In vitro and in vivo analysis of human cell-based immunotherapies for acute myeloid leukemia

Conventional cancer therapies like surgery, radiation and chemotherapy help to eliminate primary tumor masses but often fail to eradicate disseminated tumor cells. However, it is such residual tumor cells that frequently underlie metastasis and relapse. Major obstacles for targeting such cells are wide spread dissemination and long-term persistence in niches that are difficult to reach. For example, many patients with acute myeloid leukemia (AML) show persistence of leukemia after chemotherapy – so-called minimal residual disease (MRD) – which confers a life-threatening risk for relapse in over 70% of patients. Arming the immune system to attack residual tumor cells has high therapeutic potential since immune cells can patrol the body to find and destroy residual tumor cells. Therapeutic approaches using the immune system - so-called immunotherapies - can take several forms. My project concentrated on preclinical studies of two strategies: 1) use of dendritic cells (DC) for therapeutic vaccination and 2) adoptive T cell therapy with lymphocytes expressing transgenic T cell receptors (TCR) specific for tumor-associated antigens (TAA). In therapeutic vaccination a highly potent vaccine is needed to induce a valid immune response in patients with cancer. Effective antitumor immunity requires mobilization of IFN-γ-producing CD4+ T cells (Th1 cells) and lymphocytes with cytotoxic function, including cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In my studies, high potency vaccines were developed using mature DC generated in 3 days (3d-mDC) that were stimulated with synthetic Toll-like receptor TLR3 and/or TLR7/8 agonists. This TLR stimulation mimics DC interaction with viruses and causes mDC to secrete the bioactive form of IL-12, supporting induction of effector cells. Characterization in vitro showed that TLR-activated 3d-mDC were superior to conventional 7d-mDC in capacity to induce Th1 cells as well as CTL. A humanized mouse model was established to verify these observations in vivo. NOD/scid IL2Rgnull mice, lacking murine T, B and NK cells, were reconstituted with human peripheral mononuclear cells and vaccinated with 3d-mDC, stimulated or not with TLR agonists, and conventional 7d-mDC. Induction of CTL was quantified ex vivo using splenocyte populations containing human lymphocytes. The in vivo results were concordant with in vitro observations, demonstrating the superior capacity of 3d-mDC that were stimulated with TLR agonists to induce CTL. Adoptive T cell therapy using TCR-modified lymphocytes represents a second powerful way to provide patients with specific antitumor immunity. Here previously isolated TCR gene sequences are introduced into activated patient-derived lymphocytes, assigning them new antigen specificities. First, T cells must be isolated with TAA specificities that express high-affinity TCR which effectively recognize tumor cells. It was contended that T cell stimulation using peptide-epitopes from TAA presented on foreign MHC would allow isolation of high-affinity TCR, since these T cells had not yet undergone negative selection in the thymus. This contention was proved in individual experiments, as described in this thesis, for the antigens tyrosinase, survivin and HMMR (hyaluronan-mediated motility receptor). Since survivin and HMMR are broadly expressed in AML, TCR specific for these TAA were isolated and subsequently transferred into recipient lymphocytes. Expression of survivin-specific TCR resulted in MHC-restricted death of transduced lymphocytes due to their elevated survivin expression after activation. This precludes use of survivin-specific TCR for therapy of AML. In contrast, transfer of an HMMR-specific TCR yielded effector lymphocytes that effectively killed AML cells in vitro. The behavior in vivo of TCR transduced lymphocytes is crucial for therapeutic outcome. To explore this capacity a xenograft mouse model was established using solid and disseminated human tumor cells injected into NOD/scid IL2Rgnull mice. Adoptive transfer of lymphocytes expressing an HMMR-specific TCR into tumor-bearing mice resulted in significant retardation of tumor outgrowth. Adoptive transfer of memory-like lymphocytes with higher proliferative potential and prolonged in vivo survival may also affect tumor growth. Analyses in vivo and in vitro showed that IL-15-induced effector memory T cells conferred the most potent antitumor immunity. In summary, this work provides evidence for potent in vivo antitumor effects by either using DC-based vaccines or adoptive transfer of TCR transduced lymphocytes, opening application of both strategies for immunotherapy of cancer

NOD/scid IL-2Rgnull mice: a preclinical model system to evaluate human dendritic cell-based vaccine strategies in vivo

<p>Abstract</p> <p>Background</p> <p>To date very few systems have been described for preclinical investigations of human cellular therapeutics <it>in vivo</it>. However, the ability to carry out comparisons of new cellular vaccines <it>in vivo </it>would be of substantial interest for design of clinical studies. Here we describe a humanized mouse model to assess the efficacy of various human dendritic cell (DC) preparations. Two reconstitution regimes of NOD/scid IL2Rg^null(NSG) mice with adult human peripheral blood mononuclear cells (PBMC) were evaluated for engraftment using 4-week and 9-week schedules. This led to selection of a simple and rapid protocol for engraftment and vaccine evaluation that encompassed 4 weeks.</p> <p>Methods</p> <p>NSG recipients of human PBMC were engrafted over 14 days and then vaccinated twice with autologous DC via intravenous injection. Three DC vaccine formulations were compared that varied generation time <it>in vitro </it>(3 days versus 7 days) and signals for maturation (with or without Toll-like receptor (TLR)3 and TLR7/8 agonists) using MART-1 as a surrogate antigen, by electroporating mature DC with <it>in vitro </it>transcribed RNA encoding full length protein. After two weekly vaccinations, the splenocyte populations containing human lymphocytes were recovered 7 days later and assessed for MART-1-specific immune responses using MHC-multimer-binding assays and functional assessment of specific killing of melanoma tumor cell lines.</p> <p>Results</p> <p>Human monocyte-derived DC generated <it>in vitro </it>in 3 days induced better MART-1-specific immune responses in the autologous donor T cells present in the humanized NSG mice. Moreover, consistent with our <it>in vitro </it>observations, vaccination using mature DC activated with TLR3 and TLR7/8 agonists resulted in enhanced immune responses <it>in vivo</it>. These findings led to a ranking of the DC vaccine effects <it>in vivo </it>that reflected the hierarchy previously found for these mature DC variations <it>in vitro</it>.</p> <p>Conclusions</p> <p>This humanized mouse model system enables comparisons among different DC vaccine types to be rapidly assessed <it>in vivo</it>. In addition, <it>ex vivo </it>analyses of human CD3⁺T cells recovered from the spleens of these mice are also possible, including studies on lymphocyte subsets, Th1/Th2 polarization, presence of regulatory T cells and the impact of DC vaccination on their functions.</p

A team effort: natural killer cells on the first leg of the tumor immunity relay race

Recent work by Böttcher and colleagues defines a new role for Natural Killer cells in the anti-tumor immune response, arriving early into the tumor microenvironment before passing the baton to DC1 dendritic cells. DC1 dendritic cells subsequently activate CD8+ T cells resulting in effective anti-tumor immunity. This work highlights the cooperative nature of anti-tumor immunity set in motion by Natural Killer cells, and immune evasion by tumors through their exclusion.National Cancer Institute (U.S.) (R00CA204595

Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

<p>Abstract</p> <p>Background</p> <p>Antigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) <it>in vitro</it>. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.</p> <p>Methods</p> <p>In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.</p> <p>Results</p> <p>Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of <it>in vitro </it>transcribed (<it>ivt</it>)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of <it>ivt</it>RNA.</p> <p>Conclusions</p> <p>This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or <it>ivt</it>RNA as sources of specific antigen.</p

Both intratumoral regulatory T cell depletion and CTLA-4 antagonism are required for maximum efficacy of anti-CTLA-4 antibodies

Anti-CTLA-4 antibodies have successfully elicited durable tumor regression in the clinic; however, long-term benefit is limited to a subset of patients for select cancer indications. The incomplete understanding of their mechanism of action has hindered efforts at improvement, with conflicting hypotheses proposing either antagonism of the CTLA-4:B7 axis or Fc effector-mediated regulatory T cell (Treg) depletion governing efficacy. Here, we report the engineering of a nonantagonistic CTLA-4 binding domain (b1s1e2) that depletes intratumoral Tregs as an Fc fusion. Comparison of b1s1e2-Fc to 9d9, an antagonistic anti-CTLA-4 antibody, allowed for interrogation of the separate contributions of CTLA-4 antagonism and Treg depletion to efficacy. Despite equivalent levels of intratumoral Treg depletion, 9d9 achieved more long-term cures than b1s1e2-Fc in MC38 tumors, demonstrating that CTLA-4 antagonism provided additional survival benefit. Consistent with prior reports that CTLA-4 antagonism enhances priming, treatment with 9d9, but not b1s1e2-Fc, increased the percentage of activated T cells in the tumor-draining lymph node (tdLN). Treg depletion with either construct was restricted to the tumor due to insufficient surface CTLA-4 expression on Tregs in other compartments. Through intratumoral administration of diphtheria toxin in Foxp3-DTR mice, we show that depletion of both intratumoral and nodal Tregs provided even greater survival benefit than 9d9, consistent with Treg-driven restraint of priming in the tdLN. Our data demonstrate that anti-CTLA-4 therapies require both CTLA-4 antagonism and intratumoral Treg depletion for maximum efficacy-but that potential future therapies also capable of depleting nodal Tregs could show efficacy in the absence of CTLA-4 antagonism

Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host\u27s response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14-15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual\u27s recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4-5, 2019

Correction to: Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Following publication of the original article [1], the author reported that an author name, Roberta Zappasodi, was missed in the authorship list