Taylor H. Schreiber

Gp96-Ig/Costimulator (OX40L, ICOSL, or 4-1BBL) Combination Vaccine Improves T-cell Priming and Enhances Immunity, Memory, and Tumor Elimination

Local anticancer therapies can increase antitumor efficacy while reducing toxicities. Cellular vaccines that locally secreted costimulation ligands produced stronger, more specific T-cell activation and tumor rejection with lower ligand concentrations than did vaccines combined with systemic antibody agonists. T-cell costimulation typically occurs in a defined microenvironment that is not recapitulated by agonistic antibody therapy. To deliver such stimulation under more favorable conditions, we investigated whether an allogeneic cell-based vaccine that secreted Fc-OX40L, Fc-ICOSL, or Fc-4-1BBL would activate and expand T cells comparably with systemically administered agonist antibodies. Among these costimulators, locally secreted Fc-OX40L provided superior priming of antigen-specific CD8+ T cells, compared with combinations with OX40 antibodies or vaccine alone. Vaccine-expressed Fc-OX40L also stimulated IFNγ, TNFα, granzyme B, and IL2 by antigen-specific CD8+ T cells similarly to OX40 antibodies, without off-target consequences such as proinflammatory cytokine induction. Vaccine-secreted Fc-OX40L increased CD127+KLRG-1− memory precursor cells during the contraction phase, resulting in improved proliferation upon secondary antigen challenge, as compared with OX40 antibody. A cell-based vaccine cosecreting gp96-Ig and Fc-OX40L led to even more pronounced tumor control, complete tumor rejection, and increased tumor antigen–specific T-cell proliferation, including in tumor-infiltrating lymphocytes, as compared with combinations of gp96-Ig vaccine and OX40 antibodies, in mice with established melanoma or colorectal carcinoma. These data suggest that local modulation of the vaccine microenvironment has unexpected advantages over systemic costimulation with agonistic antibodies, which may simplify the clinical translation of such combination immunotherapies into humans. Cancer Immunol Res; 4(9); 766–78. ©2016 AACR.

Phase I study of patients with non-muscle invasive bladder cancer (NMIBC) treated with vesigenurtacel-L (HS-410) after Bacillus Calmette-Guérin (BCG)

Meeting abstracts Vesigenurtacel-L (HS-410), consists of an allogeneic bladder cancer cell line, selected for high expression of a series of tumor antigens that are known to be shared by a high proportion of bladder tumors. The cell line secretes gp96-Ig, a modified heat shock protein which

A Phase I/II study of vesigenurtacel-l (hs-410) or placebo in combination with Bacillus Calmette-Guérin (BCG) in patients with non-muscle invasive bladder cancer (NMIBC)

Meeting abstracts Vesigenurtacel-L is an allogeneic cell-based therapeutic cancer vaccine that was selected based on its expression of a range of tumor antigens (surviving, LAGE-1, MAGE-A3, MAGE-A11, PRAME and others) that are known to be expressed amongst a high proportion of primary bladder

Abstract 5567: Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy

Current attempts at combination immunotherapy with bispecific antibodies, linked scFv9s or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule. This is likely because these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. SIRPα-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of SIRPα (CD172a) and the ECD of CD40L, adjoined by a central Fc domain. The SIRPα end of the ARC binds immobilized CD47 at 3.59 nM affinity and binds CD47 on the surface of human tumor cells both in vitro and in vivo, but does not bind human platelets or RBCs. Further, no hemolytic activity was observed with the human SIRPα-Fc-CD40L ARC in vitro as compared to CD47 mAbs. The CD40L end of the ARC binds immobilized CD40 at 756 pM affinity and binds CD40 on primary macrophages. The SIRPα-Fc-CD40L ARC stimulates functional activity (independent of Fc receptor cross-linking) in NFκB-luciferase reporter cells (CD40 driven activation of NFκB). Addition of SIRPα-Fc-CD40L to an ex vivo super-antigen (SEB) cytokine release assay stimulated secretion of IL2 and TNFα from human PBMC. Furthermore, time-lapse high content live cell imaging has shown that when activated human macrophages were co-cultured with CD47 positive human tumor cells, SIRPα -Fc-CD40L was shown to enhance phagocytosis. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established murine MC38 and CT26 tumors was superior to either CD47 blocking antibody, CD40 agonist antibody or combination antibody therapy. Notably, the superior anti-tumor immunity observed with SIRPα-Fc-CD40L was not accompanied by significant mucosal inflammation and weight loss stimulated by murine CD40 agonist antibodies. Finally, treatment of cynomolgus macaques with SIRPα-Fc-CD40L was safe, and no evidence of hemolysis or thrombocytopenia was observed. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule. Signal replacement of CD47 by CD40L may uniquely poise macrophages in the tumor microenvironment for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis. Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5567.

Abstract 1405: Vesigenurtacel-L stimulates tumor infiltration of unique polyclonal T cell clones in non-muscle invasive bladder cancer patients

Non-muscle invasive bladder cancer affects approximately 50,000 Americans each year, requires chronic medical management with a combination of surgical resection and intravesical BCG instillations for treatment. While BCG improves recurrence-free survival in this population, there remains a significant unmet medical need for therapeutics in patients who have a particularly high risk of recurrence and/or progression to muscle-invasive disease. Vesigenurtacel-L is an allogeneic, cell based, vaccine engineered to secrete the heat shock protein, gp96-Ig, which facilitates cross-presentation of cell-derived antigens on MHC I to activate CD8+ T cell responses across MHC barriers. We recently reported that in patients treated with Vesigenurtacel-L in a Phase I clinical trial, a minimum of 15 antigens were shared between the vaccine and individual patient tumors. Although no patient received a complete course of maintenance BCG, 70% of patients remained recurrence free beyond 12 months. Baseline biopsies were collected from all patients, and serial biopsies for several patients when clinically indicated. Analysis of these tissues by RNA Sequencing revealed that recurring patient tumors expressed higher levels of B7-H4, GITR and KIR3 at baseline, while non-recurring patient tumors expressed an immune profile consistent with an interferon defense signature. In addition, patients with progressive elimination of residual disease in repeat biopsies showed increased levels of CD3, CD8, IL-23A and OX40. Deep sequencing of peripheral blood T cells and total tumor DNA for T cell receptors was performed at baseline and at several post-treatment time points. These data demonstrated polyclonal expansion of TCR clones that were either absent, or present at low frequencies at baseline in the peripheral blood. Remarkably, approximately 40% of these clones could also be found expanded within the tumor. These data correlate positively with the absolute increase in tumor infiltrating lymphocyte percentages observed by immunohistochemistry. ELISPOT assays were performed to demonstrate the specificity of expanded T cell clones toward shared tumor antigens, since the antigens in both the patients tumors and Vesigenurtacel-L were known. These data provide exciting clinical evidence of patient benefit derived from an allogeneic immunotherapy vaccine and compelling evidence for a vaccine therapy converting ‘cold’ tumor to (predominantly CD8+) TIL-rich tumors, with a corresponding analysis of the antigenic determinants of the response. Citation Format: Taylor H. Schreiber, George Fromm, Xin Xu, Eric Yusko, Brandon Early, Melissa Price. Vesigenurtacel-L stimulates tumor infiltration of unique polyclonal T cell clones in non-muscle invasive bladder cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1405.

Abstract 2353: Combination immunotherapy: T-cell costimulation (OX40L, TL1A, 4-1BBL and ICOSL) secreted locally by Gp96-Ig vaccines, elicits robust antigen-specific, memory T cell responses and tumor elimination

Combination cancer immunotherapy incorporating T cell costimulation, vaccination, and checkpoint inhibition is anticipated to broaden clinical response compared to any single agent. Because T cell costimulation occurs at the site of immunization, we asked whether the delivery of a costimulator by a gp96-Ig secreting allogeneic vaccine would provide comparable costimulation to systemically administered agonist antibodies. As proof of concept, we engineered gp96-Ig vaccines that locally secrete Fc-OX40L and demonstrate that the priming of antigen-specific CD8+ T cells (peak of 13.3% of total CD8+) is significantly higher when compared to combinations with OX40 antibodies (8.4%) or vaccine alone (5.6%). Vaccine-expressed Fc-OX40L was associated with increased CD127+KLRG-1- memory precursor cells and antigen-specific CD4+ proliferation, with reduced off-target inflammation. Importantly, vaccine-expressed Fc-OX40L stimulated IFNγ+, TNFα+, granzyme-b+ and IL-2+ by antigen-specific CD8+ T cells, which enhanced rejection of established CT26 and B16.F10 tumors. We have subsequently expanded our repertoire of combination vaccines to secrete gp96-Ig along with either Fc-tagged TL1A, 4-1BBL or ICOSL. Each costimulator secreting vaccine cell line has a unique functionality, without the negative consequences of off-target inflammation associated with systemic administration of its agonist antibody counterpart. For example, costimulation with a TNFRSF25 (receptor for TL1A) agonist antibody synergized with gp96-Ig vaccination and generated a robust antigen-specific CD8+ T cell response. However, TNFRSF25 treatment also lead to a significant accumulation of FOXP3+ regulatory T cells (Treg). A gp96-Ig vaccine co-secreting Fc-TL1A resulted in similar antigen-specific CD8+ T cell production with no activation of the Treg compartment. Additionally, combining two separate gp96-Ig vaccines (one secreting Fc-OX40L and the other Fc-TL1A) leads to an additive increase in memory precursor production (CD127+KLRG1-), which may play a vital role in maintaining effective anti-tumor immunity. Together, we demonstrate that the magnitude and specificity of vaccination can be enhanced by locally secreted costimulatory molecules when delivered within a single product. This may simplify clinical translation and importantly, provide significant patient benefit by improving safety and lowering costs. Citation Format: George J. Fromm, Suresh de Silva, Louise Giffin, Jason Rose, Aditi Goyal, Taylor H. Schreiber. Combination immunotherapy: T-cell costimulation (OX40L, TL1A, 4-1BBL and ICOSL) secreted locally by Gp96-Ig vaccines, elicits robust antigen-specific, memory T cell responses and tumor elimination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2353.

Abstract 567: In vivo intra-tumoral electroporation of Gp96-Ig/Fc-OX40L stimulates CD8+ T cell cross-priming to tumor specific neoantigens

Cancer immunotherapy relies on presentation of shared- and neo- antigens from a patient9s tumor cells for recognition and clearance by the immune system. However, the tumor microenvironment deploys multiple strategies to evade immune recognition and often remains non-immunogenic, which is one of the challenges that need to be addressed when designing new therapies. Among the strategies to increase a tumor9s immunogenicity is their genetic manipulation in situ via expression of molecular chaperones, T cell costimulators and/or pro-inflammatory genes using DNA/RNA vectors packaged in oncolytic viruses, lipid based components or through electroporation. In vivo electroporation-mediated gene transfer of IL-12 triggers tumor regression and systemic anti-tumor immune responses in experimental mouse models and in patients, demonstrating the feasibility of this intratumoral (IT) gene-transfer technology. We set out to test whether intratumoral electroporation of Gp96-Ig/Fc-OX40L, a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression, would generate a robust anti-neoantigen CD8+ T cell response. Gp96-Ig/Fc-OX40L is a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression. To assess antigen-specific CD8+ expansion, mice were adoptively transferred with OT-I cells after B16.F10-ovalbumin cells were injected to generate primary and contralateral melanotic tumors. Contralateral tumors were monitored to assess whether a systemic CD8+ T cell response could be elicited following primary tumor electroporation. IT electroporation of DNA expressing Gp96-Ig/Fc-OX40L in the primary tumor triggered a significant expansion of antigen-specific OT-I cells, which was absent in control mice. Remarkably, increases in antigen-specific OT-I cells correlated with regression of both the treated primary and untreated contralateral tumors. We further validated our findings in a CT26 mouse colorectal cancer tumor model, in which the expression of Gp96-Ig/Fc-OX40L from electroporated DNA stimulated an expansion of antigen-specific CD8+ T cells and again led to regression of both the treated primary and untreated contralateral tumor. Our findings demonstrate that in situ manipulation of intratumoral cells to express Gp96-Ig/Fc-OX40L stimulates potent antigen-specific cross priming to tumor specific neoantigens that culminates in robust systemic anti-tumor response. These findings provide exciting proof-of-principal and warrant further investigation into the direct delivery of molecular chaperones such as Gp96-Ig/Fc-OX40L and/or pro-inflammatory molecules for elevating the immunogenicity of tumors for a potent anti-tumor CD8+ T cell response. Citation Format: Suresh de Silva, George Fromm, Jamil Haque, Jean S. Campbell, Robert H. Pierce, Taylor H. Schreiber. In vivo intra-tumoral electroporation of Gp96-Ig/Fc-OX40L stimulates CD8+ T cell cross-priming to tumor specific neoantigens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 567.

Locally secreted Fc-OX40L is superior to systemic, antibody mediated, OX40 costimulation for combination immunotherapy.

The dramatic clinical success of checkpoint inhibitory therapy (anti-CTLA-4 and anti-PD-1) in a small percentage of patients has highlighted the need to identify combination approaches that may increase the frequency of responders. Two immunotherapy modalities that are proposed to synergize both with each other, and with checkpoint inhibitors are therapeutic vaccines and T cell co-stimulators. To prioritize which T cell co-stimulators enhance the efficacy of an allogeneic, gp96-Ig secreting, cell-based vaccine, we investigated the activity of agonistic antibodies targeting OX40, 4-1BB and ICOS administered together with gp96-Ig vaccines (ImPACT). These data demonstrated that antigen-specific CD8+ T cell expansion is significantly enhanced by OX40, but not 4-1BB or ICOS stimulation. Because T cell co-stimulation occurs at the site of immunization, we then asked when co-expression of Fc-OX40L by the gp96-Ig secreting allogeneic vaccine cells (ComPACT) would provide comparable co-stimulation to systemically administered OX40 agonist antibodies (Figure A). Interestingly, these data demonstrated that locally secreted Fc-OX40L provided superior priming of antigen-specific CD8+ T cells (peak of 14.05% of total CD8+) as compared to combinations with OX40 antibodies (8.9%) or vaccine alone (5.2%) (Figure B). The reason for the beneficial response was related to more potent activation of CD127+KLRG-1- memory precursor cells by the Fc-OX40L expressing vaccine. Importantly, antigen-specific CD8+ cells stimulated by vector-encoded Fc-OX40L demonstrated a protracted contraction phase after priming, and remained at high levels in both tissues and peripheral blood for 3 weeks after priming (Figure C). Systemic administration of OX40 antibodies also led to proliferation of antigen non-specific CD4+ T cells, Treg and systemic increases in IL-4, IL-5, IL-6, TNFα and IFNα. Importantly, vaccine-expressed Fc-OX40L led to high frequencies of IFNα+, TNFα+, granzyme-b+ and IL-2+ antigen-specific CD8+ T cells at both priming and boosting, which enhanced rejection of established CT26 tumors. These data demonstrate that vaccination and co-stimulation can be approached with a single cell-based product, and that locally delivery of a vaccine and T cell co-stimulator enhances the primary and memory responses of highly activated CD8+ T cells as compared to co-stimulatory antibodies. This may lead to more rapid development of combination immunotherapeutics with reduced toxicity, as a result of increased specificity, and a simpler cost structure than combining multiple independent biologic medicines. Figure 1

Abstract A24: Cancer-testis antigen expression is shared among gynecological tumors

Objective: Immunotherapy has become one of the novel therapeutic strategies under investigation for several cancers including ovarian cancer. Although antigenic targets for the immune system exist in the form of tumor-associated antigens, no tumor-specific antigens have been identified for gynecological cancers. Expression of cancer-testis (CT) antigens in ovarian cancer has been described. CT antigens are attractive targets for cancer immunotherapy due to their restricted expression in testis, germ cells and tumors. Current data from gene expression profiles in many types of tumors suggests that antigen sharing between allogeneic tumors may be a generalizable phenomenon. We hypothesize that there will be antigen sharing between gynecological cancers. The aim of our study was to evaluate the expression of a panel of 20 CT antigens in gynecological tumor cell lines, and to determine if antigen sharing occurs among different gynecologic cancers. Methods: RNA was isolated from 16 gynecological cancer cell lines (9 epithelial ovarian cancer, 8 endometrial cancer, 1 cervical cancer and 1 leiomyosarcoma cell line). Real time-PCR analysis was performed to determine the expression level of 20 CT antigens, including NY-ESO-1, MAGE-A3/A4, GAGE, LAGE, PIWIL-2, CT-45. Results: Expression of CT antigens was identified in gynecologic cancer cell lines. Among 20 evaluated antigens 90% (18/20) expressed in ovarian and endometrial cancer cell lines, while only 30% (6/20) expressed in cervical cancer and leyomyosarcoma cell lines. The most frequently expressed CT antigens were SP-17(93.8% of cell lines), MAGE-A4 (87.5%), OYTES (68.8%), BAGE (62.5%),CT-45 (62.5%), PIWIL-2 (56.3%) and GAGE (56.3%). CT45, SP-17 and MAGE-A4 were expressed among all gynecological cancers. We found that different gynecological cancers shared the expression of CT antigens, with the mean number of antigens shared among cancer types ranging from 3 to 4.6 antigens. There was sharing of CT antigens between cell lines of the same tumor type and with cell lines of other gynecological tumor types. The mean number of antigens shared among endometrial cell lines (5.5) was higher than the one shared by the ovarian cancer cell lines (3.8) with their corresponding tumor type (p-value= 0.0122). Conclusion: Cancer-testis antigens are expressed in gynecological cancers. No single antigen was noted universally expressed among all gynecological cancer cell lines, however most expressed SP-17. Utilizing a multiepitope immunotherapy approach could provide a universal therapeutic option for gynecological cancers. Citation Format: Arlene E. Garcia-Soto, Tulay Koru-Sengul, Feng Miao, Taylor Schreiber, Wilberto Nieves-Neira, Eckhard Podack. Cancer-testis antigen expression is shared among gynecological tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A24.

Abstract 5037: Immunotherapy meets epigenetics: GP96-Ig cellular vaccine genetic signature is modulated by the inhibition of histone modifications

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA A number of novel cancer treatment strategies have been identified and developed over the last decade with hopes of eradicating a diverse range of cancers, however the molecular mechanisms behind patient immune responses and tumor microenvironment signaling are complex, and have often hindered the translation of these ideas into successful patient treatments. Two modalities at the forefront of these potential blockbuster strategies are combinatorial immunotherapy and epigenetic manipulation. These approaches are supported by compelling clinical data, but have been encumbered by a number of factors including: adverse event profiles, complicated manufacturing and regulatory issues, toxicity due to non-specific small molecule interactions, limited overlap in patient/therapy antigen presentation, and exorbitant cost of biological treatments. To circumvent a number of these limitations, we have taken a novel approach by engineering allogeneic cellular vaccines tailored to specific cancers, that secrete heat-shock protein gp96-Ig and achieve high-frequency polyclonal CD8+ T cell responses to femto-molar concentrations of tumor antigens through antigen cross-priming in vivo. Using genomics (RNA-sequencing) we identified differential mRNA expression levels genome-wide between normal bladder tissue, our proprietary bladder cancer cellular vaccine Vesigenurtacel-L or HS410, and patient bladder tumor biopsy samples, to generate a genomic signature of Bladder Cancer Altered Genes, or BCAGs. Next, we examined a number of small molecule inhibitors of epigenetic modifications (i.e. histone deacetylases or HDACs), and determined their effect on BCAG expression and antigen secretion in HS410 cells. We find that the inhibition of certain HDACs results in decreased proliferation, dampened secretion of gp96-Ig, and attenuated expression of tumor specific genes. Together our work has defined the pattern of gene dysregulation genome-wide in bladder cancer and shown that epigenetic modifiers are capable of modulating the expression of many of these genes, which may be important for the efficacy of our cancer vaccine strategy. Therefore, epigenetic manipulation may present an attractive means to fine-tune gene expression programs and maximize antigen secretion thereby increasing vaccine potency and the ability of a single vaccine to target a variety of cancer types. Citation Format: George J. Fromm, Neal Schilling, Taylor H. Schreiber. Immunotherapy meets epigenetics: GP96-Ig cellular vaccine genetic signature is modulated by the inhibition of histone modifications. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5037. doi:10.1158/1538-7445.AM2015-5037