Chemical Strategies to Enhance the Therapeutic Efficacy of Toll-like Receptor Agonist Based Cancer Immunotherapy.

Recent developments in the fields of biomedical chemistry and immune bioengineering have enabled innovative therapeutic approaches that can enhance the efficacy, accuracy, and safety of cancer immunotherapy. Among the numerous strategies utilized in cancer immunotherapy, Toll-like receptor (TLR) agonist-based approaches have been studied for a long time since they trigger the innate immune system and generate antigen-specific T cell responses to fight against tumors. In addition to these immunostimulatory functions, TLR agonists also contribute to the reprogramming of immune suppressive tumor microenvironments. Although TLR agonists are now being intensively studied in clinical trials due to their substantial immunomodulatory properties, they still show a low therapeutic index. Nonspecific and random stimulation of various immune cells produces excess levels of proinflammatory cytokines, resulting in cytokine storms and chronic diseases. Therefore, the development of chemical strategies to enhance the therapeutic efficacy as well as the safety of TLR agonist-based immunotherapy is essential and in high demand.In this Account, we summarize and discuss recent developments in biomedical chemistry and bioengineering techniques for the immunomodulation of TLR agonists that have addressed the limitations in current cancer immunotherapy. Immunomodulation of TLR agonists can be classified into two different approaches: (1) molecular modulation via chemical structure modification and (2) macroscopic modulation via an engineered drug delivery system. In molecular modulation, based on prodrug and antedrug principles, activity is modulated (active or inactive) through immolative chemical linkers that can respond to extrinsic or intrinsic biological stimulation and the plasmatic environment, respectively. To increase the effectiveness of TLR agonists as immunostimulatory agents, researchers have conjugated TLR agonists with other immunotherapeutic moieties (antigen, antibody, other TLR agonist, etc.). For macroscopic modulation, bioengineering of delivery carriers differing in size or with albumin hitchhiking moieties has been utilized to increase the efficiency of the targeting of these carriers to secondary lymphoid organs (lymph nodes (LNs) and spleen). The conjugation of specific targeting ligands and incorporation of stimulus-triggering moieties can promote the delivery of TLR agonists into specific cells or intracellular compartments. Implantable porous scaffolds for specific immune cell recruitment and in situ depot-forming gel systems for controlled release of immunomodulatory drugs can increase the therapeutic efficacy of TLR agonists while reducing systemic toxicity. Taken together, these findings show that well-designed and precisely controlled chemical strategies for the immunomodulation of TLR agonists at both the molecular and macroscopic levels are expected to play key roles in improving the therapeutic efficacy of cancer immunotherapy while minimizing immune-related toxicity.

Molecular Masking of Synthetic Immunomodulator Evokes Antitumor Immunity With Reduced Immune Tolerance and Systemic Toxicity by Temporal Activity Recovery and Sustained Stimulation.

Activation of the innate immune system counteracts tumor-induced immunosuppression. Hence, small molecule-based toll-like receptor 7/8 agonists (TLR7/8a), which can modulate immunosuppression in the tumor microenvironment along with the activation of innate immunity, are emerging as essential components of cancer immunotherapy. However, the clinical application of synthetic TLR7/8a therapies is limited by systemic immune-associated toxicity and immune tolerance induced by uncontrolled stimulatory activities and repeated treatments. To address these limitations, a dynamic immunomodulation strategy incorporating masking and temporal recovery of the activity of TLR7/8a through prodrug-like TLR7/8a (pro-TLR7/8a) at the molecular level and a sustained and controlled release of active TLR7/8a from nanoliposome (pro-TLR7/8a) (NL(pro-TLR7/8)) in a macroscale depot are designed. Immunization with cationic NL(pro-TLR7/8) and anionic antigens triggers robust activation of innate immune cells as well as antigen-specific T cell responses, eliciting reprogramming of immunosuppressive cells into tumor-suppressive cells, with decreased systemic adverse effects and immune tolerance. Combination treatment with NL(pro-TLR7/8a) and immune checkpoint inhibitors (anti-CTLA-4 plus anti-PD-L1) or nanoliposomes (Doxorubicin) has synergistic effects on antitumor immunity in various tumor models. The concept of pro-TLR7/8a suggested herein may facilitate the advancement of small-molecule-based immunomodulators for clinical translation and safe and effective cancer immunotherapy.

Bioconjugated Antibody-Trojan Immune Converter Enhance Cancer Immunotherapy with Minimized Toxicity by Programmed Two-Step Immunomodulation of Myeloid Cells.

Current immune checkpoint blockade therapy (ICBT) predominantly targets T cells to harness the antitumor effects of adaptive immune system. However, the effectiveness of ICBT is reduced by immunosuppressive innate myeloid cells in tumor microenvironments (TMEs). Toll-like receptor 7/8 agonists (TLR7/8a) are often used to address this problem because they can reprogram myeloid-derived suppressor cells (MDSCs) and tumor-associated M2 macrophages, and boost dendritic cell (DC)-based T-cell generation; however, the systemic toxicity of TLR7/8a limits its clinical translation. Here, to address this limitation and utilize the effectiveness of TLR7/8a, this work suggests a programmed two-step activation strategy via Antibody-Trojan Immune Converter Conjugates (ATICC) that specifically targets myeloid cells by anti-SIRPα followed by reactivation of transiently inactivated Trojan TLR7/8a after antibody-mediated endocytosis. ATICC blocks the CD47-SIRPα ("don't eat me" signal), enhances phagocytosis, reprograms M2 macrophages and MDSCs, and increases cross-presentation by DCs, resulting in antigen-specific CD8+ T-cell generation in tumor-draining lymph nodes and TME while minimizing systemic toxicity. The local or systemic administration of ATICC improves ICBT responsiveness through reprogramming of the immunosuppressive TME, increased infiltration of antigen-specific CD8+ T cells, and antibody-dependent cellular phagocytosis. These results highlight the programmed and target immunomodulation via ATICC could enhance cancer immunotherapy with minimized systemic toxicities.

Transformable Gel-to-Nanovaccine Enhances Cancer Immunotherapy via Metronomic-Like Immunomodulation and Collagen-Mediated Paracortex Delivery.

The generation of non-exhausted effector T-cells depends on vaccine's spatiotemporal profile, and untimely delivery and low targeting to lymph node (LN) paracortex by standard bolus immunization show limited efficacy. By recapitulating the dynamic processes of acute infection, a bioadhesive immune niche domain (BIND) is developed that facilitates the delivery of timely-activating conjugated nanovaccine (t-CNV) in a metronomic-like manner and increased the accumulation and retention of TANNylated t-CNV (tannic acid coated t-CNV) in LN by specifically binding to collagen in subcapsular sinus where they gradually transformed into TANNylated antigen-adjuvant conjugate by proteolysis, inducing their penetration into paracortex through the collagen-binding in LN conduit and evoking durable antigen-specific CD8+ T-cell responses. The BIND combined with t-CNV, mRNA vaccine, IL-2, and anti-PD-1 antibody also significantly enhanced cancer immunotherapy by the dynamic modulation of immunological landscape of tumor microenvironment. The results provide material design strategy for dynamic immunomodulation that can potentiate non-exhausted T-cell-based immunotherapy.

Protective Efficacy and Immunogenicity of Rv0351/Rv3628 Subunit Vaccine Formulated in Different Adjuvants Against Mycobacterium tuberculosis Infection.

Bacillus Calmette-Guerin (BCG) vaccine is the only licensed vaccine for tuberculosis (TB) prevention. Previously, our group demonstrated the vaccine potential of Rv0351 and Rv3628 against Mycobacterium tuberculosis (Mtb) infection by directing Th1-biased CD4+ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the lungs. Here, we assessed immunogenicity and vaccine potential of the combined Ags (Rv0351/Rv3628) formulated in different adjuvants as subunit booster in BCG-primed mice against hypervirulent clinical Mtb strain K (Mtb K). Compared to BCG-only or subunit-only vaccine, BCG prime and subunit boost regimen exhibited significantly enhanced Th1 response. Next, we evaluated the immunogenicity to the combined Ags when formulated with four different types of monophosphoryl lipid A (MPL)-based adjuvants: 1) dimethyldioctadecylammonium bromide (DDA), MPL, and trehalose dicorynomycolate (TDM) in liposome form (DMT), 2) MPL and Poly I:C in liposome form (MP), 3) MPL, Poly I:C, and QS21 in liposome form (MPQ), and 4) MPL and Poly I:C in squalene emulsion form (MPS). MPQ and MPS displayed greater adjuvancity in Th1 induction than DMT or MP did. Especially, BCG prime and subunit-MPS boost regimen significantly reduced the bacterial loads and pulmonary inflammation against Mtb K infection when compared to BCG-only vaccine at a chronic stage of TB disease. Collectively, our findings highlighted the importance of adjuvant components and formulation to induce the enhanced protection with an optimal Th1 response.

Development and Validation of an Artificial Intelligence-Based Motion Analysis System for Upper Extremity Rehabilitation Exercises in Patients with Spinal Cord Injury: A Randomized Controlled Trial.

In this study, we developed an AI-based real-time motion feedback system for patients with spinal cord injury (SCI) during rehabilitation, aiming to enhance their interest and motivation. The effectiveness of the system in improving upper-limb muscle strength during the Thera band exercises was evaluated. The motion analysis program, including exercise repetition counts and calorie consumption, was developed using MediaPipe, focusing on three key motions (chest press, shoulder press, and arm curl) for upper extremity exercises. The participants with SCI were randomly assigned to the experimental group (EG = 4) or control group (CG = 5), engaging in 1 h sessions three times a week for 8 weeks. Muscle strength tests (chest press, shoulder press, lat pull-down, and arm curl) were performed before and after exercises. Although both groups did not show significant differences, the EG group exhibited increased strength in all measured variables, whereas the CG group showed constant or reduced results. Consequently, the computer program-based system developed in this study could be effective in muscle strengthening. Furthermore, these findings may serve as a valuable foundation for future AI-driven rehabilitation exercise systems.

A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances cancer immunotherapy and reduces immune cell exhaustion.

Although conventional innate immune stimuli contribute to immune activation, they induce exhausted immune cells, resulting in suboptimal cancer immunotherapy. Here we suggest a kinetically activating nanoadjuvant (K-nanoadjuvant) that can dynamically integrate two waves of innate immune stimuli, resulting in effective antitumour immunity without immune cell exhaustion. The combinatorial code of K-nanoadjuvant is optimized in terms of the order, duration and time window between spatiotemporally activating Toll-like receptor 7/8 agonist and other Toll-like receptor agonists. K-nanoadjuvant induces effector/non-exhausted dendritic cells that programme the magnitude and persistence of interleukin-12 secretion, generate effector/non-exhausted CD8+ T cells, and activate natural killer cells. Treatment with K-nanoadjuvant as a monotherapy or in combination therapy with anti-PD-L1 or liposomes (doxorubicin) results in strong antitumour immunity in murine models, with minimal systemic toxicity, providing a strategy for synchronous and dynamic tailoring of innate immunity for enhanced cancer immunotherapy.

Effects of Resistance Circuit Training on Health-Related Physical Fitness in People With Paraplegia: A Pilot Randomized Controlled Trial.

OBJECTIVE: To evaluate the efficacy and safety of 8 weeks of resistance circuit training in people with paraplegia due to spinal cord injury. METHODS: Participants were randomized into experimental and control groups. Although the intensity and sequence of movements of the exercise programs were identical in both groups, the resting time between sets was limited to 1 minute in the experimental group. In the control group, the participants were allowed to rest until they were comfortable. Both groups received 8 weeks of training twice per week. Before and after the program, muscle mass, body fat percentage, fat mass, blood pressure, heart rate, muscle strength and muscular endurance were evaluated, and 6-minute propulsion test was conducted. Additionally, the safety of the program was assessed. RESULTS: Twenty-two individuals with paraplegia were enrolled (11 in each group). After the training program, the experimental group showed a significant decrease in the resting blood pressure and improvement in the upper extremity muscle mass, strength, and endurance (p<0.05). Each variable showed significant inter-group differences (p<0.05). Furthermore, none of the participants showed autonomic adverse events, musculoskeletal side effects, or discomfort. CONCLUSION: The results show that resistance circuit training programs with short resting intervals are superior to the usual resistance exercise programs in improving the blood pressure and physical strength and are safe for people with upper thoracic level injuries at T6 or higher.

Overcoming Chemoimmunotherapy-Induced Immunosuppression by Assemblable and Depot Forming Immune Modulating Nanosuspension.

The deficiency of antigen-specific T cells and the induction of various treatment-induced immunosuppressions still limits the clinical benefit of cancer immunotherapy. Although the chemo-immunotherapy adjuvanted with Toll-like receptor 7/8 agonist (TLR 7/8a) induces immunogenic cell death (ICD) and in situ vaccination effect, indoleamine 2,3-dioxygenase (IDO) is also significantly increased in the tumor microenvironment (TME) and tumor-draining lymph node (TDLN), which offsets the activated antitumor immunity. To address the treatment-induced immunosuppression, an assemblable immune modulating suspension (AIMS) containing ICD inducer (paclitaxel) and supra-adjuvant (immune booster; R848 as a TLR 7/8a, immunosuppression reliever; epacadostat as an IDO inhibitor) is suggested and shows that it increases cytotoxic T lymphocytes and relieves the IDO-related immunosuppression (TGF-ß, IL-10, myeloid-derived suppressor cells, and regulatory T cells) in both TME and TDLN, by the formation of in situ depot in tumor bed as well as by the efficient migration into TDLN. Local administration of AIMS increases T cell infiltration in both local and distant tumors and significantly inhibits the metastasis of tumors to the lung. Reverting treatment-induced secondary immunosuppression and reshaping "cold tumor" into "hot tumor" by AIMS also increases the response rate of immune checkpoint blockade therapy, which promises a new nanotheranostic strategy in cancer immunotherapy.

Lyophilizable and Multifaceted Toll-like Receptor 7/8 Agonist-Loaded Nanoemulsion for the Reprogramming of Tumor Microenvironments and Enhanced Cancer Immunotherapy.

The low therapeutic efficacy of current cancer immunotherapy is related to nonimmunogenic and immunosuppressive tumor microenvironments (TMEs). To overcome these limitations, both the immune priming of antitumoral lymphocytes and the reprogramming of immunosuppressive factors in TMEs are essential. Here, we suggest a nanoemulsion (NE)-based immunotherapeutic platform that can not only modulate tumor-induced suppression but also induce an effective cell-mediated immune response for T cell proliferation. Multifunctional NEs can be fabricated by integrating the efficacy of NEs as delivery systems and the multifaceted immunomodulation characteristics (i.e., immunostimulation and reprogramming of immunosuppression) of small molecule-based Toll-like receptor 7/8 agonists. Local in situ vaccination of melanoma and cervical tumor models with tumor antigens (protein and peptide) adjuvanted with NE loaded with TLR7/8 agonists [NE (TLR7/8a)] induced the recruitment and activation of innate immune cells, infiltration of lymphocytes, and polarization of tumor-associated M2 macrophages, which resulted in inhibition of tumor growth and prolonged survival in both primary and rechallenged tumor models. Antibody-depletion experiments also suggested that macrophages, type I IFN (IFN-α and IFN-ß), CD8+ T cells, and NK1.1+ cells contributed to the antitumor effect of NE (TLR7/8a). The combination of antitumoral lymphocytes and reprogramming of immunosuppressive TMEs induced by NE (TLR7/8a) treatment evoked a synergistic antitumor immune response with immune checkpoint blockade therapy (anti-PD-1 and anti-PD-L1).

Syringeable immunotherapeutic nanogel reshapes tumor microenvironment and prevents tumor metastasis and recurrence.

The low response rate of current cancer immunotherapy suggests the presence of few antigen-specific T cells and a high number of immunosuppressive factors in tumor microenvironment (TME). Here, we develop a syringeable immunomodulatory multidomain nanogel (iGel) that overcomes the limitation by reprogramming of the pro-tumoral TME to antitumoral immune niches. Local and extended release of immunomodulatory drugs from iGel deplete immunosuppressive cells, while inducing immunogenic cell death and increased immunogenicity. When iGel is applied as a local postsurgical treatment, both systemic antitumor immunity and a memory T cell response are generated, and the recurrence and metastasis of tumors to lungs and other organs are significantly inhibited. Reshaping of the TME using iGel also reverts non-responding groups to checkpoint blockade therapies into responding groups. The iGel is expected as an immunotherapeutic platform that can reshape immunosuppressive TMEs and synergize cancer immunotherapy with checkpoint therapies, with minimized systemic toxicity.