Adenoviral CD40 Ligand Immunotherapy in 32 Canine Malignant Melanomas-Long-Term Follow Up

Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. Gene therapy using adenoviruses encoding the immunostimulatory gene CD40L (AdCD40L) has shown promise in initial clinical trials enrolling human patients with various malignancies including melanoma. We report a study of local AdCD40L treatment in 32 cases of canine melanoma (23 oral, 5 cutaneous, 3 ungual and 1 conjunctival). Eight patients were World Health Organization (WHO) stage I, 9 were stage II, 12 stage III, and 3 stage IV. One to six intratumoral injections of AdCD40L were given every seven days, combined with cytoreductive surgery in 20 cases and only immunotherapy in 12 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response based on result of immunotherapy included 7 complete responses, 5 partial responses, 5 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 285 days (range 20-3435 d). Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is ongoing.Peer reviewe

Strategic use of an adenoviral vector for rapid and efficient ex vivo-generation of cytomegalovirus pp65-reactive cytolytic and helper T cells

Cytomegalovirus (CMV) reactivation can cause severe complications for transplant patients. Such patients can be protected against CMV-associated diseases through reconstitution of donor-derived CMV-reactive cytolytic and helper T cells. We have developed a strategic protocol for efficient simultaneous generation of CMV-reactive CD8+ and CD4+ T cells ex vivo. The protocol uses peripheral blood lymphocytes (PBLs), antigen-modified mature dendritic cells (DCs) generated in only 3 days and an adenoviral vector encoding the CMV pp65 antigen (Adpp65) both as an endogenous and exogenous source of antigen. PBLs stimulated once with Adpp65-transduced DCs (endogenously expressed pp65) resulted in preferential activation and expansion of pp65-specific CD8+ T cells while PBLs stimulated with DCs pulsed with cell lysate from Adpp65-transduced autologous monocytes (exogenously expressed pp65) yielded pp65-specific CD4+ T cells. Stimulation with double-modified DCs efficiently activated and expanded cytolytic and helper T cells simultaneously. The frequency of T cells producing interferon (IFN)-γ in response to pp65 increased after one stimulation on average 9.6-fold to 4.3% for CD8+ T cells and 25.8-fold to 6.5% for CD4+ T cells. This implies that sufficient number of pp65-specific cytolytic and helper T cells for adoptive transfer may be obtained in only two weeks

Characterization of human prostate and breast cancer cell lines for experimental T cell-based immunotherapy

BACKGROUND. In order to develop experimental immunotherapy for prostate and breast cancer it is of outmost importance to have representative target cell lines that through human leukocyte antigen (HLA) class I molecules present relevant levels of peptides from tumor-associated antigens for cytotoxic T lymphocyte (CTL) recognition. METHODS. We sequenced the HLA-A and HLA-B loci of eight commonly used prostate and breast cancer cell lines and analyzed the surface expression of HLA-ABC, HLA-DR, CD40, CD80, CD86, and CD54 by flow cytometry. We also analyzed the cell lines for mRNA expression from 25 genes reported to be specifically or preferentially expressed by prostate cells. RESULTS. Among the analyzed cell lines we found that LNCaP, PC-346C and MCF-7 are HLA-A*0201 positive. However, the HLA-A2 expression level is low and only MCF-7 upregulates HLA-A2 in response to IFN-γ stimulation. MCF-7 also expresses high levels of CD54, which further improve its value as a CTL target cell line. On the other hand, LNCaP and PC-346C express 25 and 23 out of 25 prostate-related genes, respectively, while MCF-7 expresses 16 out of 25 genes. CONCLUSIONS. None of the analyzed prostate cancer cell lines are optimal CTL target cells. However, MCF-7 could in many cases be used as a complement to HLA-A*0201 positive prostate cancer cells. The LNCaP and PC-346C cell lines are rich sources of prostate-related antigens that may be valuable for cancer vaccine development

High frequency of prostate antigen-directed T cells in cancer patients compared to healthy age-matched individuals

BACKGROUND. In order to obtain a sustained cytotoxic T lymphocyte (CTL) response against cancer cells it is preferable to have CTLs directed against multiple peptide epitopes from numerous tumor-associated antigens. METHODS. We used a flow cytometry-based interferon (IFN)-g secretion assay to analyze whether CD8+ T cells directed against any of 24 HLA-A*0201-binding peptides from 15 prostate-associated proteins can be found in the peripheral blood of patients with localized prostate cancer. We also investigated whether multiple prostate antigen-specific CD8+ T cells can be generated simultaneously, from a naïve T cell repertoire. In that case, dendritic cells (DCs) from peripheral blood of healthy donors were divided in six portions and separately pulsed with six peptides. The peptide-pulsed DCs were then pooled and used to stimulate autologous T cells. The T cells were re-stimulated with peptide-pulsed monocytes. RESULTS. We found prostate antigen-restricted CD8+ T cells in the peripheral blood in 48 out of 184 (26.1%) analyzed samples from 25 cancer patients. This is significantly higher than 17 out of 214 analyzed samples (7.9%) from 10 healthy age-matched male individuals (p = 0.0249). In the cases when antigen-specific T cells could not be detected, we were able to generate IFN-g-producing CD8+ T cells specific for up to three prostate antigens simultaneously from a naïve T cell repertoire. CONCLUSIONS. CD8+ T cells directed against prostate antigen peptides can be found in, or generated from, peripheral blood. This indicates that such T cells could be expanded ex vivo for adoptive transfer to prostate cancer patients

Local CTLA4 blockade effectively restrains experimental pancreatic adenocarcinoma growth in vivo.

Antibody-mediated blockade of CTLA4 has been shown to be effective in treating a select group of patients with late-stage melanoma. The precise mechanism underlying the clinical activity of CTLA4 immunotherapy is poorly understood, although recent experimental findings indicate that antibody-mediated depletion of regulatory T cells (Tregs) in the tumor microenvironment plays a key role in efficacious antitumor responses. In the current study, we used an experimental model of pancreatic adenocarcinoma to compare the antitumor efficacy of peritumoral low-dose anti-CTLA4 monoclonal antibody (mAb) administration to that of a commonly utilized systemic high-dose anti-CTLA4 regimen. We selected pancreatic adenocarcinoma as it presents a particular challenge to clinicians due to its aggressive behavior, metastatic spread and limited treatment options. Furthermore, Fc gamma receptor (FcγR)-dense myeloid cells commonly infiltrate pancreatic tumors, such that these tumor types exhibit increased susceptibility to CTLA4 antibody-targeted Treg depletion via antibody-dependent cell-mediated cytotoxicity (ADCC). Locally administered anti-CTLA4 mAb effectively reduced tumor growth at a low dose and no additional anti-tumor effects were apparent when increasing the dose or number of injections. No significant difference in overall survival was seen when comparing locally administered low-dose with standard systemic high-dose CTLA4 blockade therapy, and both delivery routes led to increased tumor-infiltrating effector T cells and reduced Treg cells. As opposed to low-dose peritumoral treatment, high-dose systemic therapy stimulated the accumulation of Tregs in secondary lymphoid organs, an effect that could potentially counteract the antitumor immunotherapeutic benefit of CTLA4 blockade. Our study confirms previous findings that local administration of low-dose anti-CTLA4 antibody generates sustained antitumor effects and provides rationale to devise ultrasound-guided intratumoral anti-CTLA4 antibody injection regimens to treat patients with pancreatic adenocarcinoma and other types of solid tumors. In support, clinical relevancy could include reduced immune-related adverse events by limiting systemic antibody spread to immune cell-dense organs

Synergistic augmentation of CD40-mediated activation of antigen-presenting cells by amphiphilic poly(γ-glutamic acid) nanoparticles.

Agonistic anti-CD40 monoclonal antibodies (mAbs) hold great potential for cancer immunotherapy. However, systemic administration of anti-CD40 mAbs can be associated with severe side effects, such as cytokine release syndrome and liver damage. With the aim to increase the immunostimulatory potency as well as to achieve a local drug retention of anti-CD40 mAbs, we linked an agonistic mAb to immune activating amphiphilic poly(γ-glutamic acid) nanoparticles (γ-PGA NPs). We demonstrate that adsorption of anti-CD40 mAb to γ-PGA NPs (anti-CD40-NPs) improved the stimulatory capacity of the CD40 agonist, resulting in upregulation of costimulatory CD80 and CD86 on antigen-presenting cells, as well as IL-12 secretion. Interestingly, anti-CD40-NPs induced strong synergistic proliferative effects in B cells, possibly resulting from a higher degree of CD40 multimerization, enabled by display of multiple anti-CD40 mAbs on the NPs. In addition, local treatment with anti-CD40-NPs, compared to only soluble CD40 agonist, resulted in a significant reduction in serum levels of IL-6, IL-10, IL-12 and TNF-α in a bladder cancer model. Taken together, our results suggest that anti-CD40-NPs are capable of synergistically enhancing the immunostimulatory effect induced by the CD40 agonist, as well as minimizing adverse side effects associated with systemic cytokine release. This concept of nanomedicine could play an important role in localized immunotherapy of cancer

Local checkpoint inhibition of CTLA-4 as a monotherapy or in combination with anti-PD1 prevents the growth of murine bladder cancer

Checkpoint blockade of CTLA-4 results in long-lasting survival benefits in metastatic cancer patients. However, patients treated with CTLA-4 blockade have suffered from immune-related adverse events, most likely due to the breadth of the induced T-cell activation. Here, we investigated the efficacy of a local low-dose anti-CTLA-4 administration for treatment of subcutaneous or orthotopic murine bladder 49 (MB49) bladder carcinoma in C57BL/6 mice. When MB49 tumors were grown s.c., peritumoral (p.t.) injection of anti-CTLA-4 treatment was equally effective as intravenous or s.c. (nontumor bearing flank) administration. Notably, p.t. injection was associated with lower circulating antibody levels and decreased IL-6 serum levels as compared to systemic treatment. Ultrasound-guided intratumoral anti-CTLA-4 antibody treatment of orthotopically growing MB49 tumors resulted in tumor regression, with more than tenfold reduction in systemic antibody levels as compared to i.v. or s.c. administration, in line with the compartmentally restrained nature of the bladder. Local anti-CTLA-4 therapy in combination with anti-PD-1 therapy resulted in complete responses, superior to each therapy alone. In addition, p.t. anti-CTLA-4 therapy was potentiated by depletion of regulatory T cells. Our results demonstrate that local anti-CTLA-4 antibody therapy is equally effective as systemic administration, but reduces systemic antibody levels and cytokine release, and enhances the response to anti-PD1 therapy