PD-1 derived CA-170 is an oral immune checkpoint inhibitor that exhibits preclinical anti-tumor efficacy.

Small molecule immune checkpoint inhibitors targeting PD-1 and other pathways may offer advantages including ease of dosing, ability to manage immune-related adverse events (irAEs) due to their shorter pharmacokinetic exposure and opportunity to target more than one pathway for improving efficacy. Here we describe the identification and characterization of CA-170, an amino acid inspired small molecule inhibitor of PD-L1 and VISTA derived from the interface of PD-1 and PD-L1. CA-170 exhibited potent rescue of proliferation and effector functions of T cells inhibited by PD-L1/L2 and VISTA with selectivity over other immune checkpoint proteins as well as a broad panel of receptors and enzymes. Observed blocking of PD-L1 signaling and binding to PD-L1 in the cellular context without preventing the assembly of PD-1:PD-L1 complex support the formation of a defective ternary complex as the mechanism of action of CA-170. Oral administration of CA-170 resulted in increased proliferation and activation of T cells in the tumor, and significant anti-tumor efficacy in a number of immunocompetent mouse tumor models either as a single agent or in combination with approved therapeutics. These results prompted the advancement of CA-170 to human clinical trials.

ODM-203, a Selective Inhibitor of FGFR and VEGFR, Shows Strong Antitumor Activity, and Induces Antitumor Immunity.

Alterations in the gene encoding for the FGFR and upregulation of the VEGFR are found often in cancer, which correlate with disease progression and unfavorable survival. In addition, FGFR and VEGFR signaling synergistically promote tumor angiogenesis, and activation of FGFR signaling has been described as functional compensatory angiogenic signal following development of resistance to VEGFR inhibition. Several selective small-molecule FGFR kinase inhibitors are currently in clinical development. ODM-203 is a novel, selective, and equipotent inhibitor of the FGFR and VEGFR families. In this report we show that ODM-203 inhibits FGFR and VEGFR family kinases selectively and with equal potency in the low nanomolar range (IC50 6-35 nmol/L) in biochemical assays. In cellular assays, ODM-203 inhibits VEGFR-induced tube formation (IC50 33 nmol/L) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC50 50-150 nmol/L). In vivo, ODM-203 shows strong antitumor activity in both FGFR-dependent xenograft models and in an angiogenic xenograft model at similar well-tolerated doses. In addition, ODM-203 inhibits metastatic tumor growth in a highly angiogenesis-dependent kidney capsule syngenic model. Interestingly, potent antitumor activity in the subcutaneous syngenic model correlated well with immune modulation in the tumor microenvironment as indicated by marked decrease in the expression of immune check points PD-1 and PD-L1 on CD8 T cells and NK cells, and increased activation of CD8 T cells. In summary, ODM-203 shows equipotent activity for both FGFR and VEGFR kinase families and antitumor activity in both FGFR and angigogenesis models.

Definition and characterization of novel HLA-*A02-restricted CD8+ T cell epitopes derived from JCV polyomavirus with clinical relevance.

Human JC and BK polyomaviruses (JCV/BKV) can establish a latent infection without any clinical symptoms in healthy individuals. In immunocompromised hosts infection or reactivation of JCV and BKV can cause lethal progressive multifocal leukoencephalopathy (PML) and hemorrhagic cystitis, respectively. Vaccination with JCV/BKV derived antigen epitope peptides or adoptive transfer of virus-specific T cells would constitute an elegant approach to clear virus-infected cells. Furthermore, donor leukocyte infusion (DLI) is another therapeutic approach which could be helpful for patients with JCV/BKV infections.So far, only few immunodominant T cell epitopes of JCV and BKV have been described and therefore is a fervent need for the definition of novel epitopes. In this study, we identified novel T cell epitopes by screening libraries of overlapping peptides derived from the major capsid protein VP1 of JCV. Virus like particles (VLPs) were used to confirm naturally processing. Two human leucocyte antigen (HLA)-A*02-restricted epitopes were characterized by fine mapping with overlapping peptides and nonamer peptide sequences were identified. Cytokine release profile of the epitope-specific T cells was analyzed by enzyme-linked immunospot (ELISPOT) assays and by flow cytometry. We demonstrated that T cell responses were of polyfunctional nature with the potential of epitope-specific killing and cross-reactivity between JCV and BKV. These novel epitopes might constitute a new potential tool to design effective diagnostic and therapeutic approaches against both polyomaviruses.

Standardization of cryopreserved peripheral blood mononuclear cells through a resting process for clinical immunomonitoring--Development of an algorithm.

Flow cytometry, as a powerful tool for immunomonitoring and quality control of peripheral blood mononuclear cells (PBMCs), is routinely used in clinical studies. However, flow cytometry based assays for cryopreserved peripheral blood mononuclear cells (cPBMCs) constitute a challenge. Down-regulation of surface and intracellular markers, as well as impairment of cell function might result from cryopreservation. Furthermore, protocols for resting cPBMCs are available but diverse. Therefore, we performed a standardization of the resting process concerning resting position, cell concentration, resting period and material of cell culture tubes as well as culture media. We further investigated the influence of resting on the phenotype and functionality of T cells comparing fresh PBMCs as gold standard to rested and non-rested cPBMCs. Polychromatic flow cytometry staining, peptide-MHC class I restricted tetramer staining and intracellular cytokine staining as major methods were used. Our results revealed that a horizontal position, a cell concentration of 2 to 5 × 10(6) cells/ml and an overnight resting phase is beneficial to eliminate dead or dying cells in cPBMCs with a mean cell loss of 14% overall cell populations. In addition, the quality and quantity of regulatory T cells and antigen specific T cells recovered upon resting. For multifunctional T cells a decrease of activation threshold in the way of a twofold mean fluorescence intensity (MFI) and increase of degranulation marker CD107a, co-stimulatory marker CD28, adhesion molecule CD62L as well as the ability to secrete antiviral cytokines like interferon gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin 2 (IL-2) comparable to fresh PBMCs were observed. However, based upon our data resting is not helpful for the flow cytometric analyses of myeloid-derived suppressor cells (MDSCs) and large/intermediate size lymphocytes which rather decreased/vanished ex vivo. Therefore, we developed an algorithm to indicate for which cell population and for which type of analyses the resting process is useful or not.

T cell-based targeted immunotherapies for patients with multiple myeloma.

Despite high-dose chemotherapy followed by autologs stem-cell transplantation as well as novel therapeutic agents, multiple myeloma (MM) remains incurable. Following the general trend towards personalized therapy, targeted immunotherapy as a new approach in the therapy of MM has emerged. Better progression-free survival and overall survival after tandem autologs/allogeneic stem cell transplantation suggest a graft versus myeloma effect strongly supporting the usefulness of immunological therapies for MM patients. How to induce a powerful antimyeloma effect is the key issue in this field. Pivotal is the definition of appropriate tumor antigen targets and effective methods for expansion of T cells with clinical activity. Besides a comprehensive list of tumor antigens for T cell-based approaches, eight promising antigens, CS1, Dickkopf-1, HM1.24, Human telomerase reverse transcriptase, MAGE-A3, New York Esophageal-1, Receptor of hyaluronic acid mediated motility and Wilms' tumor gene 1, are described in detail to provide a background for potential clinical use. Results from both closed and on-going clinical trials are summarized in this review. On the basis of the preclinical and clinical data, we elaborate on three encouraging therapeutic options, vaccine-enhanced donor lymphocyte infusion, chimeric antigen receptors-transfected T cells as well as vaccines with multiple antigen peptides, to pave the way towards clinically significant immune responses against MM.

Modulation of lymphocyte subpopulations by extracorporeal photopheresis in patients with acute graft-versus-host disease or graft rejection.

Extracorporeal photopheresis (ECP) constitutes a promising treatment for patients with steroid-refractory acute graft-versus-host disease (aGvHD) after allogeneic stem cell transplantation and for patients with graft rejection after solid organ transplantation (SOT). There is an increasing body of evidence that modulation of lymphocyte subsets might play a crucial role in the mechanism of action in ECP. We therefore analyzed immunological effects concomitantly with clinical findings in patients under ECP therapy using multicolor flow cytometry. In a patient with steroid-refractory aGvHD and a patient with progressive bronchiolitis obliterans syndrome (BOS) after double-lung transplantation, clinical responses to ECP therapy were paralleled by an increase of CD4 + CD25hiFoxP3 + regulatory T cells and a decrease of T(EMRA) (CD3 + CD8+ CD45RA+ CD62L+ effector memory T) cells as well as of natural killer (NK)T cells. In summary, immunomonitoring of T cell subsets can elucidate the mechanism of action in ECP.

Cellular immunotherapy for patients with reactivation of JC and BK polyomaviruses after transplantation.

Immunosuppression of patients after hematopoietic stem cell or kidney transplantation potentially leads to reactivation of JC and BK polyomaviruses. In hematopoietic stem cell transplantation, the reactivation rate of BKV can be up to 60%, resulting in severe complications of the urogenital tract, particularly hemorrhagic cystitis and renal dysfunction. After kidney transplantation, BKV reactivation can cause a loss of the graft. JCV can cause progressive multifocal leukoencephalopathy, a lethal disease. Adoptive transfer of donor-derived polyomavirus-specific T cells is an attractive and promising treatment that restores virus-specific cellular immunity. Pioneering work in the early 1990s on the reconstitution of cellular immunity against cytomegalovirus and recent development in the field of monitoring and isolation of antigen-specific T cells paved the way toward a personalized T-cell therapy. Multimer technology and magnetic beads are available to produce untouched T cells in a single-step, good manufacturing practice-compliant procedure. Another exciting aspect of T-cell therapy against polyomaviruses is the fact that both JCV and BKV can be targeted simultaneously because of their high sequence homology. Finally, "designer T cells" can be redirected to recognize polyomavirus antigens with high-affinity T-cell receptors. This review summarizes the state-of-the art technologies and gives an outlook of future developments in the field.

Biosimilar G-CSF based mobilization of peripheral blood hematopoietic stem cells for autologous and allogeneic stem cell transplantation.

The use of granulocyte colony stimulating factor (G-CSF) biosimilars for peripheral blood hematopoietic stem cell (PBSC) mobilization has stimulated an ongoing debate regarding their efficacy and safety. However, the use of biosimilar G-CSF was approved by the European Medicines Agency (EMA) for all the registered indications of the originator G-CSF (Neupogen (®) ) including mobilization of stem cells. Here, we performed a comprehensive review of published reports on the use of biosimilar G-CSF covering patients with hematological malignancies as well as healthy donors that underwent stem cell mobilization at multiple centers using site-specific non-randomized regimens with a biosimilar G-CSF in the autologous and allogeneic setting. A total of 904 patients mostly with hematological malignancies as well as healthy donors underwent successful autologous or allogeneic stem cell mobilization, respectively, using a biosimilar G-CSF (520 with Ratiograstim®/Tevagrastim, 384 with Zarzio®). The indication for stem cell mobilization in hematology patients included 326 patients with multiple myeloma, 273 with Non-Hodgkin's lymphoma (NHL), 79 with Hodgkin's lymphoma (HL), and other disease. 156 sibling or volunteer unrelated donors were mobilized using biosimilar G-CSF. Mobilization resulted in good mobilization of CD34+ stem cells with side effects similar to originator G-CSF. Post transplantation engraftment did not significantly differ from results previously documented with the originator G-CSF. The side effects experienced by the patients or donors mobilized by biosimilar G-CSF were minimal and were comparable to those of originator G-CSF. In summary, the efficacy of biosimilar G-CSFs in terms of PBSC yield as well as their toxicity profile are equivalent to historical data with the reference G-CSF.

Suppression of cytomegalovirus-specific CD8(+)T cells by everolimus.

Abstract Everolimus (RAD-001) has recently been used as an immunosuppressive drug to treat patients after hematopoietic stem cell transplant (HSCT), thereby reducing cyclosporine-related nephrotoxicity. We studied the immunomodulatory effect of everolimus on mitogen-stimulated and particularly cytomegalovirus (CMV)-specific cytotoxic T cells. Proliferation of CD4(+) and CD8(+) T cells stimulated with staphylococcal endotoxin B and phytohemagglutinin was strongly inhibited at very low doses. Proliferation of CMV-specific CD8(+) T cells could be completely suppressed. Similarly, the frequency of CMV-specific, cytokine-secreting and CD137-expressing CD8(+) T cells decreased in a dose-dependent manner. However, interferon-γ (IFN-γ) secretion by CMV-specific CD8(+) T cells remained unchanged, as could be demonstrated by intracellular cytokine staining. As reactivation of CMV plays a pivotal role in the outcome of patients after HSCT, attention must be paid to early detection and preemptive treatment of CMV reactivity in patients treated with everolimus.

Therapeutical doses of temozolomide do not impair the function of dendritic cells and CD8+ T cells.

The median survival of patients with glioblastoma multiforme (GBM) remains poor. Innovative immunotherapies with dendritic cell (DC) vaccination might be combined with standard temozolomide (TMZ) treatment. Here, we evaluated the influence of TMZ on the phenotype and function of DCs and CD8+ T cells. DCs were generated from the peripheral blood of healthy volunteers (HVs) and GBM patients. DCs were analyzed by light microscopy and flow cytometry. Phagocytic activity was tested by FITC-dextran engulfment. Mixed lymphocyte peptide cultures were followed by enzyme-linked immunospot (ELISPOT) and flow cytometry assays. TMZ was added to DC and T cell cultures at concentrations up to 500 µM. Mature DCs were generated from HVs and GBM patients. Cells displayed a typical DC morphology and a mature DC phenotype. Expression of CD209 was even higher in DCs generated from patients under therapy than from HVs (75.2 vs. 51.1%). In contrast, CD40 (1.1 vs. 13.5%) and BDCA4 (26.5 vs. 52.9%) were lower expressed in GBM patients at time of diagnosis. Immature DCs showed high phagocytic activity. Addition of TMZ at concentrations up to 50 µM did neither impair the phenotype nor the function of DCs. In ELISPOT and flow cytometry assays, no impairment of CD8+ T cell responses to viral antigens could be observed. Taken together, TMZ does not impair the function of either DCs or the CD8+ T cells.

Activation of anti-tumor immune response and reduction of regulatory T cells with Mycobacterium indicus pranii (MIP) therapy in tumor bearing mice.

BACKGROUND: Role of immune system in protecting the host from cancer is well established. Growing cancer however subverts immune response towards Th2 type and escape from antitumor mechanism of the host. Activation of both innate and Th1 type response is crucial for host antitumor activity. In our previous study it was found, that Mycobacterium indicus pranii (MIP) also known as M. w induces Th1 type response and activates macrophages in animal model of tuberculosis. Hence, we studied the immunotherapeutic potential of MIP in mouse tumor model and the underlying mechanisms for its antitumor activity. METHODOLOGY AND PRINCIPAL FINDINGS: Tumors were implanted by injecting B16F10 melanoma cells subcutaneously into C57BL/6 mice. Using the optimized dose and treatment regimes, anti-tumor efficacy of heat killed MIP was evaluated. In MIP treated group, tumor appeared in only 50-60% of mice, tumor growth was delayed and tumor volume was less as compared to control. MIP mediated immune activation was analysed in the tumor microenvironment, tumor draining lymph node and spleen. Induction of Th1 response and higher infiltration of immune cells in the tumor microenvironment was observed in MIP treated mice. A large fraction of these immune cells were in activated state as confirmed by phenotypic and functional analysis. Interestingly, percentage of Treg cells in the tumor milieu of treated mice was less. We also evaluated efficacy of MIP along with chemotherapy and found a better response as compared to chemotherapy alone. CONCLUSION: MIP therapy is effective in protecting mice from tumor. It activates the immune cells, increases their infiltration in tumor, and abrogates tumor mediated immune suppression.

Streptamer-based selection of WT1-specific CD8+ T cells for specific donor lymphocyte infusions.

OBJECTIVE: Donor lymphocyte infusions may generate a desirable graft-versus-leukemia effect, but also elicit a noxious graft-versus-host disease. A positive selection of leukemia (antigen)-specific T cells would be highly desirable. In this study, we focused on the immunogenic leukemia antigen Wilms' Tumor gene 1 (WT1). MATERIALS AND METHODS: We employed the technology of streptamers available at good manufacturing practice level to first determine the frequency of human leukocyte antigen-A2 restricted WT1-specific CD8(+) T cells. Then, specific cells were labeled with streptamers and selected by magnetic cell separation. Purity and immunophenotype of selected cells were analyzed. RESULTS: Twenty-one of 40 healthy donors had naïve WT1-specific CD8(+) T-cell frequencies of >0.5%, and 8 of 40 even >1.0% of all CD8(+) T cells. In 7 of 10 acute myeloid leukemia patients, the frequencies were 0.5% to 3.65%. After positive selection by magnetic cell separation, a 60-fold increase with a purity of up to 17.79% in the lymphocyte gate and 86.18% in the CD8(+) T-cell gate could be achieved for CD8(+)WT1 streptamer(+)CD28(+/-)CD45RA(+)CCR7(-) effector T cells. CONCLUSIONS: Streptamer technology allows selection of pure WT1-specific effector T cells. This is a prerequisite for clinical applications targeting tumor-specific antigens, such as adoptive T-cell transfer.

Comparison of susceptibility to opsonic killing by in vitro human immune response of Enterococcus strains isolated from dairy products, clinical samples and probiotic preparation.

The genus Enterococcus like other LAB has also been featured in food and probiotic industry for decades due to its specific biochemical traits and beneficial health claims. At the same time, some enterococcal strains present an emerging pool of opportunistic pathogens for humans and are frequently armed with potential virulence factors. Thus, there is a need to assure the safety of enterococci before their use in food and probiotic preparations. Opsonophagocytic assay is an important test for the safety assessment of enterococci. In the present study comparative safety assessment of the different enterococcal strains isolated from dairy products, faeces, clinical samples and a commercial probiotic preparation was carried out by in vitro opsonophagocytic assay. Eleven strains of Enterococcus spp. were tested for their susceptibility to killing by opsonophagocytic assay. Among them, six isolates (Enterococcus faecium strain) were from our previous study (isolated from dairy products and faecal sample), four were from clinical samples and one from a probiotic preparation. Five out of six previous isolates and the isolate from probiotic preparation showed higher susceptibility to killing in contrast to the clinical isolates. The difference in the susceptibility to opsonic killing among the clinical and non-clinical Enterococcus isolates may be attributed to the presence of a capsule in the former, which protect them against the opsonophagocytic killing. Thus, these susceptible E. faecium strains may be designated as safe. However, certain other virulence traits must be evaluated prior to their exploitation in food and probiotic preparations.

Immunogenicity and protective efficacy of "Mycobacterium w" against Mycobacterium tuberculosis in mice immunized with live versus heat-killed M. w by the aerosol or parenteral route.

As the disease caused by Mycobacterium tuberculosis continues to be a burden, there is a concerted effort to find new vaccines to combat this problem. One of the important vaccine strategies is whole bacterial vaccines. This approach relies on multiple antigens and built-in adjuvanticity. Other mycobacterial strains which share cross-reactive antigens with M. tuberculosis have been considered as alternatives to M. bovis for vaccine use. One such strain, "Mycobacterium w", had been evaluated for its immunomodulatory properties in leprosy. A vaccine against leprosy based on killed M. w is approved for human use, where it has resulted in clinical improvement, accelerated bacterial clearance, and increased immune responses to Mycobacterium leprae antigens. M. w shares antigens not only with M. leprae but also with M. tuberculosis, and initial studies have shown that vaccination with killed M. w induces protection against tuberculosis in Mycobacterium bovis BCG responder, as well as BCG nonresponder, strains of mice. Hence, we further studied the protective potential of M. w and the underlying immune responses in the mouse model of tuberculosis. We analyzed the protective efficacy of M. w immunization in both live and killed forms through the parenteral route and by aerosol immunization, compared with that of BCG. Our findings provide evidence that M. w has potential protective efficacy against M. tuberculosis. M. w activates macrophage activity, as well as lymphocytes. M. w immunization by both the parenteral route and aerosol administration gives higher protection than BCG given by the parenteral route in the mouse model of tuberculosis.