Variations in “rescuability” of immunoglobulin molecules from different forms of human lymphoma: implications for anti-idiotype vaccine development

Idiotypic (Id) vaccination has shown promising results in patients with follicular lymphoma (FL). However, it still remains unclear whether the same approach might be suitable for the treatment of other B-cell malignancies. For this reason, we recently performed an interim analysis of patients proposed to receive this treatment at our center. The feasibility of employing idiotype vaccines was evaluated for five different B-cell malignancies in their first relapse, both in terms of induction and fusion, as well as overall treatment. Our data suggest that, unlike follicular lymphoma (87%), this approach is not feasible to treat other B-cell malignancies (0–20%) such as mantle cell, small lymphocytic, diffuse large cell and Burkitt’s lymphoma (P < 0.01). The main difficulties encountered were technical problems related to the survival of idiotype-producing hybridomas (83%) and the early loss of idiotype production by growing hybridomas (17%). However, it remains possible that an idiotype vaccine might still be produced through molecular means for most, if not all cases of relapsing B-cell malignancies

Active immunotherapy in the treatment of haematological neoplasias

Abstract The continuous search for therapeutic approaches that improve the conventional treatments of neoplasms, together with an improved understanding of the immune system, has led in recent years to the development of Immunotherapy. Basically, a distinction can be made between two forms of immunotherapy: passive immunotherapy, which consists in the transfer of antibodies or cells previously generated in vitro that are directed against the tumour, and active immunotherapy, which attempts to activate in vivo the immune system and induce it to elaborate a specific response against the tumor antibodies. Hematological neoplasms, specifically some B lymphomas, express in their membrane an immunoglobulin that is considered a specific antigen of the tumour, which is why these diseases have become the ideal target for immunotherapy treatments. There are many alternatives, ranging from protein vaccines, which have already shown clinical benefits, to those of the second generation, which make use of the new techniques of molecular biology to increase the efficacy of the vaccines and obtain their production in a quicker and less costly way, but with which there are not yet definitive clinical results

Clinical implications of antigen transfer mechanisms from malignant to dendritic cells: Exploiting cross-priming

Expansion and activation of cytolytic T lymphocytes bearing high-affinity T-cell receptors specific for tumor antigens is a major goal of active cancer immunotherapy. Physiologically, T cells receive promitotic and activating signals from endogenous professional antigen-presenting cells (APC) rather than directly from malignant cells. This phenomenon fits with the broader concept of cross-presentation that earlier was demonstrated for minor histocompatibility and viral antigens. Many mechanisms have been found to be capable of transferring antigenic material from malignant cells to APC so that it can be processed and subsequently presented by MHC class I molecules expressed on APC. Dendritic cells (DC) are believed to be the most relevant APC mediating cross-presentation because they can take up antigens from apoptotic, necrotic, and even intact tumor cells. There exist specific molecular mechanisms that ensure this transfer of antigenic material: 1) opsonization of apoptotic bodies; 2) receptors for released heat shock proteins carrying peptides processed intracellularly; 3) Fc receptors that uptake immunocomplexes and immunoglobulins; and 4) pinocytosis. DC have the peculiar capability of reentering the exogenously captured material into the MHC class I pathway. Exploitation of these pieces of knowledge is achieved by providing DC with complex mixtures of tumor antigens ex vivo and by agents and procedures that promote infiltration of malignant tissue by DC. The final outcome of DC cross-presentation could be T-cell activation (cross-priming) but also, and importantly, T-cell tolerance contingent upon the activation/maturation status of DC. Artificial enhancement of tumor antigen cross-presentation and control of the immune-promoting status of the antigen-presenting DC will have important therapeutic implications in the near future

Mitochondrial Morphological and Functional Reprogramming Following CD137 (4-1BB) Costimulation.

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798-811. ©2018 AACR.This project was supported by imCORE Network (Roche Genentech), MINECO (SAF2014-52361-R, 2017-83267-C2-1-R; I. Melero), European Commission VII Framework and Horizon 2020 programs (IACT and PROCROP), Fundacion de la Asociaci on Espa nola Contra el C ~ ancer (AECC), and Fundacion BBVA. A. Teijeira receives support from a Juan de la Cierva contract, MINECO. Electron and light microscopy CIMA services as well as Flow Cytometry CIMA facilities (Diego Alignani) and personnel at blood bank of Navarra are acknowledged for their technical support. We are also grateful to Drs. Lasarte and HervasStubbs for scientific discussion and to Dr. Paul Miller for editing the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.S

Acquired potential N-glycosylation sites within the tumor-specific immunoglobulin heavy chains of B-cell malignancies

Background and Objectives. Among B-cell malignancies, follicular lymphomas (FL) more frequently show acquired, potential N-glycosylation sites (AGS) within tumor-specific immunoglobulin. The aim of this study was to extend this observation and to evaluate the pattern of presentation of AGS within five different forms of B-cell lymphoma. Design and Methods. We sequenced the tumor-specific immunoglobulin heavy chain variable region fragment, including complementarity-determining regions 2 and 3, of forty-seven consecutive patients with a B-cell malignancy enrolled in idiotype vaccine clinical trials. This sequencing approach is known to allow the identification of most AGS. We then statistically analyzed differences in presentation pattern, in terms of tumor histology, immunoglobulin isotype, AGS location and amino acid composition. Results. All twenty-four FL cases presented with at least one AGS, whereas the vast majority of four B-cell lymphoma types other than FL did not. The non- FL group of tumors included four cases of Burkitt’s lymphoma, six of diffuse large cell lymphoma, seven mantle cell lymphomas and six small lymphocytic lymphomas. Most IgM-bearing follicular lymphoma cases featured their AGS within complementarity-determining region 2, as opposed to those bearing an IgG, which mostly displayed the AGS within complementarity- determining region 3. The vast majority of AGS located within either complementarity- determining region ended with a serine residue, whereas those located within framework regions mostly featured threonine as the last amino acid residue. Interpretation and Conclusions. In our series, all cases of FL had AGS within their tumor-specific immunoglobulin heavy chain variable regions. In contrast, most B-cell malignancies other than FL did not. Further studies are warranted in order to establish the possible meaning of these findings in terms of disease pathogenesis, their diagnostic value in doubtful cases and their potential implications for immunotherapy

Identification of the B-cell tumor-specific molecular fingerprint using non-radiolabelled PCR consensus primers

Abstract BACKGROUND: The complementarity determining region 3 (CDR3) of the immunoglobulin (Ig) heavy chain variable region (VH) is the most reliable molecular fingerprint for most if not all human B cells. The nucleotide sequence encoding for any B-cell tumor-specific VH CDR3 is currently identified by PCR sequencing based on procedures involving the usage of either radioactive materials, patient/family-specific primers, or bacterial cloning. PATIENTS AND METHODS: In six consecutive patients with follicular lymphoma we assessed the feasibility of a method that allows for identification of the tumor-specific VH CDR3 using consensus primers while avoiding both radioactive materials and bacterial cloning procedures. RESULTS: The tumor-specific VH CDR3 was successfully identified in all six patients in nearly half the time typically required by any other method currently utilized. The feasibility of the proposed method was not significantly affected either by the tumor-specific Ig isotype, or by the tumor infiltration in the original biopsy specimen. In the three patients for whom tumor specimen-derived hybridomas were available, the tumor-specific VH CDR3 was also found in at least 8 of 10 of them. CONCLUSIONS: The proposed method allows the ability to quickly identify the B-cell tumor-specific VH CDR3 using consensus primers while avoiding radioactive materials and bacterial cloning procedures

Imatinib inhibits proliferation of Ewing tumor cells mediated by the stem cell factor/KIT receptor pathway, and sensitizes cells to vincristine and doxorubicin-induced apoptosis

Purpose and Experimental Design: The stem cell factor/ KIT receptor loop may represent a novel target for molecular- based therapies of Ewing tumor. We analyzed the in vitro impact of KIT blockade by imatinib in Ewing tumor cell lines. Results: KIT expression was detected in 4 of 4 Ewing tumor cell lines and in 49 of 110 patient samples (44.5%) by immunohistochemistry and/or Western blot analysis. KIT expression was stronger in Ewing tumors showing EWSFLI1 nontype 1 fusions. Despite absence of c-kit mutations, constitutive and ligand-inducible phosphorylation of KIT was found in all tumor cell lines, indicating an active receptor. Treatment with KIT tyrosine kinase inhibitor imatinib (0.5–20 M) induced down-regulation of KIT phosphorylation and dose response inhibition of cell proliferation (IC50, 12–15 M). However, imatinib administered alone at doses close to IC50 for growth inhibition (10 M) did not induce a significant increase in apoptosis. We then analyzed if blockade of KIT loop through imatinib (10 M) was able to increase the antitumor in vitro effect of doxorubicin (DXR)and vincristine (VCR), drugs usually used in Ewing tumor treatment. Addition of imatinib decreased in 15–20 and 15–36% of the proliferative rate of Ewing tumor cells exposed to DXR and VCR, respectively, and increased in 15 and 30% of the apoptotic rate of Ewing tumor cells exposed to the same drugs. Conclusions: Inhibition of Ewing tumor cell proliferation by imatinib is mediated through blockade of KIT receptor signaling. Inhibition of KIT increases sensitivity of these cells to DXR and VCR. This study supports a potential role for imatinib in the treatment of Ewing tumor

Enhancement of antibody-dependent cellular cytotoxicity of cetuximab by a chimeric protein encompassing interleukin-15

Enhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8+ T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8+ T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo. The EGFR+ human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2-/-γc-/- mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1-/- mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens

Improved Endpoints for Cancer Immunotherapy Trials

Unlike chemotherapy, which acts directly on the tumor, cancer immunotherapies exert their effects on the immune system and demonstrate new kinetics that involve building a cellular immune response, followed by changes in tumor burden or patient survival. Thus, adequate design and evaluation of some immunotherapy clinical trials require a new development paradigm that includes reconsideration of established endpoints. Between 2004 and 2009, several initiatives facilitated by the Cancer Immunotherapy Consortium of the Cancer Research Institute and partner organizations systematically evaluated an immunotherapy-focused clinical development paradigm and created the principles for redefining trial endpoints. On this basis, a body of clinical and laboratory data was generated that supports three novel endpoint recommendations. First, cellular immune response assays generate highly variable results. Assay harmonization in multicenter trials may minimize variability and help to establish cellular immune response as a reproducible biomarker, thus allowing investigation of its relationship with clinical outcomes. Second, immunotherapy may induce novel patterns of antitumor response not captured by Response Evaluation Criteria in Solid Tumors or World Health Organization criteria. New immune-related response criteria were defined to more comprehensively capture all response patterns. Third, delayed separation of Kaplan–Meier curves in randomized immunotherapy trials can affect results. Altered statistical models describing hazard ratios as a function of time and recognizing differences before and after separation of curves may allow improved planning of phase III trials. These recommendations may improve our tools for cancer immunotherapy trials and may offer a more realistic and useful model for clinical investigation