Tumor Lysate Vaccine Research Articles

Abstract 6750: Validation of a platform to develop new dendritic cell vaccines for cancer immunotherapy

Abstract Osteosarcoma is the most common malignant form of bone cancer that generally causes patients to die of pulmonary metastasis. If detected before the tumor has metastasized, osteosarcoma has a 75% survival at 5 years after the conventional therapy with surgery and chemotherapy. However, most osteosarcomas are high-grade tumors that grow quickly and spread before being detected, lowering patient survival to 20%. Our unique protocol using the human platelet lysate PLTMax® allows establishing tumor cell lines without genetically modifying cells and with minimal genetic drift. The use of PLTMax® and allogeneic tumor cell cultures has proven to be a major advantage for the generation of large amounts of rich tumor antigen libraries for dendritic cell (DC) vaccines. This protocol was used in an autologous dendritic cell/allogeneic tumor lysate vaccine developed at the Mayo Clinic and licensed to Mill Creek Life Sciences (MCLS) to treat newly diagnosed glioblastoma (GBM), which normally has a poor survival rate at only 2%. The technology was used in a phase I clinical trial (MC1272) performed at the Mayo Clinic. The clinical outcomes proved over a 10 time increase in survival rate of patients that received the DC vaccine after the conventional therapy, providing over 20% three-year survival rate. Our technology differs from other clinically used DC vaccines in three primary ways: a) Highly efficient cell line establishment: The unique method for generating tumor cell lines uses Mill Creek Life Sciences’ PLTMax®, a media supplement derived from human platelets. PLTMax® more closely mimics the native microenvironment of tumors and is used to reduce genetic drift and senescence. b) High genetic fidelity: GBM tumor cells expanded via PLTMax® maintain genetic fidelity and TAA expression—a feat that has not been previously achieved by any other GBM cell culture method. c) Reduction of treatment delays and inclusion of all patients that qualify for treatment: Because we will use allogeneic tumor cell lines, we will be able to avoid delays in treatment caused by the time required to culture autologous tumor cell lines, and all patients may receive treatment without exclusion. Through our preliminary work we transferred the technology from a labor-intensive, small-scale approach, to an automated scalable system. Our ultimate goal is to apply this strategy to not only osteosarcoma, but also several other tumor types to validate it as a platform. Like osteosarcoma, there are other therapy-refractory cancers that we believe can benefit from the use of our platform technology, through the use of DC vaccines that offer an immune response against a wider panel of tumor antigens. Citation Format: Vanesa Alonso-Camino, William Mirsch. Validation of a platform to develop new dendritic cell vaccines for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6750.

Tumor-derived autophagosome vaccines combined with immune adjuvants mediate antitumor immune responses via the neoantigen pathway.

Vaccines composed of autophagosomes derived from tumor cells called DRibbles (DRiPs-containing blebs) are involved in the cross-presentation of tumor antigens, thus inducing cross-reactive T-cell responses against the tumor. Compared with traditional tumor lysate vaccines, autophagosome vaccines were found to be better sources of multiple tumor-associated antigens (TAAs) that activate antigen-specific T-cells. However, the involvement of tumor neoantigens in the immune responses of autophagosome vaccines remains unclear. The present study showed that exogenous autophagosome vaccines (DRibbles) combined with immune adjuvants (anti-OX40 antibody and ATP) can effectively activate functional T cells in vitro. Importantly, the combination of exogenous tumor-derived autophagosome vaccines and immune adjuvants was found to induce tumor regression in B16F10 and 4T1 tumor-bearing mice. The combination of autophagosome-enriched DRibbles with anti-OX40 antibody and ATP also exhibited optimal immune stimulation and antitumor efficiency in vivo. The effectiveness of exogenous DRibble vaccines was mainly due to their enhancement of tumor immunogenicity by increasing the presentation and release of tumor neoantigens. These findings suggest that this immunotherapeutic method may be effective in the treatment of cancer.

Abstract P2-20-05: Personalized tumor vaccine can suppress tumor growth and metastasis in the syngeneic mouse breast cancer model

Abstract Objective Immunotherapy using the tumor-specific antigens (TSAs) is a promising strategy in breast cancer. Studies have suggested that the in vivo exposures to certain tumors can induce adaptive anti-tumor immunity in syngeneic tumor models. In this study, we show the efficacy of the tumor lysate vaccine and peptide-based vaccine against tumor neoantigen in suppressing tumor growth and metastasis in 4T1 syngeneic tumor models. Method We used BALB/c mice and its syngeneic tumor cell lines to evaluate the anti-tumor effect induced by the transient exposure to the tumor cells. For tumor vaccines, we synthesized the tumor lysate vaccine by the freeze-thaw method or synthetic peptide against the selected tumor neoantigens identified by exome sequencing. We systemic and local immune remodeling was investigated by using immunohistochemistry, flow cytometry, and single cell RNA sequencing. Results We observed a significant reduction of tumor growth and metastasis in 4T1 syngeneic tumors when the mice were previously exposed to the same cells (pre-exposure group). This anti-tumor effect induced by the exposures to the tumor was cell line-specific. The 4T1 tumor lysate vaccines administered prior to the tumor cell injection also showed significant inhibitory effect on tumor growth and metastasis. T lymphocytes, isolated from the tumor tissues of the 4T1 pre-exposure mice and lysate vaccine-treated mice, showed higher levels of TNF-ɑ and IFN-ɣ when compared to the control those from the control tumors. The lysate vaccine treatment resulted in a substantial remodeling of tumor microenvironment including reduction of myeloid-derived suppressor cells and M2 tumor-associated macrophages. On the other hand, the numbers of M1 tumor-associated macrophages and effector memory CD8+ T cells were increased by the lysate vaccine. While the peptide vaccine showed no inhibitory effect on the primary tumor growth, it also suppressed spontaneous lung metastasis. Finally, we administered lysate tumor vaccine after the tumor establishment to determine the therapeutic effect. The lysate vaccine significantly suppressed the tumor growth and lung metastasis of the syngeneic 4T1 tumors. Conclusion Tumor lysate vaccine can suppress the tumor growth and metastasis in the 4T1 syngeneic mouse models by inducing substantial remodeling of tumor immune microenvironment. Additionally, tumor lysate vaccine can elicit similar anti-tumor immune response when administered after the establishment of the primary tumor suggesting a potential therapeutic value. Citation Format: Hyeong-Gon Moon, Hye Youn Son, Woo Hang Heo, Mingji Quan, SONGBIN LI, Haritonova Valentina, Hamin Jeong, Wonshik Han, Han-Byoel Lee, YUJEONG HER, Ju Hee Kim. Personalized tumor vaccine can suppress tumor growth and metastasis in the syngeneic mouse breast cancer model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-05.

Glucomannan as a Dietary Supplement for Treatment of Breast Cancer in a Mouse Model.

Konjac glucomannan (KGM) is a water-soluble polysaccharide derived from the Amorphophallus’s tuber and, as herbal medicine has shown, can suppress tumor growth or improve health. However, there has been no investigation into the effects of KGM on breast tumor-bearing mice. Therefore, in two cohort experiments, we assessed the effect of glucomannan at daily doses of 2 and 4 mg for 28 days as a dietary supplement and also glucomannan in combination with tumor lysate vaccine as an adjuvant. Tumor volume was monitored twice weekly. In addition, TNF-α cytokines and granzyme B (Gr–B) release were measured with ELISA kits, and IL-2, IL-4, IL-17, and IFN-γ were used as an index for cytotoxic T lymphocyte activity. Moreover, TGF-β and Foxp3 gene expression were assessed in a real-time PCR test. The results show that glucomannan as a dietary supplement increased the IFN-γ cytokine and Th1 responses to suppress tumor growth. Glucomannan as a dietary supplement at the 4 mg dose increased the IL-4 cytokine response compared to control groups. In addition, cell lysate immunization with 2 or 4 mg of glucomannan suppressed tumor growth. As an adjuvant, glucomannan at both doses showed 41.53% and 52.10% tumor suppression compared with the PBS group. Furthermore, the administration of glucomannan as a dietary supplement or adjuvant reduced regulatory T cell response through decreasing TGF-β and Foxp3 gene expression in the tumor microenvironment. In conclusion, glucomannan as a dietary supplement or adjuvant enhanced the immune responses of tumor-bearing mice and decreased immune response suppression in the tumor milieu, making it a potentially excellent therapeutic agent for lowering breast tumor growth.

PD-1 blockage combined with vaccine therapy can facilitate immune infiltration in tumor microenvironment of Lynch syndrome colon cancer.

Background: Lynch syndrome is a genetic disease resulting from mismatch repair gene mutation. Vaccine therapy can enhance the immunogenicity of Lynch syndrome and improve the therapeutic efficacy of immunotherapy. However, there is no approved Lynch syndrome vaccine coming onto the market. Methods: Herein, we used gene knockdown method to construct Lynch syndrome cell model, paving way for us to develop Lynch syndrome tumor lysate vaccine. Then the isograft technique was employed for constructing the tumor-bearing mouse model of Lynch syndrome. And this isograft model was treated with PD-1 monoclonal antibody and tumor vaccine, respectively. Flow cytometry was used for detecting the proportion of immune cells and immunosuppressive cells, and ELISA was used for detecting the contents of chemokines and cytokines in the blood circulation system and tumor tissues of mice. Finally, IHC was used to detect the effects of tumor vaccines as well as PD-1 antibody on tumor tissue proliferation and angiogenesis. Results: The results demonstrated that tumor vaccine could prolong the overall survival of mice, and improve the disease-free survival rate of mice. The vaccine could increase the proportion of inflammatory cells and decrease the proportion of anti-inflammatory cells in the blood circulation system of mice. In addition, tumor vaccine could also improve inflammatory infiltration in the tumor microenvironment and reduce the proportion of immunosuppressive cells. The results of IHC showed that tumor vaccine could inhibit angiogenesis and tumor cell proliferation in mouse tumor tissues. Conclusion: In colon cancer associated with Lynch syndrome, tumor vaccine can hinder the growth of tumor cells, and assist immunotherapy whose therapeutic effect on this kind of cancer is thus enhanced.

Abstract CT245: First in human CD200 activation receptor ligand and tumor lysate vaccine immunotherapy for recurrent glioblastoma in adults

Abstract Background: Immunotherapy has revolutionized clinical management of malignancies rendering cancers that previously had a dismal prognosis more manageable. Unfortunately, these therapies can cause serious immune-related adverse events, including death. In CNS cancers, currently available immunotherapies have limited to no efficacy. Our work in glioblastoma (GBM), which is considered one of the most aggressive cancers as well as being universally fatal, is focused on the CD200 immune checkpoint that modulates the immune system through the inhibitory receptor (CD200R1) and activation receptor (CD200AR). The peptide checkpoint ligand (CD200AR-L) was developed to target the CD200AR that downregulates the inhibitory PD-1/PD-L1, CTLA4 and CD200R immune checkpoints, permitting immune system activation that is less hampered by the inhibitory effects of GBM. Following a positive clinical trial in dogs that spontaneously developed high-grade gliomas, we initiated a first-in-human Phase 1 Study of the CD200 Activation Receptor Ligand (CD200AR-L) and Allogeneic Tumor Lysate Vaccine (GBM6-AD) immunotherapy for Recurrent Glioblastoma (NCT04642937). Methods: This is a single-center, first-in-human, dose-escalation Phase 1 clinical trial utilizing a 3+3 design at three dose levels; 3.75micrograms/kg, 5micrograms/kg, and 7.25micrograms/kg. Adults 18 years and old with recurrent GBM and no pre-exiting immune-related condition plus adequate performance score were eligible. Accrual started in December 2020 at dose level 1 with a second cohort having received treatment on dose level 2. CD200AR-L is administered on days 1 and 2 by suprascapular intradermal injection following the application of topical imiquimod. On day 2, a fixed dose of the allogeneic vaccine is injected intradermally at the same site. The “Induction Phase” consists of this injection series weekly for 4 weeks with monitoring for dose-limiting toxicity through Day28. The “Maintenance Phase” doses the vaccine series monthly for 2 months, then the “Booster Phase” doses every eight weeks for 24 months or until end of treatment. Patients are monitored for adverse events, including immune-mediated intracranial edema and other immune-related complications, and their neurological function is scored per NANO criteria. Correlative studies include immune monitoring (including expression of CD200R1, PD1/PD-L1, and CTLA-4) and MRI imaging assessment per iRANO criteria. Citation Format: Michael R. Olin, Elizabeth C. Neil, Anne Eaton, Shannon Lunn, Christopher L. Moertel. First in human CD200 activation receptor ligand and tumor lysate vaccine immunotherapy for recurrent glioblastoma in adults [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 CT245.

Phase I trial of adjuvant mature autologous dendritic cell/allogeneic tumor lysate vaccines in combination with temozolomide in newly diagnosed glioblastoma.

BackgroundGlioblastoma (GBM) has poor prognosis despite aggressive treatment. Dendritic cell (DC) vaccines are promising, but widespread clinical use has not been achieved, possibly reflecting manufacturing issues of antigen choice and DC potency. We previously optimized vaccine manufacture utilizing allogeneic human GBM tumor cell lysate and potent, mature autologous DCs. Here, we report a phase I study using this optimized DC vaccine in combination with standard therapy.MethodsFollowing surgical resection and radiation with concurrent temozolomide (TMZ), newly diagnosed adult GBM patients received intradermal DC vaccines plus TMZ. Primary endpoints were safety and feasibility. Immune and treatment responses were recorded.ResultsTwenty-one patients were enrolled in this study. One progressed between leukapheresis and vaccine manufacture. Twenty patients received treatment per protocol. Vaccine doses (≥15) were generated following a single leukapheresis for each patient. No dose-limiting vaccine toxicities were encountered. One patient had symptomatic, histologically proven pseudoprogression. Median progression-free survival was 9.7 months. Median overall survival was 19 months. Overall survival was 25% at 2 years and 10% at 4 years. One patient remains progression-free 5 years after enrollment. Specific CD8 T-cell responses for the tumor-associated antigen gp100 were seen post-vaccination. Patients entered the trial with a leukocyte deficit compared to healthy donors which partly normalized over the course of therapy.ConclusionsThis vaccine platform is safe and highly feasible in combination with standard therapy for newly diagnosed patients. Imaging, histological, survival, and immunological data suggest a positive biological response to therapy that warrants further investigation.

Abstract P100: Use of a novel checkpoint inhibitor peptide ligand in a first-in-human phase 1 trial for adults with recurrent glioblastoma

Abstract Cancer immunotherapy has revolutionized clinical management for many systemic malignancies by generating durable control and rendering a significant improvement on an otherwise dismal prognosis. Unfortunately, these therapies often elicit adverse events including autoimmunity and death. In addition, these therapies are not successful in all solid tumors and show no effect on cancers of the central nervous system. Our research is on the CD200 checkpoint that modulates the immune system through the inhibitory receptor (CD200R1) and activation receptors (CD200ARs). We demonstrated that targeting CD200ARs with our checkpoint inhibitor peptide ligand (CD200AR-L) activates the immune system and renders it impervious to the inhibitory effects of the tumor-associated protein, CD200. In a preclinical trial in pet dogs with spontaneous high-grade glioma (n=31), CD200AR-L added to tumor lysate vaccines increased the median OS and PFS compared to TL alone. We suggest these results were due to the ability of CD200AR-L to modulate multiple immune checkpoints. During the characterization of CD200AR-L, we discovered signaling molecules shared by the CD200 and PD-1/PD-L1 pathways, suggesting that these immune checkpoints are interconnected. We initiated a first-in-human, single-center dose-escalation phase 1 clinical trial (NCT04642937) utilizing a 3+3 design. Induction therapy consisted of injection of CD200AR-L (3.75 ug/kg BW) with an allogeneic-brain allogeneic brain tumor vaccine (GBM6-AD) weekly for 4 weeks while monitoring for dose-limiting toxicities. Six patients enrolled in this first cohort; 5 patients were at first recurrence and 1 at second, 5 had MGMT-promoter unmethylated cancers. Although all patients completed induction, one experienced a dose-limiting toxicity of grade III encephalopathy. Non-dose limiting toxicities included lymphopenia (n=1) and immunotherapy-related cerebral edema (n=2) that was mitigated with a bevacizumab rescue protocol. There were no local injection site reactions or other grade I-II toxicities. Three patients were taken off study for radiographic disease progression that was confirmed by histopathology (n=1) or by progressive neurological decline (n=2). The patient with 2 prior recurrences remains stable with no progression on protocol-directed maintenance therapy. . Immune stimulation, completed in 3 of 6 patients, correlated with increases in CD4/CD8 T cells, NK and NKT cells between weeks 2 and 4 post vaccination. A reduction in immunosuppression was documented by decreases in the expression of CD200R1, PD-1, PD-L1 and CTLA4 on CD4/8 T-cells, CD14, CD11c and MDSC populations compared to basline (n=5). The ability of PBMCs to overcome immunosuppression was shown by increases in IL-2, IL-6 and TNF alpha between weeks 2 and 4 after restimulation with PHA. Immune memory was suggested in 2 patients by increases in IFNg and TNFa. CONCLUSION: Therapy with CD200AR-L in combination with GBM6-AD was well -tolerated with early evidence of an immunological response. Dose escalation enrollment in the Phase 1 Trial continues. Citation Format: Michael R. Olin, G. Elisabeth Pluhar, Ingunn Stromnes, Anne Eaton, Shannon Lynn, Emily Greengard, Christopher L. Moertel. Use of a novel checkpoint inhibitor peptide ligand in a first-in-human phase 1 trial for adults with recurrent glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P100.

IMMU-10. USE OF A SINGLE PEPTIDE CHECKPOINT INHIBITOR FOR TREATMENT OF CENTRAL NERVOUS SYSTEM TUMORS

Cancer immunotherapy has revolutionized clinical management of malignancies by generating long-term, durable control of tumors. Unfortunately, these therapies often cause serious immune-related adverse events. In addition, only a small percentage of solid tumors respond to these therapies and there is little efficacy in CNS tumors. Our research is focused on the CD200 immune checkpoint, which modulates the immune system through the inhibitory receptor (CD200R1) and activation receptors (CD200AR). We demonstrated that targeting the CD200AR with a checkpoint peptide ligand (CD200AR-L) activates the immune system and renders it impervious to the inhibitory effects of CD200. In a pre-clinical canine spontaneous high-grade glioma trial, CD200AR-L, with autologous tumor lysate vaccination, resulted in a 20% two-year progression-free survival; no toxicities or adverse effects were observed. We suggest this result was due to the ability of the CD200AR-L to modulate multiple immune checkpoints. During the characterization of the CD200AR-L, we discovered signaling molecules are shared by the CD200 and PD-1/PD-L1 checkpoint pathways, suggesting these immune checkpoints are connected. Our preliminary studies demonstrated that the inhibitory CD200R1 and PD-1 mediate immune checkpoint signaling activities through the SHIP1/2. Moreover, CD200AR-L overpowers the suppressive effects of CD200 and PD-L1, which are both shed by tumors, by downregulating the inhibitory CD200R1 and PD-1 on both antigen-presenting cells (APC) and T-cells. In addition, CD200AR-L downregulates PD-1 on APCs and inhibits the upregulation of PD-L1 and CTLA4. These studies led to the discovery that the novel peptide modulates the CD200, PD-1/PD-L1 and CTLA-4 pathways, providing the basis for the translatable development of a CD200-directed peptide for clinical use against multiple tumors including gliomas. These studies led to FDA approval of this peptide for the first in human phase I single center, open-label, dose-escalation clinical trial (NCT04642937) in adult and pediatric trial for children with recurrent malignant brain tumors.

CTIM-31. USE OF A SINGLE PEPTIDE CHECKPOINT INHIBITOR FOR TREATMENT OF CENTRAL NERVOUS SYSTEM TUMORS

Abstract Cancer immunotherapy has revolutionized clinical management of malignancies by generating long-term, durable control of tumors. Unfortunately, these therapies often cause serious immune-related adverse events. In addition, only a small percentage of solid tumors respond to these therapies and there is little efficacy in CNS tumors. Our research is focused on the CD200 immune checkpoint, which modulates the immune system through the inhibitory receptor (CD200R1) and activation receptors (CD200AR). We demonstrated that targeting the CD200AR with a checkpoint peptide ligand (CD200AR-L) activates the immune system and renders it impervious to the inhibitory effects of CD200. In a pre-clinical canine spontaneous high-grade glioma trial, CD200AR-L, with autologous tumor lysate vaccination, resulted in a 20% two-year progression-free survival; no toxicities or adverse effects were observed. We suggest this result was due to the ability of the CD200AR-L to modulate multiple immune checkpoints. During the characterization of the CD200AR-L, we discovered signaling molecules are shared by the CD200 and PD-1/PD-L1 checkpoint pathways, suggesting these immune checkpoints are connected. Our preliminary studies demonstrated that the inhibitory CD200R1 and PD-1 mediate immune checkpoint signaling activities through the SHIP1/2. Moreover, CD200AR-L overpowers the suppressive effects of CD200 and PD-L1, which are both shed by tumors, by downregulating the inhibitory CD200R1 and PD-1 on both antigen-presenting cells (APC) and T-cells. In addition, CD200AR-L downregulates PD-1 on APCs and inhibits the upregulation of PD-L1 and CTLA4. These studies led to the discovery that the novel peptide modulates the CD200, PD-1/PD-L1 and CTLA-4 pathways, providing the basis for the translatable development of a CD200-directed peptide for clinical use against multiple tumors including gliomas. These studies led to FDA approval of this peptide for the first in human phase I single center, open-label, dose-escalation clinical trial in adult and pediatric trial for children with recurrent malignant brain tumors.

PD-1 Immune Checkpoint Blockade Promotes Therapeutic Cancer Vaccine to Eradicate Lung Cancer.

(1) Background: Targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the activation of immune responses against cancer. Therapeutic vaccination, which induces specific immune responses against tumor antigens (Ags), is an attractive option. (2) Methods: Utilizing a K-RasG12Dp53null murine lung cancer model we determined tumor burden, tumor-infiltrating T cell (TIL) cytolysis, immunohistochemistry, flow cytometry, and CD4 and CD8 depletion to evaluate the efficacy of PD-1 blockade combined with CCL21-DC tumor lysate vaccine. (3) Results: Anti-PD-1 plus CCL21-DC tumor lysate vaccine administered to mice bearing established tumors (150 mm3) increased expression of perforin and granzyme B in the tumor microenvironment (TME), increased tumor-infiltrating T cell (TIL) activity, and caused 80% tumor eradication. Mice with treatment-induced tumor eradication developed immunological memory, enabling tumor rejection upon challenge and cancer-recurrence-free survival. The depletion of CD4 or CD8 abrogated the antitumor activity of combined therapy. PD-1 blockade or CCL21-DC tumor lysate vaccine monotherapy reduced tumor burden without tumor eradication. (4) Conclusion: Immune checkpoint blockade promotes the activity of the therapeutic cancer vaccine. PD-1 blockade plus CCL21-DC tumor lysate vaccine therapy could benefit lung cancer patients.

CD200 Checkpoint Reversal: A Novel Approach to Immunotherapy.

Advances in immunotherapy have revolutionized care for some patients with cancer. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor (CD200AR-L) overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the CD200AR-L in a trial in companion dogs with spontaneous high-grade glioma. Addition of the peptide to autologous tumor lysate vaccines significantly increased the median overall survival to 12.7 months relative to tumor lysate vaccines alone, 6.36 months. This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitor. We developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional response. Using proteomics, we developed humanized CD200AR-L to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in human CD14+ cells. GMP-grade peptide was further validated for activity. We demonstrated that the CD200AR-L specifically targets a CD200AR complex. Moreover, we developed and validated a humanized CD200AR-L for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhanced an antigen-specific response, and determined that the use of the CD200AR-L downregulated the expression of CD200 inhibitory and PD-1 receptors. These results support consideration of a CD200AR-L as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.

SCIDOT-51. OVERPOWERING MULTIPLE INHIBITORY IMMUNE CHECKPOINTS WITH A SINGLE PEPTIDE INHIBITOR ENHANCED SURVIVAL IN A SPONTANEOUS HIGH-GRADE GLIOMA CANINE PILOT-TRIAL

Abstract Given the modest effects current immune checkpoint-inhibitors have on patients with CNS tumors, we focused our studies on the novel CD200 immune checkpoint. The CD200 checkpoint suppresses the immune system through binding of the inhibitory CD200 protein to the inhibitory receptor (CD200R1) expressed on immune cells. In addition to CD200R1, the CD200 checkpoint includes activation receptors (CD200AR), which we are targeting with a peptide ligand (CD200AR-L) to reverse the inhibitory effects of the CD200 protein. We showed that spontaneous high-grade glioma dogs receiving a canine specific CD200AR-L in combination with autologous tumor lysate vaccine had an increased median overall survival of 9 months, compared to 6.3 months with lysate alone, resulting in an overall 36-month survival of 21%. However, we have a 45% event-free (progression-free) group of dogs that died of non-tumor related deaths with an extended median survival of 18 months. Six dogs remain on trial, 87% of the dogs receiving tumor lysate only died of tumor recurrence within 6 months. Serum chemistry profiles and physical examinations showed that the peptide did not induce any systemic toxicity. To determine how we obtained these unprecedented results, we focused on the mechanism of CD200AR-L/CD200AR binding. We discovered DAP10/12 signaling pathways and CD200ARs are upregulated, while the inhibitory CD200R1 is downregulated. We discovered the molecular connection between CD200, PD-1 and CTLA4. We demonstrated that our murine and human CD200AR-L downregulates the inhibitory PD-1 and PD-L1 and inhibits the upregulation of CTLA4. Therefore, we attribute the success of CD200AR-L to the ability to concurrently modulate multiple immune checkpoints. We have now developed human GMP grade CD200AR-L peptides to develop an immunotherapy approach for patients with CNS tumors; we will conduct a Phase I trial utilizing the peptide ligand concomitantly with an allogeneic GBM6-AD cell line as a tumor vaccine and the adjuvant imiquimod.

MHC class I dysfunction of glioma stem cells escapes from CTL-mediated immune response via activation of Wnt/β-catenin signaling pathway.

Glioma stem cells (GSCs) decrease T cells cognition and evade systemic immunosurveillance via downregulations or defects of major histocompatibility complex class I (MHC-I) molecule and antigen-processing machinery (APM) components. Improvement of tumor surface antigens of GSCs may be effective strategy to trigger an adaptive immune response and activate cytotoxic T cells (CTLs) to eliminate glioma. In this study, our data indicated that downregulations of MHC-I and APM components expressions were associated with Wnt pathway activation in GSCs. Histone deacetylases (HDAC) inhibition improved MHC-I and APM components expressions, which could be partly reverted by Wnt pathway activation. Blocking CTLs-mediated killing decreased the anti-tumor effect of tumor lysate vaccine. The enhancement of T cells immune response resulting from HDAC inhibition was dependent on CTLs cognition on tumor antigens presented by upregulated MHC-I molecule in GSCs. These data suggest that suppression of stemness pathway may be effective for GSCs-based immunotherapy against immune-escaped tumors.

Treatment Combining CD200 Immune Checkpoint Inhibitor and Tumor-Lysate Vaccination after Surgery for Pet Dogs with High-Grade Glioma.

Recent advances in immunotherapy have included inhibition of immune checkpoint proteins in the tumor microenvironment and tumor lysate-based vaccination strategies. We combined these approaches in pet dogs with high-grade glioma. Administration of a synthetic peptide targeting the immune checkpoint protein, CD200, enhanced the capacity of antigen-presenting cells to prime T-cells to mediate an anti-glioma response. We found that in canine spontaneous gliomas, local injection of a canine-specific, CD200-directed peptide before subcutaneous delivery of an autologous tumor lysate vaccine prolonged survival relative to a historical control treated with autologous tumor lysate alone (median survivals of 12.7 months and 6.36 months, respectively). Antigen-presenting cells and T-lymphocytes primed with this peptide suppressed their expression of the inhibitory CD200 receptor, thereby enhancing their ability to initiate immune reactions in a glioblastoma microenvironment replete with the immunosuppressive CD200 protein. These results support consideration of a CD200 ligand as a novel glioblastoma immunotherapeutic agent.

Histone deacetylase inhibition up-regulates MHC class I to facilitate cytotoxic T lymphocyte-mediated tumor cell killing in glioma cells.

Background: Immune cells recognize tumor antigens presented on major histocompatibility complex class I (MHC-I) molecule. Increase of MHC-I molecular expression makes tumor cells more susceptible to lysis by immune cells.Methods: Tumor lysate vaccine was prepared to damage glioma cells including cell lines and primary cultured cells from surgical samples. The enhanced effect of histone deacetylase inhibitors (HDACi) to tumor lysate vaccine was observed. The expressions of MHC-I pathway molecules were detected by flow cytometry and western blot after HDACi treatment. Cell apoptosis and cell lysis were measured following blocking cytotoxic T lymphocyte (CTL) pathway. Tumor size and mice survival were analyzed in combinative treatment with HDACi and tumor lysate.Results: HDACi up-regulated the expressions of MHC-I pathway molecules, and enhanced the recognition and killing of immune cells, which was activated by tumor lysate. Activated antigen specific immune responses regulated CTL activity, and HDACi promoted immune response through cytotoxic effect of CTL. Anti-tumor effect of tumor lysate pulse immunogenicity in vivo was elevated by HDACi due to up-regulation of antigen presentation.Conclusions: Our study showed that HDACi enhanced recognition of glioma cell by immune cells and sensitivity of tumor immunotherapy, and improved the anti-tumor effect of tumor lysate vaccine through activating CTL immune response. These pharmacological molecular mechanisms of increasing immune recognition suggest that epigenetic modulation is a promising strategy for sensitizing immunotherapy for glioma treatment.

Abstract 732: A practical strategy to pancreatic cancer immunotherapy using resected human tumor lysate vaccines remodeled to express α-gal epitopes

Abstract Cancer immunotherapy is a potential treatment for pancreatic ductal adenocarcinoma (PDAC) patients. As the predominant natural Ab found in humans, anti-Gal accounts for approximately 1% of immunoglobulins. The ligand of anti-Gal, “α-gal epitope (Galα1-3Galβ1-4GlcNAc-R),” is a major carbohydrate antigen, expressed by non-primate mammals, New World monkeys. One useful application of anti-Gal abundance is the enhancement of the immunogenicity of tumor-associated antigens (TAAs) that promote effective uptake by antigen-presenting cells (APCs). To develop an effective vaccine-based immunotherapy for PDAC, we hypothesized that resected tumor tissue lysates from patients might be an attractive source of PDAC-associated TAAs vaccination. This study presents a novel immunotherapy expressing α-gal epitopes using freshly obtained human PDAC tumor tissue homogenates from patients. Tumor and normal pancreatic tissue specimens were obtained from 10 patients at the time of surgical exploration for primary PDAC. To synthesize α-gal epitopes on either tumor membranes or normal pancreatic tissue membranes, we employed recombinant α1,3 galactosyltransferase (α1,3GT). PDAC membranes or normal pancreatic tissues were homogenized, and were incubated with UDP-Gal and α1,3GT. α1,3GT KO mice were immunized with pig tissues to produce anti-Gal Ab. The high anti-Gal KO mice were vaccinated by i.p. injection with unprocessed or processed PDAC tumor lysate (α-gal(-) PDAC-ly or α-gal(+) PDAC-ly). The high anti-Gal KO mice were also vaccinated with unprocessed normal or processed normal pancreatic tissue lysate (α-gal(-) N-ly or α-gal(+) N-ly). Effective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates in Western blot analysis. α-gal(+) PDAC-ly vaccines elicited significant antibody production against multiple TAAs, assessed by ELISPOT (anti-MUC1; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=28.7 vs. 151.8 spots [P=0.0008], anti-mesothelin; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=36.5 vs. 97.2 spots [P=0.029]) and activated multiple tumor-specific T cells, assessed by ELISPOT (MUC1; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=146.0 vs. 828.0 spots [P=0.0009], mesothelin; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly= 250.7 vs. 988.0 spots [P=0.021], α-gal(-) N-ly and α-gal(+) N-ly groups did not display significant spots.). To demonstrate in vivo effectiveness, splenocytes from vaccinated KO mice were prepared, and these isolated cells were transferred by i.p. injection into NOD/SCID mice. Then, live PANC1 cells were challenged with s.c. injection. The survival time of NOD/SCID mice received from α-gal(+) PDAC-ly groups was significantly extended (95.0 days) compared with α-gal(-) PDAC-ly groups (45 days, p<0.01). We conclude that α-gal(+) PDAC-ly vaccination may be a practical and effective new immunotherapeutic approach for treating PDAC. Citation Format: Masahiro Tanemura, Kenta Furukawa, Eiji Miyoshi, Hidetoshi Eguchi, Hiroaki Nagano, Katsuyoshi Matsunami, Satoshi Nagaoka, Manabu Mikamori, Kentaro Kishi, Hiroki Akamatsu, Masaki Mori, Yuichiro Doki. A practical strategy to pancreatic cancer immunotherapy using resected human tumor lysate vaccines remodeled to express α-gal epitopes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 732.

A practical approach to pancreatic cancer immunotherapy using resected tumor lysate vaccines processed to express α-gal epitopes.

ObjectivesSingle-agent immunotherapy is ineffective against poorly immunogenic cancers, including pancreatic ductal adenocarcinoma (PDAC). The aims of this study were to demonstrate the feasibility of production of novel autologous tumor lysate vaccines from resected PDAC tumors, and verify vaccine safety and efficacy.MethodsFresh surgically resected tumors obtained from human patients were processed to enzymatically synthesize α-gal epitopes on the carbohydrate chains of membrane glycoproteins. Processed membranes were analyzed for the expression of α-gal epitopes and the binding of anti-Gal, and vaccine efficacy was assessed in vitro and in vivo.ResultsEffective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates from 10 different patients, and tumor lysates readily bound an anti-Gal monoclonal antibody. α-gal(+) PDAC tumor lysate vaccines elicited strong antibody production against multiple tumor-associated antigens and activated multiple tumor-specific T cells. The lysate vaccines stimulated a robust immune response in animal models, resulting in tumor suppression and a significant improvement in survival without any adverse events.ConclusionsOur data suggest that α-gal(+) PDAC tumor lysate vaccination may be a practical and effective new immunotherapeutic approach for treating pancreatic cancer.

Immunological consequences of immunization with tumor lysate vaccine and propranolol as an adjuvant: A study on cytokine profiles in breast tumor microenvironment

In this study, we sought out to study the anti-cancer effects of propranolol (Pro) in combination with tumor lysate vaccine on lymphocyte proliferation activities as well as on IL-2, IL-4, IL-10, IL-12, IL-17 and IFN-γ cytokine concentration in the tumor microenvironment (TME). A tumor model was established in inbred Balb/C mice using transplantation of tumor to the flank of native mice. Tumor-bearing mice were immunized with lysate tumor cells (vaccine), a combination of Pro/Vaccine (Vac). Control groups consisted of tumor-bearing mice receiving only propranolol or PBS three times with one week interval via subcutaneous (s.c) injection. One week after the last immunization, tumor was removed, homogenate was prepared and the levels of IL-12, IL-17, 1L-2, IL-10, IL-4, IFN-γ cytokine concentrations were evaluated by commercial ELISA kits. In addition, spleen cell suspension was used for the lymphocyte proliferation assay using the BrdU method. Results from this study indicated that Pro/Vac had the ability to significantly increase lymphocyte proliferation, and to suppress tumor growth. Administration of breast tumor lysates with propranolol increased the concentration of IL-12, IL-17, 1L-2 and IFN-γ cytokines in tumor microenvironment. This study has proved the efficiency of propranolol as an adjuvant in combination with the tumor vaccine model on tumor suppression via cytokine pattern modulation in tumor microenvironment.

Abstract 2355: Strong immune responses to pancreatic cancer cells induced by human tumor lysate vaccine remodeled to express α-gal epitopes and their implication for a universal vaccine

Abstract Pancreatic cancer (PC) is a lethal disease that remains one of the most resistant to traditional therapies. We are in urgent need of other therapies. Immunotherapy designed to target tumor-associated antigens (TAAs) is a promising treatment approach for PC. But vaccination against a single TAA seems to be insufficient. In our previous study, we showed effectiveness vaccination by whole PC cells, engineered to express α-gal epitopes (Cancer Res, 2010). Subsequently we investigated strong immune response of tumor lysate vaccine engineered to express α-gal epitopes (Int J Oncol, 2015). In this study, for developing clinical application of this immunotherapy, we investigated the effects of vaccination with human PC tumor lysate obtained from PC patients, expressing α-gal epitopes. Tumor specimens were obtained from 11 PC patients at the time of surgical resection. To express α-gal epitopes, we cloned the α1,3galactosyltransferas (α1,3GT) from a New World Monkey and expressed it in a soluble form in the yeast expression system of Pichia pastoris. α1,3GT KO mice were immunized with pig tissue to generate anti-Gal Ab in their sera. These high anti-Gal KO mice were vaccinated intraperitoneally by either unsynthesized (control group; group C) or α-gal tumor lysate (α-gal group; group A). Vaccination with α-gal PC tumor lysate elicited strong immune responses of anti-PC cell IgG and anti-PC TAAs, including MUC1 and Mesothelin. Productions of anti-PC cell IgG in group A were 8∼32-fold higher than those of group C. Furthermore, productions of anti-MUC1 and of ant-Mesothelin Ab in group A were 4∼8-fold higher than those of group C. Expansion of TAAs (MUC1 and Mesothelin)-specific B cells was significantly higher [MUC1: spots of group A vs. C = 151 vs. 28 (p<0.001), Mesothelin: 97 vs. 36 (p = 0.03)]. The number of spots of IFN-γ secreting T cells in the presence of MUC1 and Mesothelin peptide stimulation was significantly higher in group A than in group C [MUC1: 828 vs. 146 (p<0.001), Mesothelin: 988 vs. 384 (p = 0.02)]. To demonstrate in vivo tumor destruction, an animal experiment was performed. Splenocytes from vaccinated KO mice were prepared, and then transferred intraperitoneally into NOD/SCID mice. Followed by transferring, mice were challenged with 1×107 of live PANC-1 cells. In mice from group A, regrowth of tumors was significantly prevented and survival period was significantly prolonged [group A vs. C = 95.0 vs. 45.0 days (p<0.001)]. In the immunohistochemical analysis, the tumor created in NOD/SCID mice from group A was shown severe central necrosis and strong infiltration of CD4+ and CD8+ T cells and macrophages into the periphery. In contrast, the tumor obtained from NOD/SCID mice of group C indicated no lymphocytic infiltration. We conclude that the use of human PC tumor lysate vaccine, remodeled to express α-gal epitopes can elicited strong immune responses to pancreatic cancer. Citation Format: Kenta Furukawa, Masahiro Tanemura, Eiji Miyoshi, Tomoki Hata, Kentaro Kishi, Hiroki Akamatsu, Hidetoshi Eguchi, Masaki Mori, Yuichiro Doki. Strong immune responses to pancreatic cancer cells induced by human tumor lysate vaccine remodeled to express α-gal epitopes and their implication for a universal vaccine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2355.