5[E1-, E2b-]-HER2/neu prevented establishment of HER2/neu-expressing tumors and significantly inhibited progression of established tumors in Ad5 naïve and Ad5 immune murine models. These data demonstrate that in vivo delivery of Ad5[E1-, E2b-]-HER2/neu can induce anti-TAA immunity and inhibit progression of HER2/neu expressing cancers.

Evidence for proteolytic cleavage of covalently bound protein A from a silica based extracorporeal immunoadsorbent and lack of relationship to treatment effects.

Studies were conducted to evaluate the potential cause for release of covalently bound Staphylococcal protein A (SpA) from a silica based extracorporeal immunoadsorbent matrix. In vitro tests revealed that SpA could be detected in human plasma, human serum, and chicken serum upon exposure to the immunoadsorbent matrix which had been treated to remove non-covalently bound SpA. In contrast, only minute quantities of SpA were detected after exposure of a physiologic mixture of purified albumin and immunoglobulin G (IgG) to the immunoadsorbent matrix. Additional tests, employing a cocktail of protease inhibitors and formalin as a general stabilizer and protease inhibitor, revealed significant inhibition of endogenous proteolytic activity present in plasma and serum. Prevention of this proteolytic activity also significantly inhibited the release of covalently bound SpA from the immunoadsorbent matrix upon contact with plasma or serum samples. Further analyses of serum samples from patients with immune thrombocytopenia, chemotherapy associated thrombotic thrombocytopenic purpura-hemolytic uremic syndrome, and breast cancer revealed a lack of association between the quantity of SpA proteolytically released and observed clinical responses or adverse effects experienced during immunoadsorption treatments. These studies indicate that SpA detected in plasma or serum after exposure to the immunoadsorbent is due to inherent endogenous proteolytic activity which cleaves protein fragments from the matrix and that these cleaved SpA fragments do not appear to contribute to the observed clinical responses or adverse effects in treated patients.

Induction and comparison of SIV immunity in Ad5 naive and Ad5 immune non-human primates using an Ad5 [E1-, E2b-] based vaccine.

The effectiveness of recombinant Adenovirus serotype 5 (Ad5) vectors to induce immune responses against targeted antigens has been limited by the presence of pre-existing or Ad5 vaccine induced anti-vector immunity. The Ad5 [E1-, E2b-] platform, a recombinant Ad5 with additional deletions, has been previously reported by us to induce immune responses in the presence of Ad5 immunity. In an Ad5 immune non-human primate (NHP) model, an Ad5 [E1-, E2b-] construct expressing HIV-1 Gag induced immune responses in the presence of pre-existing Ad5 immunity. In the present study we expand on these prior observations by comparing the cell mediated immune (CMI) responses induced by Ad5 [E1-, E2b-]-SIV-gag/nef in Ad5 naïve and Ad5 immune NHP. Additionally, NHP were immunized with an Ad5 [E1-, E2b-]-HIV-pol construct following two homologous administrations of Ad5 [E1-, E2b-]-SIV-gag/nef to determine if an immune response could be induced against a third antigen in the presence of vaccine induced Ad5 immunity. Positive CMI responses, as assessed by interferon-gamma (IFN-γ) secreting lymphocytes, were induced against all three antigens. These CMI responses increased over a course of multiple immunizations and the response profiles observed in Ad5 naïve and Ad5 immune NHP were similar. No influence of the major histocompatibility complex on CMI responses was observed. These data indicate that the new Ad5 [E1-, E2b-] platform based vaccine could be used for homologous vaccination regimes to induce robust CMI responses in the presence of Ad5 vector immunity.

Blocking (suppressor) factors, immune complexes, and extracorporeal immunoadsorption in tumor immunity

Some animal neoplasms, particularly many of those induced by chemical carcinogens, have tumor-specific transplantation antigens. These are detected by their ability to induce the rejection of tumors grafted onto properly immunized syngeneic hosts (Prehn and Main, 1957; Old and Boyse, 1964; Sjogren, 1965; K. E. Hellstrom and Brown, 1979), and the tumors expressing the antigens are commonly referred to as immunogenic. It is being debated whether “spontaneous” tumors, like most human tumors, have antigens that can be immunogenic in the native host (Klein and Klein, 1977; Hewitt et al., 1976; Hellstrom et al., 1983).