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V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family member highly expressed on circulating and intra-tumoral myeloid cells. It is a negative checkpoint inhibitor that inhibits anti-tumor T cell response. In patients, VISTA is associated with poor overall survival in multiple tumor indications and is also a potential mediator of resistance to anti-CTLA-4 and anti-PD1 therapies. Therefore, VISTA is a unique target for cancer immunotherapy. Kineta has developed a fully human monoclonal antibody targeting VISTA, KVA12123, that is currently being evaluated in a Phase 1/2 clinical trial in cancer patients with advanced solid tumors. This trial also includes a combination arm with pembrolizumab. In order to inform which patients may be susceptible to respond to our anti-VISTA antibody, we hypothesized that the best responders should be associated with a high expression of the target in the tumor microenvironment (TME). Therefore, after assay validation of VISTA labelling by immuno-histochemistry, we analyzed a large set of tumor samples and showed that VISTA was highly expressed on tumor infiltrating immune cells. This was particularly true for patients with non-small cell lung cancers, colorectal cancers, head and neck squamous cell carcinomas, hepatocellular carcinomas, melanomas and squamous cell carcinoma of the skin, and cervical cancers as well as ovarian cancers. VISTA expression was detected mostly on CD163 positive macrophages infiltrating the tumor. These macrophages potentially promote immunosuppression present in the TME and contribute to treatment failure with current immune checkpoint inhibitors like anti-PD1/PD-L1 and CTLA-4. While previous studies reported VISTA expression on cancer cells, we were not able to confirm these results. In all tumor tissues tested, only infiltrating immune cells were labelled for VISTA. We have investigated in parallel the expression level of soluble VISTA in the serum collected from cancer patients independent of this clinical trial. Sera were screened for patients with multiple tumor types. Patients of diverse sex and age were compared to healthy donors. After validation of the ELISA assay, we showed that sera derived from cancer patients exhibit high levels of soluble VISTA, and these levels tend to correlate with age. More data are needed to confirm that high levels of soluble VISTA are associated with advanced disease. In the ongoing Phase 1/2 clinical trial, tumor tissues and serum samples will be collected from cancer patients prior to treatment with KVA12123 to inform the possible significance of these biomarkers. This work could help to better understand the response to KVA12123 in relation to the expression level of VISTA in cancer tissues as well as in the blood. Citation Format: Neda Kabi, Chen Katz, Remington Lance, Jessica Cross, Nathan Eyde, Emily Frazier, Kurt Lustig, Yulia Ovechkina, David Peckham, Shaarwari Sridhar, Carla Talbaux, Isabelle Tihista, Mei Xu, Shawn Iadonato, Thierry Guillaudeux. VISTA expression in patients with advanced solid tumors: A potential biomarker in VISTA-101 clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 972.

CD27 is a member of the TNF-receptor superfamily, highly expressed on CD4+ and CD8+ T cells as well as on NK and NKT cells. It plays a key role on T cell proliferation and differentiation after stimulation with its ligand CD70. The co-stimulatory signal of CD27 on T cell is mediated via the NFκB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B. CD27/CD70 co-stimulation has the potential to boost immunity by T-cell activation, clonal expansion and enhanced differentiation into antigen specific cytotoxic and memory T cells. CD27/CD70 also influences the innate immune response via a direct activation of the NK cells and a subsequent secretion of interferon-gamma (IFN-γ). Therefore, CD27 signaling promotes cytotoxic T cell based anti-tumor immunity. With its central role in an immunological response, CD27 is a promising target for antitumor therapy. Previous works have demonstrated the efficacy of an agonistic CD27 antibody in controlling tumor growth and metastasis in different mice models including melanoma, renal cell carcinoma, breast cancer and lymphomas. This anti-tumor effect is mediated in part by an effective recruitment of IFN-γ producing CD8+ T cells within the tumor. Moreover, CD27 stimulation of Tumor Infiltrating Lymphocytes (TILs) can lower their threshold of activation and provide a broader repertoire of Ag-reactive T cells within the tumor. We have selected a lead therapeutic antibody from our library of 147 fully human anti-CD27 monoclonal antibodies generated in the Trianni mice. After confirming its binding potency and selectivity as well as its cross-reactivity with Non-Human Primate (NHP)-CD27 but not with the mouse-CD27, this lead candidate demonstrated strong agonistic proprieties. This was shown by its ability to induce a strong NFκB signal as well as to induce T cell proliferation and activation with secretion of pro-inflammatory cytokines. This antibody demonstrated agonistic proprieties without cross-linking confirming its potency. T cell activation observed after treatment with our anti-CD27 antibody only occurs in the presence of TCR engagement, preventing the risk of spontaneous activation of naïve T cells in vivo. The ability of our CD27 monoclonal antibody to increase an immune response was confirmed in a Mixed Lymphocyte Reaction assay with multiple donors. The role played by the NK cells and their activation via CD27 antibody was also demonstrated. To evaluate the anti-tumor functions of our lead antibody as a single agent or in combination with other immuno-therapies in vivo we have used human CD27 transgenic mice. We have demonstrated in the MB49 bladder tumor model as well as the EG7 thymoma model that our lead antibody induces a strong single agent anti-tumor activity and these tumors were totally controlled in combination with an anti-PD1 antibody. We are now analyzing the pharmacokinetic and pharmacodynamic of our antibody as well as its safety in a NHP model Citation Format: Thierry Guillaudeux, Yulia Ovechkina, Shaarwari Sridhar, David Peckham, Jessica Cross, Nathan Eyde, Emily Frazier, Neda Kabi, Remington Lance, Kurt Lustig, Mei Xu, Tarcha Eric, Shawn Iadonato. CD27 a new immuno-oncology target shaping innate and adaptive anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4261.

Background V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family ligand expressed on circulating and intratumoural myeloid cells as well as Treg and NK cells. It has been shown to inhibit T cell responses in vitro and in preclinical models. In patients, VISTA is also a potential mediator of resistance to anti-CTLA-4 and anti-PD1 therapies and therefore is a valuable new target for cancer immunotherapy. Methods Kineta has analyzed 107 fully human ScFv antibodies directed against VISTA. Results Our lead candidates exhibit high potencies in the subnanomolar range and are also characterized by a long kDis. They specifically target human and cynomolgus monkey VISTA on a singular unique epitope. In a Staphylococcus Enterotoxin B T-cell activation assay, Kineta’s anti-VISTA antibodies efficiently induce IFNg secretion. They also promote strong maturation of Antigen Presenting Cells with an increase of CD80 and HLA-DR surface expression as well as CXCL10 secretion. The mechanism of action is mediated in part by NK cells. We demonstrated that myeloid cells acquire a high level of VISTA expression during MDSC or M2 differentiation in vitro and that Kineta’s anti-VISTA antibodies prevent the differentiation of MDSC as well as their immunosuppressive activity against T cells. Anti-VISTA antibodies mediate single-agent antitumor effects in syngeneic tumor models in wild-type mice and show enhanced activity in combination with anti-PD1 and anti-CTLA-4 treatment. Candidate anti-VISTA antibodies have also been evaluated in exploratory tolerability and PK studies in cynomolgus monkey. These studies demonstrated that multiple weekly doses of antibodies are well-tolerated with appropriate PK for lead selection and optimization. Conclusions Our results strongly favor further characterization and continued development of selected lead antibodies for the potential treatment of colder, less immunogenic tumors. Ethics Approval Study approved by the Institutional Animal Care and Use Committee PHS Assurance # D16-00885 and D16-00114

V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is an immune checkpoint regulator of the B7 family. VISTA can be found on the cell surface of some tumor types, however for the majority of cancers, VISTA is highly expressed in the immunological myeloid cell compartment in the tumor microenvironment (TME). VISTA has been shown, in vitro and in vivo, to inhibit T cell activation and prevent T cell recruitment into tumors. In patients, high VISTA expression is associated with poor prognosis and is also a potential mediator of resistance to anti-CTLA-4 and anti-PD-(L)1 therapies. Therefore, VISTA is a very attractive new target for cancer immunotherapy. Kineta has selected a lead candidate anti-VISTA monoclonal antibody after a deep screen of 107 fully human and highly diverse antibodies directed against the VISTA extracellular domain. The candidate exhibits high potency in the subnanomolar range and is characterized by a long constant of dissociation evaluated by ELISA and Octet binding. It targets human and cynomolgus monkey VISTA on a unique epitope. Cross reactivity against other B7 family members has also been evaluated, and the lead candidate demonstrates high specificity against VISTA. The candidate antibody also efficiently induces T cell activation, proliferation and IFNg secretion on a Staphylococcal EnterotoxinB assay, as well as in a coculture experiment with a cell line overexpressing VISTA. The candidate promotes maturation of Antigen Presenting Cells with an increase of CD80 and HLA-DR surface expression as well as CXCL10 secretion in a monocyte activation assay. The mechanism of action is mediated in part by NK cells. This anti-VISTA antibody also prevents the immunosuppressive function of differentiated MDSCs in vitro against T cells. In Knock-In-human VISTA mice, anti-VISTA antibody treatment mediates single-agent antitumor activity in vivo in multiple syngeneic tumor models and shows enhanced efficacy in combination with either anti-PD-(L)1 or anti-CTLA-4 treatment. Finally, anti-VISTA antibody treatment was well-tolerated in exploratory toxicology studies in cynomolgus monkey and has a half-life consistent with other monoclonal antibodies. Our results strongly support the continued development of our anti-VISTA antibody for the treatment of colder, less immunogenic tumors. Citation Format: Thierry Guillaudeux, Eric Tarcha, Robert Bader, Benjamin Dutzar, Nathan Eyde, Emily Frazier, David Jurchen, Remington Lance, Cristina Loomis, Kurt Lustig, Yulia Ovechkina, David Peckham, Jeff Posakony, Shaarwari Sridhar, Mei Xu, Shawn Iadonato. A fully human anti-vista antibody as a promising therapy against poorly immunogenic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1637.

V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family ligand expressed on circulating and intratumoural myeloid cells as well as Treg and NK cells. It has been shown to inhibit T cell responses in vitro and in preclinical models. In patients, VISTA is also a potential mediator of resistance to anti-CTLA-4 and anti-PD1 therapies and therefore is a valuable new target for cancer immunotherapy. Kineta has analyzed 107 fully human ScFv antibodies directed against VISTA. Our lead candidates exhibit high potencies in the subnanomolar range and are also characterized by a long kDis. They specifically target human and cynomolgus monkey VISTA on a singular unique epitope. In a Staphylococcus Enterotoxin B T-cell activation assay, Kineta’s anti-VISTA antibodies efficiently induce IFNg secretion. They also promote strong maturation of Antigen Presenting Cells with an increase of CD80 and HLA-DR surface expression as well as CXCL10 secretion. The mechanism of action is mediated in part by NK cells. We demonstrated that myeloid cells acquire a high level of VISTA expression during MDSC or M2 differentiation in vitro and that Kineta’s anti-VISTA antibodies prevent the differentiation of MDSC as well as their immunosuppressive activity against T cells. Anti-VISTA antibodies mediate single-agent antitumor effects in syngeneic tumor models in wild-type mice and show enhanced activity in combination with anti-PD1 and anti-CTLA-4 treatment. Candidate anti-VISTA antibodies have also been evaluated in exploratory tolerability and PK studies in cynomolgus monkey. These studies demonstrated that multiple weekly doses of antibodies are well-tolerated with appropriate PK for lead selection and optimization. Our results strongly favor further characterization and continued development of selected lead antibodies for the potential treatment of colder, less immunogenic tumors. This abstract is also being presented as PO035. Citation Format: Thierry Guillaudeux, Eric Tarcha, Robert Bader, Benjamin Dutzar, Nathan Eyde, Emily Frazier, David Jurchen, Remington Lance, Cristina Loomis, Kurt Lustig, Yulia Ovechkina, David Peckham, Jeff Posakony, Shaarwari Sridhar, Mei Xu, Shawn Iadonato. Highly potent fully human anti-VISTA antibodies – A new target checkpoint inhibitor against immunosuppressive myeloid cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR005.

SUMMARY Periodontal disease is a chronic inflammatory condition induced by tooth-associated microbial biofilms that induce a host immune response. Therapeutic control of progressive tissue destruction in high-risk patients is a significant challenge in therapy. Soluble protein delivery of antagonists to tumor necrosis factor alpha (TNF-α) inhibits alveolar bone resorption due to periodontitis. However, protein therapy raises several concerns, such as recurrence of disease activity after treatment cessation and repeated dosing regimens. In this study, we used pseudotyped adeno-associated virus vector based on serotype 1 (AAV2/1) to deliver the TNF receptor-immunoglobulin Fc (TNFR:Fc) fusion gene to rats subjected to experimental Porphyromonas gingivalis (Pg)-lipopolysaccharide (LPS)-mediated bone loss. Animals received Pg-LPS delivered to the gingivae thrice weekly for 8 weeks, vehicle alone, Pg-LPS and intramuscular delivery of pseudotyped AAV2/1-TNFR:Fc vector (1×1011 DNase I-resistant particles) or AAV2/1-TNFR:Fc vector delivered to naïve animals. AAV2/1-TNFR:Fc therapy led to sustained therapeutic levels of serum TNFR protein and protected against Pg-LPS-mediated loss of bone volume and density. Furthermore, AAV2/1-TNFR:Fc administration reduced local levels of multiple pro-inflammatory cytokines and osteoclast-like cells at the periodontal lesions. These findings suggest that delivery of AAV2/1-TNFR:Fc may be a viable approach to modulate periodontal disease progression.

We previously reported that administration of an adeno-associated virus 2 (AAV2) vector encoding a rat tumor necrosis factor (TNF) receptor-immunoglobulin Fc (TNFR:Fc) fusion gene to rats with streptococcal cell wall-induced arthritis resulted in suppression of joint inflammation and cartilage and bone destruction, as well as expression of joint proinflammatory cytokines. In this study, we used an alternate rat model of arthritis to compare the serum levels and duration of TNFR:Fc protein expression following intramuscular administration of pseudotyped AAV-TNFR:Fc vectors based on serotypes 1, 2, and 5. All three pseudotyped AAV-TNFR:Fc vectors led to sustained expression of serum TNFR:Fc protein for at least one year. Serum TNFR:Fc protein levels in rats administered intramuscularly with AAV2/1-TNFR:Fc vector were up to 100- and 10-fold higher than in rats administered the AAV2-TNFR:Fc or AAV2/5-TNFR:Fc vectors, respectively. A single intramuscular administration of AAV2/1-TNFR:Fc vector at vector doses ranging from 10(10) to 10(12) DNase-resistant particles (DRP) per animal, resulted in complete and long-term suppression of recurrent joint inflammation for at least 150 days. Our results establish a proof of concept for administration of an AAV2/1-TNFR:Fc vector to the muscle to achieve long-term, sustained and therapeutically relevant levels of TNFR:Fc protein to treat chronic systemic inflammatory joint diseases.

Top of pageAbstract Rheumatoid arthritis is a chronic autoimmune disease characterized by synovial inflammation, leading to the destruction of cartilage and bone. Previously, we demonstrated that a single intra-articular administration of AAV2-TNFR:Fc vector to rats with experimental arthritis resulted in suppression of disease. For treatment of chronic inflammatory arthritis using intra-articular delivery of AAV2-TNFR:Fc vector, intra-articular re-administrations may be required. In this study, we evaluated if serum anti-AAV2 capsid neutralizing antibodies, elicited after intra-articular administration of AAV2-TNFR:Fc vector to rat joints, affect joint transduction following intra-articular re-administration of an AAV2-FlagLuc vector. AAV2-FlagLuc was administered approximately one, three, nine and twelve months after injection of either vehicle or AAV2-TNFR:Fc vector. Two weeks post intra-articular injection of the AAV2-FlagLuc vector, we measured and compared luciferase enzyme activity in joint tissues of vehicle-treated animals with animals treated first with AAV2-ratTNFR:Fc vector. Intra-articular administration of AAV2-TNFR:Fc vector to rat joints elicited serum anti-AAV2 capsid neutralizing antibody response. Following re-administration with AAV2-FlagLuc, the anti- AAV2 capsid neutralizing antibody levels in the serum were up to 10-fold higher compared with those detected prior to vector re-administration. Luciferase enzyme activity, measured following re- administration of AAV2-FlagLuc vector to vehicle-treated animals, was 25-fold higher compared with the assay background in normal joints, and 12-fold higher in inflamed joints. In contrast, luciferase enzyme activity was not detected in normal or arthritic rat joints that were injected first with AAV2-ratTNFR:Fc and then re-administered with AAV2-FlagLuc. However, using RT-PCR analyses, we observed sustained expression of TNFR:Fc for at least 378 days. These results demonstrate that in rats the levels of serum anti-AAV2 capsid neutralizing antibodies, elicited one to twelve months after AAV2 vector administration to the rat joint, are sufficiently high to inhibit transduction following intra-articular re-administration with a vector of the same serotype. The evidence that TNFR:Fc is expressed in the joints for more than a year suggests that frequent repeat administrations to the joints may not be needed.

TNF-|[alpha]| antagonists such as anti-TNF-|[alpha]| monoclonal antibodies or soluble tumor necrosis factor receptor have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. In the current study, we employed a monoarticular arthritis model in rats that allowed us to evaluate the long-term effect of TNFR:Fc expression on recurrence of joint inflammation. Monoarticular arthritis was initiated by injection of a small dose of peptidoglycan-polysaccharide polymers (PG-APS) isolated from cell walls of group A streptocci into the hind ankle joint. This resulted in a flare of inflammation that resolved within five days. Intramuscular administration of AAV-TNFR:Fc vectors performed on day seven. To assess the long-term effect of TNFR:Fc expressions on suppression of arthritis in this model, animals were evaluated for four rounds of remission and reactivation of joint inflammation. Reactivation of joint inflammation was induced by intravenous injection of PG-APS. Animals that were treated with vehicle, 109, 5x109 DRP of AAV2/1-TNFR:Fc or 1012 DRP of AAV2-TNFR:Fc developed a significant flare of inflammation in their ankle joints. In these animals, levels of TNFR:Fc protein in the circulation were below 0.75 |[mu]|g/mL. In contrast, when animals were treated with 1010, 1011or 1012 DRP of AAV2/1-TNFR:Fc, levels of TNFR:Fc protein in the circulation were 1.27|[ndash]|8.35 |[mu]|g/mL, 6.8|[ndash]|27.4 |[mu]|g/mL and 19.6|[ndash]|61 |[mu]|g/mL, respectively. The inflammatory response in these animals, as measured by joint volume, was completely suppressed. To increase the transduction efficiency of AAV2/2- TNFR:Fc following administration of 1012 DRP, we employed treatment with proteosome inhibitor that has been shown to enhance AAV transduction. In these animals, levels of TNFR:Fc protein in the circulation increased up to 3.65 |[mu]|g/mL, and led to significant decrease in joint inflammation.

The Ad/AAV hybrid vector is an E1 gene-deleted adenovirus containing a transgene cassette flanked on its 5' and 3' ends by AAV ITR vector sequences. After coinfection of Ad/AAV and wt Ad5 of cells containing the rep-cap genes, the AAV ITR-containing transgene cassette is excised from the Ad/AAV, amplified, and then packaged into AAV particles.

This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-immunoglobulin G1 (IgG1) Fc fusion (TNFR:Fc) gene to the lung by single and repeat administrations of multiple pseudotyped adeno-associated virus (AAV) vectors as a means for achieving systemic distribution of the soluble TNFR:Fc protein. A single endotracheal administration of AAV[2/5]cytomegalovirus (CMV)-TNFR:Fc vector (containing the AAV2 inverted terminal repeats and AAV5 capsid) to the rat lung resulted in long-term, high levels of serum TNFR:Fc protein that gradually declined over a period of 8 months. Endotracheal delivery of AAV[2/1]CMV-TNFR:Fc resulted in serum TNFR:Fc protein levels that were detectable for at least 4 months but were 10-fold lower than that of the AAV[2/5] vector. In contrast, secretion of the TNFR:Fc protein following pulmonary delivery of AAV[2/2]CMV-TNFR:Fc vector was very inefficient, and the protein was detected in the blood only when an airway epithelial cell-specific promoter, CC10, was substituted for the CMV enhancer/promoter to control transgene expression. In the context of AAV[2/5], the CC10 promoter was as efficient as CMV enhancer/promoter in generating similar levels of systemic TNFR:Fc protein, suggesting that this protein is secreted primarily from the airway epithelium. In mice, comparable long-term secretion of TNFR:Fc protein was demonstrated after AAV[2/2] and AAV[2/5] delivery, although the kinetics of transduction appeared to be different. All pseudotyped AAV vectors elicited serum anti-AAV capsid-neutralizing antibody responses, but these did not prevent lung transduction and efficient secretion of TNFR:Fc protein to the circulation following readministration with AAV[2/5]. These results highlight the potential utility of AAV vectors containing serotype 5 capsid to deliver and redeliver genes of secreted proteins to the lung to achieve long-term systemic protein expression.

Although periodontitis is initiated by specific oral pathogens that colonize and invade the oral tissues, the host inflammatory response is a major factor in disease progression. Soluble protein delivery of antagonists to tumor necrosis factor alpha (TNF-receptor:Fc fusion protein) inhibit alveolar bone resorption. However, the clinical use of recombinant p75-TNFR:Fc fusion protein raises several concerns, such as reoccurrence of disease activity after cessation of therapy. Objectives: This pilot study used adeno-associated virus (AAV) as a mode to deliver the TNFR:Fc transgene to periodontal lesions in an experimental model of P. gingivalis LPS-mediated bone loss. Methods: Three groups of Sprague-Dawley rats received one of three treatments: LPS delivered to the maxillary interdental gingivae thrice weekly for 8 weeks; vehicle alone (PBS) or LPS plus the single local delivery of pseudotyped AAV[2/5]-TNFR:Fc vector [6 sites|[times]|15uL of 8|[times]|1011 DNase I-resistant particles (DRP)] at baseline. Microcomputed tomography (MicroCT), systemic blood levels of TNFR:Fc protein and cytokine profiles were performed at various timepoints ranging from baseline to 8 weeks following TNFR:Fc transgene delivery. Results: TNFR protein serum levels were low to undetectable in all groups over the entire observation period indicating that local vector delivery did not result in systemic distribution of the soluble TNFR:Fc protein. Real-time PCR showed higher expression of both TNFR1 and TNFR2 than controls (p

TNF-alpha antagonists such as anti-TNF-alpha monoclonal antibodies or soluble tumor necrosis factor receptors have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. Recently, we have demonstrated that local (intra-articular) or systemic (intramuscular) administration of an AAV2-ratTNFR:Fc vector, encoding the rat tumor necrosis factor-a receptor: immunoglobulin (IgG1) Fc fusion gene, to rats with experimental arthritis led to suppression of disease as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. In the current study we employed a monoarticular arthritis model in rats that allow us to evaluate the long-term effect of TNFR:Fc expression on recurrence of joint inflammation. Monoarticular arthritis was initiated by injection of a small dose of peptidoglycan-polysaccharide polymers (PG-APS) isolated from cell walls of group A streptocci into the hind ankle joints. This resulted in a flare of inflammation that resolved within 5 to 7 days. Thirty-two days later, reactivation of joint inflammation was induced only in the joints previously injured by intravenous injection of subarthropathic dose of PG-APS. Twenty-six days later, rats were administered intramuscularly with either vehicle or with AAV-TNFR:Fc vector pseudotyped with AAV type 1 capsid at a dose of 1E12 DNase-resistant particles (DRP). Four weeks post-vector administration, the mean levels of circulating TNFR:Fc protein was 21.5 μg/mL. PG-APS was then administered intravenously to induce a recurring flare of joint inflammation. As expected, animals that were treated with vehicle developed a significant flare of inflammation in their ankle joints. In contrast, the inflammatory response, measured by joint volume, was completely suppressed in AAV2/1-TNFR:Fc-treated animals. To assess the long-term effect of TNFR:Fc expression on suppression of arthritis in this model, animals are currently being evaluated for another round of remission and reactivation of joint inflammation.

Recently, we have demonstrated that local (intraarticular) or systemic (intramuscular) administration of an AAV-ratTNFR:Fc vector, encoding the rat tumor necrosis factor-a receptor: immunoglobulin (IgG1) Fc fusion gene, to rats with experimental arthritis led to suppression of disease as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. Last year we began to evaluate the serum expression levels, in rats, of TNFR:Fc fusion protein following either endotracheal, intramuscular or intravenous administration of AAV-ratTNFR:Fc vectors pseudotyped with capsids from AAV serotypes 1, 2 or 5. Intramuscular delivery proved to be the most efficient route of administration and resulted in very high and sustained serum levels of TNFR:Fc protein for over a year. AAV2/1 was 100- to 1000-fold more efficient than AAV2/5 and AAV2/2, while AAV2/5 was up to 5-fold more efficient than AAV2/2. All animals developed anti-capsid neutralizing antibodies. For AAV2/1, these neutralizing antibodies reduced but not eliminated transduction following repeat intramuscular administration. Following a single intravenous administration, AAV2/1 demonstrated an over a 100-fold enhanced expression compared with AAV2/5 and AAV2/2, and AAV2/5 was up to 10-fold more efficient than AAV2/2. All animals developed anti-capsid neutralizing antibodies. Biodistribution of AAV2/1 vector DNA to perfused organs as well as TNFR:Fc mRNA expression is currently being assessed and data will be presented. Following a single pulmonary delivery of AAV2/5, maximum expression was achieved at 6-weeks post-administration, then gradually declined but remained detectable over a period of more than 250 days. Anti-AAV type 5 capsid neutralizing antibodies were elicited but did not prevent transduction and renewed expression of circulating TNFR:Fc protein for additional 6 months following a second AAV2/5 administration. The serum levels of TNFR:Fc protein were below the limit of detection (< 2 ng/mL) over 9-weeks post-AAV2/2 administration to the lung, although anti-AAV2 neutralizing antibodies were clearly evident. These had no apparent impact on transduction following repeat administration with an AAV2/5 vector. After more than 400 days, AAV2/5-transduced lungs demonstrated 100-fold more TNFR:Fc mRNA than animals administered with AAV2/2. AAV2/1 administration to the lung resulted in secretion of low but detectable TNFR:Fc protein that was almost 10-fold less efficient than AAV2/5. AAV2/1-treated animals developed anti-AAV type 1 capsid neutralizing antibodies. Re-administration of AAV2/5 to AAV2/1-treated animals resulted in renewed high levels of circulating TNFR:Fc protein which declined over a period of 6 months. These results demonstrate the utility of AAV vectors of alternate serotypes for long-term systemic delivery of secreted soluble TNFR:Fc protein and highlight their potential to treat systemic TNF-alpha-mediated autoimmune diseases such as reumatoid arthritis and psoriasis.