Candidate For Cancer Immunotherapy Research Articles

Natural killer cells and engagers: Powerful weapons against cancer.

Natural killer (NK) cells are innate immune effectors whose functions rely on receptors binding cytokines, recognizing self-molecules, or detecting danger signals expressed by virus-infected or tumor cells. The potent cytotoxic potential makes NK cells promising candidates for cancer immunotherapy. To enhance their activity strategies include cytokine administration, blocking of immune checkpoints, and designing of antibody-based NK cell engagers (NKCEs). NKCEs represent a cutting-edge approach to cancer therapy: they strengthen the NK-to-target cell interactions and optimize tumor killing, possibly overcoming the immunosuppressive tumor microenvironment. NK cells belong to the innate lymphoid cells (ILCs) and are categorized into different subsets also including cells with a memory-like phenotype: this complexity needs to be explored in the context of cancer immunotherapy, particularly when designing NKCEs. Two strategies to enhance NK cell activity in cancer patients can be adopted: activating patients' own NK cells versus the adoptive transfer of exvivo activated NK cells. Furthermore, the capability of NKCEs to activate γδ T cells could have a significant synergistic effect in immunotherapy.

Characterization of HIF-1α Knockout Primary Human Natural Killer Cells Including Populations in Allogeneic Glioblastoma.

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Natural killer cells (NK) are major innate effector cells with broad cytotoxicity against tumors. Accordingly, NK cells are ideal candidates for cancer immunotherapy, including glioblastoma (GBM). Hypoxia is a common feature of solid tumors, and tumor cells and normal cells adapt to the tumor microenvironment by upregulating the transcription factor hypoxia-inducible factor (HIF)-1α, which can be detrimental to anti-tumor effector immune cell function, including that of NK cells. We knocked out HIF-1α in human primary NK cells using clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9). Then, cellular characterizations were conducted in normoxic and hypoxic conditions. Electroporating two HIF-1α-targeting guide RNA-Cas9 protein complexes inhibited HIF-1α expression in expanded NK cells. HIF-1α knockout human NK cells, including populations in hypoxic conditions, enhanced the growth inhibition of allogeneic GBM cells and induced apoptosis in GBM-cell-derived spheroids. RNA-sequencing revealed that the cytotoxicity of HIF-1α knockout NK cells could be related to increased perforin and TNF expression. The results demonstrated that HIF-1α knockout human NK cells, including populations, enhanced cytotoxicity in an environment mimicking the hypoxic conditions of GBM. CRISPR-Cas9-mediated HIF-1α knockout NK cells, including populations, could be a promising immunotherapeutic alternative in patients with GBM.

Nucleic Acid Materials-Mediated Innate Immune Activation for Cancer Immunotherapy.

Abnormally localized nucleic acids (NAs) are considered as pathogen associated molecular patterns (PAMPs) in innate immunity. They are recognized by NAs-specific pattern recognition receptors (PRRs), leading to the activation of associated signaling pathways and subsequent production of type I interferons (IFNs) and pro-inflammatory cytokines, which further trigger the adaptive immunity. Notably, NAs-mediated innate immune activation is highly dependent on the conformation changes, especially the aggregation of PRRs. Evidence indicates that the characteristics of NAs including their length, concentration and even spatial structure play essential roles in inducing the aggregation of PRRs. Therefore, nucleic acid materials (NAMs) with high valency of NAs and high-order structures hold great potential for activating innate and adaptive immunity, making them promising candidates for cancer immunotherapy. In recent years, a variety of NAMs have been developed and have demonstrated significant efficacy in achieving satisfactory anti-tumor immunity in multiple mouse models, exhibiting huge potential for clinical application in cancer treatment. This review aims to discuss the mechanisms of NAMs-mediated innate immune response, and summarize their applications in cancer immunotherapy.

Abstract LB354: A novel oncolytic vaccinia virus with intravenous compatibility evaded complement- and neutralizing antibody-mediated inhibition and provided superior anti-tumor activity to intra-tumoral administration in immunocompetent mice

Abstract Oncolytic viruses (OVs) are potent cancer therapeutics that can selectively kill tumor cells and promote anti-tumor immunity. mSJ-650 (Wyeth, K2L-, TK-, murine GM-CSF, human CD55) is the Wyeth strain of vaccinia virus with human complement regulatory protein CD55 incorporated on the intracellular mature virion membrane to evade complement-mediated attack during circulation. The virus also expresses mGM-CSF in replace of thymidine kinase to increase tumor selectivity and enhance immunogenicity. We previously reported that a single dose of mSJ-650 effectively evades complement and shows prominent anti-tumor efficacy in A549 and HCT116 xenograft model. Here, we further investigated the capability of mSJ-650 in promoting anti-tumor response after multiple systemic administration in immunocompetent breast cancer mouse model. In an orthotopic EMT6 breast cancer model, mSJ-650 or control mSJ-612 devoid of CD55 was intravenously injected at low dose (1x106 pfu) and high dose (3x106 pfu), twice weekly. mSJ-650 treatment dose-dependently suppressed tumor growth, showing superior antitumor efficacy compared to treatment of control mSJ-612. Moreover, multiple systemic administration of mSJ-650 was well-tolerated with no apparent signs of toxicity in serum tested via AST and ALT assay. To further elucidate whether mSJ-650 can evade neutralization by antibody and maintain its antitumor potency in vivo, we designed two experimental models. Firstly, endogenous vaccinia virus specific neutralizing antibody was generated by intravenous injection of mSJ-650 prior to tumor cell inoculation in tumor-bearing mouse, followed by systemic administration of mSJ-650 twice weekly. Second, serum containing vaccinia virus specific neutralizing antibody was intravenously injected 24 hours prior to mSJ-650 administration in tumor-bearing mouse. In both experimental models, mSJ-650 treated group significantly inhibited tumor growth despite the presence of neutralizing antibody, while control mSJ-612 treated group failed to suppress tumor growth. These results imply that mSJ-650 is capable of evading neutralization and maintaining its antitumor efficacy. Moreover, we performed multi-color flow cytometry analysis of tumor-infiltrating leukocytes to evaluate immunogenicity of mSJ-650. As a result, we observed a significant increase in recruitment of activated CD4+ and CD8+ T cells by 2-fold and high expression level of cytotoxic molecules (perforin, TNF-α, IFN-γ) in tumor-infiltrating T cells. In summary, multiple systemic administration of mSJ-650 markedly evaded neutralization and suppressed tumor growth by promoting anti-tumor response in immunocompetent breast cancer mouse model, thus implying that mSJ-650 is a promising candidate for cancer immunotherapy. Citation Format: Yeonsoo Yang, Solchan Won, Namhee Lee, Eunhye Kim, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. A novel oncolytic vaccinia virus with intravenous compatibility evaded complement- and neutralizing antibody-mediated inhibition and provided superior anti-tumor activity to intra-tumoral administration in immunocompetent mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB354.

Targeted and Self-Adjuvated Nanoglycovaccine Candidate for Cancer Immunotherapy.

Advanced-stage solid primary tumors and metastases often express mucin 16 (MUC16), carrying immature glycans such as the Tn antigen, resulting in specific glycoproteoforms not found in healthy human tissues. This presents a valuable approach for designing targeted therapeutics, including cancer glycovaccines, which could potentially promote antigen recognition and foster the immune response to control disease spread and prevent relapse. In this study, we describe an adjuvant-free poly(lactic-co-glycolic acid) (PLGA)-based nanoglycoantigen delivery approach that outperforms conventional methods by eliminating the need for protein carriers while exhibiting targeted and adjuvant properties. To achieve this, we synthesized a library of MUC16-Tn glycoepitopes through single-pot enzymatic glycosylation, which were then stably engrafted onto the surface of PLGA nanoparticles, generating multivalent constructs that better represent cancer molecular heterogeneity. These glycoconstructs demonstrated affinity for Macrophage Galactose-type Lectin (MGL) receptor, known to be highly expressed by immature antigen-presenting cells, enabling precise targeting of immune cells. Moreover, the glycopeptide-grafted nanovaccine candidate displayed minimal cytotoxicity and induced the activation of dendritic cells in vitro, even in the absence of an adjuvant. In vivo, the formulated nanovaccine candidate was also nontoxic and elicited the production of IgG specifically targeting MUC16 and MUC16-Tn glycoproteoforms in cancer cells and tumors, offering potential for precise cancer targeting, including targeted immunotherapies.

Abstract 4484: Discovery of a potential first-in-class Za targeted small molecule inhibitor of ADAR1 p150 which demonstrates strong anti-tumor efficacy

Abstract Background: Adenosine deaminase, RNA specific (ADAR1), catalyzes the hydrolytic deamination of adenosine (A) to inosine (I) in double-stranded (ds) RNAs. ADAR1 has two isoforms: p110 in the nucleus and p150 in the cytoplasm. Both isoforms alter the coding and non-coding sections of self-dsRNA. The ADAR1 p150 isoform is expressed from an interferon (IFN)-response promoter and has a Z-DNA/Z-RNA binding domain at the N-terminus. ADAR1 has been implicated in promoting cell survival and resistance to immune response in several cancer types like multiple myeloma, breast, lung, liver, and skin cancer. Inhibition of ADAR1 p150 has the potential to enhance anti-tumor efficacy as monotherapy and in combination with other therapeutic modalities. The Zα domain of ADAR1 p150 provides exclusivity to p150 compared to the other isoforms. Herein, we outline the discovery of a potential first-in-class Zα targeted ADAR1 p150 inhibitor and degrader for cancer immunotherapy. Methods: AVA-ADR-001 was identified through a high throughput p110 knockout cell-based assay. The ability of AVA-ADR-001 to induce interferons was confirmed in various cell lines. The anti-tumor efficacy of AVA-ADR-001 was evaluated in B16F10 syngeneic melanoma mice model as monotherapy and in combination with anti-PD-1. PROTAC AVA-ADR-703 was used to demonstrate selective degradation of the ADAR1 p150 protein. Results: We have discovered AVA-ADR-001, a novel small molecule inhibitor of ADAR1 which significantly increases the IFN response in vitro in an MDA5-dependent manner. In vitro binding studies confirm that AVA-ADR-001 binds to the Zα domain of ADAR1 with low nM potency. AVA-ADR-001 demonstrates anti-tumor efficacy 1.5X superior to anti-PD1 ICB non-responsive tumor models. In addition, the AVA-ADR-001 monotherapy and combination groups showed a significant upregulation of numerous interferon-stimulated genes, including IFIH1, IFN-β, and CXCL-10. Finally, targeted protein degradation of ADAR1 p150 while sparing ADAR1 p110 was confirmed when cells were treated with AVA-ADR-703 with nM DC50, the PROTAC variant of AVA-ADR-001. Conclusions: To our knowledge, AVA-ADR-001 and AVA-ADR-703 are the first selective Zα-targeted small molecule inhibitor and degrader of ADAR1 p150 respectively to be disclosed. As a powerful and targeted first-in-class ADAR1 inhibitor, AVA-ADR-001 has demonstrated a strong induction of interferon (IFN) in a number of cancer cell lines and in vivo. AVA-ADR-001 has also demonstrated strong in vivo anti-tumor efficacy as monotherapy and in combination with anti-PD1. Furthermore, AVA-ADR-703 demonstrates selective and highly efficient degradation of ADAR1 p150 while sparing ADAR1 p110. Given that ADAR1 has a pro-metastatic and immune-suppressive role, AVA-ADR-001 and AVA-ADR-703 have significant potential to emerge as strong cancer immunotherapy candidates. Citation Format: Avijit Goswami, Sandeep Goyal, Kawaljit Singh, Princy Khurana, Barnali Deb, Aditya Kulkarni. Discovery of a potential first-in-class Za targeted small molecule inhibitor of ADAR1 p150 which demonstrates strong anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4484.

Recent Biomaterial-Assisted Approaches for Immunotherapeutic Inhibition of Cancer Recurrence.

Biomaterials possess distinctive properties, notably their ability to encapsulate active biological products while providing biocompatible support. The immune system plays a vital role in preventing cancer recurrence, and there is considerable demand for an effective strategy to prevent cancer recurrence, necessitating effective strategies to address this concern. This review elucidates crucial cellular signaling pathways in cancer recurrence. Furthermore, it underscores the potential of biomaterial-based tools in averting or inhibiting cancer recurrence by modulating the immune system. Diverse biomaterials, including hydrogels, particles, films, microneedles, etc., exhibit promising capabilities in mitigating cancer recurrence. These materials are compelling candidates for cancer immunotherapy, offering in situ immunostimulatory activity through transdermal, implantable, and injectable devices. They function by reshaping the tumor microenvironment and impeding tumor growth by reducing immunosuppression. Biomaterials facilitate alterations in biodistribution, release kinetics, and colocalization of immunostimulatory agents, enhancing the safety and efficacy of therapy. Additionally, how the method addresses the limitations of other therapeutic approaches is discussed.

Structural insight into the dual-antagonistic mechanism of AB928 on adenosine A2 receptors.

The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A2AR complexed with AB928 and a selective A2AR antagonist 2-118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2-118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy.

Targeting the activin receptor 1C on CD4+ T cells for cancer immunotherapy

ABSTRACT Activins, members of the TGF-beta superfamily, have been isolated and identified in the endocrine system, but have not been substantially investigated in the context of the immune system and endocrine-unrelated cancers. Here, we demonstrated that tumor-bearing mice had elevated systemic activin levels, which correlated directly with tumor burden. Likewise, cancer patients have elevated plasma activin levels compared to healthy controls. We observed that both tumor and immune cells could be sources of activins. Importantly, our in vitro studies suggest that activins promote differentiation of naïve CD4+ cells into Foxp3-expressing induced regulatory T cells (Tregs), particularly when TGF-beta was limited in the culture medium. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that activin receptor 1c (ActRIC) was uniquely expressed on Tregs and that both ActRIC and ActRIIB (activin receptor 2b) were highly upregulated during iTreg differentiation. ActRIC-deficient naïve CD4+ cells were found to be defective in iTreg generation both in vitro and in vivo. Treg suppression assays were also performed, and ActRIC deficiency did not change the function or stability of iTregs. Mice lacking ActRIC or mice treated with monoclonal anti-ActRIC antibody were more resistant to tumor progression than wild-type controls. This phenotype was correlated with reduced expression of Foxp3 in CD4+ cells in the tumor microenvironment. In light of the information presented above, blocking activin-ActRIC signaling is a promising and disease-specific strategy to impede the accumulation of immunosuppressive iTregs in cancer. Therefore, it is a potential candidate for cancer immunotherapy.

A Novel Anti-CD47 Nanobody Tetramer for Cancer Therapy.

CD47 acts as a defense mechanism for tumor cells by sending a "don't eat me" signal via its bond with SIRPα. With CD47's overexpression linked to poor cancer outcomes, its pathway has become a target in cancer immunotherapy. Though monoclonal antibodies offer specificity, they have limitations like the large size and production costs. Nanobodies, due to their small size and unique properties, present a promising therapeutic alternative. In our study, a high-affinity anti-CD47 nanobody was engineered from an immunized alpaca. We isolated a specific VHH from the phage library, which has nanomolar affinity to SIRPα, and constructed a streptavidin-based tetramer. The efficacy of the nanobody and its derivative was evaluated using various assays. The new nanobody demonstrated higher affinity than the monoclonal anti-CD47 antibody, B6H12.2. The nanobody and its derivatives also stimulated substantial phagocytosis of tumor cell lines and induced apoptosis in U937 cells, a response confirmed in both in vitro and in vivo settings. Our results underscore the potential of the engineered anti-CD47 nanobody as a promising candidate for cancer immunotherapy. The derived nanobody could offer a more effective, cost-efficient alternative to conventional antibodies in disrupting the CD47-SIRPα axis, opening doors for its standalone or combinatorial therapeutic applications in oncology.

Dual regulation effect and mechanism of human myeloid-derived suppressor cells on anticolorectal cancer cells activity of Vγ9Vδ2 T cells.

Myeloid-derived suppressor cells (MDSC) are a group of immature inhibitory cells of bone marrow origin. Human γδ T cells (mainly Vγ9Vδ2 T cells) have emerged as dominant candidates for cancer immunotherapy because of their unique recognition pattern and broad killing activity against tumor cells. Intestinal mucosal intraepithelial lymphocytes are almost exclusively γδ T cells, so it plays an important role in inhibiting the development of colorectal cancer. In this study, we investigated the effects and molecular mechanism of human MDSC on anticolorectal cancer cells activity of Vγ9Vδ2 T cells. Our results suggested that MDSC can reduce the NKG2D expression of Vγ9Vδ2 T cells through direct cell-cell contact, which is associated with membrane-type transforming growth factor-β. In contrast, MDSC can increase Vγ9Vδ2 T cells activation and production of IFN-γ, perforin, Granzyme B through direct cell-cell contact. This may be related to the upregulation of T-bet in Vγ9Vδ2 T cells by MDSC. However, MDSC had a dominant negative regulatory effect on the anticolorectal cancer cells activity of Vγ9Vδ2 T cells. Our study provides a theoretical basis for the immune regulatory function of human MDSC on γδ T cells. This will be conducive to the clinical development of a new antitumor therapy strategy.

A lipid-based LMP2-mRNA vaccine to treat nasopharyngeal carcinoma

This paper focuses on developing a lipid-based vaccine targeting nasopharyngeal carcinoma (NPC), a highly severe and invasive epithelial malignancy associated with the Epstein-Barr virus (EBV). The researchers selected EBV latent membrane protein 2 (LMP2) as the preferred antigen for the vaccine. They synthesized full-length LMP2 using an in vitro transcription method and encapsulated it into cationic liposomes based on (2,3-dioleacyl propyl) trimethylammonium chloride (DOTAP) to create the mRNA vaccine (LPX-mLMP2). Cell assays in the study demonstrated that antigen-presenting cells efficiently took up LPX-mLMP2 and expressed LMP2. This led to the formation of peptide-major histocompatibility complexes (pMHC) for presentation. Moreover, the proliferation of antigen-specific T cells at the tumor site indicated the promising potential for mRNA vaccines in combating virus-induced cancers, such as NPC. The researchers concluded that the newly developed mRNA vaccine encoding the antigen offered advantages in the context of NPC and highlighted the attractiveness of mRNA vaccines as candidates for cancer immunotherapy.

In Situ Vaccination with An Injectable Nucleic Acid Hydrogel for Synergistic Cancer Immunotherapy.

Recently, therapeutic cancer vaccines have emerged as promising candidates for cancer immunotherapy. Nevertheless, their efficacies are frequently impeded by challenges including inadequate antigen encapsulation, insufficient immune activation, and immunosuppressive tumor microenvironment. Herein, we report a three-in-one hydrogel assembled by nucleic acids (NAs) that can serve as a vaccine to in situ trigger strong immune response against cancer. Through site-specifically grafting the chemodrug, 7-ethyl-10-hydroxycamptothecin (also known as SN38), onto three component phosphorothioate (PS) DNA strands, a Y-shaped motif (Y-motif) with sticky ends is self-assembled, at one terminus of which an unmethylated cytosine-phosphate-guanine (CpG) segment is introduced as an immune agonist. Thereafter, programmed cell death ligand-1 (PD-L1) siRNA that performs as immune checkpoint inhibitor is designed as a crosslinker to assemble with the CpG- and SN38-containing Y-motif, resulting in the formation of final NA hydrogel vaccine. With three functional agents inside, the hydrogel can remarkably induce the immunogenic cell death to enhance the antigen presentation, promoting the dendritic cell maturation and effector T lymphocyte infiltration, as well as relieving the immunosuppressive tumor environment. When inoculated twice at tumor sites, the vaccine demonstrates a substantial antitumor effect in melanoma mouse model, proving its potential as a general platform for synergistic cancer immunotherapy.

In Situ Vaccination with An Injectable Nucleic Acid Hydrogel for Synergistic Cancer Immunotherapy

AbstractRecently, therapeutic cancer vaccines have emerged as promising candidates for cancer immunotherapy. Nevertheless, their efficacies are frequently impeded by challenges including inadequate antigen encapsulation, insufficient immune activation, and immunosuppressive tumor microenvironment. Herein, we report a three‐in‐one hydrogel assembled by nucleic acids (NAs) that can serve as a vaccine to in situ trigger strong immune response against cancer. Through site‐specifically grafting the chemodrug, 7‐ethyl‐10‐hydroxycamptothecin (also known as SN38), onto three component phosphorothioate (PS) DNA strands, a Y‐shaped motif (Y‐motif) with sticky ends is self‐assembled, at one terminus of which an unmethylated cytosine‐phosphate‐guanine (CpG) segment is introduced as an immune agonist. Thereafter, programmed cell death ligand‐1 (PD‐L1) siRNA that performs as immune checkpoint inhibitor is designed as a crosslinker to assemble with the CpG‐ and SN38‐containing Y‐motif, resulting in the formation of final NA hydrogel vaccine. With three functional agents inside, the hydrogel can remarkably induce the immunogenic cell death to enhance the antigen presentation, promoting the dendritic cell maturation and effector T lymphocyte infiltration, as well as relieving the immunosuppressive tumor environment. When inoculated twice at tumor sites, the vaccine demonstrates a substantial antitumor effect in melanoma mouse model, proving its potential as a general platform for synergistic cancer immunotherapy.

Identification and optimization of peptide inhibitors to block VISTA/PSGL-1 interaction for cancer immunotherapy.

Developing new therapeutic agents for cancer immunotherapy is highly demanding due to the low response ratio of PD-1/PD-L1 blockade in cancer patients. Here, we discovered that the novel immune checkpoint VISTA is highly expressed on a variety of tumor-infiltrating immune cells, especially myeloid derived suppressor cells (MDSCs) and CD8+ T cells. Then, peptide C1 with binding affinity to VISTA was developed by phage displayed bio-panning technique, and its mutant peptide VS3 was obtained by molecular docking based mutation. Peptide VS3 could bind VISTA with high affinity and block its interaction with ligand PSGL-1 under acidic condition, and elicit anti-tumor activity invivo. The peptide DVS3-Pal was further designed by d-amino acid substitution and fatty acid modification, which exhibited strong proteolytic stability and significant anti-tumor activity through enhancing CD8+ T cell function and decreasing MDSCs infiltration. This is the first study to develop peptides to block VISTA/PSGL-1 interaction, which could act as promising candidates for cancer immunotherapy.

Small molecule inhibitors for cancer immunotherapy and associated biomarkers - the current status.

Following the success of cancer immunotherapy using large molecules against immune checkpoint inhibitors, the concept of using small molecules to interfere with intracellular negative regulators of anti-tumor immune responses has emerged in recent years. The main targets for small molecule drugs currently include enzymes of negative feedback loops in signaling pathways of immune cells and proteins that promote immunosuppressive signals within the tumor microenvironment. In the adaptive immune system, negative regulators of T cell receptor signaling (MAP4K1, DGKα/ζ, CBL-B, PTPN2, PTPN22, SHP1), co-receptor signaling (CBL-B) and cytokine signaling (PTPN2) have been preclinically validated as promising targets and initial clinical trials with small molecule inhibitors are underway. To enhance innate anti-tumor immune responses, inhibitory immunomodulation of cGAS/STING has been in the focus, and inhibitors of ENPP1 and TREX1 have reached the clinic. In addition, immunosuppressive signals via adenosine can be counteracted by CD39 and CD73 inhibition, while suppression via intratumoral immunosuppressive prostaglandin E can be targeted by EP2/EP4 antagonists. Here, we present the status of the most promising small molecule drug candidates for cancer immunotherapy, all residing relatively early in development, and the potential of relevant biomarkers.

Caffeine-folic acid-loaded-chitosan nanoparticles combined with methotrexate as a novel HepG2 immunotherapy targeting adenosine A2A receptor downstream cascade

BackgroundMethotrexate (MTX) is a common chemotherapeutic drug that inhibits DNA synthesis and induces apoptosis. Treatment with MTX increased CD73 expression, which leads to higher levels of extracellular adenosine. Adenosine levels are also high in the tumor microenvironment through Cancer cells metabolism. That promotes the survival of cancer cells and contributes to tumor immune evasion through the Adenosine 2a Receptor. A2A receptor antagonists are an emerging class of agents that treat cancers by enhancing immunotherapy, both as monotherapy and in combination with other therapeutic agents. Caffeine is an adenosine receptor antagonist. Herein, we demonstrate the ability of a novel well prepared and characterized nano formula CAF-FA-CS-NPs (D4) for A2aR blockade when combination with MTX to improve its antitumor efficacy by enhancing the immune system and eliminating immune suppression.MethodsCAF-FA-CS-NPs (D4) were prepared and characterized for particle size, loading efficiency, and release profile. Molecular docking was used to validate the binding affinity of caffeine and folic acid to A2A receptor. The effects of the nano formula were evaluated on human liver cancer cells (HepG2), breast cancer cells (MCF-7), and MDA-MB-231, as well as normal human cells (WI-38). Different combination ratios of MTX and D4 were studied to identify the optimal combination for further genetic studies.ResultsMolecular docking results validated that caffeine and folic acid have binding affinity to A2A receptor. The CS-NPs were successfully prepared using ionic gelation method, with caffeine and folic acid being loaded and conjugated to the nanoparticles through electrostatic interactions. The CAF loading capacity in D4 was 77.9 ± 4.37% with an encapsulation efficiency of 98.5 ± 0.37. The particle size was optimized through ratio variations. The resulting nanoparticles were fully characterized. The results showed that (D4) had antioxidant activity and cytotoxicity against different cancer cells. The combination of D4 with MTX (IC50 D4 + 0.5 IC50 MTX) resulted in the downregulation of Bcl-2, FOXP3, CD39, and CD73 gene expression levels and upregulation of Bax and A2AR gene expression levels in HepG2 cells.ConclusionsThis study suggests that CAF-FA-CS-NPs (D4) in combination with MTX may be a promising candidate for cancer immunotherapy, by inhibiting A2aR signaling and leading to improved immune activation and anti-tumor activity of MTX.

Discovery of Anti-CD47 Peptides as Innate Immune Checkpoint Inhibitors.

Cancer immunotherapy targeting adaptive immune cells has been attracting considerable interest due to its great success in treating multiple cancers. Recently, there is also increasing interest in agents that can stimulate innate immune cell activities. Immune checkpoint inhibitors targeting innate immune cells can block inhibitory interactions ('don't eat me' signals) between tumor cells and phagocytes. CD47 is a transmembrane protein overexpressed in various cancers and acts as a potent 'do not eat me' signal that contributes to the immune evasion of cancer cells. Anti-CD47 peptides that can bind to CD47 and block CD47/SIRPα interaction were discovered using a novel phage display biopanning strategy. Anti-CD47 peptides enhanced the macrophage-mediated phagocytosis of NCI-H82 tumor cells in vitro. Unlike anti-CD47 antibodies, these peptides do not induce the agglutination of RBCs. Moreover, anti-CD47 peptides exhibit high specificity for MC-38 cancer cells expressing CD47. CMP-22 peptide showed the ability to increase the antitumor activity of doxorubicin and extends the survival of CT26 tumor-bearing mice. The discovered anti-CD47 peptides can be considered potential candidates for cancer immunotherapy by blocking the CD47/SIRPα interaction, especially in combination with chemotherapy, to elicit synergistic effects.

Development of a Functional Nanobody Targeting Programmed Cell Death Protein-1 as Immune Checkpoint Inhibitor.

Programmed cell death protein 1 (PD-1) is a membrane receptor that is expressed on the surface of various immune cells, such as T cells, B cells, monocytes, natural killer T cells, and dendritic cells. In cancer, the interaction between PD-1 and its ligand PD-L1 suppresses the activation and function of T lymphocytes, leading to the impairment and apoptosis of tumor-specific T cells. This mechanism allows cancer cells to evade the immune response and promotes tumor progression. Recombinant PD-1 protein was produced and used to immunize a camel. A nanobody library was generated from the camel's peripheral blood lymphocytes and screened for PD-1 binding. A specific nanobody (3PD9) was selected and characterized by affinity measurement, western blotting, and flow cytometry analysis. The ability of the selected nanobody to block the inhibitory signal of PD-1 in peripheral blood mononuclear cells (PBMCs) was evaluated by measuring the level of interleukin-2 (IL-2). The selected nanobody showed high specificity and affinity for human PD-1. Western blot and flow cytometry analysis confirmed that 3PD9 could recognize and bind to human PD-1 on the cell surface. It was demonstrated that the level of IL-2 was significantly increased in PBMCs treated with 3PD9 compared to the control group, indicating that the nanobody could enhance the T cell response by disrupting the PD-1/PD-L1 interaction. The results suggested that the anti-PD-1 nanobody could be a promising candidate for cancer immunotherapy.

Aptamer-Based Immune Drug Systems (AptIDCs) Potentiating Cancer Immunotherapy

Aptamers are artificial oligonucleotides with excellent molecule-targeting ability. Compared with monoclonal antibodies, aptamers have the advantages of low cost, no batch effect, and negligible immunogenicity, making them promising candidates for cancer immunotherapy. To date, a series of aptamer agonists/antagonists have been discovered and directly used to activate immune response, such as immune checkpoint blockade, immune costimulation, and cytokine regulation. By incorporating both tumor- and immune cell-targeting aptamers, multivalent bispecific aptamers were designed to pursue high tumor affinity and enhanced immune efficacy. More importantly, benefiting from feasible chemical modification and programmability, aptamers can be engineered with diverse nanomaterials (e.g., liposomes, hydrogels) and even living immune cells (e.g., NK cells, T cells). These aptamer-based assemblies exhibit powerful capabilities in targeted cargo delivery, regulation of cell–cell interactions, tumor immunogenicity activation, tumor microenvironment remodeling, etc., holding huge potential in boosting immunotherapeutic efficacy. In this review, we focus on the recent advances in aptamer-based immune drug systems (AptIDCs) and highlight their advantages in cancer immunotherapy. The current challenges and future prospects of this field are also pointed out in this paper.