1 Abstract Adoptive cell therapy using tumor antigen-targeting T cell receptors (TCRs) offers a compelling approach to treat both hematological cancers and solid tumors due to broad antigen accessibility and the ability to target cancer-specific neoantigens. However, unlike clinically validated second generation CAR-T cells bearing built-in co-stimulatory signaling modules (i.e. 41BB or CD28), TCR-T cells receive little to no co-stimulation within most tumor microenvironments leading to attenuated cellular responses. Additionally, CD4+ TCR-T cells engineered to express HLA-Class I restricted TCRs possess minimal T-helper cell activity and thus do not effectively mobilize CD8+ TCR-T cells or host anti-tumor immune responses. To address these limitations, we used CRISPR-Cas9 to engineer TCR-T cells with targeted integration of chimeric CD8 constructs containing intracellular co-stimulatory domains. We found that expression of wild-type CD8αβ, but not CD8αα, could promote CD4+ T cell activities in HLA-Class I restricted TCR-T cells. However, this was insufficient to drive durable anti-tumor responses in challenging tumor mouse models when using a high-affinity WT1-directed TCR. To address this, several CD8 co-stimulatory fusion constructs containing CD28 or 41BB intracellular domains were designed and screened, identifying two CD8-41BB based chimeras that substantially increased TCR-T cell activity relative to wild-type CD8αβ. WT1-TCR-T cells co-expressing the CD8-41BB fusions demonstrated not only enhanced CD4+ activity including strong and polarized Th1-type cytokine secretion, but also enhanced the proliferation, cytokine release, and cytotoxicity of CD8+ CTLs. Remarkably, when combined with TGFBR2 gene disruption, WT1-TCR-T cells co-expressing CD8-41BB receptors were able to completely regress established cell line-derived ovarian tumors, showed robust in vivo expansion and persistence, and provided long-term protection from tumor rechallenge. Importantly, the specificity profile of the WT1-TCR including its HLA-A*02:01 restriction and WT1 peptide recognition motif was preserved upon expression of CD8-41BB. To simplify cell engineering processes for clinical applications, we configured a homology directed repair (HDR) cassette to allow for efficient CRISPR-Cas9-based insertion of both the TCR and CD8-41BB transgenes in the TRAC locus in a single step with >80% efficiency. Lastly, the enhanced activity conferred by CD8-41BB expression was validated with a second clinically relevant TCR targeting PRAME, suggesting this platform can be a universal approach for enhancing the therapeutic potential of TCR-based cell therapies.

BACKGROUND AIMS:Autologous T-cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily as the result of their individualized manufacturing requirements. METHODS:To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select human leukocyte antigens (HLA) class I and II alleles (HLA-A, HLA-B and the class II transactivator [CIITA]) while retaining HLA-C to protect from natural killer (NK) cell rejection. RESULTS AND CONCLUSION:Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus template allowed for a high localized insertion rate while simultaneously removing the endogenous TCR and preventing graft-versus-host disease. Using an optimized T-cell engineering process involving orthogonal CRISPR/Cas9 cleavage and base editors coupled with lipid nanoparticle delivery, we achieved efficient production of Allo-CAR T cells with high editing rates and cell expansion in a scalable manner. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene-editing approach significantly minimized the occurrence of chromosomal aberrations. This promising allogeneic approach also has been applied to induced pluripotent stem cells (iPSCs) with triple edits targeting HLA-A, HLA-B and CIITA (TKO). Pancreatic progenitor cells or cardiomyocytes derived from TKO iPSCs were protected from host peripheral blood mononuclear cell-mediated rejection when matched for HLA-C, suggesting potential applications in regenerative medicine applications.

Autologous T cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily due to their individualized manufacturing requirements. To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select HLA Class I and II alleles ( HLA-A , HLA-B , and the class II transactivator ( CIITA )), while retaining HLA-C to protect from NK cell rejection. Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus (AAV) template allowed for high localized insertion rate while simultaneously removing the endogenous TCR and preventing GvHD. Using a lipid nanoparticle (LNP)-based delivery system of the editing components enabled a robust cell engineering process, achieving high editing rates and cell expansion. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene editing approach generated cells with minimal chromosomal aberrations. The Allo strategy has also been applied to induced pluripotent stem cells (iPSCs), suggesting potential applications in regenerative medicine applications.

Background The success of T cell therapies for the treatment of solid tumors has been limited. Factors limiting efficacy in solid tumors are poor infiltration, exhaustion, and an immunosuppressive tumor microenvironment (TME). Methods To address these issues, we developed multiple mouse syngeneic tumor models to conduct in vivo CRISPR screening to identify Immune Enhancing Edits (IEEs) that augment CD8+ T cell function across TMEs. Several IEEs were validated in multiple mouse syngeneic solid tumor models, displaying significant in vivo tumor control. To better understand the therapeutic potential in human T cells, we engineered Wilms Tumor 1 (WT1)-specific TCR-T cells with these IEEs and tested them against WT1-expressing human solid tumor xenografts. Results WT1-specific TCR-T cells with either single IEE or a combination of IEEs induced tumor regression across multiple human tumor models. These IEE targets were also able to enhance CAR-based T cell therapies against solid tumors, adding to the broad applicability of this platform. Conclusions Coupling CRISPR-engineered immune enhancements with our allogeneic platform, Intellia is creating next-generation cell therapies for the treatment of solid tumors. Ethics Approval The mouse studies described here have been performed in compliance with protocols approved by Intellia Therapeutics' IACUC (Institutional Animal Care and Use Committee). (Mainly protocol number IT008)

T cell receptor (TCR)-based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01-restricted TCRs, three that were specific to the less explored immunodominant WT137-45 and two that were specific to the noncanonical WT1-78-64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR β constant (TRBC) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137-45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.

Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of “off-the-shelf” T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a “missing-self” signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed.

Adoptive cell therapy using T cells expressing transgenic (tg) tumor antigen-targeting T cell receptors (TCRs) has become an attractive modality to treat hematological and solid cancers due to a broader array of accessible targets relative to CAR-T cell therapies. However, high-avidity TCRs specific for shared oncogenic antigens are difficult to identify. In addition, manufacturing of TCR-redirected T cells with single TCR specificity is desired to avoid mispairings and competition with endogenous chains, which can negatively impact T cell specificity and TCR expression levels. This can be achieved with CRISPR/Cas9-mediated replacement of the endogenous TCR α and β chains, by knocking out the TRAC and TRBC genes and inserting the tgTCR into the TRAC locus. While CRISPR/Cas9 genome editing has been demonstrated to be highly efficient, simultaneous edits in different loci could result in increased translocations, potentially impairing the quality and safety of the cell product. Moreover, existing cell engineering technology negatively impacts T cell quality and yield. Here, we focused on engineering T cells with specificity for Wilms' Tumor 1 (WT1), a transcription factor overexpressed by a wide range of hematological and solid tumors, that has both, restricted expression on healthy tissues and a strong correlation with oncogenesis. By applying rapid isolation technologies of WT1-specific T cells from healthy donors, we identified a lead TCR to the WT137-45 epitope, restricted to the common human leukocyte antigen, HLA-A*02:01. T cells expressing this tgTCR showed nM avidity and killed leukemia cell lines and primary acute myeloid leukemia (AML) blasts at low effector-to-target cell ratios. Epitope specificity evaluation by alanine scanning suggested that the minimal peptide recognition sequence for this TCR is restricted to WT1. Further, the lead TCR was able to activate CD8+ and CD4+ T cells, which may be beneficial for T cell persistence. By developing an improved T cell engineering process, we have achieved multiple sequential gene edits in primary human T cells, leading to knockout of the endogenous TCR with up to 99% efficiency and insertion of tgTCRs into 55-80% of the cells. This cell engineering process is scalable, adaptable to a closed system, and results in marked improvements in T cell expansion, yield, stem cell memory phenotype and T cell polyfunctionality, such as cytotoxicity, cytokine release and proliferation in response to WT1+ target cells. Additionally, the high viability profile of the process readily allows for sequential CRISPR/Cas9 gene knockout in T cells, leading to near-complete endogenous TCR removal while limiting TRAC/TRBC translocation to levels close to those found in untreated cells. T cells engineered to express the lead TCR using this process resulted in potent anti-tumor activity in vivo. Disseminated primary AML patient derived xenograft and acute lymphoblastic leukemia (ALL) cell line models were established by intravenous injection of the tumor cells in NSG or NOG mice. Animals were treated subsequently with WT1-specific or control T cells. Almost complete tumor growth inhibition in the blood and bone marrow was noted in the primary AML model. In the fast growing ALL model, WT1-T cells significantly reduced tumor burden and increased survival compared to control groups, which could be further boosted in human IL-15-expressing NOG mice vs. standard NOG mice. No signs of graph-versus-host disease (GvHD) were observed during the course of the study, which is consistent with removal of the endogenous TCR. NTLA-5001 is being advanced into clinical development for AML immunotherapy. Given the expression of WT1 in many solid tumors, engineered WT1 TCR-T cells are being further explored in those indications. Disclosures Liu: Intellia Therapeutics: Current Employment. Prodeus:Intellia Therapeutics: Current Employment. Becker:Intellia Therapeutics: Current Employment. Foisey:Intellia Therapeutics: Current Employment. Balwani:Intellia Therapeutics: Current Employment. Dutta:Intellia Therapeutics: Current Employment. Zhang:Intellia Therapeutics: Current Employment. Arredouani:Intellia Therapeutics: Current Employment. McKee:Intellia Therapeutics: Current Employment. Ciceri:Intellia Therapeutics: Membership on an entity's Board of Directors or advisory committees. Sepp-Lorenzino:Intellia Therapeutics: Current Employment. Bonini:Kiadis: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Molmed: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Intellia Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Schultes:Intellia Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Sorafenib and sunitinib are multiple tyrosine kinase inhibitors. Both of them have been approved by the US FDA in the treatment of patients with malignancies. In order to develop an effective and clinically useful chemoimmunotherapy modality against hepatocellular cancer (HCC), we investigate their tumoricidal and immune modulatory effect in the setting of HCC. In vitro experiments suggested that sunitinib and sorafenib both induced HCC cell apoptosis at an equivalent level, but stronger suppressive function to cell proliferation was detected in sorafenib. Correspondingly, treatment of tumor-bearing mice with sorafenib led to the suppression of tumor growth to a larger extent than sunitinib. Flow cytometry showed that treatment with sunitinib, not sorafenib, significantly reduced the frequency of regulatory T cells (Tregs) and myeloid-derived suppressive cells (MDSCs) in tumor-bearing mice; and allowed splenic lymphocytes to produce equivalent levels of IFN-γ and TNF-α in response to vaccination as that in wild type mice. This activation was not detected in control and sorafenib-treated tumor mice. In addition, treatment of tumor-bearing mice with sunitinib followed by adoptive transfer of tumor antigen-specific CD8+ T cells and immunization resulted in the additional suppression to tumor growth compared to sunitinib monotherapy. These results imply treatment with sunitinib, not sorafenib, is able to prevent tumor-induced immunotolerance and activate antitumorimmunity. Our data suggest that sunitinib may be a preferable chemotherapeutic agent to use in combination with immunotherapy for the treatment of HCC.

BACKGROUND & AIMS: Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. METHODS: Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachloride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8(+) T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8(+) T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. RESULTS: Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8(+) T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8(+) T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. CONCLUSIONS: In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8(+) T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

Successful development of immunotherapeutic strategies for hepatocellular cancer (HCC) has been impeded by limited understanding of tumor-induced profound tolerance and lack of a clinically faithful HCC model. Recently, we developed a novel model that recapitulates typical features of human HCC. Using this clinically relevant model, we demonstrate that tumor growth impairs host immunity and causes a profound exhaustion of tumor antigen-specific (TAS) CD8+ T cells. Increase in frequency and suppressive function of regulatory T cells (Tregs) is critically involved in this tumor-induced immune dysfunction. We further demonstrate that sunitinib suppresses Tregs and prevents tumor-induced immune tolerance, allowing TAS immunization to activate endogenous CD8+ T cells. As a result, this combinational strategy delays tumor growth. Importantly, the additional integration of exogenous naïve TAS CD8+ T cells by adoptive cell transfer (ACT) leads to the elimination of the established tumors without recurrence and promotes long-term survival of the treated mice. Mechanistically, sunitinib treatment primes the antitumor immune response by significantly decreasing Treg frequency, reducing TGF-β and IL-10 production by Tregs, and also protecting TAS CD8+ T cells from tumor-induced deletion in the setting of HCC. Taken together, sunitinib quantitatively and qualitatively modifies Tregs to overcome tumor-induced immune deficiency, suggesting the potential of sunitinib as a therapeutic immune activator for HCC control.

Antibody therapy targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA4) elicited survival benefits in cancer patients; however, the overall response rate is limited. In addition, anti-CTLA4 antibody therapy induces a high rate of immune-related adverse events. The underlying factors that may influence anti-CTLA4 antibody therapy are not well defined. We report the impact of a cancer-derived immune modulator, the human-soluble natural killer group 2D (NKG2D) ligand sMIC (solublemajor histocompatibility complex I chain-relatedmolecule), on the therapeutic outcome of anti-CTLA4 antibody using anMIC transgenic spontaneousTRAMP(transgenic adenocarcinomaof the mouse prostate)/ MIC tumor model. Unexpectedly, animals with elevated serum sMIC (sMIC(hi)) responded poorly to anti-CTLA4 antibody therapy, with significantly shortened survival due to increased lung metastasis. These sMIChi animals also developed colitis in response to anti-CTLA4 antibody therapy. Coadministration of an sMIC-neutralizing monoclonal antibody with the anti-CTLA4 antibody alleviated treatment-induced colitis in sMIChi animals and generated a cooperative antitumor therapeutic effect by synergistically augmenting innate and adoptive antitumor immune responses. Our findings imply that a new combination therapy could improve the clinical response to anti-CTLA4 antibody therapy. Our findings also suggest that prescreening cancer patients for serum sMIC may help in selecting candidates who will elicit a better response to anti-CTLA4 antibody therapy.

Manipulation of immune system toward the rejection of established cancers has become the standard of care in some patients. Here we propose the development of an in situ autologous cancer vaccine, inCVAX, for the treatment of hepatocellular cancer (HCC). inCVAX is based on the induction of local immunogenic cancer cell death combined with local dendritic cell stimulation by intratumoral injection of the immune-activator N-dihydro-galacto-chitosan (GC). In a first set of experiments, cellular and molecular studies were performed to investigate the effect of inCVAX on immune activation in a murine model of HCC that we previously developed. Once large tumors were formed in mice, the tumor is surgically exposed and a laser fiber was inserted into the center of an individual tumor mass. Using a 10 mm diffuser tip, laser irradiation of 1.5 W was applied to heat the tumor at different durations (6-10 min) to assess tolerability of photothermal application at different temperatures. The laser application was followed by immediate injection of GC, and each mouse received one laser treatment and one GC injection. ELISA was used to assess the level of cytokines; immunohistochemical staining was conducted to analyze the effect of inCVAX on immune cell tumor-filtration and expression of tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). Results indicate that survival correlated to thermal exposure. At lower temperatures the photothermal effect was sufficient to induce tumor necrosis, but without obvious complication to the mice, although at these temperatures the treatment didn't alter the level of TSAs and TAAs, so further optimization is suggested. Nevertheless, in response to the inCVAX treatment, cytotoxic cytokine IFN-γ was significantly increased, but suppressive cytokine TGF-β was dramatically reduced. Furthermore, inCVAX prompted tumor infiltration of CD3+, CD4+, and CD8+ T cells; but modulated macrophage subsets differently. In conclusion, while the protocol needs further optimization, it would appear that inCVAX for the treatment of HCC activates an immune response in tumor-bearing mice, which in turn may have potential for the treatment of HCC.

Abstract Radiofrequency Ablation (RFA) is one of current clinical option in the treatment of hepatocellular cancer (HCC); however, RFA monotherapy is often not curative as a consequence of tumor micro-metastases and recurrence. Thus, induction of systemic response with additive treatment is required. In the present study, we are exploring the combination of RFA and sunitinib, an FDA-approved multiple tyrosine kinase inhibitor, in the treatment of HCC. Using our established strategy a clinically relevant murine model of HCC is prepared which mimics human disease and reflect its typical features. The follow-up magnetic resonance imaging (MRI) is applied to monitor tumor progression until the cumulative tumor volume reaches 2000 mm3. Size-matched tumor-bearing mice are assigned into 3 groups and receive the following treatments: no treatment, RFA alone with our improved machine and optimized parameters, combination of sunitinib and RFA. The therapeutic efficacy is evaluated by measuring tumor volume with MRI every twice weeks. We demonstrate that tumors grow substantially in the mice without treatment. RFA alone effectively destroys tumors, but unable to prevent tumor recurrence and micro-metastasis. In contrast, > 50% size reduction in tumor size is detected in the combined group compared to that in control mice, even no detectable tumors are observed in some treated mice. In conclusion, combination of sunitinib and RFA powerfully suppress tumor growth and could be translate into clinic practice. Citation Format: Xiaoqiang Qi, Guangfu Li, Dai Liu, Kevin F Staveley-O’Carroll, Eric Kimchi. Combination of radiofrequency ablation and Sunitinib in the treatment of experimental hepatocellular cancer. [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 5188.

Hepatocellular carcinoma (HCC) is the third most deadly cancer in the world. New treatment strategies are desperately needed due to limited standard therapies. Activation of the Erk, Akt, and STAT3pathways is implicated in the prognosis of HCC. The Se,Se′-1,4-phenylenebis(1,2-ethanediyl) bisisoselenourea (PBISe), is a selenium-containing MAPK and PI3 kinase inhibitor, effectively inhibit tumorigenesis in a variety of experimental models. The aim of our study is to demonstrate the potential role of PBISe in the treatment of HCC. The anti-proliferative and pro-apoptotic ability of PBISe is studied in vitro in four human HCC cell lines and in vivo in a spontaneous murine HCC model. Inhibition of cancer growth was performed by cell viability assay and apoptosis by caspase 3/7, PARP cleavage, annexin-V, and TUNEL assays. Role of PBISe on PI3 kinase, MAPK and STAT3 signaling is determined by Western blotting. In vivo effects of PBISe on tumor sizes were monitored using MRI in a spontaneous murine HCC. Liver tissues from the PBISe-treated mice are analyzed for angiogenesis, proliferation, and signaling pathway markers. Overall, PBISe activated caspase-3/7 and increased DNA fragmentation, which is positively correlated with the increased PARP cleavage. PBISe promoted apoptosis by inhibiting PI3K, MAPK, and STAT3 signaling with significant reduction in the tumor sizes (p < 0.007). PBISe-treated tumors reduced survival marker PCNA, and angiogenesis markers Vegf-A, Vegf-R3 and CD34. These results demonstrate the chemotherapeutic effects of PBISe, by inhibiting tumor growth and facilitating tumor apoptosis for HCC treatment.

Background & Aims: We have established a clinically relevant animal model of hepatocellular cancer (HCC) in immune competent mice to elucidate the complex dialog between host immunity and tumors during HCC initiation and progression. Mechanistic findings have been leveraged to develop a clinically feasible anti-tumor chemoimmunotherapeutic strategy.Methods: Intraperitoneal injection of carbon tetrachloride and intrasplenic inoculation of oncogenic hepatocytes were combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. Immunization and adoptive cell transfer (ACT) were used to dissect the tumor antigen-specific immune response. The ability of the tyrosine kinase inhibitor sunitinib to enhance immunotherapy in the setting of HCC was evaluated.Results: This new mouse model mimics human HCC and reflects its typical features. Tumor-antigen-specific CD8(+) T cells maintained a naive phenotype and remained responsive during early-stage tumor progression. Late tumor progression produced circulating tumor cells, tumor migration into draining lymph nodes, and profound exhaustion of tumor-antigen-specific CD8(+) T cells associated with accumulation of programmed cell death protein 1 (PD-1)(hi) CD8(+) T cells and regulatory T cells (Tregs). Sunitinib-mediated tumoricidal effect and Treg suppression synergized with antibody-mediated blockade of PD-1 to powerfully suppress tumor growth and activate anti-tumor immunity.Conclusion: Treg accumulation and upregulation of PD-1 provide two independent mechanisms to induce profound immune tolerance in HCC. Chemoimmunotherapy using Food and Drug Administration-approved sunitinib with anti-PD-1 antibodies achieved significant tumor control, supporting translation of this approach for the treatment of HCC patients.Lay summary: In the current study, we have established a clinically relevant mouse model which mimics human liver cancer. Using this unique model, we studied the response of the immune system to this aggressive cancer. Findings from this trial have led to the development of an innovative and clinically feasible chemoimmunotherapeutic strategy. 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Abstract We have established a clinically relevant murine model to reveal tumor-induced immunotoerance in hepatocellular carcinoma (HCC). Critical factors are targeted to develop immune-based therapies for HCC control. Intrasplenic (ISPL) inoculation of oncogenic hepatocytes and intraperitoneal (IP) injection of carbon tetrachloride (CCl4) are combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. We characterize the features of this model, examine tumor-antigen-specific (TAS) immunity during tumor initiation and progression, and identify the critical factors in tumor-induced immune tolerance. The established murine model recapitulates human HCC and reflects its typical features. TAS CD8+ T cells initially maintain a naive phenotype and function in early-stage tumor-bearing mice, then become profound exhaustion with the tumor progression to the advanced stage. The deep immunosuppression is associated with the significant upregulation of Programmed cell death protein 1(PD-1), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the increase of Tregs. While the changes in Tregs and PD-1 are systemic, they are particularly pronounced in the tumor microenvironment. Sunitinib-mediated reduction of Treg together with antibody-mediated blockade of PD-1 synergistically suppress tumor growth and activate anti-tumor immunity. Our data provide evidence that oncogenic hepatocytes escape immune surveillance during tumor initiation via immune ignorance, while later-stage established HCCs evade immune destruction via tumor-induced immunotolerance. Synergy of sunitinib and anti-PD-1 Ab generates favorable effect on suppressing tumor growth and activating anti-tumor immunity. This combinational strategy has high translational potential in HCC treatment. Citation Format: Dai Liu, Guangfu Li, Timothy Cooper, Eric Kimchi, Xiaoqiang Qi, Ningfei Li, Don Rockey, Todd Schell, Kevin Staveley-O’Carroll. Development of a successful combination therapy for hepatocellular cancer by targeting Treg and PD-1. [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 4004.

Abstract Purpose: The human tumor-derived soluble MHC I-chain–related molecule (sMIC) is highly immune suppressive in cancer patients and correlates with poor prognosis. However, the therapeutic effect of targeting sMIC has not been determined, due to the limitation that mice do not express homologs of human MIC. This study is to evaluate the therapeutic effect of a monoclonal antibody (mAb) targeting sMIC in a clinically relevant transgenic animal model. Experimental Design: We treated the engineered MIC-expressing “humanized” TRAMP/MIC bitransgenic mice at advanced disease stages with a sMIC-neutralizing nonblocking anti-MIC mAb and assessed the therapeutic efficacy and associated mechanisms. Results: A sMIC-neutralizing nonblocking anti-MIC mAb effectively induced regression of primary tumors and eliminated metastasis without inducing systemic toxicity. The therapeutic effect is conferred by revamping endogenous antitumor immune responses, exemplified by restoring natural killer (NK) cell homeostasis and function, enhancing susceptibility of MIC+-tumor cells to NK cell killing, reviving and sustaining antigen-specific CD8 T-cell responses, augmenting CD4 T cells to Th1 responses, priming dendritic cells for antigen presentation, and remodeling tumor microenvironment to be more immune reactive. Conclusions: Therapy with a sMIC-neutralizing nonblocking anti-MIC mAb can effectuate antitumor immune responses against advanced MIC+ tumors. Our study provided strong rationale for translating sMIC-neutralizing therapeutic mAb into clinics, either alone or in combination with current ongoing standard immunotherapies. Clin Cancer Res; 21(21); 4819–30. ©2015 AACR.

RFA is used in treatment of patients with hepatocellular cancer (HCC); however, tumor location and size often limit therapeutic efficacy. The absence of a realistic animal model and a radiofrequency ablation (RFA) suitable for small animals presents significant obstacles in developing new strategies. To establish a realistic RFA platform that allows the development of effective RFA-integrated treatment in an orthotopic murine model of HCC, a human cardiac radiofrequency generator was modified for murine use. Parameters were optimized and RFA was then performed in normal murine livers and HCCs. The effects of RFA were monitored by measuring the ablation zone and transaminases. The survival of tumor-bearing mice with and without RFA was monitored, ablated normal liver and HCCs were evaluated macroscopically and histologically. We demonstrated that tissue-mimicking media was able to optimize RFA parameters. Utilizing this information we performed RFA in normal and HCC-bearing mice. RFA was applied to hepatic parenchyma and completely destroyed small tumors and part of large tumors. Localized healing of the ablation and normalization of transaminases occurred within 7 days post RFA. RFA treatment extended the survival of small tumor-bearing mice. They survived at least 5 months longer than the controls; however, mice with larger tumors only had a slight therapeutic effect after RFA. Collectively, we performed RFA in murine HCCs and observed a significant therapeutic effect in small tumor-bearing mice. The quick recovery of tumor-bearing mice receiving RFA mimics observations in human subjects. This platform provides us a unique opportunity to study RFA in HCC treatment.