Systemic Tumor-specific Immune Responses Research Articles

Abstract 4206: In situ vaccination with CCL21-DC sensitizes non-responsive murine NSCLCs to anti-PD-1 therapy and generates systemic tumor-specific immune memory

Abstract Decresased responses to immune checkpoint inhibitors (ICIs) in NSCLC are associated with low tumor mutational burden (TMB), impaired tumor antigen presentation, decreased baseline CD8+ T lymphocytes tumor infiltration and low PD-L1 expression in the tumor microenvironment (TME). One approach to augment the efficacy of ICIs in NSCLC is to enhance anti-tumor T cell responses by utilizing in situ vaccination (ISV) with professional antigen-presenting dendritic cells (DCs). In preclinical studies as well as a phase I clinical trial, we have shown that ISV with gene-modified DCs expressing CCL21 (CCL21-DC) promotes effector T lymphocyte infiltration into the tumor, PD-L1 upregulation, and systemic tumor-specific immune responses. We hypothesized that in situ vaccination with CCL21-DC could promote T cell priming and activation, thereby sensitizing non-responsive NSCLC tumors to ICI. Utilizing multiple syngeneic murine models of NSCLC with distinct driver mutations [KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (K)] and varying TMB, we observed that CCL21-DC ISV synergizes with anti-PD-1 therapy resulting in the eradication of a subset of tumors. Evaluation of KPL tumors by flow cytometry and scRNA-seq revealed that combination therapy reprogrammed the myeloid compartment to be less immunosuppressive, and induced T cell infiltration, Th1 polarization, as well as the enrichment of stem-like TCF1+ T cells within the TME. Intratumoral (IT) CCL21-DC enhanced CD103+DC migration to the local lymph node as determined by flow cytometry. Addition of ICI to IT CCL21-DC resulted in enhanced T cell proliferation and INF-γ secretion within the TME and the spleen. Whole exome sequencing (WES) of tumors revealed immunoediting of tumor subclones after IT CCL21-DC and ICI combination therapy. Tumor rechallenge studies demonstrated the establishment of systemic tumor-specific immunity in a subset of mice that were cured of disease.In conclusion, tumor vaccination with CCL21-DC sensitizes immune-resistant murine NSCLC to anti-PD-1 therapy. Combination therapy enhances anti-tumor T cell responses, resulting in immunoediting of tumor subclones and the generation of systemic tumor-specific immunity. In an ongoing clinical trial, we are evaluating the safety and efficacy of ISV with CCL21-DC in combination with anti-PD-1, pembrolizumab, in patients with advanced NSCLC who have progressed on front-line therapy. Citation Format: Ramin Salehi-Rad, Raymond J. Lim, Linh M. Tran, Yushen Du, Camelia Dumitras, Tianhao Zhang, William Crosson, Jensen Abascal, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination with CCL21-DC sensitizes non-responsive murine NSCLCs to anti-PD-1 therapy and generates systemic tumor-specific immune memory [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 4206.

Abstract CT219: Phase I trial of in situ vaccination with autologous CCL21 gene-modified dendritic cells (CCL21-DC) combined with pembrolizumab for advanced non-small cell lung cancer (NSCLC)

Abstract Background: Although immune checkpoint blockade (ICB) targeting Programmed Death-1/Programmed Death-Ligand 1 (PD-1/PD-L1) alone or in combination with chemotherapy is now a first-line option for patients with advanced NSCLC, the majority of patients do not benefit from anti-PD-1/PD-L1 monotherapy, and the combination with chemotherapy is often associated with toxicity. For patients who initially respond to PD-1/PD-L1 inhibition, many relapse after an initial response and are in need for innovative strategies to overcome the resistance to ICB. One possible approach is to utilize in situ vaccination with functional chemokine gene-modified antigen presenting cells (APCs) that take advantage of the full repertoire of tumor antigens in the tumor microenvironment (TME) in order to restore tumor antigen presentation, promote tumor infiltration of immune cells driven by chemokine gradient, and facilitate tumor-specific T cell activation, both locally and systemically. The chemokine CCL21 is a therapeutic candidate due to its ability to promote infiltration and co-localization of naïve T cells and antigen-experienced dendritic cells (DCs) and facilitate T cell activation. In preclinical studies as well as a phase I clinical trial, we observed that intratumoral (IT) injection of CCL21-DC induces the infiltration of autologous DC and T cells into the TME, and generates systemic tumor-specific immune responses against multiple tumor antigens. We also observed tumoral PD-L1 upregulation following CCL21-DC injection, which may hinder T cell function. Similarly, the lack of efficacy of PD-1/PD-L1 inhibitors could potentially be combated by enhanced T cell infiltration and augmented APC function in the TME following in situ vaccination with CCL21-DC. Therefore, we are currently evaluating the safety and efficacy of combining IT CCL21-DC and intravenous (IV) pembrolizumab in advanced NSCLC patients following progression on ICB or tyrosine kinase inhibitor (TKI) therapy in a phase I trial (NCT03546361). Methods: Phase I, dose-escalating, multi-cohort trial followed by dose expansion. Maximum of 24 patients (9-12 escalation + 12 expansion) with stage IV NSCLC will be evaluated who have tumors accessible for IT injection and are either EGFR/ALK wild-type after progression on a PD-(L)1 inhibitor or EGFR/ALK mutant after progression on TKI. Three IT injections of autologous CCL21-DC (days 0, 21, 42) will be concurrently administered with pembrolizumab, followed by q3wk pembrolizumab up to 1 year. Primary objective of dose escalation is safety and determination of maximum tolerated dose (MTD) of IT CCL21-DC when combined with pembrolizumab. Primary objective of dose expansion is objective response rate at MTD. Secondary objectives include adverse event profiling and immune monitoring studies. [B.L. and A.L. contributed equally to this work as first authors.] Citation Format: Bin Liu, Aaron Lisberg, Ramin Salehi-Rad, Jay Lee, Linh M. Tran, Kostyantyn Krysan, Raymond Lim, Camelia Dumitras, Zhe Jing, Michael Oh, Fereidoun Abtin, Robert Suh, Scott Genshaft, Scott Oh, Gregory Fishbein, Ciara M. O'Higgins, Anita Kaul, Kanwarpal Kahlon, Shahryar Ashouri, Jonathan Goldman, David Elashoff, Edward Garon, Steven Dubinett. Phase I trial of in situ vaccination with autologous CCL21 gene-modified dendritic cells (CCL21-DC) combined with pembrolizumab for advanced non-small cell lung cancer (NSCLC) [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 CT219.

Framework Nucleic Acid Immune Adjuvant for Transdermal Delivery Based Chemo-immunotherapy for Malignant Melanoma Treatment.

Despite the tremendous progresses of cancer immunotherapy, its current clinical responses rate in melanoma remains to be improved. Here, we have reported a skin penetrating tetrahedral framework nucleic acid immune adjuvant (FNAIA) to transdermally deliver chemotherapy drugs into melanoma to induce the immunogenic death of tumor cells and expose tumor antigens, which with assistance of CpG oligodeoxynucleotide incorporated in FNAIA could trigger systemic tumor-specific immune responses. Compared with free CpG, FNAIA could penetrate deeper into subcutaneous tumor tissues and more effectively stimulate dendritic cell maturation. Notably, doxorubicin-loaded FNAIA locally applied on the intact skin above the melanoma could effectively inhibit the growth of mouse B16F10 melanoma and increase tumor CD8+ T cell infiltration. Moreover, combined with immune checkpoint inhibitor, the growth of distant tumors could also be effectively inhibited, suggesting that this strategy could induce systemic immune responses. Therefore, this work provides a new idea for non-invasive treatment of skin cancer.

Abstract 1798: CTLA-4 photoimmunotherapy depletes intratumoral regulatory T cells (Tregs) to augment local and systemic immunity for anticancer activity

Abstract Introduction: Photoimmunotherapy is an investigational cancer treatment platform that utilizes an antibody conjugated to a light activated dye IRDye® 700DX (IR700) combined with illumination with a non-thermal, red light for selective cell killing. CTLA-4 is a lymphocyte exhaustion marker targeted by anti-CTLA-4 checkpoint inhibitors to induce anticancer immune response and is highly expressed on intratumoral regulatory T cells (Tregs). In this study we used anti-CTLA-4-IR700 photoimmunotherapy (CTLA-4 photoimmunotherapy), which involves intratumoral Treg depletion with checkpoint inhibition, in syngeneic mouse models, including models refractory to immune checkpoint inhibition, to assess anticancer activity and immune response. Methods: Mice inoculated with CT26, LL/2, MCA205, or 4T1 tumors were treated with CTLA-4 photoimmunotherapy. Tumor volume was determined by caliper measurements in target (illuminated) and distal (non-illuminated) tumors. Intratumoral immune responses were characterized by flow cytometry. To evaluate the activation of systemic immune responses, cytotoxic T lymphocyte assays were conducted with splenocytes derived from mice treated with CTLA-4 photoimmunotherapy. Tumor re-challenge studies were conducted to assess for tumor-specific immune memory responses. Results: CTLA-4 photoimmunotherapy induced notable anticancer responses including complete responses (CRs), and enhanced survival of mice in multiple tumor models, including tumors resistant to anti-PD-1 or anti-CTLA-4 treatment. CTLA-4 photoimmunotherapy resulted in a reduction of intratumoral Tregs, an increase of the CD8/Treg ratio, and an increase of intratumoral NK and CD8 T cell activation in comparison to control groups. All CR mice (n=7) rejected new tumor inoculations, suggesting enhancement of systemic immune memory. Splenocytes derived from CTLA-4 photoimmunotherapy-treated mice displayed an expansion of tumor antigen-specific lymphocytes in comparison to control groups. In mice bearing bilateral CT26 tumors, tumor regression was observed in target and distal tumors after CTLA-4 photoimmunotherapy, demonstrating systemic anticancer immunity and potential abscopal effects. Conclusion: CTLA-4 photoimmunotherapy elicited anticancer responses in a variety of tumor models, including tumors resistant to anti-PD-1 or anti-CTLA-4 treatment. Depletion of intratumoral Tregs by CTLA-4 photoimmunotherapy resulted in the local activation of CD8 T cell-mediated anticancer activity and systemic tumor specific immune response. These results indicate that treatment with CTLA-4 photoimmunotherapy can induce anticancer responses in checkpoint resistant mouse tumor models by combining rapid Treg depletion with anti-CTLA-4 checkpoint inhibition into a single method of treatment. Citation Format: C. Daniel de Magalhaes Filho, Stephanie M. Okamura, Daniele M. Bergeron, Ahiram Rodriguez, Abram Lozano, Jerry J. Fong, Miguel Garcia-Guzman. CTLA-4 photoimmunotherapy depletes intratumoral regulatory T cells (Tregs) to augment local and systemic immunity for anticancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1798.

Cryoablation and Immunotherapy for Breast Cancer: Overview and Rationale for Combined Therapy.

Cryoablation is a well-tolerated outpatient procedure that has been used to treat metastatic sites as well as small breast cancers in patients who are considered poor candidates for surgery. Recent studies suggest that cell disruption caused by cryoablation may increase the expression and immunogenicity of tumor neoantigens, which could enhance the ability of the immune system to recognize and attack cancer cells at both local and distant sites. Such an approach might broaden the role of immunotherapy for the treatment of breast cancer, which has previously demonstrated limited response to these agents, likely owing to the modest immunogenicity of most breast cancer subtypes. If cryoablation can induce a systemic tumor-specific response, it could enhance tumor susceptibility to immunotherapy agents. This review briefly summarizes the necessary components for generating an immune response against tumor cells, reviews the tumor microenvironment of breast cancer, describes the rationale for and limitations of immune checkpoint inhibition, highlights the potential for cryoablation to induce a systemic tumor-specific immune response, and describes the rationale for combining cryoablation and immune checkpoint inhibitors for the treatment of breast cancer. Keywords: Ablation Techniques, Breast, Neoplasms-Primary, Percutaneous, Tumor Microenvironment, Tumor Response, Ultrasonography © RSNA, 2021.

A35 Dendritic Cell in Situ Vaccination Potentiates Anti-PD-1 Efficacy and Induces Immunoediting in a Murine Model of NSCLC

Studies reveal that responses to checkpoint blockade in non-small cell lung cancer (NSCLC) are associated with high tumor mutational burden (TMB), preexisting CD8+ T-cell infiltration, and high baseline PD-L1 expression within the tumor microenvironment (TME). In contrast, co-occurring KRAS/LKB1 mutation is associated with primary resistance to PD-1 blockade and decreased overall survival. In preclinical studies as well as a phase I clinical trial, we have discovered that intratumoral (IT) vaccination with gene-modified dendritic cells expressing CCL21 (CCL21-DC) promotes tumor effector T-lymphocyte infiltration, PD-L1 upregulation, and systemic tumor-specific immune responses. We hypothesized that in situ vaccination with CCL21-DC could restore tumor antigen presentation and promote T-cell priming and activation, thereby sensitizing nonresponsive NSCLC tumors to checkpoint blockade. Although genetically engineered murine models (GEMMs) of NSCLC bear driver mutations of the disease, recent studies reveal that these GEMMs possess low mutational burden. We established novel GEMMs of NSCLC [KrasG12D (K), KrasG12DP53-/- (KP), KrasG12DP53+/-Lkb1-/- (KPL)] bearing common driver mutations and varying mutational loads by in vitro exposure of tumor cell lines to the carcinogen N-methyl-N-nitrosourea (MNU). Our preclinical KPL model with high TMB recapitulates the immunologic phenotype of human disease, and contains a predominance of myeloid-derived suppressor cells (MDSC), low tumor-infiltrating lymphocytes (TILs), and low PD-L1 expression within the TME. As anticipated, the KPL tumors are resistant to anti-PD-1 therapy, even with increased mutational load. We evaluated IT CCL21-DC combined with anti-PD-1 therapy in immunocompetent mice bearing KPL tumors with high TMB, and observed that IT CCL21-DC vaccination induces infiltration of autologous T lymphocytes and conventional type I DCs (cDC1s) into the TME and sensitizes the tumors to anti-PD-1 therapy. Combination therapy also reprogrammed the myeloid compartment, resulting in a significant reduction of MDSCs and a concurrent increase in CD11b+Ly6GhiLy6Clo monocyte/myeloid population. Whole-exome sequencing (WES) of tumors revealed immunoediting and selective depletion of tumor subclones post IT CCL21-DC and anti-PD1 combination therapy. Future studies will evaluate the evolution of the T-cell receptor (TCR) repertoire in response to the combination treatment and define functional responses to neoepitopes. These studies will enhance our understanding of the molecular mechanisms of tumor vaccination and facilitate the development of rational combination strategies.

A minimal modeling framework of radiation and immune system synergy to assist radiotherapy planning

Recent evidence indicates the ability of radiotherapy to induce local and systemic tumor-specific immune responses as a result of immunogenic cell death. However, fractionation regimes routinely used in clinical practice typically ignore the synergy between radiation and the immune system, and instead attempt to completely eradicate tumors by the direct lethal effect of radiation on cancer cells. This paradigm is expected to change in the near future due to the potential benefits of considering radiation-induced antitumor immunity during treatment planning. Towards this goal, we propose a minimal modeling framework based on key aspects of the tumor-immune system interplay to simulate the effects of radiation on tumors and the immunological consequences of radiotherapy. The impacts of tumor-associated vasculature and intratumoral oxygen-mediated heterogeneity on treatment outcomes are ininvestigated. The model provides estimates of the minimum radiation doses required for tumor eradication given a certain number of treatment fractions. Moreover, estimates of treatment duration for disease control given predetermined fractional radiation doses can be also obtained. Although theoretical in nature, this study motivates the development and establishment of immune-based decision-support tools in radiotherapy planning.

PH and Thermal Dual-Sensitive Nanoparticle-Mediated Synergistic Antitumor Effect of Immunotherapy and Microwave Thermotherapy.

Cationic anticancer peptides, which can induce tumor cell immunogenic death and further promote systemic tumor-specific immune responses, have offered a promising solution to relieve the tumor immunosuppressive microenvironment. However, peptide drugs are easily degraded and lack of targeting ability when administered systemically, leading to limitations in their applications. Herein, we report a pH and thermal dual-sensitive bovine lactoferricin-loaded (one of the most widely studied cationic anticancer peptides) nanoparticles, which simultaneously exhibited antitumor and immune cell activated effects when applied with microwave thermotherapy, an auxiliary method of immunotherapy. The bovine lactoferricin could be delivered to the tumor site by nanoparticles, be immediately released from nanoparticles in the acidic environment of lysosomes and the thermal condition caused by microwave radiation, and ultimately induce tumor apoptosis with the release of damage-associated molecular patterns (DAMPs). It is worth noting that the strategy of bovine lactoferricin-loaded nanoparticles intravenous injection combined with local microwave thermotherapy not only showed excellent efficacy in relieving tumor growth but also resulted in strong antitumor immunities, which was due to the released bovine lactoferricin under stimulating conditions, and the pool of tumor-associated antigens generated by tumor destruction. In conclusion, this work presents a strategy for tumor treatment based on dual-sensitive bovine lactoferricin-loaded nanoparticles combined with microwave thermotherapy, which may provide a solution for cationic anticancer peptides delivery and improving antitumor immune responses.

Recombinant Poxvirus and the Tumor Microenvironment: Oncolysis, Immune Regulation and Immunization

Oncolytic viruses (OVs) are being extensively studied for their potential roles in the development of cancer therapy regimens. In addition to their direct lytic effects, OVs can initiate and drive systemic antitumor immunity indirectly via release of tumor antigen, as well as by encoding and delivering immunostimulatory molecules. This combination makes them an effective platform for the development of immunotherapeutic strategies beyond their primary lytic function. Engineering the viruses to also express tumor-associated antigens (TAAs) allows them to simultaneously serve as therapeutic vaccines, targeting and amplifying an immune response to TAAs. Our group and others have shown that vaccinating intratumorally with a poxvirus that encodes TAAs, in addition to immune stimulatory molecules, can modulate the tumor microenvironment, overcome immune inhibitory pathways, and drive both local and systemic tumor specific immune responses.

Abstract A56: Intratumoral injection of IMO-2055, a novel Toll-like receptor 9 agonist, with ipilimumab induces a systemic tumor-specific immune response

Abstract Through multiple pathways, tumors suppress immune activity during all phases of growth and dissemination, and enhancement of antitumor immunity is a major goal of cancer treatment. Attractive immunotherapy targets include Toll-like receptors, which activate the innate immune system and play an important role in adaptive immune responses, and immune-checkpoint pathways, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), which regulate T cell activity. Agents directed against these targets alone or in combination have the potential to break immune tolerance and enhance antitumor immune responses. IMO-2055 is a potent and selective TLR 9 agonist designed to stimulate an immune response, and in previous studies it demonstrated synergistic or additive effects when combined with tyrosine-kinase inhibitors and monoclonal antibodies (mAb). In multiple clinical studies, IMO-2055 has been generally well tolerated in more than 300 healthy volunteers and patients with renal, lung, colon, or head and neck cancer. In the present studies, we studied IMO-2055 alone and in combination with ipilimumab, an approved mAb against CTLA-4, in a CT26 colon carcinoma model to evaluate antitumor immune activity and effects on treated and distant tumors. In the first study, BALB/c mice were s.c. implanted with 106 CT26 and CT26.CL25 cells, a subclone of CT26 expressing a model antigen β-galactosidase (β-gal), on right and left flanks, respectively. Treatment was initiated when tumor nodules reached 200 mm3. Treated mice received 2.5 mg/kg IMO-2055 by intratumoral injections only in CT26 implanted right tumor twice per week for total 5 times. A similar experiment with IMO-2055 was conducted in the MB-49 bladder cancer mouse model. In the second study, BALB/c mice were s.c. implanted with 7.5 x 105 CT26 on right flank and the same mice were i.v. implanted with 4 x 105 CT26.CL25 to generate lung metastases. Coinjections of 2.5 mg/kg IMO-2055 and 0.5 mg/kg ipilimumab into CT26 solid tumors on the right flank were initiated on day 8 when tumor nodules reached 200 to 300 mm3. IMO-2055 was given on days 8, 11, 13 and 15 while ipilimumab was administered on days 8 and 13. IMO-2055 monotherapy resulted in tumor growth inhibition in both treated and distant tumors. Distant tumors grew when treatment stopped, indicating the possible role of immune tolerance. Similar results were observed in the MB-49 bladder cancer model. In situ IMO-2055 injections elicited statistically significant differences in tumor-specific cytotoxic T cells against CT26 associated antigen AH1 (p<0.0001) and β-gal presented only in remote CT26.CL25 tumors (p=0.0003) versus PBS control. IMO-2055 and ipilimumab combination therapy resulted in improved tumor growth inhibition versus IMO-2055 or ipilimumab alone. The cytotoxic T cells against β-gal presented in the systemic lung metastasis sites were dramatically increased (p<0.01) compared to either monotherapy alone. The antitumor immune response generated by the combination of IMO-2055 and ipilimumab eradicated disseminated lung metastases in 6 of 8 tumor-bearing mice. In conclusion, IMO-2055 monotherapy and IMO-2055 and ipilimumab combination therapy showed potent antitumor activity in preclinical models. In addition, combination therapy overcame immune tolerance and demonstrated improved antitumor activity versus monotherapy with either agent, supporting further evaluation of combination regimens with IMO-2055 and CTLA-4 mAbs or possibly other checkpoint inhibitors. Citation Format: Daqing Wang, Dong Yu, Sudhir Agrawal. Intratumoral injection of IMO-2055, a novel Toll-like receptor 9 agonist, with ipilimumab induces a systemic tumor-specific immune response. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A56.

Oncolytic virus immunotherapy for melanoma.

Melanoma is a type of skin cancer arising from melanocytes and is increasing in incidence. Although complete surgical excision of early stage lesions may be curative, metastatic melanoma continues to be a major therapeutic challenge. Advances in understanding the molecular pathways that promote tumorigenesis and the interactions between melanoma cells and the immune system have resulted in the approval of several newly targeted agents and immunotherapy strategies for the treatment of advanced disease. Oncolytic virus immunotherapy is a new approach that uses native or attenuated live viruses to selectively kill melanoma cells and induce systemic tumor-specific immune responses. A variety of viruses are now in clinical development with the attenuated oncolytic herpesvirus encoding granulocyte-macrophage colony stimulating factor, known as talimogene laherparepvec, recently demonstrating an improvement in durable response rate in patients with advanced melanoma compared with granulocyte-macrophage colony stimulating factor alone. A major advantage of talimogene laherparepvec and related agents is the limited toxicity and ability to use each individual tumor as a source of antigen to generate a highly specific antitumor immune response. These agents are easily administered in the out-patient setting and may be a reasonable option for patients with limited metastatic tumor burden, those with a good performance status and without extensive prior treatment, and in those who cannot tolerate more difficult therapeutic regimens. Further investigation into the impact on overall survival as monotherapy and combination of oncolytic virus immunotherapy with other forms of immunotherapy merit high priority for further clinical application of these novel agents for the treatment of melanoma and perhaps other cancers as well.

Intralesional Injection of Rose Bengal Induces a Systemic Tumor-Specific Immune Response in Murine Models of Melanoma and Breast Cancer

Intralesional (IL) injection of PV-10 has shown to induce regression of both injected and non-injected lesions in patients with melanoma. To determine an underlying immune mechanism, the murine B16 melanoma model and the MT-901 breast cancer model were utilized. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions. In a murine model of melanoma, B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL PV-10 led to regression of the injected lesion as well as the distant B16 melanoma lung metastases. Anti-tumor immune responses were measured in splenocytes collected from mice treated with IL PBS or PV-10. Splenocytes isolated from tumor bearing mice treated with IL PV-10 demonstrated enhanced tumor-specific IFN-gamma production compared to splenocytes from PBS-treated mice in both models. In addition, a significant increase in lysis of B16 cells by T cells isolated after PV-10 treatment was observed. Transfer of T cells isolated from tumor-bearing mice treated with IL PV-10 led to tumor regression in mice bearing B16 melanoma. These studies establish that IL PV-10 therapy induces tumor-specific T cell-mediated immunity in multiple histologic subtypes and support the concept of combining IL PV10 with immunotherapy for advanced malignancies.

584 THERAPEUTIC EFFECT OF INTRAVESICAL ADMINISTRATION OF A NOVEL NANOTRANSPORTER FOR PACLITAXEL IN AN ORTHOTOPIC BLADDER CANCER MODEL

was to examine the potential of intravesical chitosan/IL-12 immunotherapy to eliminate established orthotopic bladder tumors and elicit adaptive, tumor-specific immunity. METHODS: C57BL/6 mice bearing orthotopic MB49 bladder tumors were treated intravesically with saline, IL-12 or chitosan/IL-12 on days 6, 9, and 12 after tumor implantation. Mice eliminating orthotopic MB49 tumors following intravesical chitosan/IL-12 treatment were rechallenged subcutaneously on the flank with MB49 cells or an irrelevant tumor (B16 melanoma). Lymphocytes isolated from cured mice were evaluated for cytotoxic T lymphocyte activity against MB49 or B16 targets. RESULTS: Intravesical chitosan/IL-12 immunotherapy was found to eliminate established tumors in 100% of mice. In addition, all cured mice were protected from a distant MB49 rechallenge, but not B16 challenge. Similarly, lymphocytes from cured mice were found to lyse MB49, but not B16 cells in vitro. Protective immunity was found to be dependent on CD4 and CD8 subsets as their depletion abrogated protection. CONCLUSIONS: These data are the first to show that an intravesical IL-12-based immunotherapy for bladder cancer can induce a systemic tumor-specific immune response. As such, this treatment may be suitable for patients with recurrent superficial disease as well as invasive and metastatic bladder cancers for which there are no effective treatment options.

Blockade of Programmed Death-1 Pathway Rescues the Effector Function of Tumor-Infiltrating T Cells and Enhances the Antitumor Efficacy of Lentivector Immunization

Despite intensive effort, the antitumor efficacy of tumor vaccines remains limited in treating established tumors regardless of the potent systemic tumor-specific immune response and the increases of tumor infiltration of T effector cells. In the current study, we demonstrated that although lentivector (lv) immunization markedly increased Ag-dependent tumor infiltration of CD8 and CD4 T cells and generated Ag-specific antitumor effect, it simultaneously increased the absolute number of myeloid-derived suppressor cells and regulatory T cells in the tumor lesions. In addition, lv immunization induced expression of programmed death-ligand 1 in the tumor lesions. Furthermore, the tumor-infiltrating CD8 T cells expressed high levels of programmed death-1 and were partially dysfunctional, producing lower amounts of effector cytokines and possessing a reduced cytotoxicity. Together, these immune-suppression mechanisms in the tumor microenvironment pose a major obstacle to effective tumor immunotherapy and may explain the limited antitumor efficacy of lv immunization. The loss of effector function in the tumor microenvironment is reversible, and the effector function of CD8 T cells in the tumor could be partially rescued by blocking programmed death-1 and programmed death-ligand 1 pathway in vitro and in vivo, resulting in enhanced antitumor efficacy of lv immunization. These data suggest that immunization alone may exacerbate immune suppression in the tumor lesions and that methods to improve the tumor microenvironment and to rescue the effector functions of tumor-infiltrating T cells should be incorporated into immunization strategies to achieve enhanced antitumor efficacy.

LIGHT protein suppresses tumor growth by augmentation of immune response

The tumor necrosis factor (TNF) superfamily member LIGHT has potent anti-tumor activities through activation of the immune response, and it is a promising candidate for use in cancer immunotherapy. However, there are no reports of the anti-tumor effects of LIGHT protein in vivo because of the lack of easy, efficient methods of manufacturing this protein. Here, we developed a method of manufacturing recombinant LIGHT protein using Escherichia coli through refolding of inclusion bodies; we then evaluated the anti-tumor activity of the protein. LIGHT protein expressed in E. coli showed the same biological activities and binding affinities to its receptors as did LIGHT expressed in mammalian cells. In addition, intratumoral injection of LIGHT significantly suppressed tumor growth, with augmentation of antigen-specific IFN-γ-producing cells in the regional lymph nodes and spleen. These results indicate that LIGHT protein efficiently evokes the systemic tumor-specific immune response, and thus induces tumor suppression.

Endobronchial Intratumoral Chemotherapy

Endobronchial Intratumoral Chemotherapy

High Number of Intraepithelial CD8+ Tumor-Infiltrating Lymphocytes Is Associated with the Absence of Lymph Node Metastases in Patients with Large Early-Stage Cervical Cancer

In a prospective study, we have examined the tumor-specific immune response in a group of 59 patients with human papillomavirus (HPV) 16-positive (HPV16(+))-induced or HPV18(+)-induced cervical cancer. Local antitumor immunity was analyzed by the enumeration of tumor-infiltrating dendritic cells and CD4+, CD8+, and regulatory T cells as well as by calculation of the ratio of CD8+/CD4+ T cells and CD8+/regulatory T cells. Systemic tumor-specific immunity was assessed by determination of the HPV E6- and/or E7-specific T-cell response in the blood of these patients. Finally, these variables were evaluated with respect to known histopathologic prognostic variables, including the absence (LN-) or presence (LN+) of lymph node metastases. Stratification according to the lymph node status of patients revealed a significantly stronger CD8+ T-cell tumor infiltration, a higher CD8+/CD4+ T-cell ratio, and higher CD8+/regulatory T-cell ratio in the group of patients in which the tumor failed to metastasize to the tumor-draining lymph node. Subdivision according to the presence (IR+) or absence (IR-) of circulating HPV-specific T cells disclosed that the highest number of tumor-infiltrating CD8+ T cells was found in the group of LN- patients displaying a concomitant systemic tumor-specific immune response (LN-IR+). CD8+ T-cell infiltration in LN-IR- patients was comparable with that of LN+ patients. In cervical cancer, the absence of lymph node metastases is strongly associated with a better prognosis. Our data indicate that, especially in a subgroup of LN- patients, a strong and effective interaction between immune system and tumor exists. This subgroup of cervical cancer patients may have the best prognosis.

Adoptive T Cell Therapy Using Antigen-Specific CD8+ T Cells for the Treatment of Patients with Cancer: A Phase I Clinical Study.

The adoptive transfer of in vitro induced and expanded tumor antigen-specific cytotoxic T lymphocytes (CTL) provides a promising approach to the immunotherpy of cancer. We have previously shown that antigen-specific CTL can be generated from HLA-A2.1+ cancer patients by 4 rounds of in vitro stimulation of purified CD8+ T cells with autologous dendritic cells pulsed with HLA-A2 binding tumor-associated peptides. Based on these results we have initiated a pilot study of adoptive T cell therapy in advanced melanoma patients demonstrating that in vitro generated Melan-A (ELAGIGILTV)-specific CTL survive intact in vivo for several weeks and localize preferentially to tumor. Here we report on the clinical results of a phase I study of 11 HLA-A2+ melanoma patients that received at least three i.v. infusions of Melan-A-specific CTL i.v. at 2-week intervals. Each T cell infusion was accompanied by a 6-day course of s.c. IL-2 (3x106 IU daily). A total of 51 T-cell infusions were administered, averaging 2.1 x108 Melan-A specific T cells per infusion, with a range from 0.11 – 13.1 x108 T cells per infusion. Clinical side effects were mild and consisted of chills and low-grade fever (WHO grade I–II) in 7 out of 11 patients that typically occurred within 6 to 8 h post infusion. Hematological effects, observed after T cell transfer, included an increase in eosinophils up to 50% in 7 out of 11 patients, peaking 24h post transfer. Clinical and immunological responses consisted of antitumor responses in 3 out of 11 patients (1 CR, 1 PR, 1 mixed response), an elevated frequency of circulating Melan-A multimer+ T cells up to 2% of total CD8+ T cells for 2 weeks post T cell infusion, suggesting long-term survival and/or proliferation of transferred CTL, and a complete loss of Melan-A expression in lymph node metastases of 2 patients after T cell transfer. Our data indicate that the adoptive transfer of antigen-specific T cells in cancer patients is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects.

Adoptive T cell therapy using antigen-specific CD8 + T cells for the treatment of patients with metastatic melanoma: a phase I clinical study

The adoptive transfer of in vitro induced and expanded tumor antigen-specific cytotoxic T lymphocytes (CTL) provides a promising approach to the immunotherpy of cancer. We have previously shown that Melan-A-specific CTL can be generated from HLA-A2.1+ melanoma patients by 4 rounds of in vitro stimulation of purified CD8+ T cells with autologous dendritic cells pulsed with a mutated HLA-A2 binding Melan-A (ELAGIGILTV) peptide. Based on these results we have initiated a pilot study of adoptive T cell therapy in advanced melanoma patients demonstrating that in vitro generated Melan-A specific CTL survive intact in vivo for several weeks and localize preferentially to tumor (Meidenbauer et al., J Immunol 2003, 170:2161, 2003). Here we report on the clinical results of a phase I study of 12 HLA-A2+ melanoma patients that received at least three i.v. infusions of MelanA-specific CTL i.v. at 2-week intervals. Each T cell infusion was accompanied by a 6-day course of s.c. IL-2 (3 × 106 IU daily). A total of 51 T-cell infusions were administered, averaging 1.48 × 108 Melan-A multimer+ T cells per infusion, with a range from 0.11–6.58 × 108 Melan-A-specific T cells per infusion. Clinical side effects were mild and consisted of chills and low-grade fever (WHO grade I-II) in 8 out of 12 patients that typically occurred within 6 to 8 h post infusion. Hematological effects, observed after T cell transfer, consisted of an increase in eosinophils up to 30% in 7 out of 12 patients, peaking 24 h post transfer. Clinical and immunological responses consisted of antitumor responses in 3 out of 12 patients (2 PR, 1 mixed response), an elevated frequency of circulating Melan-A multimer+ T cells up to 2% of total CD8+ T cells up to 14 days post transfer, suggesting long-term survival and/or proliferation of transferred CTL, and a complete loss of Melan-A expression in lymph node metastases of 2 patients after T cell transfer. Our data indicate that the adoptive transfer of antigen-specific T cells in melanoma patients is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects. from Association for Immunotherapy of Cancer: Cancer Immunotherapy – 2nd Annual Meeting Mainz, Germany, 6–7 May 2004

Phase I study in melanoma patients of a vaccine with peptide-pulsed dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34+ peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-α. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1+ or HLA-A2+ patients received at least 4 i.v. infusions of 5 × 106 to 5 × 107 DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I–II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2+ patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects. Int. J. Cancer 86:385–392, 2000. © 2000 Wiley-Liss, Inc.