Marieke F. Fransen

CD40 stimulation leads to effective therapy of CD40(-) tumors through induction of strong systemic cytotoxic T lymphocyte immunity.

Adequate spontaneous activation of tumor-specific T lymphocytes in tumor-bearing hosts is rare, despite the expression of tumor antigens that are potentially highly immunogenic. For example, failure of the immune system to raise competent responses against established tumors expressing the human adenovirus E1A-antigen allows this tumor to grow in immunocompetent mice. We show that systemic in vivo administration of agonistic anti-CD40 antibodies into tumor-bearing mice results in tumor eradication mediated by CD8+ T cells. Treatment resulted in a strong expansion and systemic accumulation of E1A-specific CTL and depended on CD40 expression on host cells, as the tumor was CD40−, and therapy failed in CD40-deficient mice. Local intratumoral administration of anti-CD40 mAb is equally effective in licensing strong, systemic CTL immunity, resulting in the clearance of distant tumor nodules. Our data indicate that the immune response after cancer–host interactions can be directed toward competence, leading to the cure of established tumors merely by delivery of a CD40-dependent “license to kill” signal.

Activation of Dendritic Cells That Cross-Present Tumor-Derived Antigen Licenses CD8+ CTL to Cause Tumor Eradication

The fate of naive CD8+ T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c+ APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8+ CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.

In Vivo Triggering Through 4-1BB Enables Th-Independent Priming of CTL in the Presence of an Intact CD28 Costimulatory Pathway

Triggering of 4-1BB, a member of the TNFR family, through in vivo administration of agonistic anti-4-1BB Ab delivers a powerful costimulatory signal to CTL. We found this signal to effectively replace the need for CD4+ T cell help in the cross-priming of tumor-specific CTL immunity. Furthermore, 4-1BB Ab can convert an otherwise tolerogenic peptide vaccine into a formulation capable of efficient CTL priming. Initial activation of naive CTL can occur in the absence of 4-1BB costimulation, but this signal permits increased survival of Ag-stimulated CTL. Because naive CTL do not express 4-1BB at their surface, susceptibility to 4-1BB triggering depends on prior up-regulation of this receptor. We show that this requires both stimulation of the TCR and CD28-dependent costimulation. Accordingly, blockade of the CD28-costimulatory pathway abrogates the capacity of agonistic anti-4-1BB Ab to trigger Th-independent CTL immunity. In conclusion, our data reveal that the 4-1BB-mediated survival signal is positioned downstream of Ag-specific TCR triggering and CD28-dependent costimulation of naive CTL. The powerful effects of 4-1BB triggering on the induction, amplification, and persistence of CTL responses provide a novel strategy for increasing the potency of vaccines against cancers.

Local Activation of CD8 T Cells and Systemic Tumor Eradication without Toxicity via Slow Release and Local Delivery of Agonistic CD40 Antibody

Purpose: Immunotherapy against tumors with anti-CD40 agonistic antibodies has been extensively studied in preclinical animal models and recently also in clinical trials. Although promising results have been obtained, antibody (Ab)-related toxicity has been a limiting factor. We reasoned that strict local activation of tumor-specific CD8 T cells through stimulation of CD40 on the dendritic cells (DC) in the tumor area while excluding systemic stimulation might be sufficient for effective tumor eradication and can limit systemic toxicity. Experimental Design: Preclinical in vivo models for immunogenic tumors were used to investigate the potential of delivering a nontoxic dose of agonistic anti-CD40 Ab to the tumor region, including draining lymph node, in a slow-release formulation (montanide). Results: The delivery of anti-CD40 monoclonal Ab, formulated in slow-release Montanide ISA-51, reprograms CTLs by inducing local but not systemic DC activation, resulting in effective tumor-specific CTL responses that eradicate local and distant tumors. Adverse side effects, assayed by organ histology and liver enzymes in the blood, were much lower after local anti-CD40 Ab delivery than systemic administration. The local delivery of anti-CD40 Ab activates only CTLs against antigens presented in the tumor-draining area, because unrelated distant tumors expressing different tumor antigens were not eradicated. Conclusions: These results establish a novel therapeutic principle that local delivery and slow release of agonistic anti-CD40 Ab to the tumor-draining area effectively activates local tumor-specific CD8 T cells to become systemic effectors without causing systemic toxicity or nonspecific CTL activation. These findings have important implications for the use of anti-CD40 therapies in patients. Clin Cancer Res; 17(8); 2270–80. ©2011 AACR.

Longevity of Antigen Presentation and Activation Status of APC Are Decisive Factors in the Balance Between CTL Immunity Versus Tolerance

Encounter of Ag by naive T cells can lead to T cell priming as well as tolerance. The balance between immunity and tolerance is controlled by the conditions of Ag encounter and the activation status of the APC. We have investigated the rules that govern this balance in case an environment that normally induces tolerance is reverted into a milieu that promotes T cell priming, using a minimal CTL epitope derived from human adenovirus type 5 E1A. Vaccination of mice s.c. with E1A peptide in IFA readily induces CTL tolerance, resulting in the inability to control E1A-expressing tumors. The present study shows that efficient CTL priming is achieved when this peptide vaccine is combined with systemic administration of APC-activating compounds like agonistic anti-CD40 mAb or polyriboinosinate-polyribocytidylate. Surprisingly, this CTL response is not long-lasting and therefore fails to protect against tumor outgrowth. Disappearance of CTL reactivity was strongly associated with systemic persistence of the peptide for >200 days. In contrast, peptide administered in PBS does not persist and generates long term CTL immunity capable of rejecting Ad5E1A-positive tumors, when combined with CD40 triggering. Thus, presentation of CTL epitopes in an appropriate costimulatory setting by activated APC, although being essential and sufficient for CTL priming, eventually results in tolerance when the Ag persists systemically for prolonged times. These observations are important for the development of immune intervention schemes in autoimmunity and cancer.

Vaccine-Induced Effector-Memory CD8+ T Cell Responses Predict Therapeutic Efficacy against Tumors

CD8+ T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8+ T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L−KLRG1+ effector-memory CD8+ T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8+ T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.

The Tumoricidal Activity of Memory CD8+ T Cells Is Hampered by Persistent Systemic Antigen, but Full Functional Capacity Is Regained in an Antigen-Free Environment

Naive T cells can be tolerized in the periphery by diverse mechanisms. However, the extent to which memory T cells are susceptible to tolerance induction is less well defined. Vaccination of mice with a minimal CTL epitope derived from human adenovirus type 5 E1A in IFA s.c. readily tolerizes naive as well as recently activated CD8+ T cells due to the overwhelming systemic and persistent presence of the peptide. We have now studied the effect of this peptide on established memory cells, which were induced at least 50 days before by virus vaccination. Memory cells did not undergo peripheral deletion and kept their ability to produce IFN-γ as well as their cytolytic activity in response to Ag directly ex vivo. However, memory CTL responses in virus vaccinated mice injected with peptide ceased to control tumor outgrowth. Interestingly, functional capacities were regained when T cells were transferred to an Ag-free environment in vivo as determined by their ability to reject an otherwise lethal tumor challenge. Together, these findings indicate that memory CTL responses can be functionally incapacitated, but are not, in contrast to naive or recently activated T cells, irreversibly tolerized by persistent systemic Ag, as memory T cells quickly regain effector function upon disappearance of the Ag.

Near-infrared labeled, ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine: In vivo tracking and evaluation of antigen-specific CD8+ T cell immune response

Particulate antigen delivery systems aimed at the induction of antigen-specific T cells form a promising approach in immunotherapy to replace pharmacokinetically unfavorable soluble antigen formulations. In this study, we developed a delivery system using the model protein antigen ovalbumin (OVA) encapsulated in nanoparticles based on the hydrophilic polyester poly(lactide-co-hydroxymethylglycolic acid) (pLHMGA). Spherical nanoparticles with size 300-400 nm were prepared and characterized and showed a strong ability to deliver antigen to dendritic cells for cross-presentation to antigen-specific T cells in vitro. Using near-infrared (NIR) fluorescent dyes covalently linked to both the nanoparticle and the encapsulated OVA antigen, we tracked the fate of this formulation in mice. We observed that the antigen and the nanoparticles are efficiently co-transported from the injection site to the draining lymph nodes, in a more gradual and durable manner than soluble OVA protein. OVA-loaded pLHMGA nanoparticles efficiently induced antigen cross-presentation to OVA-specific CD8+ T cells in the lymph nodes, superior to soluble OVA vaccination. Together, these data show the potential of pLHMGA nanoparticles as attractive antigen delivery vehicles.

Polymeric nanoparticles for co-delivery of synthetic long peptide antigen and poly IC as therapeutic cancer vaccine formulation

The aim of the current study was to develop a cancer vaccine formulation for treatment of human papillomavirus (HPV)-induced malignancies. Synthetic long peptides (SLPs) derived from HPV16 E6 and E7 oncoproteins have been used for therapeutic vaccination in clinical trials with promising results. In preclinical and clinical studies adjuvants based on mineral oils (such as incomplete Freunds adjuvant (IFA) and Montanide) are used to create a sustained release depot at the injection site. While the depot effect of mineral oils is important for induction of robust immune responses, their administration is accompanied with severe adverse and long lasting side effects. In order to develop an alternative for IFA family of adjuvants, polymeric nanoparticles (NPs) based on hydrophilic polyester (poly(d,l lactic-co-hydroxymethyl glycolic acid) (pLHMGA)) were prepared. These NPs were loaded with a synthetic long peptide (SLP) derived from HPV16 E7 oncoprotein and a toll like receptor 3 (TLR3) ligand (poly IC) by double emulsion solvent evaporation technique. The therapeutic efficacy of the nanoparticulate formulations was compared to that of HPV SLP+poly IC formulated in IFA. Encapsulation of HPV SLP antigen in NPs substantially enhanced the population of HPV-specific CD8+ T cells when combined with poly IC either co-encapsulated with the antigen or in its soluble form. The therapeutic efficacy of NPs containing poly IC in tumor eradication was equivalent to that of the IFA formulation. Importantly, administration of pLHMGA nanoparticles was not associated with adverse effects and therefore these biodegradable nanoparticles are excellent substitutes for IFA in cancer vaccines.

Intraocular Tumor Antigen Drains Specifically to Submandibular Lymph Nodes, Resulting in an Abortive Cytotoxic T Cell Reaction

Ocular immune privilege is considered essential in the protection against sight-threatening immune responses, as illustrated by the ability of the ocular environment to permit the growth of tumors that are rejected when implanted at other sites. Although several studies indicate that soluble Ag can drain directly into the spleen when injected into the anterior chamber, the primary site of intraocular tumor Ag presentation to tumor-specific CTLs has not been studied. To gain a better understanding of the mechanism involved in ocular immune privilege, we examined to which lymphoid organs anterior chamber tumor Ags primarily drain. Our data show that intraocular tumor Ag drains exclusively to the submandibular lymph nodes, resulting in activation of tumor-specific CTLs, whereas no Ag drainage was found in spleen. However, these tumor-specific CTLs do not distribute systemically and, as a consequence, intraocular tumor growth is unhampered. A similar lack of CTL efficacy has been observed in mice bearing s.c. tumors, which is converted to a systemic tumoricidal CTL response by administration of agonistic anti-CD40 mAb. In contrast, systemic anti-CD40 treatment of eye tumor-bearing mice did not result in mobilizing tumor-specific CTLs or tumor eradication. Together, these results show that intraocular tumor Ag drains to regional lymph nodes for activation of tumor-specific CTLs. However, the induced tumor-specific immunity is insufficient for tumor clearance, even combined with otherwise highly effective immune intervention protocols.