CT26 Tumor Model Research Articles

Palliative Radiofrequency Ablation Accelerates the Residual Tumor Progression Through Increasing Tumor-Infiltrating MDSCs and Reducing T-Cell-Mediated Anti-Tumor Immune Responses in Animal Model.

Previous studies showed that radiofrequency ablation (RFA) has a favorable treatment efficacy for hepatocellular carcinoma (HCC) or colorectal liver metastases (CRLMs). Palliative RFA (pRFA) resulting from larger HCC or multiple CRLMs further accelerated the progression of potential residual tumor, yet its mechanism was still unknown. This study investigated the influence of myeloid-derived suppressor cells (MDSCs) on T-cell immune responses and tumor recurrence after pRFA. CT26 tumor models were used. The percentage of MDSCs in peripheral blood was analyzed by flow cytometry after pRFA. The level of Th1 and Th2 cytokines were measured by ELISA through different treatments (n = 4/group). The tumor-infiltrating MDSCs, dendritic cells, and intracellular cytokines level were analyzed by IHC staining after different treatments. The functional CD8+ T cells were confirmed by the co-localization immunofluorescence staining. The long-term outcomes were also evaluated through CT26 and 4T1 tumor models. The results showed that tumor models treated with pRFA displayed significant increases in the percentage of MDSCs of peripheral blood and tumor infiltration. The expression level of TGF-β and IL-6 after pRFA was higher than that before pRFA by ELISA and IHC staining. After depleting MDSCs by combining with Abs, the pRFA + Abs group achieved a higher level of Th1 cytokines and greatly enhanced the percentage of tumor-infiltrating functional CD8+ T cells when compared with pRFA alone. The depletion of MDSCs through combination with Abs also resulted in tumor regression. In conclusion, pRFA accelerates the residual tumor progression through increasing tumor-infiltrating MDSCs and reducing T-cell-mediated anti-tumor immune responses, which could provide a potential approach for delaying tumor recurrence caused by pRFA.

Abstract 6679: Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment

Abstract Model selection for evaluation of immuno-oncology agents is often aided by both response to therapy and changes in immune cell subsets following treatment. To identify underlying factors that influence differences in therapeutic response, CT26 colorectal and 4T1-Luc (4T1) breast tumor models, which are immunologically warm and cold, respectively, were treated with anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4). Flow cytometry and transcriptome analysis were used to describe treatment-related changes in immune cell infiltrate, TIL activation status, cytokine/granzyme B production, and overall changes in gene expression. Response to anti-CTLA-4 therapy was distinct between these models. Tumor growth inhibition was observed in CT26 (74%), but not 4T1. In CT26, CD8+ and NK T cell infiltration increased, while M2 macrophages decreased. In 4T1 tumors, only increased NKT cell infiltration was noted after treatment, and no demonstrable changes were observed in any other cell type or activation marker. Several pharmacodynamic changes in immune cell activation were observed in CT26. Notably, CT26-derived CD4+, CD8+, and NK T cell subsets had increased IFNγ production (70%, 25%, and 17%, respectively, versus control). An increase in polyfunctional T cells producing both IFNγ and TNFα was also noted for CT26 and was correlated with slower tumor growth. Granzyme B was reduced and CD107a was increased in CT26-derived NK T cells, suggesting functionally active T cells. This was also supported by upregulation of genes in the cytotoxicity, antigen presentation, and NFkappaB pathways. Following CT26 treatment with anti-CTLA-4, transcriptome analysis with the nCounter® Mouse PanCancer IO 360 Panel (NanoString) showed an 8-10 fold induction of several T cell-related genes as well as an 8-14 fold induction of several chemokines and their receptors. These data complement the flow cytometry data and support stimulation of T cell activation and recruitment to CT26 tumors following anti-CTLA-4 treatment. In contrast, analysis of 4T1 tumors indicated only a 2 to 3-fold induction of CCL4, CCL11, and Granzyme A after treatment. Interestingly, genes in the matrix remodeling pathway were upregulated in 4T1 tumors upon anti-CTLA-4 treatment. In summary, CT26 tumors were responsive to anti-CTLA-4 treatment, showing increased polyfunctional T cell recruitment, NK T cell activity, and reduced M2 macrophage infiltration, as well as marked upregulation of several genes involved in T cell recruitment and activation in tumor. In contrast, 4T1 tumors did not display any remarkable response to therapy, changes to TIL infiltrates, and only limited changes to gene expression with treatment. Collectively, these results suggest tumor profiles following treatment can provide insight into potential targets of interest and are important for preclinical model selection. Citation Format: Sheri R. Barnes, David Draper, Sarah E. Church, Maryland Rosenfeld Franklin. Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6679.

Abstract 4572: mONCR-177 oncolytic virotherapy stimulates anti-tumor and anti-viral immunogenicity

Abstract Oncolytic viruses represent a promising class of therapeutic agents for the treatment of cancers that poorly respond to immune checkpoint inhibitors. Oncorus is developing ONCR-177, an oncolytic herpes simplex virus engineered for potent oncolysis and stimulation of antitumor immunity. ONCR-177 is replication competent in tumor cells, but its replication and neuropathic activities are attenuated in healthy cells using microRNA-dependent degradation of viral transcripts and by mutations in UL37 that prevent retrograde transport in neurons. In addition, ONCR-177 encodes five immune-stimulatory transgenes: IL-12, CCL4, FLT3LG, a PD-1 humanized antagonist VHH nanobody, and a CTLA4 blocking monoclonal antibody. Transgenes were chosen to efficiently recruit effector T (CD8 and CD4 Th1), NK, and classical dendritic cells to the tumor, and to counter upregulation of immune checkpoints. The individual and collective mechanisms of action of the transgenes are designed to augment local and distant innate and adaptive anti-tumor immune responses. Tumor-specific response was evaluated using mONCR-177, a mouse functional surrogate for ONCR-177, that is built on the same base vector ONCR-159 but expresses the immune transgenes suitable for murine studies. In a CT26 bilateral colon carcinoma tumor model and a MC38 colon adenocarcinoma model, immune responses elicited by intratumoral injection of mONCR-177 and ONCR-159 were compared. Intratumoral T cell responses were evaluated for a tumor-associated antigen (TAA) and an HSV specific antigen in the MC38 model. In the CT26 tumor model, treatment with mONCR-177 resulted in greater immune infiltration with a higher activation status and an improved CD8:Treg ratio. Treatment with mONCR-177 elicited significantly more tumor antigen specific IFNγ and TNFα producing, intratumoral CD8T cells. The increase in anti-tumor response with mONCR-177 treatment was further evidenced by the increase in polyfunctional CD8 T cells producing IFNγ, TNFα, and/or IL-2 in response to TAA stimulation. In the MC38 tumor model, treatment with mONCR-177 resulted in greater immune infiltration in both the injected and non-injected tumor. The CD8:Treg ratio was improved and PD-1 expression was decreased. Tumor antigen specific cytokine production was higher with mONCR-177 treatment. Additionally, a significant increase in HSV-specific polyfunctional CD8+ T cells was observed. mONCR-177 effectively stimulates an intratumoral immune response to both tumor and viral antigens. Treatment results in increased infiltrating immune cells, activation, and polyfunctional CD8+ T cells. These results highlight the dual modality mechanism of action of mONCR-177 treatment through direct tumor cell killing and immune stimulation through the antiviral response, and their enhancement by the immune stimulatory transgenes. Citation Format: Melissa Hayes, Agnieszka Denslow, Jacqueline Hewett, Daniel Wambua, Lingxin Kong, Jacob Spinale, Peter Grzesik, Jennifer Lee, Terry Farkaly, Edward M. Kennedy, Lorena Lerner, Christophe Queva, Brian Haines, Sonia Feau. mONCR-177 oncolytic virotherapy stimulates anti-tumor and anti-viral immunogenicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4572.

Abstract LB-396: Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of CXCL9 synergizes with IL-12 gene therapy (TAVO) to elicit robust anti-tumor immunity

Abstract Clinical and preclinical studies have demonstrated that plasmid IL-12 (tavokinogene telseplasmid; TAVO) delivered intratumorally via electroporation drives IFN-γ expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our IL-12/anti-PD-1 clinical trial has identified that clinical responses are closely tied to intratumoral CXCR3 levels. While all patients had a similar frequency of activated CD8+ T cells in the periphery, responding patients had a significant increase of intratumoral CXCR3 transcripts post-treatment (p=0.03) compared to nonresponding patients (p=0.4), underscoring the clinical relevance of tumor-infiltrating CXCR3+ immune cells. Since the IFN-γ/CXCL9/CXCR3 axis is known to increase sensitivity to anti-PD-1 therapies, we hypothesized that combining intratumoral TAVO with a DNA-encoded locally secreted CXCL9 (cognate ligand for CXCR3) would further augment/restore this axis and ‘license' a robust anti-PD-1 response beyond the treated lesion. An appropriate CXCL9 gradient can productively modulate frequencies of tumor infiltrating tumor-reactive CXCR3+ T cells. We have previously demonstrated that intratumoral electroporation of plasmid IL-12 and CXCL9 elicits a robust antitumor immune response evidenced by increased systemic antigen-specific CD8+ T cells and improved regression of both treated and contralateral CT26 tumors. In the current study, we demonstrate that an enhanced CXCL9 gradient via intratumoral electroporation leads to efficient trafficking of CXCR3+ CD8+ T cells into CT26 tumors. We further explored how DNA-encodable IL-12/CXCL9 can work together to improve checkpoint inhibitor response. We demonstrated that intratumoral TAVO rapidly drives a population of CXCR3+ CD8+ T cells in the lymph node and importantly, depletion of these CXCR3+ immune cells abrogated an IL-12-mediated anti-tumor response. Furthermore, upon electroporation of IL-12 and CXCL9, transcriptomic analysis of the tumor microenvironment revealed an enrichment of genes associated with immune-related pathways (IFN-γ signaling, interleukin signaling, GPCR signaling), antigen presentation machinery, and TCR signaling, indicating that this combination therapy augments anti-tumor immunity. Lastly, leveraging the partially responsive anti-PD-1 CT26 tumor model, we demonstrated that intratumoral electroporation of plasmid IL-12 with CXCL9 significantly improved anti-PD1 response, providing a strong rationale for filing an Investigational New Drug application based on this intratumoral DNA-encodable combination approach.Intratumoral CXCL9 with IL12 Citation Format: Anandaroop Mukhopadhyay, Bianca Nguyen, Jack Y. Lee, Mia Han, Jon Salazar, Reneta Hermiz, Lauren Svenson, Erica Browning, Daniel J. O'Connor, David A. Canton, Daniel Fisher, Joseph Skitzki, Christopher G. Twitty. Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of CXCL9 synergizes with IL-12 gene therapy (TAVO) to elicit robust anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-396.

Abstract 4548: SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity

Abstract CD112R (PVRIG) is a recently identified inhibitory receptor of the PVR gene family, which includes TIGIT, CD96, and CD226, that is expressed on NK cells and T cells. CD112R suppresses immune cell activation through its association with the cell adhesion molecule CD112 (PVRL2), a ligand that it competes for with the activating receptor CD226. CD112 binding to CD112R induces downstream signaling via recruitment of SHP-1, SHP-2, and SHIP-1 phosphatases to the ITIM in the cytoplasmic tail, resulting in dampened effector cell activation. As CD112 is commonly upregulated on cancer cells and is also expressed on tumor infiltrating myeloid cells, it was hypothesized that disruption of the CD112:CD112R interaction would promote anti-tumor immune responses. Phenotypic screening of a panel of antibodies generated against CD112R led to the identification of SRF813, a high affinity, fully human CD112R antibody that blocks the interaction of CD112R with CD112. To explore the therapeutic potential of CD112R blockade, the ability of SRF813 to increase immune cell activation was evaluated in human peripheral blood mononuclear cells. In vitro assays showed that SRF813 treatment enhanced NK cell activation in response to multiple tumor cell lines. This activation was characterized by upregulation of several cell surface markers, including 4-1BB and ICAM-1, in addition to increased cytokine production and degranulation. The activity of SRF813 was highly dependent on the antibody isotype and Fc receptor engagement. Mechanistically, the enhanced immunologic responses observed in NK cells treated with SRF813 could be reversed in the presence of a CD226 blocking antibody, confirming the role of CD112R in regulating CD226 mediated signaling. Blockade of CD112R in mouse syngeneic tumor models using a murine surrogate of SRF813 demonstrated reduced tumor growth with an accompanying increase in TIL activation following treatment. In the CT-26 tumor model, anti-CD112R treatment resulted in complete tumor regression in a subset of treated mice. These mice rapidly rejected tumors upon re-challenge, signifying the development of an immunologic anti-tumor memory response in these animals. Additional depletion studies highlighted that the anti-tumor efficacy was dependent on both CD8 T cells and NK cells. Moreover, the combination of anti-CD112R with PD-1 blockade led to greater inhibition of tumor growth than either treatment alone. Collectively, these preclinical data demonstrated that CD112R is a negative regulator of immune responses and that CD112R blockade can potentiate anti-tumor responses in cancers that express CD112. Citation Format: Marisella Panduro, Roy M. Dornbrook, Kshama A. Doshi, Jing Hua, Jamie Strand, Vito J. Palombella, Pamela M. Holland, Jonathan A. Hill, James F. Mohan. SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4548.

Abstract 5566: Discovery and functional validation of a rationally selected, orally administered, live biotherapeutic consortium of commensal bacteria for the treatment of solid tumors

Abstract Recent evidence supports the role of gut commensal microbes in mediating patient response to immune checkpoint inhibitor therapies (ICT) and chemotherapy. Fecal transplants from ICT responder or non-responder patients into mice modulate syngeneic tumor growth and response to checkpoint antibody treatment, recapitulating the response patterns seen in the human donors. These and other data support the conclusion that certain gut commensal bacteria can promote a systemic immune response to tumors distant from the gut mucosa. We hypothesize that a well-defined consortium of bacteria, rationally selected based on disease-relevant immunological mechanisms, could provide meaningful clinical benefit upon oral administration and delivery to the intestine of cancer patients. To this end, we have identified candidate live biotherapeutic microbes from a library of commensal bacteria isolated from well-curated healthy human stool samples. Identities of purified bacterial strains were established by 16s and whole genome sequencing. Phenotypic parameters, including C- and N- source utilization and antibiotic resistance were determined. Individual strains were characterized based on function by co-incubating live bacteria with human cells in a panel of validated in vitro assays with endpoints measuring disease-relevant, immune-stimulatory mechanisms for a cancer therapeutic. Bacterial strains that exhibited reproducible immune-stimulatory functions in vitro, such as induction of pro-inflammatory cytokines from dendritic cells or PBMCs, T-cell activation and/or M1 macrophage shift, were selected for in vivo testing by oral administration in CT26 and B16F10 syngeneic mouse tumor models. Results of in vivo testing demonstrated reproducible anti-tumor activities by several bacterial strains, as measured by reduced tumor volume over time compared to vehicle control. Bacteria promoting an anti-tumor response also induced hallmarks of immune activation, such as increased inflammatory cytokines in the tumors and changes in immune cell subsets. Further syngeneic tumor studies are underway to test efficacious bacterial strains in combination with each other as consortia as well as with checkpoint antibodies to ultimately select the best candidate therapeutic consortia for clinical development. Using well-defined robust GMP processes, candidate therapeutic consortia will be prepared as lyophilized formulations confirmed to retain viability and function in the preclinical assays and models, then encapsulated for oral administration and evaluation in human subjects. Citation Format: Linh Nguyen, Peyman Akbari, Jason Hudak, Jun Ma, Kiran Dhami, Karin Ku, Ali Akin, Zuelay Rosario, Jackie Papkoff. Discovery and functional validation of a rationally selected, orally administered, live biotherapeutic consortium of commensal bacteria for the treatment of solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5566.

Abstract 6529: GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models

Abstract Introduction: Immune checkpoint blockades (ICBs) have revolutionized cancer treatment and broadened clinical applicability. However, the majority of patients still fail to respond to standard ICBs. To overcome such unmet needs in a clinical study, we designed GI-101, combining the extracellular domain of CD80 serve as a CTLA-4 blockade and an IL-2 variant that preferentially binds the IL-2 receptor β subunit (IL-2Rβ) together. The harmonizing mechanisms of action are projected to translate into improved clinical benefits for this first-in-class immune checkpoint inhibitor fusion protein, even in non-inflamed “cold” tumors. Methods: Binding affinity of GI101 to IL2Rs, CTLA4, and CD28 was determined by SPR. Immune cell proliferation was analyzed by CFSE assay. In vivo anti-tumor efficacy was tested by single or combination treatment on CT26, MC38 and B16F10 syngeneic tumor models. To elucidate the involvement of GI101 on tumor microenvironment (TME), immune cell population was analyzed by flow cytometry from tumor. Tumor specific T cells (surrogate marker, gp70) were measured by splenocyte proliferation assay and IFN-γ ELISPOT assay. RNA sequencing was performed to elucidate immune mechanism of GI-101. Results: GI101 highly binds to CTLA-4 (Kd, 2.9 nM) which leads to the reinforcement of endogenous CD80 and CD28 interaction resulting in the activation of T cells. Bivalent IL-2 variant of GI101 triggers both CD8+ T and NK cells proliferation in vitro and in vivo without Tregs proliferation. GI101 has no evidence for toxicity associated with IL-2 activity including vascular leakage syndrome and cytokine storm in non-GLP monkey studies whereas isolated mortality was observed in the anti-PD-1 and anti-CTLA4 combination treatment group. GI101 elicits restoration of immune functions in vitro settings using mouse splenocytes co-cultured with different PDL-1 and CTLA-4 expression level tumor cells. A dose-dependent (3 to 12 mg/kg) inhibition of tumor growth was observed in CT26 syngeneic models without toxicity. Immune profiling of tumor samples also revealed that a robust increment of M1 macrophages, CD8+ central memory T cells (Tcm) and Ki-67+ proliferating T cells but not Tregs in TME (p < 0.05). Tumor specific T cells were strongly proliferated when stimulated with CT26 neoantigens (gp70, RSPWFTTLI and MGPLIVLLL) in splenocyte. IFN-γ+ cells were significantly increased in draining lymph nodes from GI101 treated mice. Furthermore, drastic tumor regression was observed in MC38 tumor-bearing mice treated with GI101 and anti-PD-1 combination. Conclusion: GI101 facilitates the dual function of checkpoint blockade and IL2 activity that enhances the proliferation and activation of T and NK cells. This novel target drug is expected to be interpreted as superior clinical efficacy and safety as indicated even in ‘cold tumor' models. GI101 is the promising immune-oncology drug to replace the first-generation ICBs by single or combining with other immunotherapies. Our findings provide a rationale for further clinical investigations. Keywords: CD80, IL-2 variant, GI101, Bispecific fusion protein, immunotherapy Citation Format: Kyoung-Ho Pyo, Young Jun Koh, Chun-Bong Synn, Jae Chan Park, Jae-Hwan Kim, Yeongseon Byeon, Sung Eun Kim, Ji Min Lee, Ha Ni Jo, Wongeun Lee, Do Hee Kim, Sungwon Park, Yoo Jeong Song, Won Jae Lee, Ji Young Kim, Hyung Nam Ji, Sang Su Park, Kyung Wha Lee, Young Gyu Cho, Young Min Oh, Bo Gie Yang, Su Youn Nam, Myoung Ho Jang, Byoung Chul Cho. GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6529.

Recombinant KRAS G12D Protein Vaccines Elicit Significant Anti-Tumor Effects in Mouse CT26 Tumor Models.

Drug development targeting the most frequently mutation G12D of KRAS has great significance. As an attractive immunotherapy, cancer vaccines can overcome binding difficulties of small molecules; however, the weak immunogenicity and production difficulties of reported KRAS mutation vaccines limit their clinical application. To improve antigen-specific immune responses and Anti-Tumor effects on tumors expressing KRAS G12D mutation, we designed recombinant proteins containing KRAS peptide (amino acids 5–21) with G12D (called SP) in two forms: DTT-SP4 and DTSP. DTT-SP4 was constructed by fusing four copies of SP to the C-terminal of the translocation domain of diphtheria toxin (DTT), and DTSP was constructed by grafting SP onto DTT. The two vaccines in combination with aluminum hydroxide (Alum) and cytosine phosphoguanine (CpG) successfully induced conspicuous SP-specific humoral and cellular immune responses, and displayed prominent protective and therapeutic Anti-Tumor effects in mouse CT26 tumor models. Surprisingly, the DTSP-treated group displayed better Anti-Tumor effects in vivo compared with the DTT-SP4-treated and control groups. Moreover, 87.5 and 50% of DTSP-treated mice in the preventive and therapeutic models were tumor free, respectively. Notably, in the DTSP-treated group, the interferon-γ (IFN-γ) expression of T cells in vitro and the T-helper 1 (Th1)–related cytokine expression in tumor tissues indicated that the activated Th1 immune response may be involved in Anti-Tumor activity. Furthermore, DTSP treatment remarkably altered the subpopulation of T cells in splenocytes and tumor-infiltrating lymphocytes. The percentage of effector CD8+ T cells increased, whereas that of immunosuppressive CD4+Foxp3+ T cells remained reduced in the DTSP group. Dramatic tumor-inhibitory effects of DTSP, which is easily prepared, make it a more attractive strategy against KRAS G12D tumors.

FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models.

With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8+ T-cell activation assays in a PD-L1-dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8+ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.

Functionalization of developed bacterial cellulose with magnetite nanoparticles for nanobiotechnology and nanomedicine applications

This paper deals with the preparation of novel magnetic materials made from tetraaza macrocyclic Schiff base bacterial cellulose ligands with magnetite nanoparticles (Fe3O4NPs) through a multi-step procedure for antimicrobial and cytotoxic activities and chemotherapy in cancer treatment. First, the 2,3-dialdehyde bacterial cellulose (DABC) was chemically modified by ethylenediamine (EDA) and benzil (Bzl) in the presence of ferrous ions. Then, the magnetite nanoparticles (Fe3O4NPs) was produced inside the complex [Fe(DABC-EDA-Bzl)Cl2] through a co-precipitation method. In nanobiotechnology, the magnetic [Fe3O4NP-INS-(DABC-EDA-Bzl)] material was showed moderate antimicrobial and cytotoxic activities against different species and cells, respectively. In particular, the magnetic [Fe3O4NP-INS-(DABC-EDA-Bzl)] material have not any cytotoxic activity towards peripheral blood mononucleocyte (PBMC) cells. Anti-tumor studies demonstrated that the magnetic [Fe3O4NP-INS-(DABC-EDA-Bzl)] material effectively inhibits the growth of the CT26 tumor model in BALB/c mice compared with other resulting materials throughout the experimental period and can be effective drug delivery in nanomedicine.

Inhibition of Aurora-A recruited CD8+ T cells infiltration via mediating IL-10 production of cancer cells

Abstract It has been shown that approximately 10% of infiltrated CD8+ T cells could be the tumor-specific intratumoral CD8+ T cells in colorectal cancer. While, inhibition of Aurora-A, a member of serine/threonine Aurora kinase family, by alisertib promoted the percentage of infiltrated intratumoral CD8+ T cells. We therefore assumed that targeting Aurora-A could be a therapeutic strategy for recruitment of intratumoral CD8+ T cells. In CT-26 tumor model, blockade of Aurora-A with alisertib slowed the tumor growth in association with an increased infiltration, activation and expansion of intratumoral CD8+ T cells. We further analyzed the role of Aurora-A in colon cancer associated to colitis using the azoxymethane/dextran sodium sulphate (AOM/DSS) colitis-associated colon cancer model, and found that AURKA deficiency significantly decreased tumor formation, in parallel with an increase in infiltration of intratumoral CD8+ T cells. Gene expression profiling indicated that Il10ra expression was obviously increased in intratumoral CD8+ T cells sorted from alisertib-treated tumors. Antibody-mediated blockade of IL-10Ra dramatically attenuated the anti-tumor ability of alisertib in association with reducing the percentage of intratumoral CD8+ T cells. IL-10Ra was the key mediator of IL-10. We herein examined the levels of IL-10 and found that the levels of IL-10 in the serum of AURKA deficiency mice treated with AOM/DSS were elevated. Additionally, IL-10 obviously promoted migration of CD8+ T cells in vitro. Blockade of Aurora-A increased the transcriptional levels of IL-10. In conclusion, inhibition of Aurora-A could be useful for recruiting infiltration of intratumoral CD8+ T cell by mediating IL-10 production.

Whole-body immunoPET imaging to monitor immune activation status in situ

Abstract Advances in immune modulation for cancer and inflammation warrant parallel development of imaging technologies to gauge in situ immune activity. We have developed a toolbox of full-length monoclonal antibody (mAb) positron emission tomography (immunoPET) tracers by desferrioxamine conjugation and 89Zr radiolabeling. Tracer uptake is reported as % injected dose/gram (%ID/g) tissue. Because mAbs to cell surface molecules often facilitate depletion, we tested enzymatic removal of the Fc glycan of CD8 mAb 2.43 to disrupt Fc:FcR interaction and prevent depletion. In the CT26 tumor model, CD8 immunoPET showed similar uptake between WT and deglycosylated tracers. However, unlike WT 2.43, the deglycosylated tracer did not deplete peripheral CD8 T cells, supporting this approach. In addition to surface markers, tracers that define cell function may have great utility. Checkpoint blockade therapy (anti-PD-1/anti-CTLA-4) in CT26-bearing mice shows IFNg immunoPET tracer uptake in 5/5 treated (16.5 ± 3.6 %ID/g) and 2/5 untreated (12.1 ± 4.8 %ID/g) tumors, possibly indicating baseline CT26 immunogenicity. To evaluate antigen presenting cell activation, an IL-12-specific tracer was also developed. Mouse mammary tumor TUBO-bearing mice showed enhanced tracer uptake after intratumoral injection of adenovirus encoding GM-CSF (2.6 ± 0.7 %ID/g) versus controls (0.4 ± 0.4 %ID/g). IL-12 PET was also tested for applications outside of cancer by mimicking inflammation with i.m. LPS injection. LPS-treated mice showed localized, elevated uptake at the site of injection (3.7 ± 0.7 %ID/g) versus controls (0.7 ± 0.1 %ID/g). These results support the use of immunoPET imaging for non-invasive monitoring of immune activity in situ. NIH R37 CA220482.

Abstract B85: Anticancer efficacy of a novel immune-stimulating oncolytic virus: VG161 in gastrointestinal cancers

Abstract Oncolytic viruses (OVs) are the wild-type or genomic modified viruses, which can specifically replicate in cancer cells but not in normal cells. They cause cancer cell lysis and more importantly, their replication in tumor cells induces an antitumor immune response from the host. As synergistic stimulation of antitumor immune response requires multiple immune-regulatory factors, OVs armed with multiple immune-modulating factors are of high potential in cancer immunotherapy. We have constructed a novel herpes simplex virus type-1 virus (HSV-1), VG161, that is armed with IL12, IL15, and a unique PDL-1 peptide blocker. The aim of this study is to investigate the anticancer efficacy of VG161 in gastrointestinal (GI) cancers including gastric, hepatic, and colorectal cancers. The immune-stimulating activities in tumor-bearing immune competent mice were also demonstrated. VG161 was given to the tumor-bearing animals by intratumor injection (5 × 106-5 × 107 PFU/animal, once a day for 3-5 days). Antitumor activity of VG161 was tested in both immune-deficient mice as well as in immune-competent mice. Oncolytic activity of VG161 was demonstrated in human gastric (SGC-7901), hepatic (patient-derived xenograft, PDX), and colorectal (LS174T) cancer-bearing nude mice as the human tumors are susceptible to HSV infection and the immune system is compromised in those models. To determine the antitumor immune response activated by VG161, colorectal cancer (CT26) syngeneic model was used in immune competent animals and T cell subpopulation analysis, ELISPOT, and tumor rechallenge assay was applied to measure specific antitumor immunity. VG161 caused complete tumor eradication in PDX and CT26 tumor models and significantly inhibited tumor growth in SGC-7901 and LS174T models. All animals treated with VG161 survived for many months till sacrificed. In the CT26 model, no tumor could be found after rechallenging with the same tumor cells. The number of CD8+ tumor infiltrated T cells was significantly increased, and systemic specific anticancer immunity was confirmed by ELISPOT assay on T cells isolated from spleen. VG161 is a novel oncolytic virus that is both strong in oncolysis and stimulating antitumor immunity for GI cancers. Intratumorally expressing multiple immune-regulatory factors by an oncolytic virus may significantly change the GI tumor immune microenvironment and stimulate anticancer immunity, to further enhance the efficacy of the oncolytic virus. Citation Format: Ronghua Zhao, Jun Ding, Tianchi Liu, Shuyun Liu, Dmitry Chouljenko, Yanal Murad, Erica Lee, Guoyu Liu, Luke Bu, William Jia. Anticancer efficacy of a novel immune-stimulating oncolytic virus: VG161 in gastrointestinal cancers [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B85.

Abstract B08: C-C chemokine receptor 4 (CCR4) antagonism enhances the effectiveness of checkpoint Inhibition in mouse tumor models

Abstract Introduction: Chemokines and their receptors influence many of the hallmark processes in cancer: they act not only on infiltrating leukocytes, but also on fibroblasts, endothelial cells, and directly on some types of tumor cells. C-C chemokine receptor 4 (CCR4) and its ligands have been found to be highly expressed in multiple types of human tumors and are associated with poor prognosis. CCR4 antagonism has been demonstrated to reduce tumor growth in various mouse tumor models. Here we assess small-molecule inhibition of CCR4 as a therapeutic agent to potentiate the effects of checkpoint inhibitors in the CT26 and KCM tumor models. Methods: The subcutaneous CT26 colon cancer model and the orthotopic KCM pancreatic cancer model were used to assess the effects of a CCR4 inhibitor, CCX6239, in combination with anti-CTLA-4 antibody. CT26 cells were implanted into the flanks of 9wk female Balb/c mice. Mice were randomized into study groups based on the tumor sizes on day 6, and dosing of both CCX6239 and anti-CTLA-4 started on day 7. For the orthotopic pancreatic cancer model, KCM cells were implanted directly into the pancreas, and dosing of CCX6239 and anti-CTLA-4 also began on day 7. CCX6239 was dosed orally twice daily at 30mg/kg, and anti-CTLA-4 was dosed IP on days 7, 11 and 15 at 100μg/mouse. Results: Blockade of CCR4 appreciably enhanced the therapeutic effects of anti-CTLA-4 in both models. Combined anti-CTLA-4/CCR4 inhibitor significantly decreased tumor size and increased the proportion of long-term survivors in the CT26 model. The antitumor response was CT26-specific; long-term survivors were resistant to reinoculation with CT26 cells (without further dosing of either drug), but 4T1 breast tumors grew well upon challenge of CT26 survivors. Mice with tumor regression exhibited a high proportion of CD8 T cells that recognized a CT26-specific neoantigen, as illustrated by AH1 peptide-MHC tetramer staining. Although long-term survival has not yet been examined in the KCM model, CCX6239 alone significantly reduced tumor burden in 3 independent studies. Anti-CTLA-4 alone provided substantial inhibition of tumor growth in this model, which was further enhanced by CCX6239. Summary: A specific CCR4 small-molecule inhibitor enhanced the effectiveness of anti-CTLA-4 in 2 preclinical models, suggesting that targeting CCR4 may be a viable clinical approach for combinatorial treatment with checkpoint blockers. Citation Format: Shijie “Chris” Li, James J. Campbell, Linda S. Ertl, Zhenhua Miao, Vicky Chhina, Alice Kumamoto, Simon Yau, Ton Dang, Penglie Zhang, Thomas J. Schall, Rajinder Singh. C-C chemokine receptor 4 (CCR4) antagonism enhances the effectiveness of checkpoint Inhibition in mouse tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B08.

Development of Tumor Cell-Based Vaccine with IL-12 Gene Electrotransfer as Adjuvant.

Tumor cell-based vaccines use tumor cells as a source of tumor-associated antigens. In our study, we aimed to develop and test a tumor vaccine composed of tumor cells killed by irradiation combined with in vivo interleukin-12 gene electrotransfer as an adjuvant. Vaccination was performed in the skin of B16-F10 malignant melanoma or CT26 colorectal carcinoma tumor-bearing mice, distant from the tumor site and combined with concurrent tumor irradiation. Vaccination was also performed before tumor inoculation in both tumor models and tumor outgrowth was followed. The antitumor efficacy of vaccination in combination with tumor irradiation or preventative vaccination varied between the tumor models. A synergistic effect between vaccination and irradiation was observed in the B16-F10, but not in the CT26 tumor model. In contrast, up to 56% of mice were protected from tumor outgrowth in the CT26 tumor model and none were protected in the B16-F10 tumor model. The results suggest a greater contribution of the therapeutic vaccination to tumor irradiation in a less immunogenic B16-F10 tumor model, in contrast to preventative vaccination, which has shown greater efficacy in a more immunogenic CT26 tumor model. Upon further optimization of the vaccination and irradiation regimen, our vaccine could present an alternative tumor cell-based vaccine.

Abstract B25: The effect of peritumoral gene electrotransfer of interleukin-12 as an adjuvant immunotherapy to electrochemotherapy varies according to immune status of treated tumors

Abstract The aim of the study was to test a new combined therapy including electrochemotherapy (ECT) and gene electrotransfer (GET) of plasmid encoding interleukin-12 (IL-12) in three immunologically different tumors. Therapeutic effectiveness of ECT in the clinics is up to 80% of local tumor control. However, systemic antitumor effect (abscopal effect) has not been observed. We proposed a combined therapy consisting of ECT with intratumoral application of cisplatin, oxaliplatin, or bleomycin with peritumoral GET of plasmid encoding IL-12. In the combination, IL-12 boosts the in situ vaccination effect of ECT and together with immunogenic cell death and danger-associated molecular patterns release recruits effector immune cells. Our hypothesis was that IL-12 potentiates the effect of ECT on a local and systemic level and may vary regarding immunologic profile of the treated tumors. The therapy was tested in three immunologically different primary murine tumor models (malignant melanoma [B16F10], mammary carcinoma [4T1], and colon carcinoma [CT26]) and on dual-flank melanoma model mimicking systemic disease. Tumor growth was followed and blood samples were collected for immunohistochemical and cytometric analysis of the tumor microenvironment and for the detection of IL-12. We demonstrated that GET of IL-12 potentiates the effect of ECT with all three chemotherapeutic drugs on local level. The most pronounced potentiation was with ECT using cisplatin, resulting in 38% of complete responses as well as an abscopal effect. The antitumor effectiveness of this treatment combination could be ascribed to the induction of the local and systemic immune response. Furthermore, the combined therapy was also tested in two other immunologically different tumor models, 4T1 and CT26. The results indicate that peritumoral GET of IL-12 inversely correlates with tumor response to ECT. We observed better responsiveness to ECT in more immunogenic tumors, where the addition of GET led to the lowest potentiation. So far, we have demonstrated an abscopal effect after the combination of ECT with cisplatin and peritumoral IL-12 GET in B16F10 tumor model. Currently, we are also testing the systemic effect of the combined therapy in 4T1 and CT26 tumor models. In conclusion, we showed that peritumoral GET of IL-12 significantly potentiates ECT in treated melanoma tumors and has notable effect on distant untreated tumors, predominantly when cisplatin was used for ECT. Potentiation of this treatment combination depends on the immunologic status of the tumors. ECT was more effective in more immunogenic tumors, but the contribution of peritumoral GET was not as pronounced as in less immunogenic tumors. Citation Format: Katja Ursic, Spela Kos, Urska Kamensek, Maja Cemazar, Gregor Sersa. The effect of peritumoral gene electrotransfer of interleukin-12 as an adjuvant immunotherapy to electrochemotherapy varies according to immune status of treated tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B25.

Abstract B77: TNFR2 blockade alone or in combination with PD-1 blockade shows therapeutic efficacy in murine cancer models

Abstract Tumor necrosis factor receptor 2 (TNFR2) is central to immune balance control in humans and mice. We created human-directed anti-TNFR2 antibodies as a therapeutic approach in cancer, with positive findings (Torrey H, et al. Sci Signal 2017). We have also identified a murine-directed surrogate antagonistic antibody to TNFR2. This antibody shares traits identified in our human-directed antibodies as critical to limiting regulatory T cell (Treg) expansion and activating T effector (Teff) cells. We now present data on this surrogate anti-TNFR2 antibody in two syngeneic mouse models of colon cancer. To study the therapeutic effects of solo and combined immunotherapy using the murine-directed anti-TNFR2 antibody, we used CT26 and MC38 colon tumor models to compare: (i) the murine-directed surrogate anti-TNFR2 antibody therapy, (ii) a commercially available anti-PD1 therapy, and (iii) anti-TNFR2/anti-PD1 combination immunotherapy. Mice were dosed bi-weekly (100ug/mouse antibody). Antigen-specific CD8 and Treg infiltrates were also studied. Mice were tested and monitored for tumor growth by either Champions Oncology (Hackensack, NJ) or a third-party pharmaceutical company in accordance with their respective animal welfare guidelines. In the CT26 model, anti-TNFR2 antagonism alone or cotreatment with anti-PD1 and anti-TNFR2 was highly efficacious (55-62% of mice cured). Anti-PD1 alone was less efficacious (25% cured). In the MC38 model, therapy with anti-TNFR2 alone showed some efficacy (20% cured), anti-PD1 alone had the least efficacy (10% cured), and anti-PD1 in combination with anti-TNFR2 yielded the best overall survival (70% cured). Sequential antibody dosing with anti-PD1 followed by anti-TNFR2 showed no synergy. In contrast, sequential treatment with anti-TNFR2 first followed by anti-PD1, or the combination of anti-TNFR2 plus anti-PD1, showed synergy and highest efficacy. Anti-TNFR2 therapy was distinct from anti-PD1 in showing pronounced Treg depletion and enhanced Teff infiltration in the tumor microenvironment, demonstrating in vivo specificity for disease-causing cells only in the tumor. Anti-TNFR2 immunotherapy provides benefits in two colon cancer models, both as a single agent and when administered in combination with anti-PD1. Anti-PD1 before anti-TNFR2 was associated with poor outcomes for survival, histology, and lack of long-term cure, suggesting that nonspecific unleashing of the immune system with anti-PD1 destroys the tumor microenvironment specificity of anti-TNFR2. These results highlight the value of anti-TNFR2 antagonism in vivo in mouse tumor models as solo therapy or as a combination therapy, administered first or concurrently with anti-PD1. This study of new immunotherapy combinations highlights the need to test both single-agent therapy and sequential combination therapy as new agents are brought forward to the clinic. Citation Format: Russell LaMontagne, Katie Case, Lisa Tran, Hui Zheng, Michael Yang, Denise Faustman. TNFR2 blockade alone or in combination with PD-1 blockade shows therapeutic efficacy in murine cancer models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B77.

CD40 Enhances Type I Interferon Responses Downstream of CD47 Blockade, Bridging Innate and Adaptive Immunity.

Disrupting the binding of CD47 to SIRPα has emerged as a promising immunotherapeutic strategy for advanced cancers by potentiating antibody-dependent cellular phagocytosis (ADCP) of targeted antibodies. Preclinically, CD47/SIRPα blockade induces antitumor activity by increasing the phagocytosis of tumor cells by macrophages and enhancing the cross-presentation of tumor antigens to CD8+ T cells by dendritic cells; both of these processes are potentiated by CD40 signaling. Here we generated a novel, two-sided fusion protein incorporating the extracellular domains of SIRPα and CD40L, adjoined by a central Fc domain, termed SIRPα-Fc-CD40L. SIRPα-Fc-CD40L bound CD47 and CD40 with high affinity and activated CD40 signaling in the absence of Fc receptor cross-linking. No evidence of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or in cynomolgus macaques. Murine SIRPα-Fc-CD40L outperformed CD47 blocking and CD40 agonist antibodies in murine CT26 tumor models and synergized with immune checkpoint blockade of PD-1 and CTLA4. SIRPα-Fc-CD40L activated a type I interferon response in macrophages and potentiated the activity of ADCP-competent targeted antibodies both in vitro and in vivo These data illustrated that whereas CD47/SIRPα inhibition could potentiate tumor cell phagocytosis, CD40-mediated activation of a type I interferon response provided a bridge between macrophage- and T-cell-mediated immunity that significantly enhanced durable tumor control and rejection.

Immune Modulation by Telomerase-Specific Oncolytic Adenovirus Synergistically Enhances Antitumor Efficacy with Anti-PD1 Antibody

The clinical benefit of monotherapy involving immune checkpoint inhibitors (ICIs) such as anti-programmed death-1 antibody (PD-1 Ab) is limited to small populations. We previously developed a telomerase-specific oncolytic adenovirus, Telomelysin (OBP-301), the safety of which was confirmed in a phase I clinical study. Here, we examined the potential of OBP-502, an OBP-301 variant, as an agent for inducing immunogenic cell death (ICD) and synergistically enhancing the efficacy of OBP-502 with PD-1 Ab using CT26 murine colon cancer and PAN02 murine pancreatic cancer cell lines. OBP-502 induced the release of ICD molecules such as adenosine triphosphate (ATP) and high-mobility group box protein 1 (HMGB1) from CT26 and PAN02 cells, leading to recruitment of CD8-positive lymphocytes and inhibition of Foxp3-positive lymphocyte infiltration into tumors. Combination therapy involving OBP-502 intratumoral administration and PD-1 Ab systemic administration significantly suppressed the growth of not only OBP-502-treated tumors but also tumors not treated with OBP-502 (so-called abscopal effect) in CT26 and PAN02 bilateral subcutaneous tumor models, in which active recruitment of CD8-positve lymphocytes was observed even in tumors not treated with OBP-502. This combined efficacy was similar to that observed in a CT26 rectal orthotopic tumor model involving liver metastases. In conclusion, telomerase-specific oncolytic adenoviruses are promising candidates for combined therapies with ICIs.

Comparative [18F]FDG and [18F]DPA714 PET imaging and time-dependent changes of brown adipose tissue in tumor-bearing mice

ABSTRACT Brown adipose tissue (BAT) is important in monitoring energy homeostasis and cancer cachexia. Different from white adipose tissue, BAT is characterized by the presence of a large number of mitochondria in adipocytes. Translocator protein 18 kDa (TSPO), a critical transporter, is expressed in the outer membrane of mitochondria. We speculated that [18F]DPA714, a specific TSPO tracer, may monitor BAT activity in tumor-bearing mice in vivo. We first analyzed the radioactive uptake of positron emission tomography (PET) tracers in BAT of CT26 xenograft mice with 18F-fluorodeoxyglucose ([18F]FDG) and [18F]DPA714. We also studied the BAT uptake of [18F]DPA714 in CT26, A549 and LLC tumor models. The dynamic distribution of [18F]FDG is quite variable among animals, even in mice of the same tumor model (%ID/g-mean: mean ± SDM, 8.61 ± 8.90, n = 16). Contrarily, [18F]DPA714 produced high-quality and stable BAT imaging in different tumor models and different animals of the same model. Interestingly, %ID/g-mean of [18F]DPA714 in BAT was significantly higher on day 26 than that on day 7 in CT26 xenograft model. Taken together, these results strongly indicate the potential feasibility of [18F]DPA714 PET imaging in investigating BAT and energy metabolism during tumor progression in preclinical and clinical study.