Integrating immunopeptidome analysis for the design and development of cancer vaccines.

The repertoire of naturally presented peptides within the MHC (major histocompatibility complex) or HLA (human leukocyte antigens) system on the cellular surface of every mammalian cell is referred to as ligandome or immunopeptidome. This later gained momentum upon the discovery of CD8 + T cells able to recognize and kill cancer cells in an MHC-I antigen-restricted manner. Indeed, cancer immune surveillance relies on T cell recognition of MHC-I-restricted peptides, making the identification of those peptides the core for designing T cell-based cancer vaccines. Moreover, the breakthrough of antibodies targeting immune checkpoint molecules has led to a new and strong interest in discovering suitable targets for CD8 +T cells. Therapeutic cancer vaccines are designed for the artificial generation and/or stimulation of CD8 +T cells; thus, their combination with ICIs to unleash the breaks of the immune system comes as a natural consequence to enhance anti-tumor efficacy. In this context, the identification and knowledge of peptide candidates take advantage of the fast technology updates in immunopeptidome and mass spectrometric methodologies, paying the way to the rational design of vaccines for immunotherapeutic approaches. In this review, we discuss mainly the role of immunopeptidome analysis and its application for the generation of therapeutic cancer vaccines with main focus on HLA-I peptides. Here, we review cancer vaccine platforms based on two different preparation methods: pathogens (viruses and bacteria) and not (VLPs, nanoparticles, subunits vaccines) that exploit discoveries in the ligandome field to generate and/or enhance anti-tumor specific response. Finally, we discuss possible drawbacks and future challenges in the field that remain still to be addressed.

Expanding the reach of commercial cell therapies requires changes at medical centers.

The clinical application of cell therapies is becoming increasingly important for the treatment of cancer, congenital immune deficiencies, and hemoglobinopathies. These therapies have been primarily manufactured and used at academic medical centers. However, cell therapies are now increasingly being produced in centralized manufacturing facilities and shipped to medical centers for administration. Typically, these cell therapies are produced from a patient's own cells, which are the critical starting material. For these therapies to achieve their full potential, more medical centers must develop the infrastructure to collect, label, cryopreserve, test, and ship these cells to the centralized laboratories where these cell therapies are manufactured. Medical centers must also develop systems to receive, store, and infuse the finished cell therapy products. Since most cell therapies are cryopreserved for shipment and storage, medical centers using these therapies will require access to liquid nitrogen product storage tanks and develop procedures to thaw cell therapies. These services could be provided by the hospital pharmacy or transfusion service, but the latter is likely most appropriate. Another barrier to implementing these services is the variability among providers of these cell therapies in the processes related to handling cell therapies. The provision of these services by medical centers would be facilitated by establishing a national coordinating center and a network of apheresis centers to collect and cryopreserve the cells needed to begin the manufacturing process and cell therapy laboratories to store and issue the cells. In addition to organizing cell collections, the coordinating center could establish uniform practices for collecting, labeling, shipping, receiving, thawing, and infusing the cell therapy.

The need for uniform and coordinated practices involving centrally manufactured cell therapies.

Cellular therapies have become an important part of clinical care. The treatment of patients with cell therapies often involves the collection of autologous cells at the medical center treating the patient, the shipment of these cells to a centralized manufacturing site, and the return of the cryopreserved clinical cell therapy to the medical center treating the patient for storage until infusion. As this activity grows, cell processing laboratories at many academic medical centers are involved with many different autologous products manufactured by several different centralized laboratories. The handling of these products by medical center-based cell therapy laboratories is complicated and resource-intensive since each centralized manufacturing laboratory has unique methods for labeling, storing, shipping, receiving, thawing, and infusing the cells. The field would benefit from the development of more uniform practices. The development of a coordinating center similar to those established to facilitate the collection, shipping, and transplantation of hematopoietic stem cells from unrelated donors would also be beneficial. In summary, the wide range of practices involved with labeling, shipping, freezing, thawing, and infusing centrally manufactured autologous cellular therapies lack efficiency and consistency and puts patients at risk. More uniform practices are needed.

The subtle interplay between gamma delta T lymphocytes and dendritic cells: is there a role for a therapeutic cancer vaccine in the era of combinatorial strategies?

Human gamma delta (γδ) T cells represent heterogeneous subsets of unconventional lymphocytes with an HLA-unrestricted target cell recognition. γδ T cells display adaptive clonally restricted specificities coupled to a powerful cytotoxic function against transformed/injured cells. Dendritic cells (DCs) are documented to be the most potent professional antigen-presenting cells (APCs) able to induce adaptive immunity and support the innate immune response independently from T cells. Several data show that the cross-talk of γδ T lymphocytes with DCs can play a crucial role in the orchestration of immune response by bridging innate to adaptive immunity. In the last decade, DCs, as well as γδ T cells, have been of increasing clinical interest, especially as monotherapy for cancer immunotherapy, even though with unpredictable results mainly due to immune suppression and/or tumor-immune escape. For these reasons, new vaccine strategies have to be explored to reach cancer immunotherapy's full potential. The effect of DC-based vaccines on γδ T cell is less extensively investigated, and a combinatorial approach using DC-based vaccines with γδ T cells might promote a strong synergy for long-term tumor control and protection against escaping tumor clones. Here, we discuss the therapeutic potential of the interaction between DCs and γδ T cells to improve cancer vaccination. In particular, we describe the most relevant and updated evidence of such combinatorial approaches, including the use of Zoledronate, Interleukin-15, and protamine RNA, also looking towards future strategies such as CAR therapies.

A pragmatic adaptive enrichment design for selecting the right target population for cancer immunotherapies.

One of the challenges in the design of confirmatory trials is to deal with uncertainties regarding the optimal target population for a novel drug. Adaptive enrichment designs (AED) which allow for a data-driven selection of one or more prespecified biomarker subpopulations at an interim analysis have been proposed in this setting but practical case studies of AEDs are still relatively rare. We present the design of an AED with a binary endpoint in the highly dynamic setting of cancer immunotherapy. The trial was initiated as a conventional trial in early triple-negative breast cancer but amended to an AED based on emerging data external to the trial suggesting that PD-L1 status could be a predictive biomarker. Operating characteristics are discussed including the concept of a minimal detectable difference, that is, the smallest observed treatment effect that would lead to a statistically significant result in at least one of the target populations at the interim or the final analysis, respectively, in the setting of AED.

An Update on Anti-CD137 Antibodies in Immunotherapies for Cancer.

The selective expression of CD137 on cells of the immune system (e.g., T and DC cells) and oncogenic cells in several types of cancer leads this molecule to be an attractive target to discover cancer immunotherapy. Therefore, specific antibodies against CD137 are being studied and developed aiming to activate and enhance anti-cancer immune responses as well as suppress oncogenic cells. Accumulating evidence suggests that anti-CD137 antibodies can be used separately to prevent tumor in some cases, while in other cases, these antibodies need to be co-administered with other antibodies or drugs/vaccines/regents for a better performance. Thus, in this work, we aim to update and discuss current knowledge about anti-cancer effects of anti-CD137 antibodies as mono- and combined-immunotherapies.

Probiotic species in the modulation of the anticancer immune response.

Mounting evidences are supporting a key role of distinct gut bacteria in the occurrence and progression of intestinal and extra-intestinal tumors. More importantly, it has been recently demonstrated that some gut bacteria strains synergize with largely-used anticancer drugs as alkylating or immune checkpoint blockade agents thus optimizing the immune response against multiple solid cancers. However, the exact role played by each gut bacterium in cancer occurrence and response to therapy is still in its infancy; and the current knowledge, although exciting, still needs to be transferred from mice models to human beings. Here, the advances in the understanding of how gut microbes and immune response shape each other in a cancer context are reviewed together with the implications of these finding for future antitumor therapy. Herein, the most important bacteria strains, able to boost the immune response triggered by anticancer drugs, together with their mechanism of action, whenever known, have been surveyed. It is reasonable to think that cocktails of beneficial bacteria together with an ad hoc diet or food supplements may be used as novel anticancer adjuvant agents in future therapeutic regimens.

Strategies to reprogram anti-inflammatory macrophages towards pro-inflammatory macrophages to support cancer immunotherapies.

Tumor-associated myeloid cells, including macrophages and myeloid-derived suppressor cells, can be highly prevalent in solid tumors and play a significant role in the development of the tumor. Therefore, myeloid cells are being considered potential targets for cancer immunotherapies. In this review, we focused on strategies aimed at targeting tumor-associated macrophages (TAMs). Most strategies were studied preclinically but we also included a limited number of clinical studies based on these strategies. We describe possible underlying mechanisms and discuss future challenges and prospects.

Erzhi pills reverse PD-L1-mediated immunosuppression in melanoma microenvironment.

Background: Cancer immunotherapies aimed at activating immune system, especially by blocking immune checkpoints, have become a successful modality for treating patients with advanced cancers. However, its clinical practice is frequently conceded by high outcomes, low initial response rates and severe side effects. New strategies are necessary to complement and advance this biological therapy. Erzhi Pills (EZP) have diverse pharmaceutical effects including immune regulation, anti-tumor and anti-senescence. We hypothesized that EZP could exert its antitumor effect through immunomodulation. Purpose: The aim of this study was to investigate the effects of EZP on anti-tumor activities, and define its molecular mechanisms. Methods: By applying melanoma model with high immune infiltrates, we determined the anti-melanoma effect of EZP. To identify whether this effect was mediated by direct targeting tumor cells, cell viability and apoptosis were examined in vitro. Network pharmacology analysis was used to predict the potential mechanisms of EZP for melanoma via immune response. Flow cytometry, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) and crystal violet (CV) experiments were performed to detect T cell infiltrations and functions mediated by EZP. The mechanism of EZP was further investigated by western blotting both in vivo and in vitro. Results: The administration of EZP significantly inhibited tumor weight and volume. EZP extract could only slightly reduce cell viability and induce melanoma apoptosis. Network pharmacology analysis predicted that JAK-STAT signaling pathway and T cell receptor signaling pathway might be involved during EZP treatment. Flow cytometry and IHC analyses showed that EZP increased the number of CD4+ T cells and enhanced the function of CD8+ T cells. In co-culture experiments, EZP elevated killing ability of T cells. Western blotting showed that EZP treatment reduced PD-L1 signaling pathway. Conclusion: These findings indicated that EZP exerted anti-melanoma effects by inducing apoptosis and blocking PD-L1 to activate T cells. EZP might represent a promising candidate drug for cancer immunotherapies.

Nucleic acid-loaded poly(beta-aminoester) nanoparticles for cancer nano-immuno therapeutics: the good, the bad, and the future.

Immunotherapy has emerged as a promising approach to cancer treatment, offering improved survival rates and enhanced patients' quality of life. However, realizing the full potential of immunotherapy in clinical practice remains a challenge, as there is still plenty of room for modulating the complexity of the human immune system in favor of an antitumor immunogenicity. Nanotechnology, with its unique properties, holds promise in augmenting the efficacy of cancer immunotherapies in biotherapeutic protection and site- and time-controlled delivery of the immune modulator biologicals. Polymeric nanoparticles are promising biomaterials among different nanocarriers thanks to their robustness, versatility, and cost-efficient design and production. This perspective paper overviews critical concepts in nanometric advanced delivery systems applied to cancer immunotherapy. We focus on a detailed exploration of the current state of the art and trends in using poly(beta-aminoester) (pBAE) polymers for nucleic acid-based antitumor immunotherapies. Through different examples of the use of pBAE polymers reported in the literature, we revise the main advantages these polymers offer and some challenges to overcome. Finally, the paper provides insights and predictions on the path toward the clinical implementation of cancer nano-immunotherapies, highlighting the potential of pBAE polymers for advancements in this field.

Cancer Immunotherapies Ignited by a Thorough Machine Learning-Based Selection of Neoantigens.

Identification of neoantigens, derived from somatic DNA alterations, emerges as a promising strategy for cancer immunotherapies. However, not all somatic mutations result in immunogenicity, hence, efficient tools to predict the immunogenicity of neoepitopes are needed. A pipeline is presented that provides a comprehensive solution for the identification of neoepitopes based on genomic sequencing data. The pipeline consists of a data pre-processing step and three machine learning predictive steps. The pre-processing step analyzes genomic data for different types of alterations, produces a list of all possible antigens, and determines the human leukocyte antigen (HLA) type and T-cell receptor (TCR) repertoire. The first predictive step performs a classification into antigens and neoantigens, selecting neoantigens for further consideration. The next step predicts the strength of binding between neoantigens and available major histocompatibility complexes of class I (MHC-I). The third step is engaged to predict the likelihood of inducing an immune response. Neoepitopes satisfying all three predictive stages are assumed to be potent candidates to ensure immunogenicity. The predictive pipeline is used in two regimes: selecting neoantigens from patients' sequencing data and generating novel neoantigen candidates. Two different techniques - Monte Carlo and Reinforcement Learning - are implemented to facilitate the generative regime.

IFNγ-Induced Bcl3, PD-L1 and IL-8 Signaling in Ovarian Cancer: Mechanisms and Clinical Significance.

IFNγ, a pleiotropic cytokine produced not only by activated lymphocytes but also in response to cancer immunotherapies, has both antitumor and tumor-promoting functions. In ovarian cancer (OC) cells, the tumor-promoting functions of IFNγ are mediated by IFNγ-induced expression of Bcl3, PD-L1 and IL-8/CXCL8, which have long been known to have critical cellular functions as a proto-oncogene, an immune checkpoint ligand and a chemoattractant, respectively. However, overwhelming evidence has demonstrated that these three genes have tumor-promoting roles far beyond their originally identified functions. These tumor-promoting mechanisms include increased cancer cell proliferation, invasion, angiogenesis, metastasis, resistance to chemotherapy and immune escape. Recent studies have shown that IFNγ-induced Bcl3, PD-L1 and IL-8 expression is regulated by the same JAK1/STAT1 signaling pathway: IFNγ induces the expression of Bcl3, which then promotes the expression of PD-L1 and IL-8 in OC cells, resulting in their increased proliferation and migration. In this review, we summarize the recent findings on how IFNγ affects the tumor microenvironment and promotes tumor progression, with a special focus on ovarian cancer and on Bcl3, PD-L1 and IL-8/CXCL8 signaling. We also discuss promising novel combinatorial strategies in clinical trials targeting Bcl3, PD-L1 and IL-8 to increase the effectiveness of cancer immunotherapies.

Current Landscape of Cancer Immunotherapy: Harnessing the Immune Arsenal to Overcome Immune Evasion.

Cancer immune evasion represents a leading hallmark of cancer, posing a significant obstacle to the development of successful anticancer therapies. However, the landscape of cancer treatment has significantly evolved, transitioning into the era of immunotherapy from conventional methods such as surgical resection, radiotherapy, chemotherapy, and targeted drug therapy. Immunotherapy has emerged as a pivotal component in cancer treatment, harnessing the body's immune system to combat cancer and offering improved prognostic outcomes for numerous patients. The remarkable success of immunotherapy has spurred significant efforts to enhance the clinical efficacy of existing agents and strategies. Several immunotherapeutic approaches have received approval for targeted cancer treatments, while others are currently in preclinical and clinical trials. This review explores recent progress in unraveling the mechanisms of cancer immune evasion and evaluates the clinical effectiveness of diverse immunotherapy strategies, including cancer vaccines, adoptive cell therapy, and antibody-based treatments. It encompasses both established treatments and those currently under investigation, providing a comprehensive overview of efforts to combat cancer through immunological approaches. Additionally, the article emphasizes the current developments, limitations, and challenges in cancer immunotherapy. Furthermore, by integrating analyses of cancer immunotherapy resistance mechanisms and exploring combination strategies and personalized approaches, it offers valuable insights crucial for the development of novel anticancer immunotherapeutic strategies.

Personalizing Oncolytic Virotherapy.

The use of oncolytic viruses has become an attractive tool in the clinics for the treatment of various tumor types. Such viruses are genetically modified to conditionally replicate in malignant cells while unharming healthy cells. This platform offers a highly specific tumor killing with exceptional safety profiles. However, the use of oncolytic viruses as sole oncolytic platforms has not achieved full tumor clearance in murine models and in the clinics. In fact, the formation of anti-tumor immune responses is attributed to the effectiveness of oncolytic viruses. In this review, we will discuss the various strategies that scientists have employed to enhance the anti-tumor immune responses driven by oncolytic viruses. Moreover, focus will be drawn into personalizing such anti-tumor responses by the addition of tumor-associated peptides.

Positioning SUMO as an immunological facilitator of oncolytic viruses for high-grade glioma.

Oncolytic viral (OV) therapies are promising novel treatment modalities for cancers refractory to conventional treatment, such as glioblastoma, within the central nervous system (CNS). Although OVs have received regulatory approval for use in the CNS, efficacy is hampered by obstacles related to delivery, under-/over-active immune responses, and the "immune-cold" nature of most CNS malignancies. SUMO, the Small Ubiquitin-like Modifier, is a family of proteins that serve as a high-level regulator of a large variety of key physiologic processes including the host immune response. The SUMO pathway has also been implicated in the pathogenesis of both wild-type viruses and CNS malignancies. As such, the intersection of OV biology with the SUMO pathway makes SUMOtherapeutics particularly interesting as adjuvant therapies for the enhancement of OV efficacy alone and in concert with other immunotherapeutic agents. Accordingly, the authors herein provide: 1) an overview of the SUMO pathway and its role in CNS malignancies; 2) describe the current state of CNS-targeted OVs; and 3) describe the interplay between the SUMO pathway and the viral lifecycle and host immune response.

mRNA-based Vaccines Targeting the T-cell Epitope-rich Domain of Epstein Barr Virus Latent Proteins Elicit Robust Anti-Tumor Immunity in Mice.

Epstein-Barr virus (EBV) is associated with various malignancies and infects >90% of the global population. EBV latent proteins are expressed in numerous EBV-associated cancers and contribute to carcinogenesis, making them critical therapeutic targets for these cancers. Thus, this study aims to develop mRNA-based therapeutic vaccines that express the T-cell-epitope-rich domain of truncated latent proteins of EBV, including truncatedlatent membrane protein 2A (Trunc-LMP2A), truncated EBV nuclear antigen 1 (Trunc-EBNA1), and Trunc-EBNA3A. The vaccines effectively activate both cellular and humoral immunity in mice and show promising results in suppressing tumor progression and improving survival time in tumor-bearing mice. Furthermore, it is observed that the truncated forms of the antigens, Trunc-LMP2A, Trunc-EBNA1, and Trunc-EBNA3A, are more effective than full-length antigens in activating antigen-specific immune responses. In summary, the findings demonstrate the effectiveness of mRNA-based therapeutic vaccines targeting the T-cell-epitope-rich domain of EBV latent proteins and providing new treatment options for EBV-associated cancers.

Bioinformatic-based genetic characterizations of neural regulation in skin cutaneous melanoma.

Background: Recent discoveries uncovered the complex cancer-nerve interactions in several cancer types including skin cutaneous melanoma (SKCM). However, the genetic characterization of neural regulation in SKCM is unclear. Methods: Transcriptomic expression data were collected from the TCGA and GTEx portal, and the differences in cancer-nerve crosstalk-associated gene expressions between normal skin and SKCM tissues were analyzed. The cBioPortal dataset was utilized to implement the gene mutation analysis. PPI analysis was performed using the STRING database. Functional enrichment analysis was analyzed by the R package clusterProfiler. K-M plotter, univariate, multivariate, and LASSO regression were used for prognostic analysis and verification. The GEPIA dataset was performed to analyze the association of gene expression with SKCM clinical stage. ssGSEA and GSCA datasets were used for immune cell infiltration analysis. GSEA was used to elucidate the significant function and pathway differences. Results: A total of 66 cancer-nerve crosstalk-associated genes were identified, 60 of which were up- or downregulated in SKCM and KEGG analysis suggested that they are mainly enriched in the calcium signaling pathway, Ras signaling pathway, PI3K-Akt signaling pathway, and so on. A gene prognostic model including eight genes (GRIN3A, CCR2, CHRNA4, CSF1, NTN1, ADRB1, CHRNB4, and CHRNG) was built and verified by independent cohorts GSE59455 and GSE19234. A nomogram was constructed containing clinical characteristics and the above eight genes, and the AUCs of the 1-, 3-, and 5-year ROC were 0.850, 0.811, and 0.792, respectively. Expression of CCR2, GRIN3A, and CSF1 was associated with SKCM clinical stages. There existed broad and strong correlations of the prognostic gene set with immune infiltration and immune checkpoint genes. CHRNA4 and CHRNG were independent poor prognostic genes, and multiple metabolic pathways were enriched in high CHRNA4 expression cells. Conclusion: Comprehensive bioinformatics analysis of cancer-nerve crosstalk-associated genes in SKCM was performed, and an effective prognostic model was constructed based on clinical characteristics and eight genes (GRIN3A, CCR2, CHRNA4, CSF1, NTN1, ADRB1, CHRNB4, and CHRNG), which were widely related to clinical stages and immunological features. Our work may be helpful for further investigation in the molecular mechanisms correlated with neural regulation in SKCM, and in searching new therapeutic targets.

Oncolytic ImmunoViroTherapy: A long history of crosstalk between viruses and immune system for cancer treatment.

Cancer Immunotherapy relies on harnessing a patient's immune system to fine-tune specific anti-tumor responses and ultimately eradicate cancer. Among diverse therapeutic approaches, oncolytic viruses (OVs) have emerged as a novel form of cancer immunotherapy. OVs are a naturally occurring or genetically modified class of viruses able to selectively kill cancer cells, leaving healthy cells unharmed; in the last two decades, the role of OVs has been redefined to act beyond their oncolytic activity. Indeed, the immunogenic cancer cell death mediated by OVs induces the release of tumor antigens that in turn induces anti-tumor immunity, allowing OVs to act as in situ therapeutic cancer vaccines. Additionally, OVs can be engineered for intratumoral delivery of immunostimulatory molecules such as tumor antigens or cytokines to further enhance anti-tumor response. Moreover, OVs can be used in combination with other cancer immunotherapeutic approaches such as Immune Checkpoint Inhibitors and CAR-T cells. The current review first defines the three main mechanisms of action (MOA) of OVs currently used in cancer therapy that are: i) Oncolysis, ii) OV-induced cancer-specific immune activation, and iii) Exploiting pre-existing anti-viral immunity to enhance cancer therapy. Secondly, we focus on how OVs can induce and/or improve anti-cancer immunity in a specific or unspecific fashion, highlighting the importance of these approaches. Finally, the last part of the review analyses OVs combined with other cancer immunotherapies, revising present and future clinical applications.

Immune-related adverse events of cancer immunotherapies targeting kinases.

Immunotherapies are designed to target a specific molecule of the immune system and emerged at the end of the last century as effective therapies the treatment of a widening spectrum of inflammatory diseases. Paradoxically, their use was quickly linked to the development of autoimmune disorders, whose treatment indications often include the very biological agent producing the adverse event. The scenario has changed dramatically in recent years due to the increasing use of immunotherapies in patients with solid cancers (mainly checkpoint inhibitors, but also tyrosine-kinase inhibitors and others). Cancer immunotherapies are broadly defined as therapies directly or indirectly targeting any component of the immune system involved in the immune response against cancer. These therapies include different molecules (monoclonal antibodies, small proteins, fusion proteins) that target specific proteins of cancer or immune cells. A key challenge that has emerged with the progressive broad implementation of these treatments in daily practice has been the collateral side effects on the immune system of treatment, which may lead to immune-related adverse events (irAEs). The cumulated number of cases of irAEs related to cancer immunotherapies has increased exponentially during this century. As the objective of cancer immunotherapy is to stimulate the immune system, these autoimmune and inflammatory irAEs were expected. The pharmacological targeting of kinases has led to a significant change in the therapeutic management of cancer. About one-third of all protein targets under research in the pharmaceutical industry are kinase inhibitors, overwhelmingly used in the treatment of malignancies. Very few studies have reviewed the broad scenario of irAEs related to kinase inhibitors, in contrast with the large number of studies published on irAEs caused by checkpoint inhibitors, with an often-fragmented view according to the specialty. The purpose of this review is to update current knowledge on the wide pharmacological and phenotypic scenario of irAEs associated with kinase inhibitors from a multidisciplinary perspective.

Circulating tumor DNA: Opportunities and challenges for pharmacometric approaches.

To support further development of model-informed drug development approaches leveraging circulating tumor DNA (ctDNA), we performed an exploratory analysis of the relationships between treatment-induced changes to ctDNA levels, clinical response and tumor size dynamics in patients with cancer treated with checkpoint inhibitors and targeted therapies. This analysis highlights opportunities for pharmacometrics approaches such as for optimizing sampling design strategies. It also highlights challenges related to the nature of the data and associated variability overall emphasizing the importance of mechanistic modeling studies of the underlying biology of ctDNA processes such as shedding, release and clearance and their relationships with tumor size dynamic and treatment effects.