Potentiating Antitumor Efficacy Through Radiation and Sustained Intratumoral Delivery of Anti-CD40 and Anti-PDL1.

PURPOSE: Mounting evidence demonstrates that combining radiation therapy (RT) with immunotherapy can reduce tumor burden in a subset of patients. However, conventional systemic delivery of immunotherapeutics is often associated with significant adverse effects, which force treatment cessation. The aim of this study was to investigate a minimally invasive therapeutics delivery approach to improve clinical response while attenuating toxicity. METHODS AND MATERIALS: We used a nanofluidic drug-eluting seed (NDES) for sustained intratumoral delivery of combinational antibodies CD40 and PDL1. To enhance immune and tumor response, we combined the NDES intratumoral platform with RT to treat the 4T1 murine model of advanced triple negative breast cancer. We compared the efficacy of NDES against intraperitoneal administration, which mimics conventional systemic treatment. Tumor growth was recorded, and local and systemic immune responses were assessed via imaging mass cytometry and flow cytometry. Livers and lungs were histologically analyzed for evaluation of toxicity and metastasis, respectively. RESULTS: The combination of RT and sustained intratumoral immunotherapy delivery of CD40 and PDL1 via NDES (NDES CD40/PDL1) showed an increase in both local and systemic immune response. In combination with RT, NDES CD40/PDL1 achieved significant tumor burden reduction and liver inflammation mitigation compared with systemic treatment. Importantly, our treatment strategy boosted the abscopal effect toward attenuating lung metastatic burden. CONCLUSIONS: Overall, our study demonstrated superior efficacy of combination treatment with RT and sustained intratumoral immunotherapy via NDES, offering promise for improving therapeutic index and clinical response.

Significant mucosal sIgA production after a single oral or parenteral administration using in vivo CD40 targeting in the chicken.

Many pathogens enter the host through mucosal surfaces and spread rapidly via the circulation. The most effective way to prevent disease is to establish mucosal and systemic immunity against the pathogen. However, current vaccination programs in poultry industry require repeated administrations of live-attenuated virus or large amounts (10 to 100µg) of antigen together with adjuvant to induce specific secretory IgA immune responses at the mucosal effector sites. In the present study, we show that a single administration of 0.4µg of oligopeptide complexed with an agonistic anti-chicken CD40 (chCD40) monoclonal antibody (Mab) effectively targets antigen-presenting cells of the bird's mucosa-associated lymphoid tissue in vivo, and induces peptide-specific secretory IgA (sIgA) in the trachea 7days post administration. Anti-chCD40 Mab-peptide complex was administered once to four-week old male Leghorns via various mucosal routes (orally, via cloacal drinking, or oculo-nasally) or via subcutaneous (s.c.) immunization. Immunization through any of the three mucosal induction routes induced significant peptide-specific mucosal sIgA responses 7 and 14days after immunization. Interestingly, s.c. injection of the complex also induced mucosal sIgA. Our data suggest in vivo targeting of CD40 as a potential adjuvant platform, particularly for the purpose of enhancing and speeding up mucosal vaccine responses in chickens, and potentially other food animals. This is the first study able to elicit specific sIgA immune responses in remote mucosal sites with a single administration of only 0.4µg of antigen.

Bystander activation and anti-tumor effects of CD8+ T cells following Interleukin-2 based immunotherapy is independent of CD4+ T cell help.

We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent "bystander-activated" (CD8(+)CD44high) T cells displaying a CD25(-)NKG2D(+) phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4(+) T cell help for antigen-specific CD8(+) T cell expansion, little is known regarding the role of CD4(+) T cells in antigen-nonspecific bystander-memory CD8(+) T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8(+) T cells upregulated PD-1 in the absence of CD4(+) T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8(+) T cells. Interestingly, compared to CD8(+) T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8(+) response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8(+) T cell expansion, CD4(+) T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8(+) T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.

Adjuvant effect of CD40 on H5N1 DNA vaccine in mice.

H5N1 viruses cause severe and often fatal disease in humans. DNA vaccines have been shown to provide some protection in many animal models against H5N1 influenza virus infection, but this protection is not complete. To enhance the immunogenicity of an H5N1 DNA vaccine, we constructed the eukaryotic expression systems pcD-CD40 and pcD-HA. The expression of pcD-HA or pcD-CD40 in transfected BHK cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR). The constructs were then used to immunize BALB/c mice intramuscularly three times at two-week intervals. The titers of serum HA-specific antibodies were determined by ELISA, and the expression levels of the cytokines IL-2, IL-4, IL-6 and TNF-α were determined by real-time PCR. The results showed that CD40 as a molecular adjuvant significantly enhanced the production of serum anti-HA antibodies and increased the levels of the Th2 cytokines IL-4 and IL-6, suggesting that co-immunization with CD40 upregulated the humoral immune responses to the DNA vaccine in BALB/c mice. This study will provide important information for the selection of adjuvants for DNA vaccines against HPAI H5N1 viruses or other subtypes of human influenza viruses.

Regression of atherosclerosis plaques in apolipoprotein E-/- mice after lentivirus-mediated RNA interference of CD40.

BACKGROUND: A role of CD40 (cluster of differentiation 40) is suggested in development of atherosclerosis plaques, especially in advanced plaques. However, the role of lentiviruses carrying small interfering RNA (siRNA) of CD40 in progression and destabilization of advanced atherosclerotic plaques remains unknown. The aim of this study was to determine whether inhibition of CD40 signaling by lentivirus-mediated RNA interference (RNAi) could inhibit progression of atherosclerotic plaques and increase collagen production. METHODS: Apolipoprotein E-deficient (ApoE-/-) mice aged 10 weeks were fed a high-fat diet and a constrictive collar was placed around right carotid arteries of these mice to induce plaques formation. The recombinant CD40-RNAi-Lentivirus (CD40-RNAi-LV) or negative control-green fluorescent protein-Lentivirus (NC-GFP-LV) were constructed and transfected into right carotid plaques respectively eight weeks after surgery. RESULTS: CD40-RNAi-LV not only prevented plaques progression but also decreased plaques content of lipid, increased plaques content of collagen 6 weeks after lentivirus transfection. This effect reflected a marked decrease in the intima/media ratios (0.31 ± 0.04 vs 0.68 ± 0.05, P<0.05) and a diminished degree of lumen stenosis (intima/lumen ratios, 0.17 ± 0.04 vs 0.33 ± 0.40, P<0.05). Moreover, real-time polymerase chain reaction (RT-PCR) analysis of CD40-RNAi-LV group downregulated expressions of proinflammatory cytokines, chemokines and matrix metalloproteinases. CONCLUSIONS: Lentivirus-mediated CD40 silencing by siRNA treatment would be a new strategy to inhibit plaques progression and to reduce local inflammation through the antiinflammatory effects.

A germinal center-independent pathway generates unswitched memory B cells early in the primary response.

Memory B cells can be produced from the classical germinal center (GC) pathway or a less understood GC-independent route. We used antigen-based cell enrichment to assess the relative contributions of these pathways to the polyclonal memory B cell pool. We identified a CD38(+) GL7(+) B cell precursor population that differentiated directly into IgM(+) or isotype-switched (sw) Ig(+) memory B cells in a GC-independent fashion in response to strong CD40 stimulation. Alternatively, CD38(+) GL7(+) B cell precursors had the potential to become Bcl-6(+) GC cells that then generated primarily swIg(+) memory B cells. These results demonstrate that early IgM(+) and swIg(+) memory B cells are products of a GC-independent pathway, whereas later switched Ig(+) memory B cells are products of GC cells.

A composite MyD88/CD40 switch synergistically activates mouse and human dendritic cells for enhanced antitumor efficacy.

The in vivo therapeutic efficacy of DC-based cancer vaccines is limited by suboptimal DC maturation protocols. Although delivery of TLR adjuvants systemically boosts DC-based cancer vaccine efficacy, it could also increase toxicity. Here, we have engineered a drug-inducible, composite activation receptor for DCs (referred to herein as DC-CAR) comprising the TLR adaptor MyD88, the CD40 cytoplasmic region, and 2 ligand-binding FKBP12 domains. Administration of a lipid-permeant dimerizing ligand (AP1903) induced oligomerization and activation of this fusion protein, which we termed iMyD88/CD40. AP1903 administration to vaccinated mice enabled prolonged and targeted activation of iMyD88/CD40-modified DCs. Compared with conventionally matured DCs, AP1903-activated iMyD88/CD40-DCs had increased activation of proinflammatory MAPKs. AP1903-activated iMyD88/CD40-transduced human or mouse DCs also produced higher levels of Th1 cytokines, showed improved migration in vivo, and enhanced both antigen-specific CD8+ T cell responses and innate NK cell responses. Furthermore, treatment with AP1903 in vaccinated mice led to robust antitumor immunity against preestablished E.G7-OVA lymphomas and aggressive B16.F10 tumors. Thus, the iMyD88/CD40 unified "switch" effectively and safely replaced exogenous adjuvant cocktails, allowing remote and sustained DC activation in vivo. DC "licensing" through iMyD88/CD40 may represent a mechanism by which to exploit the natural synergy between the TLR and CD40 signaling pathways in DCs using a single small molecule drug and could augment the efficacy of antitumor DC-based vaccines.

Stimulation through CD40 on mouse and human renal cell carcinomas triggers cytokine production, leukocyte recruitment, and antitumor responses that can be independent of host CD40 expression.

CD40, a member of the TNFR superfamily, is expressed on a variety of host immune cells, as well as some tumors. In this study, we show that stimulation of CD40 expressed on both mouse and human renal carcinoma cells (RCCs) triggers biological effects in vitro and in vivo. Treatment of the CD40+ Renca mouse RCC tumor cells in vitro with an agonistic anti-CD40 Ab induced strong expression of the genes and proteins for GM-CSF and MCP-1, and induced potent chemotactic activity. Similarly, administration of alphaCD40 to both wild-type and CD40-/- mice bearing Renca tumors resulted in substantial amounts of TNF-alpha and MCP-1 in the serum, increased the number of total splenocytes and MHC class II+ CD11c+ leukocytes, and when combined with IFN-gamma, inhibited the progression of established Renca tumors in vivo in both wild-type and CD40-/- mice. Similarly, treatment of CD40+ A704 and ACHN human RCC lines with mouse anti-human CD40 Ab induced strong expression of genes and proteins for MCP-1, IL-8, and GM-CSF in vitro and in vivo. Finally, in SCID mice, the numbers of ACHN pulmonary metastases were dramatically reduced by treatment with species-specific human CD40 Ab. These results show that CD40 stimulation of CD40+ tumor cells can enhance immune responses and result in antitumor activity.

Induction of antigen-dependent interleukin-12 production by negatively charged liposomes encapsulating antigens.

The aim of this study was to clarify the mechanism of adjuvant action induced by negatively charged liposomes composed of phosphatidylserine (PS-liposomes). Coculture of mouse splenic adherent cells with ovalbumin (OVA)-specific Th1 clone (42-6A) cells in the presence of PS-liposomes encapsulating OVA resulted in high levels of IL-12 and IFN-gamma productions compared with those of positively charged or neutral liposomes. IL-12 and IFN-gamma productions were dose-dependent on antigens in PS-liposomes. The expression of CD40L on 42-6A cells was enhanced by treatment with PS-liposomes encapsulating OVA. IL-12 production, but not IFN-gamma, was completely inhibited by the addition of anti-CD40L mAb. These results indicate that PS-liposomes encapsulating antigens could enhance antigen-dependent IL-12 production through the interaction between CD40 on macrophage/dendritic cells and CD40L on 42-6A cells.

CD40-mediated immune-nonimmune cell interactions induce mucosal fibroblast chemokines leading to T-cell transmigration.

BACKGROUND AND AIMS: The CD40 pathway is a key mediator of inflammation and autoimmunity. We investigated cell adhesion molecule (CAM) up-regulation and chemokine production by CD40-positive human intestinal fibroblasts (HIF) and microvascular endothelial cells (HIMEC) induced by CD40 ligand (CD40L)-positive T cells and soluble CD40L and their effect on T-cell adhesion and transmigration. METHODS: Expression of CD40, CD40L, and CAM was assessed by immunohistochemistry, confocal microscopy and flow cytometric analysis, and chemokine production using enzyme-linked immunosorbent assay. Calcein-labeled T cells were used to assay HIF adhesion and Transwell HIMEC transmigration. RESULTS: Ligation of CD40-positive HIF and HIMEC by CD40L-positive T cells or soluble CD40L induced up-regulation of CAM expression as well as interleukin-8 and RANTES production. The specificity of these responses was shown by inhibition with a CD40L blocking antibody and by CD40 signaling-dependent p38 mitogen-activated protein kinase phosphorylation. On CD40 ligation, HIF increased their T-cell binding capacity and generated chemoattractants able to induce T-cell migration through HIMEC monolayers. CONCLUSIONS: Activation of the CD40/CD40L system in the gut mucosa may trigger a self-sustaining loop of immune-nonimmune cell interactions leading to an antigen-independent influx of T cells that contributes to chronic inflammation.

Co-administration of CD40 agonistic antibody and antigen fails to overcome the induction of oral tolerance.

T-cell stimulation in the absence of a second, costimulatory signal can lead to anergy or deletion. There is growing evidence that peripheral tolerance to an exogenous antigen might be caused by the lack of costimulatory molecules on antigen-presenting cells (APCs). In the present study, we examined whether tolerance against orally administered antigen could be reversed by maturation of APCs via CD40 signalling. Monoclonal antibody (mAb) to CD40 efficiently induced costimulatory molecules on APCs. Treatment with anti-CD40 mAb potentiated the division of ovalbumin-specific T cells in response to oral ovalbumin in secondary lymphoid organs. However, such treatment did not prolong the presentation of oral ovalbumin on APCs. Surprisingly, treatment of anti-CD40 mAb at the time of oral administration of ovalbumin did not reverse the induction of tolerance to ovalbumin in either the high- or low-dose regimens. Furthermore, the induction of oral tolerance in our model is not the result of negative signalling by cytotoxic T-lymphocyte antigen-4. These results indicate that tolerance for oral antigen could be established regardless of APC maturation by a CD40-specific mAb, suggesting that there could be a unique mechanism to regulate immunity versus tolerance to encountered antigen in the gut-associated lymphoid tissue.

A novel approach for modifying tumor cell-derived plasma membrane vesicles to contain encapsulated IL-2 and engrafted costimulatory molecules for use in tumor immunotherapy.

The genetic modification of tumor cells and delivery of cytokines have been proposed as useful strategies in the development of anti-tumor vaccines; however, a number of factors limit their use in clinical settings. To facilitate vaccine development, we explored the possibility of modifying plasma membrane vesicles (PMV) by using a novel chelator lipid, nitrilotriacetic acid ditetradecylamine (NTA-DTDA). Our analyses by flow cytometry show that NTA-DTDA can be incorporated into PMV prepared from murine P815 mastocytoma and that the incorporated NTA-DTDA permits anchoring or "engraftment" onto the vesicle surface of hexahistidine-tagged proteins such as recombinant forms of the costimulatory molecules B7.1 and CD40. The engrafted PMV also can incorporate and deliver the immunostimulatory cytokine Interleukin-2 (IL-2). Our results show that modified PMV derived from P815 cells bind the murine T cell clone D10 in a receptor-ligand dependent manner, inducing cell adhesion and promoting cell survival in vitro. The modified PMV can bind syngeneic T cells, stimulating T cell proliferation and cytotoxic T cell responses. Moreover, when used as vaccines in syngeneic animals, the modified vesicles induce significant protection against challenge with the native P815 tumor. The results indicate that PMV modified by engraftment of recombinant forms of B7.1 and CD40 and incorporation of IL-2 can be used to modulate immune responses, which provides a novel approach for the development of anti-tumor vaccines and cancer immunotherapies.