An HIV cure remains elusive, and many individuals develop comorbidities; therefore, new synergistic strategies to combat HIV are needed. Enhanced natural killer (NK) cell function associates with HIV elite control, primarily through antibody-dependent cellular cytotoxicity (ADCC). We generated tri-specific killer engagers (TriKEs) to enhance NK cell responses against HIV-infected cells. HIV TriKEs, one using the VRC01/07 antibody domains (VRC TriKE) and one using the CD4 ectodomain 1 (CD4 TriKE), promote NK cell function and killing of HIV target cells. NK cells expressing both CD38 and CD57 are major mediators of this function. We performed a dose-escalation study in SIV-uninfected rhesus macaques to assess the safety and pharmacodynamics of our CD4 TriKE. The TriKE is safe and effective at expanding peripheral and lymphoid NK cells in vivo. These findings support TriKEs as a promising safe immunotherapy to recognize and kill HIV-infected cells, with potential for combination cure strategies.

We present an efficient method for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to create a library of functional ionizable lipopolymers. Here we show 155 polymers, formulated into polyplexes, to establish structure-activity relationships essential for endosomal escape and transfection. A lead structure is identified, and lipopolymer-lipid hybrid nanoparticles are developed to deliver mRNA to lung endothelium and immune cells, including T cells, with low in vivo toxicity. These nanoparticles show significant improvements in mRNA delivery to the lung compared to in vivo-JetPEI® and demonstrate effective delivery of therapeutic mRNA(s) of various sizes. IL-12 mRNA-loaded nanoparticles delay Lewis Lung cancer progression, while human CFTR mRNA restores CFTR protein function in CFTR knockout mice. Additionally, we demonstrate in vivo CRISPR-Cas9 mRNA delivery, achieving gene editing in lung tissue and successful PD-1 knockout in T cells in mice. These results highlight the platform's potential for systemic gene therapy delivery.

Immunotherapies are limited by a variety of complex suppressive signals within the tumor microenvironment, including restriction of activating anti-tumor signaling through alteration of the activating receptor repertoire. One such mechanism involves A Disintegrin And Metalloprotease 17 (ADAM17)-mediated shedding of CD16, a potent activating receptor on natural killer (NK) cells that mediates antibody dependent cellular cytotoxicity (ADCC). Our group has been exploiting ADCC for immunotherapy, pre-clinically and clinically, through a Tri-specific Killer Engager (TriKE) platform, which leverages an anti-CD16 arm, an IL-15 moiety, and an anti-tumor antigen arm (B7H3 in this case, expressed in various solid tumors). While the TriKEs display robust pre-clinical activity, in certain tumor settings (like mesothelioma and ovarian cancer) CD16 expression could be limiting, affecting the overall efficacy of the therapy. To bypass this potential issue, we created a Poly-Antigen Cytokine Complex (PACC), consisting of interleukin-15 receptor alpha (IL-15Rα ) linked to an ADAM17-blocking antibody-fragment arm, to noncovalently bind IL-15 in the TriKE molecule, as a backpack, and enhance CD16-mediated NK cell-directed anti-tumor responses against B7H3-expressing solid tumors. Our data shows that the TriKE-PACC molecule complex can prevent CD16 shedding and improve anti-tumor activity in models of mesothelioma and ovarian cancer, indicating the potential translational value of this complex. This work was supported by US Department of Defense HT9425-24-1-0706; NIH/NCI P01 CA111412 and R35 CA283892; and the Randy Shaver Community Fund. Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

155 Background: Recombinant human (rh) IL-15, the homeostatic factor for natural killer (NK) cells, is being clinically developed, but it has little antitumor activity alone. B7H3 (CD276) is an immune checkpoint inhibitor that is associated with poorer prognosis and is highly-expressed on prostate cancer. NK cells can be given as allogeneic products and, unlike T cells, do not induce cytokine-release syndrome or neurotoxicity. Here we developed a B7H3 targeting Tri-Specific Killer Engager (TriKE) as a novel dual camelid (cam) TriKE containing WT IL-15 and two cam engagers targeting CD16 on NK cells and B7H3 on tumor targets, making NK cells antigen specific. We have previously demonstrated that NK cells infiltrate prostate cancer tumors. As proof of concept, a clinical trial of a CD33-targeted TriKE for AML (NCT03214666) induced endogenous NK cell expansion and activation in refractory AML patients. Methods: Prostate cancer cell lines or patient-derived xenografts (PDX) were incubated with healthy donor or prostate cancer patient NK cells with or without B7H3 TriKE. PDX were propagated in NSG mice and then homogenized for in vitro assays. NK cell function was measured by flow cytometry and IncuCyte live tumor imaging assays. Castration-sensitive and -resistant prostate cancer (CSPC and CRPC, respectively) patient or normal donor peripheral blood mononuclear cells (PBMC) were immunophenotyped using 42-marker NK specific or broad immune cytometry time-of-flight (CyToF) panels. Results: B7H3 TriKE resulted in a dose-dependent proliferation of NK cells, but not T cells. This was in marked contrast to rhIL-15, which stimulated both cell types. camB7H3 was broadly expressed on prostate, head and neck, ovarian and glioblastoma cancers as well as multiple myeloma. We observed a B7H3 TriKE dose-dependent increase in CD107a degranulation and inflammatory cytokines to all B7H3 positive targets that was highly specific, with no response seen with B7H3 negative hematologic targets and CRISPR KO controls. Compared to rhIL-15, B7H3 TriKE given at molar equivalent dosing induced B7H3+ target killing in in a dose-dependent manner above that seen with rhIL-15 induced natural cytotoxicity. Using CSPC and CRPC patient PBMC (n=11-15), we demonstrated that there is no significant loss in NK cell degranulation/interferon gamma production or target cytotoxicity compared to healthy age- and sex-matched donors when treated with B7H3 TriKE. CyToF analysis of CSPC and CRPC patient PBMC is ongoing. In vivo activity in xenogeneic models of human tumor is underway. Conclusions: B7H3 TriKE delivers an NK cell specific IL-15 signal to expand NK cells and is highly specific against B7H3+ prostate cancer cell lines and PDX. Clinical-grade B7H3 TriKE is undergoing validation and a Phase 1/2 clinical trial is planned to open in the 3rd Quarter of 2024 for CRPC patients progressing on one or more therapies the CRPC setting.

PDF file - 198K, Figure 1. Characterization of the anti-OX40 mAb (9B12). Figure 2. Total peripheral lymphocyte counts. Figure 3. Pharmacokinetics of CD134 (anti-OX40) mAb in patients. Figure 4. Direct ex vivo detection of the murine anti-OX40 mAb bound to T cells in treated patients. Figure 5. Regression of a pulmonary metastasis in a patient with renal carcinoma enrolled in cohort 1. Figure 6. Changes in Ki-67 expression within CD4+ and CD8+ T cell subsets examined over time after anti-OX40 from patients in cohorts 1 and 2. Figure 7. Increased proliferation of CD4+ Foxp3- T cells and CD8+ T cells correlates with a decrease or stabilization of tumor burden. Figure 8. Ki-67 expression by monkey CD4+ and CD8+ memory T cells after the administration of anti-OX40 , mouse Immunoglobulin or monkey OX40L:Ig. Figure 9. Determination of the Endpoint Titer for anti-KLH Ab.

(1) Background: The objective of this analysis was to evaluate the device usage rates and patterns of use regarding Tumor-Treating Fields (TTFields) for patients with malignant pleural mesothelioma (MPM) throughout the US. (2) Methods: We evaluated de-identified data from 33 patients with MPM enrolled in FDA-required HDE protocols at 14 institutions across the US from September 2019 to March 2022. (3) Results: The median number of total TTFields usage days was 72 (range: 6-649 days), and the total treatment duration was 160 months for all patients. A low usage rate (defined as less than 6 h per day, 25%) was observed in 34 (21.2%) months. The median TTFields usage in the first 3 months was 12 h per day (range: 1.9-21.6 h), representing 50% (range: 8-90%) of the potential daily duration. The median TTFields usage after 3 months decreased to 9.1 h per day (range: 3.1-17 h), representing 38% (range: 13-71%) of the daily duration, and was lower than usage in the first 3 months (p = 0.01). (4) Conclusions: This study represents the first multicenter analysis of real-world TTFields usage based on usage patterns for MPM patients in clinical practice. The real-world usage level was lower than the suggested daily usage. Further initiatives and guidelines should be developed to evaluate the impact of this finding on tumor control.

PDF file - 68K, Details clinical trial, flow cytometry and monkey experiments referred to in the manuscript.

Recent preclinical and clinical studies have highlighted the improved outcomes of combination radiotherapy and immunotherapy. Concurrently, the development of high‐Z metallic nanoparticles as radiation dose enhancers has been explored to widen the therapeutic window of radiotherapy and potentially enhance immune activation. In this study, folate‐modified hafnium‐based metal–organic frameworks (HfMOF‐PEG‐FA) are evaluated in combination with imiquimod, a TLR7 agonist, as a well‐defined interferon regulatory factor (IRF) stimulator for local antitumor immunotherapy. The enhancement of radiation dose deposition by HfMOF‐PEG‐FA and subsequent generation of reactive oxygen species (ROS) deregulates cell proliferation and increases apoptosis. HfMOF‐PEG‐FA loaded with imiquimod (HfMOF‐PEG‐FA@IMQ) increases DNA double‐strand breaks and cell death, including apoptosis, necrosis, and calreticulin exposure, in response to X‐ray irradiation. Treatment with this multipronged therapy promotes IRF stimulation for subsequent interferon production within tumor cells themselves. The novel observation is reported that HfMOF itself increases TLR7 expression, unexpectedly pairing immune agonist and receptor upregulation in a tumor intrinsic manner, and supporting the synergistic effect observed with the γ H2AX assay. T‐cell analysis of CT26 tumors following intratumoral administration of HfMOF‐PEG‐FA@IMQ with radiotherapy reveals a promising antitumor response, characterized by an increase in CD8 + and proliferative T cells.

BackgroundNatural killer (NK) cells are being leveraged in the clinic due to their safety profile and their ability to mediate tumor killing without prior priming. However, lack of antigen-specific targeting, decreased numbers, and suppressive signals derived from the tumor microenvironment (TME) of Prostate Cancer (PCa), can impact NK cell efficacy. To bypass this issue, we designed a novel tri-specific killer engager (TriKE®) molecule that consists of three parts: an arm that engages with CD16, an activating receptor of NK cells, an arm that binds to tumor antigens express in PCa (PSMA or B7H3), and an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, priming and motility (figure 1A).MethodsTriKE molecules were generated in mammalian (Expi293) manufacturing systems. Peripheral Blood Mononuclear Cells (PBMCs) from normal donors or prostate cancer patients were either used directly or magnetically enriched for NK cells in assays co-culturing cells with PCa cell lines in the presence or absence of PSMA TriKE or B7H3 TriKE. Flow cytometry-based readouts evaluated NK cell activation and tumor killing while impedance and imaging assays were also used to dynamically measure tumor killing. Hypoxic (1% oxygen) culture condition and cytokine-induced myeloid-derived suppressor cells (MDSC) were also incorporated in some assays. Pre-clinical xenogeneic mouse models were also used to evaluate efficacy of TriKEs in vivo.ResultNormal donor and PCa patient NK cells display better, specific, degranulation against PCa cell lines in the presence of PSMA (figure 1B) or B7H3 TriKEs. NK cell cytotoxicity is also improved, even in the presence of enzalutamide resistant lines (figure 1C), hypoxia (figure 1D), or Myeloid Derived Suppressor Cells. Finally, the TriKE molecules display improved tumor control, compared to IL-15 control or no treatment, in xenogeneic models of prostate cancer.ConclusionsOur findings indicate that TriKE molecules improve PCa control in several systems and in the presence of varied TME-specific stresses. These pre-clinical studies highlight the potential for using TriKE molecules in the setting of metastatic Prostate Cancer and pave the way for future, targeted, NK cell immunotherapeutic interventions in this setting.AcknowledgementsThis work was carried out using funds from DoD grant W81XWH-20–1-0659. We would like to acknowledge the Translational Therapy Laboratory (TTL), the Data and Biospecimen Utilization Committee (DBUC), and the Clinical Informatics Shared Services (CISS) at the University of Minnesota for their excellent service in helping access de-identified Prostate Cancer patient samples from the Solid Tumor Cancer Specimen Bank (2016NTLS035).Ethics ApprovalPeripheral blood mononuclear cells (PBMCs) from the blood of deidentified healthy donors were obtained, after participants gave informed consent, from Memorial Blood Centers (Minneapolis, Minnesota, USA) and used in compliance with the Committee on the Use of Human Subjects in Research (IRB# 9709 M00134) and in accordance with the Declaration of Helsinki. The in vivo mouse studies were conducted in accordance with the Institutional Animal Care and Use Committee at the University of Minnesota (IACUC# 1908–37330A).

In breast cancer, progression to invasive ductal carcinoma (IDC) involves interactions between immune, myoepithelial, and tumor cells. Development of IDC can proceed through ductal carcinoma in situ (DCIS), a non-obligate, non-invasive stage, or IDC can develop without evidence of DCIS and these cases associate with poorer prognosis. Tractable, immune-competent mouse models are needed to help delineate distinct mechanisms of local tumor cell invasion and prognostic implications. To address these gaps, we delivered murine mammary carcinoma cell lines directly into the main mammary lactiferous duct of immune-competent mice. Using two strains of immune-competent mice (BALB/c, C57BL/6), one immune-compromised (severe combined immunodeficiency; SCID) C57BL/6 strain, and six different murine mammary cancer cell lines (D2.OR, D2A1, 4T1, EMT6, EO771, Py230), we found early loss of ductal myoepithelial cell differentiation markers p63, α-smooth muscle actin, and calponin, and rapid formation of IDC in the absence of DCIS. Rapid IDC formation also occurred in the absence of adaptive immunity. Combined, these studies demonstrate that loss of myoepithelial barrier function does not require an intact immune system, and suggest that these isogenic murine models may prove a useful tool to study IDC in the absence of a non-obligatory DCIS stage-an under-investigated subset of poor prognostic human breast cancer.

Stereotactic body radiotherapy (SBRT) demonstrates excellent local control in early stage lung cancer, however a quarter of patients develop recurrence or distant metastasis. Transforming growth factor-beta (TGF-β) supports metastasis and treatment resistance, and angiotensin receptor blockade (ARB) indirectly suppresses TGF-β signaling. This study investigates whether patients taking ARBs while undergoing SBRT for early stage lung cancer exhibited improved overall survival (OS) or recurrence free survival (RFS) compared to patients not taking ARBs. This was a single institution retrospective analysis of 272 patients treated with SBRT for early stage lung cancer between 2009 and 2018. Patient health data was abstracted from the electronic medical record. OS and RFS were assessed using Kaplan–Meier method. Log-rank test was used to compare unadjusted survival between groups. Univariable and multivariable Cox proportional hazard regression models were used to estimate hazard ratios (HRs). Of 247 patients analyzed, 24 (10%) patients took ARBs for the duration of radiotherapy. There was no difference in mean age, median tumor diameter, or median biologic effective dose between patients taking ARBs or not. Patients taking ARBs exhibited increased OS (ARB = 96.7 mo.; no ARB = 43.3 mo.; HR = 0.25 [95% CI: 0.10 to 0.62, P = .003]) and increased RFS (median RFS, ARB = 64.3 mo.; No ARB = 35.1 mo.; HR = 0.26 [95% CI: 0.10 to 0.63, P = .003]). These effects were not seen in patients taking angiotensin converting enzyme inhibitors (ACEIs) or statins. ARB use while undergoing SBRT for early stage lung cancer may increase OS and RFS, but ACEI use does not show the same effect.

Purpose/Objective(s)Radiation therapy (RT) reduces tumor burden through both intrinsic initiation of tumor cell death and enhancement of anti-tumor immunity. However, radiation treatments are not universally efficacious due to variation in radiosensitivity and anti-tumor immunity. Low proliferative rate, hypoxia, and T cell effector dysfunction can significantly degrade response to RT. Here we evaluate a rational combination therapy pairing radiosensitization and immune activation in conjunction with standard of care RT. Recent studies demonstrated the sumoylation inhibitor, TAK-981, has intrinsic capacity to reduce the proliferation of tumor cells in vitro as a monotherapy and decrease tumor volumes in syngeneic murine colon carcinoma models (CT26 and MC38) in combination with PD-1 or CTLA-4 checkpoint blockade. In these models a dendritic cell dependent type I interferon pathway was implicated to mediate the anti-tumor immune response and enhanced in vivo tumor control in TAK-981 and checkpoint blockade combination therapy, similar to previously elucidated mechanisms of immune activation following hypofractionated radiotherapy. Additionally, multiple intrinsic and extrinsic protein mediators (Myc, IRF3, p21) of tumor cell response to radiation are regulated by the post-translational modification of sumoylation. Consequently, we evaluated whether combining RT with TAK-981 resulted in enhanced tumor radiosensitivity in vitro and improved local tumor control in vivo, consistent with enhanced anti-tumor immune response in murine tumor models.Materials/MethodsWe utilized a longitudinal real time microscopic proliferation assay to evaluate RT response. Titrations of TAK-981 were added to established in vitro tumor cell cultures 4 hours prior to increasing doses of single fraction photon RT. Impacts of RT, TAK-981 or the combination treatment were assessed based upon cell proliferation for 72 hours. We assessed efficacy of this novel combination in vivo using the MC38 colorectal carcinoma model. C57BL6 mice with established tumors (> 50mm2) received TAK981 (15mg/kg) alone three times weekly or in combination with single fraction 8 Gy RT and compared to RT alone to evaluate local tumor control and impacts on overall survival.ResultsOur data demonstrates a synergistic and dose dependent reduction in MC38 colon carcinoma in vitro proliferation in response to combination RT and TAK-981. In vivo MC38 tumor models demonstrate no effect of TAK-981 alone compared to vehicle treated mice while co-administration with a single 8 Gy fraction of radiotherapy synergistically enhanced tumor control and overall survival compared to RT alone.ConclusionOur preliminary data support potential dual synergistic roles for the sumoylation inhibitor TAK-981 with radiotherapy, functioning as both an intrinsic radiosensitizer and immunomodulator.

Background Natural killer (NK) cell effector function is suppressed in the tumor microenvironment (TME) of metastatic castration-resistant prostate cancer (mCRPC), the lethal form of prostate cancer. This is largely due to the inherently 'cold' nature of the TME of mCRPC that is immunosuppressive, hypoxic and lacks cytolytic lymphocyte infiltration. To improve NK cell anti-tumor responses against mCRPC in the TME, we designed a novel tri-specific killer engager (TriKE®) molecule that consists of three parts: an arm that engages with CD16, an activating receptor of NK cells, an arm that binds to prostate-specific membrane antigen (PSMA) that is highly and specifically expressed on mCRPC, and an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, priming and motility (figure 1). Methods Flow cytometry-based functional and dye dilution proliferation assays were used to compare activation and proliferation of NK cells treated with PSMA TriKE or IL-15. NK cell cytolytic capacity against C4-2, a PSMA-expressing prostate cancer cell line, was measured using IncuCyte live cell imaging. In various assays, hypoxic (1% oxygen) culture condition and cytokine-induced myeloid-derived suppressor cells (MDSC) were incorporated to better examine PSMA TriKE function in the physiological setting of mCRPC. Results PSMA TriKE significantly enhanced expansion of peripheral blood NK cells derived from healthy donors up to 10 folds (N=9). This effect was specific to NK cells and not T cells. Additionally, PSMA TriKE induced NK cell degranulation (up to 60%) and intracellular IFNγ and TNFα buildup (up to 50%) when compared to IL-15 and no treatment groups after incubation with C4-2 (N=6). This result was not observed when PSMA knockout C4-2 were used as target cells. NK cell incubation in hypoxia for 7 days severely impacts cytotoxicity, some of which can be improved with IL-15. However, PSMA TriKE treatment markedly improved NK cell cytolytic capacity against C4-2 in hypoxia (N=5) (figure 2). Similarly, MDSCs suppressed NK cell degranulation in the presence of IL-15 alone but not with PSMA-TriKE treatment (N=4) (figure 3). Conclusions PSMA-TriKE induces specific NK cell proliferation and activation against PSMA+ tumor cells. Efficient delivery of IL-15 to NK cells by PSMA-TriKE robustly relieves NK cells from suppression induced by hypoxia and MDSCs. These results demonstrate promising potential of PSMA TriKE in overcoming suppression of NK cells in the TME of mCRPC. Acknowledgements This work was funded by the Department of Defense (DoD). Figure 1 was made using Biorender.com.

Radioenhancing nanoparticles (NPs) are being evaluated in ongoing clinical trials for various cancers including head and neck, lung, esophagus, pancreas, prostate, and soft tissue sarcoma. Supported by decades of preclinical investigation and recent randomized trial data establishing clinical activity, these agents are poised to influence future multimodality treatment paradigms involving radiotherapy. Although the physical interactions between NPs and ionizing radiation are well characterized, less is known about how these agents modify the tumor microenvironment, particularly regarding tumor immunogenicity. In this review, we describe the key multidisciplinary considerations related to radiation, surgery, immunology, and pathology for designing radioenhancing NP clinical trials. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Given the significant lack of effective therapies for malignant pleural mesothelioma (MPM), Tumor Treating Fields (TTFields) was made available for use under an FDA-approved Humanitarian Device Exemption (HDE) protocol in 2019. In the phase 2 STELLAR study, a 68% median device usage rate (16.3 hours/day) was reported for MPM patients treated with TTFields along with platinum-based chemotherapy in the first 3 months. However, there is no reported real-world usage rate of TTFields for unresectable MPM to date and patterns of use may differ in clinical practice.

Recent interest in cancer immunotherapy has largely been focused on the adaptive immune system, particularly adoptive T-cell therapy and immune checkpoint blockade (ICB). Despite improvements in overall survival and progression-free survival across multiple cancer types, neither cell-based therapies nor ICB results in durable disease control in the majority of patients. A critical component of antitumor immunity is the mononuclear phagocyte system and its role in both innate and adaptive immunity. The phagocytic functions of these cells have been shown to be modulated through multiple pathways, including the CD47-SIRPα axis, which is manipulated by cancer cells for immune evasion. In addition to CD47, tumors express a variety of other “don’t eat me” signals, including beta-2-microglobulin and CD24, and “eat me” signals, including calreticulin and phosphatidylserine. Therapies targeting these signals can lead to increased phagocytosis of cancer cells; however, because “don’t eat me” signals are markers of “self” on normal cells, treatment can result in negative off-target effects, such as anemia and B-cell depletion. Recent preclinical research has demonstrated the potential of nanocarriers to synergize with prophagocytic therapies, address the off-target effects, improve pharmacokinetics, and codeliver chemotherapeutics. The high surface area-to-volume ratio of nanoparticles paired with preferential size for passive targeting allows for greater accumulation of therapeutic cargo. In addition, nanomaterials hold promise as molecular imaging agents for the detection of phagocytic markers. This mini review highlights the unique capabilities of nanotechnology to expand the application and efficacy of immunotherapy through recently discovered phagocytotic checkpoint therapies.

Overexpression of O6-methylguanine DNA methyltransferase (MGMT) contributes to resistance to chemo-radiation therapy (CRT) in brain tumors. We previously demonstrated that non-ablative radiation improved delivery of anti-MGMT morpholino oligonucleotides (AMONs) to reduce MGMT levels in subcutaneous tumor xenografts. We evaluate this approach to enhance CRT efficacy in rat brain tumor xenograft models. The impact of radiation on targeted delivery was evaluated using fluorescent oligonucleotides (f-ON). In vitro, f-ON was localized to clathrin-coated vesicles, endosomes, and lysosomes using confocal microscopy in T98G glioma cells. In vivo, fluorescence was detected in pre-radiated, but not non-radiated Long Evans (non-tumor bearing) rat brains. Cranial radiation (2 Gy) followed by AMONs (intravenous, 10.5 mg/kg) reduced MGMT expression by 50% in both orthotopic cerebellar D283 medulloblastoma and intracerebral H460 non-small cell lung carcinoma (NSCLC) xenograft models. To evaluate the efficacy, AMONs concurrent with CRT (2 Gy radiation plus oral 20 mg/kg temozolomide ×4 days) reduced tumor volumes in the medulloblastoma model (p = 0.012), and a similar trend was found in the NSCLC brain metastasis model. We provide proof of concept for the use of non-ablative radiation to guide and enhance the delivery of morpholino oligonucleotides into brain tumor xenograft models to reduce MGMT levels and improve CRT efficacy.