Post-tumor Challenge Research Articles

Abstract PO5-20-12: A Case of Bilateral Breast Cancer in a patient on Ocrelizumab

Abstract Background: Ocrelizumab is an anti-CD20 B cell depleting monoclonal antibodies that was FDA approved in 2017 for Relapsing-remitting multiple sclerosis and primary progressive multiple sclerosis based on two large phase III trials (OPERA I and ORATORIO)1. In these trials, patients being treated with Ocrelizumab developed more malignancies compared to placebo. Among these cancers, breast cancer was prominent occurring in about 0.5-0.8% of the patients in the Ocrelizumab group as opposed to 0% in the placebo group. Here we present a case of bilateral breast cancer in a patient with Multiple Sclerosis (MS) treated with Ocrelizumab. Case Presentation A 66-year-old woman with a long-standing history of relapsing-remitting multiple sclerosis (MS) presented to us with concerns of bilateral masses on a screening mammogram. She was diagnosed with MS 30 years prior to presentation and was initially treated with multiple immunomodulators. However, she had progression of disease and was started on Ocrelizumab two years prior to presentation. She was up to date with her screening mammograms which had been unrevealing till now. Family history was significant for breast cancer in her grandmother in her 70s. Her annual mammogram screening showed bilateral irregular masses. A subsequent right breast ultrasound showed three breast masses(1cm, 0.5cm and 0.5cm respectively) and her left breast showed a 0.5 cm mass. Biopsy from the right breast showed invasive ductal and lobular carcinoma(ER+, PR+, HER2-) and that from the left breast showed invasive lobular carcinoma(ER+, PR+, HER2-).She underwent bilateral mastectomies and sentinel lymph node biopsies. The surgical margins and lymph nodes were negative for malignancy hence radiation was not warranted. Genetic testing revealed one pathogenic variant in MSH3 but was negative for BRCA1 and 2. Since her Oncotype dx score was low, adjuvant chemotherapy was not offered. She was started on aromatase inhibitor and her Ocrelizumab was permanently discontinued. Discussion Ocrelizumab is one of the immunomodulator drugs for MS with a probable mechanism being B-cell depletion. The mechanism of how it causes breast cancer is unknown. However, suggested theory is that B cell inhibitors can increase the risk of breast cancer by inhibiting the potentially protective role of the B cells. The B cells are thought to play a role in the tumor micro-environments as antigen presenting cells and activators of natural killer and cytolytic T cells targeted toward lysis of tumors. Also, B cell presence, specifically CD20 B cells, is linked to a better prognosis in breast cancer. One study has also pointed out the importance of timing of anti-b cell therapy in relation to cancer morbidity. Treatment of mice with Anti-CD20 therapy prior to tumor challenge did not result in cancer metastasis. Whereas post tumor challenge, there was increased tumor cell survival and metastasis. Our patient although had a family history of breast cancer, did not have a genetic mutation that could have contributed to her breast cancer. Her breast cancer developed after her therapy with Ocrelizumab hence raising suspicion for causation. With rise in the use of immunosuppressive therapies, further data is needed to delineate pathogenesis. Women with MS have lower all-cause survival after breast cancer diagnosis than women without MS. Hence it is important to identify any risk factors or therapies that might be associated with increased risk of cancer. Citation Format: Kriti Mehra, Mohamad Hejazi. A Case of Bilateral Breast Cancer in a patient on Ocrelizumab [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-20-12.

Evaluation of the anti-tumor effects of an anti-Human Epidermal growth factor receptor 2 (HER2) monoclonal antibody in combination with CD11b+/Gr-1+ myeloid cells depletion using a recombinant peptibody in 4T1-HER2 tumor model.

Clinical efficacy of Human Epidermal growth factor Receptor 2 (HER2) targeted strategies is limited due to impaired anti-tumor responses negatively regulated by immunosuppressive cells. We thus, investigated the inhibitory effects of an anti-HER2 monoclonal antibody (1T0 mAb) in combination with CD11b+/Gr-1+ myeloid cells depletion in 4T1-HER2 tumor model. BALB/c mice were challenged with human HER2-expressing 4T1 murine breast cancer cell line. A week post tumor challenge, each mouse received 50µg of a myeloid cells specific peptibody every other day, or 10mg/kg of 1T0 mAb two times a week, and their combination for two weeks. The treatments effect on tumor growth was measured by calculating tumor size. Also, the frequencies of CD11b+/Gr-1+ cells and T lymphocytes were measured by flow cytometry. Peptibody treated mice indicated tumor regression and 40% of the mice eradicated their primary tumors. The peptibody was capable to deplete notably splenic CD11b+/Gr-1+ cells as well as intratumoral CD11b+/Gr-1+ cells (P<0.0001) and led to an increased number of tumor infiltrating CD8+ T cells (3.3 folds) and also that of resident tumor draining lymph nodes (TDLNs) (3 folds). Combination of peptibody and 1T0 mAb resulted in enhanced expansion of tumor infiltrating CD4+and CD8+ T cells which was associated with tumor eradication in 60% of the mice. Peptibody is able to deplete CD11b+/Gr-1+ cells and increase anti-tumoral effects of the 1T0 mAb in tumor eradication. Thus, this myeloid population have critical roles in development of tumors and their depletion is associated with induction of anti-tumoral responses.

Abstract 6422: Combined TGFβ/PD-L1 blockade enhances systemic antitumor immunity in head and neck cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) unrelated to human papillomavirus (HPV) confers poor prognosis, with half of patients experiencing disease relapse in advanced stages. Response rates to traditional immunotherapies are low and novel approaches such as the bifunctional fusion protein Bintrafusp alfa (BA) that concurrently blocks PD-L1 signaling and neutralizes TGFβ are actively under investigation. Our group showed that when HNSCC patients received neoadjuvant BA therapy, it reversed exhaustion of tumor-infiltrating lymphocytes (TILs) by promoting antigen-mediated signaling. Additionally, we observed that BA promoted recirculation of TILs into peripheral blood possibly through decreased expression of the TGF-β-driven tissue-retention marker CD103. We hypothesize that addition of TGFβ neutralization to PD-L1 blockade can enhance systemic anti-tumor immunity compared to PD-L1 blockade alone. We assessed immune responses to BA in wild-type mice bearing established mouse oral cancer (MOC)-1 tumors that were randomized into the following groups: 1) vehicle; 2) anti-PD-L1 antibody (Avelumab); 3) TGFβ neutralizing construct with a mutated PD-L1 antibody (BA-M); 4) TGFβ neutralizing construct with a functional PD-L1 antibody (BA). Additionally, since our previous data suggests that BA responsiveness depends on CD8 T cell activity, we included two extra groups 5) CD8 depleting antibody alone or 6) in combination with BA. Three days after completing treatment, MOC-1 tumor cells were injected into the contralateral flank and engraftment and progression of secondary challenge tumors was analyzed. Primary tumor progression was significantly inhibited by BA and to a lesser extent avelumab compared to control or BA-M. Engraftment or progression of secondary challenge tumors was significantly inhibited by BA and to a lesser extent avelumab compared to control or BA-M. The growth inhibitory effects of BA were abrogated by CD8 depletion in both the primary and challenge tumors. Flow cytometry analysis of tissue collected on day 7 post-tumor challenge showed that BA-mediated clearance of the tumors was associated with a proportional increase of CD4+ and CD8+ TILs. We also observed that the frequency of CD103 expression among CD4+ TILs was reduced both in the primary and challenge tumors. Although TGFβ neutralization alone fails to induce anti-tumor immunity, the addition of TGFβ neutralization to PD-L1 blockade provides greater control of primary tumor growth and protection from engraftment of a challenge tumor compared to PD-L1 blockade alone. Through adoptive transfer of tumor-specific T cell receptor-engineered T cells, ongoing experiments aim to determine if TGFβ-driven CD103 expression is required for T cell tumor residency and if neutralization of TGFβ in the setting of PD-L1 immune checkpoint blockade allows for recirculation of tumor-specific T cells into the periphery. Citation Format: Magdalena Rainey, Marco Craveiro, Yvette Robbins, Cem Sievers, Clint T. Allen. Combined TGFβ/PD-L1 blockade enhances systemic antitumor immunity in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6422.

Preclinical Assessment of Immunogenicity and Protectivity of Novel ROR1 Fusion Proteins in a Mouse Tumor Model.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a new tumor associated antigen (TAA) which is overexpressed in several hematopoietic and solid malignancies. The present study aimed to produce and evaluate different fusion proteins of mouse ROR1 (mROR1) to enhance immunogenicity and protective efficacy of ROR1. Four ROR1 fusion proteins composed of extracellular region of mROR1, immunogenic fragments of TT as well as Fc region of mouse IgG2a were produced and employed to immunize Balb/C mice. Humoral and cellular immune responses and anti-tumor effects of these fusion proteins were evaluated using two different syngeneic murine ROR1+ tumor models. ROR1-specific antibodies were induced in all groups of mice. The levels of IFN-γ, IL-17 and IL-22 cytokines in culture supernatants of stimulated splenocytes were increased in all groups of immunized mice, particularly mice immunized with TT-mROR1-Fc fusion proteins. The frequency of ROR1-specific CTLs was higher in mice immunized with TT-mROR1-Fc fusion proteins. Finally, results of tumor challenge in immunized mice showed that immunization with TT-mROR1-Fc fusion proteins completely inhibited ROR1+ tumor cells growth in two different syngeneic tumor models until day 120 post tumor challenge. Our preclinical findings, for the first time, showed that our fusion proteins could be considered as a potential candidate vaccine for active immunotherapy of ROR1-expressing malignancies.

Targeting C3d in Combination with Daratumumab:a Strategy to Enhance Therapeutic Potential in Multiple Myeloma

The monoclonal antibody (mAb) daratumumab (DARA) targeting CD38 can induce minimal residual disease negative remissions in patients with multiple myeloma (MM). However, majority of patients subsequently relapse or become refractory to treatment (RRMM). Rapid reduction of surface CD38 levels in MM cells following DARA infusion due to trogocytosis by monocytes and granulocytes leads to reduced complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC), effector functions that rely on antigen density. High expression of complement regulatory proteins, CD55 and CD59 on MM cell surface can also impede CDC activity. Therefore, modalities to overcome these limitations and improve clinical outcome for patients with RRMM are needed. Daratumumab is a strong activator of complement. While a subset of cells may be lysed by the membrane attack complex, all cells reacted with daratumumab are opsonized by C3d, the terminal component of C3. C3d remains covalently attached at the cell surface. Thus, the newly acquired membrane bound C3d constitutes a target for additional intervention, particularly when the cell surface CD38 is lost or low following treatment with DARA. Using phage display technology, we generated and characterized recombinant rabbit/human chimeric mAbs that selectively bind C3 fragments including C3d but not full length C3. We selected two high affinity anti-C3d mAbs (clones 5S8 and d301 with kD values 3.35 nM and 0.99 nM, respectively) that showed reactivity against both human and mouse C3d. To deposit C3d on myeloma cell lines (MM1-S, MM1-R, RPMI8226, NCI-H929, KMS-12-PE) without activating CDC, the cells were reacted with DARA in C5 depleted normal human serum. Both 5S8 and d301 bound to the C3d opsonized cells. Based on SPR analysis these two mAbs do not compete for C3d binding suggesting that they recognize distinct epitopes. In normal human serum, the anti-C3d mAbs enhanced killing by CDC of DARA-opsonized target cells compared to DARA alone (P<0.0001). The non-targeting IgG1 isotype control mAb trastuzumab (TRA) did not bind C3d or enhance DARA-mediated killing. To test the combination of DARA with anti-C3d mAbs in vivo, we used Balb/c-SCID mice bearing subcutaneous (s. c.) MM1-R tumors. First, using clones 5S8 and d301, we demonstrated in vivo binding of mouse C3d on MM1-R tumor cells harvested 24 h after a single intraperitoneal (i. p.) injection of DARA. Next, we challenged the mice s. c. with MM1-R cells and randomly divided them into the following treatment groups: 20 mg/kg DARA single agent (n=7), or combination treatments of 10 mg/kg of each of two mAbs: DARA + TRA (n=10), DARA + 5S8 (n=13), DARA + d301 (n=13). The mAbs were given via i. p. injections weekly starting on day 7 post tumor challenge and continued for 6 weeks. Palpable tumors were noticed in mice between days 7 and 14, and tumor growth was monitored by caliper measurements twice weekly. Mice were euthanized when tumors reached pre-defined limits. All the mice that received DARA + TRA showed progressive tumor growth with median survival of 38 days and all mice had to be euthanized by day 50. Doubling the dose of DARA to 20mg/kg slightly increased median survival to 43 days (P = 0.12 vs DARA+TRA). Two of 7 mice that received DARA alone (at 20 mg/kg) survived to day 175. Median survival for mice treated with DARA (10mg/kg) plus an anti-C3d mAb was 162 days (P = 0.006 for comparison to DARA 20mg/kg), and 13 of 26 (50%) mice in this cohort survived tumor-free beyond 200 days. There was no statistically significant difference between groups treated with 5S8 or d301 in combination with DARA (P = 0.23). All the surviving mice showed no evidence of tumor, weight loss or other morbidity indicating that they are essentially tumor-free. In summary, these results demonstrate that combination treatment of DARA with anti-C3d mAbs improves efficacy over single agent DARA leading to long-term survival in the MM.1R xenograft model. The combination of C3d targeting mAbs with DARA could improve clinical efficacy and overcome resistance to single agent therapy.

Abstract 626: Personalized immunotherapeutic platform with evidence of clinical activity in glioblastoma (IGV-001) protects mice against other lethal solid tumor challenges

Abstract Background: IGV-001 is a novel immunotherapy that combines irradiated patient-derived glioblastoma tumor cells and an antisense oligonucleotide against insulin-like growth factor type 1 receptor (IMV-001) in biodiffusion chambers (BDC). We recently reported that IGV-001-treated newly diagnosed glioblastoma patients with methylated O6-methylguanine-DNA methyl-transferase promoter had a median progression free survival of 38.4 months compared with 8.3 months in historical standard-of-care-treated patients (P=0.0008). We have now found activity with the equivalent murine approach in multiple cancer models, highlighting the transformative potential of this immunotherapeutic platform beyond glioblastoma. Methodology: We utilized several murine models including the ID8-luciferase (-Luc) intraperitoneal ovarian cancer and Hepa1-6-Luc hepatocellular carcinoma orthotopic model. BDC containing saline or 1x106 IMV-001-treated tumor cells (hereon IOV-001 and IHV-001, respectively) were implanted in flanks of C57BL/6 mice and explanted 48 h later, as per glioblastoma clinical protocol. Tumor challenge for all models was conducted 28d after chamber implantation. Mice were monitored for survival and tumor growth, as determined by bioluminescence intensity. Cytokine assays and immunophenotyping were conducted. Results: 60% of IOV-001-treated mice were alive 58d post-tumor challenge, compared to only 19% of mice in the saline group (MST=37d, p=0.004). In the Hepa1-6 model, 50% of IHV-001-treated mice were alive by 100d post-tumor challenge (MST = 60.5d). In comparison, there were no survivors in the saline group beyond 28d (MST = 18d; p = 0.004). Most of the long-term survivors had undetectable tumor; a fraction showed some level of tumor burden which rose and fell, demonstrating immune control. Circulating IFNγ was significantly higher in IOV-001-treated mice compared to controls on 1d post-tumor challenge (p&amp;lt;0.001) and trended higher in those receiving IHV-001 14d after tumor challenge. Positive correlations were found between percentages of CD8+PD1+and CD4+TIM3+, and tumor burden in IOV-001-treated mice. On 14d, PD1+ expression in both CD4+ and CD8+ T cell subsets was significantly lower in surviving mice treated with IHV-001. Improved survival using this approach was also observed in the Pan02 orthotopic pancreatic model. Latest data will be presented. Conclusions: These data support the antitumor activity of this novel immunotherapeutic platform in multiple cancers beyond glioblastoma. Results suggest that efficacy is associated with a systemic immunological response, resulting in generation of Th1 antitumor cytotoxic T cells. Future studies are seeking to resolve the factors triggering good v. poor responses using phenotypic evaluation of T cell activation/exhaustion markers and Th1/Th2 cytokine production. Citation Format: Jenny Zilberberg, Amelia Zellander, Christopher Uhl, Christopher Cultrara, Kenneth kirby, Essam Elazaq, Charles B. Scott, David W. Andrews, Mark Exley. Personalized immunotherapeutic platform with evidence of clinical activity in glioblastoma (IGV-001) protects mice against other lethal solid tumor challenges [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 626.

Abstract 3273: CMP-001, a virus-like particle containing immunostimulatory CpG-A, for treatment of peritoneal carcinomatosis of gastrointestinal and pancreatic cancers

Abstract Peritoneal carcinomatosis is a common form of metastasis occurring in approximately 10% of gastrointestinal and pancreatic cancers with life expectancy often less than 6-12 months. Due to the limited options for treatment of peritoneal carcinomatosis, there is a significant need for novel therapeutic strategies. We explored the therapeutic potential of CMP-001, a novel virus-like particle composed of the Qβ bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide (CpG-A ODN) in this cancer. CpG-A ODN is a known activator of toll-like receptor 9 (TLR9), which is expressed by human and murine plasmacytoid dendritic cells (pDCs). Initial studies evaluated the effect of CMP-001 in vitro on cells obtained from the ascitic fluid of patients with peritoneal carcinomatosis. These studies demonstrated that pDCs are present in the ascitic fluid and produce IFN-α in response to CMP-001 stimulation. A mouse model of peritoneal carcinomatosis was used to further explore the therapeutic potential of CMP-001. C57BL/6 mice were challenged intraperitoneally (i.p.) with Panc02 mouse pancreatic cancer cells. On days 5, 9, and 13 post-tumor challenge mice were treated i.p. with either saline or 100 μg CMP-001. Mice treated with CMP-001 had a median survival of 35 days compared to mice treated with saline alone with a median survival of 28 days (n = 10 per group, p = 0.028). Examination of the immune response demonstrated CMP-001 induced an influx of dendritic cells (CMP- 001 8.99% ± 0.95 vs saline 4.57% ± 0.7, p = 0.0015), NK cells (CMP-001 0.26% ± 0.052 vs saline 0.087% ± 0.0085, p = 0.006), and CD4+ T cells (CMP-001 3.29% ± 0.85 vs saline 0.95% ± 0.097, p = 0.0168) into the ascitic fluid. In addition, CMP-001 induced a significant increase in antigen-experienced, effector and memory, CD11ahiCD44hi CD4+ T cells (CMP-001 54.55% ± 6.52 vs saline 28.12% ± 3.68, p = 0.0028) and CD11ahiCD8αlo CD8+ T cells (CMP-001 58.49% ± 4.47 vs saline 23.04% ± 2.86, p &amp;lt; 0.0001) in the ascites. These data demonstrate that CMP-001 treatment activates pDCs in the peritoneal fluid of patients with carcinomatosis. In addition, CMP-001 stimulates a robust immune response in the peritoneal cavity of mice with peritoneal carcinomatosis. This robust immune response likely contributes to the enhanced survival and decreased disease progression in these mice. Collectively, these promising preclinical results suggest that CMP-001 may have potential as an immunotherapy for the treatment of patients with peritoneal carcinomatosis and is worthy of further evaluation. Citation Format: Ann M. Miller, Caitlin Lemke-Miltner, Sue Blackwell, Ann Tomanek-Chalkley, Kristen Coleman, George Weiner, Carlos Chan. CMP-001, a virus-like particle containing immunostimulatory CpG-A, for treatment of peritoneal carcinomatosis of gastrointestinal and pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3273.

Unbiased testing of all single nucleotide variants specific to a tumor for anti-tumor CD8+ T cell immune response

Abstract Neoepitopes presented in the context of MHC render tumors immunogenic. However, not all potential neoepitopes elicit immunity, and the attributes of immunogenic neoepitopes are yet to be fully defined. Based on the premise that regressing tumors must elicit T cells that recognize the true anti-tumor neoepitopes, we have developed and used here an ELISPOT based high-throughput assay that allows testing of the immunogenicity of a large number of candidate neoepitopes. Naïve C57BL/6 mice were challenged with the colorectal tumor line MC38 and treated, starting day 5 post-tumor challenge, with a checkpoint-blocking antibody, leading to the beginning of tumor regression. MC38-specific CD8+ T cell response was observed in tumor-bearing hosts with regressing tumors. Exome and transcriptome sequencing of MC38 and accompanying bioinformatic analyses have revealed &amp;gt;800 single nucleotide variations (SNVs) in MC38. To identify the SNVs contributing to the immunogenicity of MC38, reactivity of the anti-MC38 CD8+ T cells isolated from mice with regressing tumors, towards individual peptides corresponding to each identified SNV, was tested. Indeed, CD8+ T cells against a number of mutant peptides were detected. These immunogenic mutant peptides are potential MHC I-restricted neoepitopes of MC38 and their role in tumor rejection is being tested.

Anti-CD19 Chimeric Antigen Receptor in Hematopoietic Stem Cells Controlled By the Suicide Gene HSV-sr39TK for Immunotherapy of B-Lineage Malignancies

Background: Patients with refractory or recurrent B-lineage hematologic malignancies have less than 50% of chance of cure despite intensive therapy and innovative approaches are needed to decrease their morbidity and mortality. Our previous published results demonstrate feasibility of stable gene modification of hematopoietic cells (HSC) with chimeric antigen receptor (CAR) directing antigen specificity of T-, NK- and myeloid cells that are continuously produced with effective anti-tumor activity in humanized mouse model. We hypothesize that gene modification of HSC with an anti-CD19 CAR will produce a multi-lineage, persistent immunotherapy against B-lineage hematological malignancies that can be controlled by the HSV-sr39TK suicide gene. Hematopoietic stem cell transplantation is frequently part of the standard of care for patients with relapsed and refractory B cell malignancies. Following HSC collection, a portion of these cells could be modified to express the CD19-specific CAR and give rise to a persistent, multi-cell lineage, HLA-independent immunotherapy, enhancing the graft-versus-malignancy activity. Based on current clinical trials of gene therapy, gene modification of a partial portion of HSC would be sufficient to give rise to a large number of antigen-specific immune cells, and ablation of such gene-modified cells would not cause myeloablation. The generation of NK and myeloid cells bearing CAR would allow earlier directed anti-tumor activity until post-transplant thymopoiesis can take place, enhancing the graft-versus-cancer activity.Methods: High-titer third-generation self-inactivating lentiviral constructs were developed to deliver second-generation CD19-specific CAR co-stimulated by CD28. These vectors also co-delivered the hyper-active herpes simplex virus thymidine kinase HSV-sr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Such vector constructs were thoroughly evaluated using Jurkat cell line and primary human T-cells. Human HSC isolated from umbilical cord blood were transduced with such lentiviral vectors and evaluated for function of both CAR and HSV-sr39TK. Gene-modified human HSC were also injected into NSG pups after irradiation for in vivo evaluation of engraftment, anti-tumor activity and suicide gene activation. In vivo ablation of gene-modified cells was performed by treatment with intraperitoneal ganciclovir 50mg/kg/day over 5 days. Persistence of gene-modified cells was assessed by flow cytometry and ddPCR of animal tissues.Results: Two promoters were compared in such vector constructs, the human elongating factor-1 short (EFS) and the retrovirus-derived MNDU3, regarding transduction efficiency of primary cells. Those vector constructs with MNDU3 as the promoter showed improved CAR expression (p=0.003) and antigen-specific cytotoxicity, despite similar integrated vector copy numbers (VCN) per cell. The addition of HSV-sr39TK to the construct did not impair CAR expression or antigen-specific cytotoxicity, and determined marked cytotoxicity to ganciclovir. Transduction of human HSC with high-titer lentiviral vectors led to satisfactory transduction efficiency with second-generation CAR and HSV-sr39TK. When tested in vivo, mice transplanted with gene-modified human HSC showed similar humanization in bone marrow and spleen (as defined by CD45+ cells) to non-modified HSC. CAR expression by flow cytometry and VCN were not significantly different between transduced cells with and without HSV-sr39TK, and expression of anti-CD19 CAR conferred anti-tumor survival advantage, with 100% of survival of mice engrafted with HSC modified with CAR and HSV-sr39TK for up to 120 days post-tumor challenge. When treated with ganciclovir, the flow cytometry detection of gene-modified cells was decreased in bone marrows (p=0.07) and spleens (p=0.05) to levels similar to background, and with significant decrease of ddPCR detection of gene-modified cells in both bone marrows (p=0.05) and spleens (p=0.01).Conclusions: The modification of HSC with second generation anti-CD19 CAR has provided specific and persistent anti-lymphoma protection. The addition of the suicide gene system HSV-sr39TK to the construct did not affect transduction efficiency, and has determined significant ablation of gene-modified cells in mouse tissues. DisclosuresNo relevant conflicts of interest to declare.

Poly (I:C)-DOTAP cationic nanoliposome containing multi-epitope HER2-derived peptide promotes vaccine-elicited anti-tumor immunity in a murine model

In the current study we aimed at developing a vaccine delivery/adjuvant system to enhance anti-tumor immunity against the natural multi-epitope HER2/Neu-derived P5 peptide. Polyriboinosinic: polyribocytidylic acid [Poly (I:C)] is a strong immunoadjuvant able to enhance specific antitumor immunity induced by peptide-based vaccines. Nevertheless, delivering the peptide and adjuvant intracellularly into their target site remains a challenging issue. We hypothesized this barrier could be overcome through the use of a cationic nanoliposome carrier system which can carry and protect the antigen and adjuvant in the extracellular environment and augment the induction of antitumor immunity. P5 was encapsulated in cationic nanoliposomes composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-Cholesterol either alone or complexed with Poly (I:C). Immunocompetent BALB/c mice were immunized with the formulations 3 times in two-week intervals and the efficiency and type of immune response were then evaluated both in vitro and in vivo. The groups immunized with Lip-P5+PIC (DOTAP-Cholestrol-P5+Poly (I:C)) and Lip+PIC (DOTAP-Cholestrol+Poly (I:C)) enhanced the release of Interferon (IFN)-γ in comparison with other groups. Flow cytometry analysis revealed that Lip-P5+PIC formulation induced the highest level of IFN-γ in CD8+ lymphocytes. Lip-P5+PIC, Lip+PIC and Lip-P5 (DOTAP-Cholestrol-P5) provided some extent of protection in terms of tumor regression in TUBO tumor mice model during the first 65days post tumor challenge but at the end only the tumors of mice immunized with Lip-P5+PIC were significantly smaller than all other groups. Furthermore, tumors of mice receiving Lip-P5+PIC grew at a significantly slower rate throughout the observation period. Our results showed that the combination of Poly (I:C) and DOTAP with the tumor antigen and without applying additional T-helper epitope induced strong antitumor responses. The observations presented here are of great interest for future vaccine studies.

Therapeutic Injection of a C-Type CpG ODN Induced an Antitumor Immune Response in C57/BL6 Mice of Orthotopically Transplanted Hepatocellular Carcinoma.

Synthetic CpG oligodeoxynucleotides (ODNs), as TLR9 agonists, have been found to play a possible role in antitumor effect. In order to determine the effect of YW002, known as a C-type CpG ODN, on the treatment of hepatocellular carcinoma (HCC), which is one of the most aggressive carcinomas, we chose to inject YW002 at the doses of 12.5 µg and 25 µg per mouse 7 days post-tumor challenge. The survival rate of mice was recorded every day. On day 14 postinjection, five mice in each group were bled and randomly sacrificed. The level of IFN-γ or TNF-α in the serum was detected and lymphocyte infiltration in the tumor tissue; the ratios of CD8+ T cells and CD4+ T cells in the spleen of mice were also analyzed. The results indicated that treatment with YW002 could raise the survival rate and delay tumor growth in the mice with orthotopically transplanted HCC. Furthermore, the treatment improved the antitumor immune response through increasing the T-cell infiltration in tumor and the ratio of CD4+, CD8+, and NK cells in the spleen. In addition, the concentration of IFN-γ was raised, and the level of TGF-β was depressed. Our data suggested that CpG ODN might be a proper medicament in a monotherapeutic regimen for treatment of HCC.

Abstract POSTER-THER-1434: Intraperitoneal anti-PD-L1 increases survival in a novel ovarian cancer model and its in vivo efficacy is influenced by baseline anti-tumor immunity of the host

Abstract Purpose: Immunotherapy with monoclonal antibodies that block inhibitory immune checkpoint molecules and enhance antitumor responses has recently showed clinical promise in advanced solid tumors. However, most of the preliminary evidence on therapeutic efficacy comes from melanoma, prostate and lung cancer. Further studies on other tumor types (including ovarian cancer), and on additional, potentially more effective regimens that take into consideration the administration route and baseline immune status of the host, are currently needed. Approach: We recently generated a triple transgenic MUC1+/-KrasG12D/+PtenloxP/loxP (MKP) mouse model that carries a conditional, oncogenic Kras mutation, Pten deletion and express human MUC1 as self. Upon AdCre injection under the ovarian bursa, the MKP mice progress to endometrioid ovarian tumors that overexpress MUC1 tumor-associated antigen. Using an orthotopic MKP ovarian tumor, we generated a new ovarian cancer cell line (2F8). MUC1.Tg mice injected IP with 8x10E5 of completely syngeneic 2F8 cells develop IP tumors. Mice wild-type for MUC1 were similarly challenged with 2F8 cells, thus creating an isogenic system, in which the hosts are syngeneic to all other tumor antigens, except for human MUC1. Results: At baseline, the cells express low PD-L1 but expression increases after in vivo growth. Anti-PDL1 was administered IP every 2 weeks (200 mg/dose) for a total of 3 doses. Treatment was started 21 days post-tumor challenge, a time point which corresponds to a late tumor stage. The anti-PD-L1 treated MUC1.Tg mice showed substantial T cell infiltration within the tumor and significantly increased survival (p= 0.001) compared to isotype control- treated mice. In contrast, wild type mice with isogenic 2F8 tumors developed spontaneous, high titer anti-MUC1 antibodies and did not respond to anti-PD-L1 therapy. However, earlier treatment (at day 11, instead of day 21) post tumor challenge, higher frequency of IP injections (weekly) and higher overall dose (seven doses, 200 μg/dose) increased treatment efficacy in these mice and led to increased survival. Anti-PDL1 therapy did not change the amplitude of MUC1 specific antibodies in responding mice. In addition to the above quantitative and qualitative changes in systemic and intratumoral T cells, broad range profiling using microarrays are ongoing and will reveal the hallmarks of response in anti-PD-L1 treated mice. Conclusions: These results demonstrate that targeting PD-L1 is a viable therapeutic strategy in ovarian cancer and can increases survival despite (1) treatment initiation at late stage, (2) low dose/low frequency of administration and (3) low PD-L1 expression on tumors. By employing an isogenic animal model that favors anti-tumor humoral immunity, we show here that tumor-bearing hosts with antibody-driven immune imbalance at time of treatment initiation are non-responsive and may require a more dose intensive regimen. This is in line with our previous findings showing inverse correlation between survival and pre-existing MUC-1 specific antibody levels in ovarian cancer patients receiving IP Interleukin -2 (IL-2). Our studies have high translational potential and support IP administration of PD-L1 blockers in ovarian cancer patients, while taking into consideration pre-existent, anti-tumor antibodies. Citation Format: Jyothi Mony, Lixin Zhang, Tejas Tirodkar, Esther Elishaev, Joan Brozick, Robert P. Edwards, Anda M. Vlad. Intraperitoneal anti-PD-L1 increases survival in a novel ovarian cancer model and its in vivo efficacy is influenced by baseline anti-tumor immunity of the host [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1434.

Anti-PD-L1 prolongs survival and triggers T cell but not humoral anti-tumor immune responses in a human MUC1-expressing preclinical ovarian cancer model.

Monoclonal antibodies that block inhibitory immune checkpoint molecules and enhance anti-tumor responses show clinical promise in advanced solid tumors. Most of the preliminary evidence on therapeutic efficacy of immune checkpoint blockers comes from studies in melanoma, lung and renal cancer. To test the in vivo potential of programmed death-ligand 1 (PD-L1) blockade in ovarian cancer, we recently generated a new transplantable tumor model using human mucin 1 (MUC1)-expressing 2F8 cells. The MUC1 transgenic (MUC1.Tg) mice develop large number of intraperitoneal (IP) tumors following IP injection of 8 × 10(5) syngeneic 2F8 cells. The tumors are aggressive and display little T cell infiltration. Anti-PD-L1 antibody was administered IP every 2 weeks (200 μg/dose) for a total of three doses. Treatment was started 21 days post-tumor challenge, a time point which corresponds to late tumor stage. The anti-PD-L1 treatment led to substantial T cell infiltration within the tumor and significantly increased survival (p = 0.001) compared to isotype control-treated mice. When the same therapy was administered to wild-type mice challenged with 2F8 tumors, no survival benefit was observed, despite the presence of high titer anti-MUC1 antibodies. However, earlier treatment (day 11) and higher frequency of IP injections restored the T cell responses and led to prolonged survival. Splenocyte profiling via Nanostring using probes for 511 immune genes revealed a treatment-induced immune gene signature consistent with increased T cell-mediated immunity. These findings strongly support further preclinical and clinical strategies exploring PD-L1 blockade in ovarian cancer.

Obesity triggers enhanced MDSC accumulation in murine renal tumors via elevated local production of CCL2.

Obesity is one of the leading risk factors for developing renal cell carcinoma, an immunogenic tumor that is treated clinically with immunostimulatory therapies. Currently, however, the mechanisms linking obesity with renal cancer incidence are unclear. Using a model of diet-induced obesity, we found that obese BALB/c mice with orthotopic renal tumors had increased total frequencies of myeloid-derived suppressor cells (MDSC) in renal tumors and spleens by d14 post-tumor challenge, relative to lean counterparts. Renal tumors from obese mice had elevated concentrations of the known myeloid cell chemoattractant CCL2, which was produced locally by increased percentages of dendritic cells, macrophages, B cells, and CD45- cells in tumors. MDSC expression of the CCL2 receptor, CCR2, was unaltered by obesity but greater percentages of CCR2+ MDSCs were present in renal tumors from obese mice. Of note, the intracellular arginase levels and per-cell suppressive capacities of tumor-infiltrating and splenic MDSCs were unchanged in obese mice relative to lean controls. Thus, our findings suggest that obesity promotes renal tumor progression via development of a robust immunosuppressive environment that is characterized by heightened local and systemic MDSC prevalence. Targeted intervention of the CCL2/CCR2 pathway may facilitate immune-mediated renal tumor clearance in the obese.

TREATMENT WITH WHITE BUTTON MUSHROOM EXTRACT ENHANCED THE ANTI-TUMOR EFFECTS AND LOWERED THE TOXICITY OF CHEMOTHERAPY IN A TUMOR MOUSE MODEL

Cyclophosphamide (CTX) is a chemotherapeutic drug used for treatment of certain solid tumors, lymphomas, and some autoimmune diseases. The antitumor effect of CTX is associated with leukopenia and liver dysfunction. There is an interest in exploring agents that can minimize the adverse effect of CTX without ameliorating its antitumor efficacy. Among the potential agents is a recent study on white button mushroom, which has been found to exert anti-toxic effects. This study was designed to assess the beneficial effects of mushroom extract on the antitumor and toxic effects of CTX. Swiss albino mice were inoculated with intraperitoneal injection (i.p.) of 0.25 x 106 (day 0) of Ehrlich ascites carcinoma (EAC) cells. Mice were treated with a single dose of CTX (200mg/kg) on day 1 or day 7 post EAC inoculation. Agaricus bisporus mushroom extract (666 mg/kg) was administrated (i.p.) on days 8-14 post tumor challenge. The numbers of leukocytes as well as liver function were assessed on day 17. Treatment with CTX on day 7 induced 46% and 37% decrease in the absolute number of monocytes and neutrophils. It had no effect on serum alanine amino transferase activity, while it induced 57% decrease in serum alkaline phosphatase activity. It induced 41% decrease in liver GSH as compared with tumor-bearing mice. Co-administration of mushroom extract with CTX induced 86% decrease in the number of EAC cells, 72% increase in the absolute number of monocytes, 37% increase in the absolute numbers of neutrophils, 57% decrease in serum ALP activity, 100% increase in GPX activity and 35% increase in GSH as compared with tumor-bearing mice. These results indicate to the beneficial effects of mushroom to enhance the antitumor effect of CTX coincided with reduction of its toxic effect.

Natural and anti-PD-L1 induced tumor immunity in a novel ovarian cancer mouse model for human mucin 1 (MUC1) (VAC3P.943)

Abstract MUC1 is a large cell surface glycoprotein overexpressed by almost all types of adenocarcinomas, including those originating in the ovaries. We recently generated a triple transgenic MKP mouse model that carry conditional Kras mutations, Pten deletion and express human MUC1 as self. Upon AdCre injection under the ovarian bursa, the MKP mice progress to endometrioid ovarian tumors. Using a freshly isolated, orthotopic ovarian tumor from a MKP mouse, we generated an ovarian cancer cell line MKP-T-2F8 (or simply 2F8) that triggers multiple MUC1 expressing intraperitoneal (IP) tumors. At baseline, the cells express low PD-L1 but expression increases after in vivo growth. Using the 2F8 transplantable tumor model, we tested in vivo the therapeutic effectiveness of anti-PD-L1 antibody (10F.9G2). MUC1.Tg or wild type mice were IP injected with 8x10E5 of 2F8 cells, followed by three doses of 200ug of anti-PD-L1 (or isotype control) antibody, administered IP every 2 weeks, starting 3 weeks post-tumor challenge. The anti-PD-L1 treated MUC1.Tg mice showed significantly increased survival (p= 0.02), with no evidence for peritoneal tumor. In contrast, wild type mice which grow 2F8 tumors and develop spontaneous anti-MUC1 antibodies in high titers, did not respond to anti-PD-L1 therapy. These results demonstrate that targeting PD-L1 is a viable therapeutic strategy in ovarian cancer; however, tumor-bearing hosts with Th2-driven immune imbalance may not benefit from PD-L1 blockade.

CD4+ T Cells Play a Critical Role in the Generation of Primary and Memory Antitumor Immune Responses Elicited by SA-4-1BBL and TAA-Based Vaccines in Mouse Tumor Models

The role of CD4+ T cells in the generation of therapeutic primary and memory immune responses in cancer diverse immunotherapy settings remains ambiguous. We herein investigated this issue using two vaccine formulations containing a novel costimulatory molecule, SA-4-1BBL, as adjuvant and HPV E7 or survivin (SVN) as tumor associated antigens (TAAs) in two mouse transplantable tumor models; the TC-1 cervical cancer expressing xenogeneic HPV E7 and 3LL lung carcinoma overexpressing autologous SVN. Single vaccination with optimized SA-4-1BBL/TAA formulations resulted in the eradication of 6-day established TC-1 and 3LL tumors in >70% of mice in both models. The in vivo depletion of CD4+ T cells one day before tumor challenge resulted in compromised vaccine efficacy in both TC-1 (25%) and 3LL (12.5%) tumor models. In marked contrast, depletion of CD4+ T cells 5 days post-tumor challenge and one day prior to vaccination did not significantly alter the therapeutic efficacy of these vaccines. However, long-term immunological memory was compromised in the 3LL, but not in TC-1 model as a significant number (85.7%) of tumor free-mice succumbed to tumor growth when rechallenged with 3LL cells 60 days after the initial tumor inoculation. Collectively, these results demonstrate the indispensable role CD4+ T cells play in the generation of therapeutic primary immune responses elicited by SA-4-1BBL/TAA-based vaccines irrespective of the nature of TAAs and establish the importance of CD4+ T cells for long-term immune memory against 3LL tumor expressing self-antigen SVN, but not TC-1 expressing xenogeneic viral antigen E7.

Intra-tumoral administration of tumor membrane vesicles expressing GPI-anchored IL-2 inhibit tumor growth in a murine breast cancer model (P2080)

Abstract Cancer is the second leading cause of death in the US with breast cancer being among the most common cancers. Tumor membrane vesicles (TMVs) used a vaccine will present a variety of tumor antigens to the immune system. TMVs due to their size and particulate nature are likely to be processed and presented by dendritic cells. To enhance their immunogenicity, TMVs were prepared from homogenized 4T07 mouse breast cancer cell transfectants expressing GPI-IL-2 Expression of GPI-IL-2 was verified by flow cytometry analysis. To ensure functionality, IL-2 expressing TMVs were co-cultured with CTLL-2 cells, an IL-2 dependent cytotoxic T cell line, for 48hrs and proliferation of the CTLL-2 cells was assessed by thymidine incorporation. Mice were then challenged with live wild-type 4T07 cells and administered 50ug of TMVs intra-tumorally on days 10 and 17 post tumor challenge. We observed that GPI-IL-2 expressing TMVs are functionally active and capable of inducing proliferation of CTLL-2 cells. More importantly, intra-tumoral injection of GPI-IL-2 expressing TMVs is capable of significantly reducing overall tumor burden in a therapeutic setting. These studies provide evidence that intra-tumoral vaccination with TMVs expressing GPI-IL-2 could potentially be a viable immunotherapeutic strategy for breast cancers that cannot be surgically removed.

Abstract 3794: Inhibition of prostate cancer bone metastases by the dickkopf-1 neutralizing antibody BHQ880

Abstract Advanced prostate carcinoma (PCa) is usually complicated by the presence of bone metastases. Current therapies targeting bone metastases are suboptimal as the metastases progress in spite of therapy. Accordingly, novel therapeutics are needed to target these lesions. The Wnt inhibitor Dickkopf-1 (DKK-1) appears to play an important role in progression of PCa bone metastases. Overexpression of DKK-1 by PCa promotes tumor growth and bone lysis in laboratory studies and DKK1 is present in clinical PCa bone metastases. Development of a fully humanized anti-DKK-1 neutralizing antibody, BHQ880 (Novartis), provides a potential therapeutic strategy to target PCa bone metastases. The goal of this study was to test if BHQ880 could impact PCa bone metastases. PC-3-luc cells (2.5x105) were injected intratibially into mice. Treatment with anti-DKK-1 or isotype control antibody (n=12 mice per group) was initiated 0, 7, or 14 days post-tumor injection. Mice were treated three times weekly by intraperitoneal injection with 200 μg/mouse (approximately 10 mg/kg) Tumor growth and bone lysis were assessed by weekly bioluminescence and bi-weekly radiography, respectively. All mice were euthanized 31 days post-tumor injection, tumor bearing limbs were formalin fixed and serum was obtained. Tumor bearing limbs were analyzed by dual energy x-ray absorptiometry (DEXA) and histology. Serum was assayed for the bone turnover biomarkers alkaline phosphatase (ALP), osteocalcin (OC), and tartate-resistant acid phosphatase 5b (TRAP5b). BHQ880 was well tolerated with no significant side effects observed. Significant inhibition of tumor growth was observed in mice where treatment was initiated 7 or 14 days post-tumor challenge when established tumors were present. Two mice from the 14-day treatment group had tumor regressions after treatment with BHQ880. Treatment with BHQ880 resulted in decreased bone lysis as determined using DEXA. Furthermore, increased serum concentrations of ALP and OC in mice treated with BHQ880 suggest increased bone production; no significant difference in TRAP5b concentration between treated and untreated mice was found. Treatment with the DKK-1 neutralizing antibody BHQ880 was found to inhibit tumor growth, tumor-induced osteolysis and increase serum bone remodeling markers associated with increased bone production in a murine model of PCa bone metastasis. These data suggest that anti-DKK-1 treatment is a plausible mechanism for inhibition of progression of PCa bone metastases. These data suggest that inhibition of DKK-1 may be an effective strategy to inhibit PCa growth in bone. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3794. doi:1538-7445.AM2012-3794

Tumor immunotherapy using adenovirus vaccines in combination with intratumoral doses of CpG ODN

The combination of viral vaccination with intratumoral (IT) administration of CpG ODNs is yet to be investigated as an immunotherapeutic treatment for solid tumors. Here, we show that such a treatment regime can benefit survival of tumor-challenged mice. C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors were therapeutically vaccinated with adenovirus type 5 encoding OVA (Ad5-OVA), and the tumors subsequently injected with the immunostimulatory TLR9 agonist, CpG-B ODN 1826 (CpG), 4, 7, 10, and 13 days later. This therapeutic combination resulted in enhanced mean survival times that were more than 3.5× longer than naïve mice, and greater than 40% of mice were cured and capable of resisting subsequent tumor challenge. This suggests that an adaptive immune response was generated. Both Ad5-OVA and Ad5-OVA + CpG IT treatments led to significantly increased levels of H-2 K(b)-OVA-specific CD8+ lymphocytes in the peripheral blood and intratumorally. Lymphocyte depletion studies performed in vivo implicated both NK cells and CD8+ lymphocytes as co-contributors to the therapeutic effect. Analysis of tumor infiltrating lymphocytes (TILs) on day 12 post-tumor challenge revealed that mice treated with Ad5-OVA + CpG IT possessed a significantly reduced percentage of regulatory T lymphocytes (Tregs) within the CD4+ lymphocyte population, compared with TILs isolated from mice treated with Ad5-OVA only. In addition, the proportion of CD8+ TILs that were OVA-specific was reproducibly higher in the mice treated with Ad5-OVA + CpG IT compared with other treatment groups. These findings highlight the therapeutic potential of combining intratumoral CpG and vaccination with virus encoding tumor antigen.