MC38 Tumor Research Articles

Development and evaluation of nanobody tracers for noninvasive nuclear imaging of the immune-checkpoint TIGIT.

T cell Ig and ITIM domain receptor (TIGIT) is a next-generation immune checkpoint predominantly expressed on activated T cells and NK cells, exhibiting an unfavorable prognostic association with various malignancies. Despite the emergence of multiple TIGIT-blocking agents entering clinical trials, only a fraction of patients responded positively to anti-TIGIT therapy. Consequently, an urgent demand arises for noninvasive techniques to quantify and monitor TIGIT expression, facilitating patient stratification and enhancing therapeutic outcomes. Small antigen binding moieties such as nanobodies, are promising candidates for such tracer development. We generated a panel of anti-human or anti-mouse TIGIT nanobodies from immunized llamas. In addition, we designed a single-chain variable fragment derived from the clinically tested monoclonal antibody Vibostolimab targeting TIGIT, and assessed its performance alongside the nanobodies. In vitro characterization studies were performed, including binding ability and affinity to cell expressed or recombinant TIGIT. After Technetium-99m labeling, the nanobodies and the single-chain variable fragment were evaluated in vivo for their ability to detect TIGIT expression using SPECT/CT imaging, followed by ex vivo biodistribution analysis. Nine nanobodies were selected for binding to recombinant and cell expressed TIGIT with low sub-nanomolar affinities and are thermostable. A six-fold higher uptake in TIGIT-overexpressing tumor was demonstrated one hour post- injection with Technetium-99m labeled nanobodies compared to an irrelevant control nanobody. Though the single-chain variable fragment exhibited superior binding to TIGIT-expressing peripheral blood mononuclear cells in vitro, its in vivo behavior yielded lower tumor-to-background ratios at one hour post- injection, indicating that nanobodies are better suited for in vivo imaging than the single-chain variable fragment. Despite the good affinity, high specificity and on-target uptake in mice in this setting, imaging of TIGIT expression on tumor- infiltrating lymphocytes within MC38 tumors remained elusive. This is likely due to the low expression levels of TIGIT in this model. The excellent affinity, high specificity and rapid on-target uptake in mice bearing TIGIT- overexpressing tumors showed the promising diagnostic potential of nanobodies to noninvasively image high TIGIT expression within the tumor. These findings hold promise for clinical translation to aid patient selection and improve therapy response.

Mixed effects modeling of radiotherapy in combination with immune checkpoint blockade or inhibitors of the DNA damage response pathway.

Dosage optimization to maximize efficacy and minimize toxicity is a potential issue when administering radiotherapy (RT) in combination with immune checkpoint blockade (ICB) or inhibitors of the DNA Damage Response Pathway (DDRi) in the clinic. Preclinical models and mathematical modeling can help identify ideal dosage schedules to observe beneficial effects of a tri-therapy. The aim of this study is to describe a mathematical model to capture the impact of RT in combination with inhibitors of the DNA Damage Response Pathway or blockade of the immune checkpoint protein - programmed death ligand 1 (PD-L1). This model describes how RT mediated activation of antigen presenting cells can induce an increase in cytolytic T cells capable of targeting tumor cells, and how combination drugs can potentiate the immune response by inhibiting the rate of T cell exhaustion. The model was fitted using preclinical data, where MC38 tumors were treated in vivo with RT alone or in combination with anti-PD-L1 as well as with either olaparib or the ataxia telangiectasia mutated (ATM) inhibitor-AZD0156. The model successfully described the observed data and goodness-of-fit, using visual predictive checks also confirmed a successful internal model validation for each treatment modality. The results demonstrated that the anti-PD-L1 effect in combination with RT was maximal in vivo and any additional benefit of DDRi at the given dosage and schedule used was undetectable. Model fit results indicated AZD0156 to be a more potent DDRi than olaparib. Simulations of alternative doses indicated that reducing efficacy of anti-PD-L1 by 68% would potentially provide evidence for a benefit of ATM inhibition in combination with ICB and increase the relative efficacy of tri-therapy.

Single-Cell RNA Sequencing Reveals Immuno-Oncology Characteristics of Tumor-Infiltrating T Lymphocytes in Photodynamic Therapy-Treated Colorectal Cancer Mouse Model.

Photodynamic therapy (PDT) has shown promise in reducing metastatic colorectal cancer (CRC); however, the underlying mechanisms remain unclear. Modulating tumor-infiltrating immune cells by PDT may be achieved, which requires the characterization of immune cell populations in the tumor microenvironment by single-cell RNA sequencing (scRNA-seq). Here, we determined the effect of Chlorin e6 (Ce6)-mediated PDT on tumor-infiltrating T cells using scRNA-seq analysis. We used a humanized programmed death-1/programmed death ligand 1 (PD-1/PD-L1) MC38 cell allograft mouse model, considering its potential as an immunogenic cancer model and in combination with PD-1/PD-L1 immune checkpoint blockade. PDT treatment significantly reduced tumor growth in mice containing hPD-1/PD-L1 MC38 tumors. scRNA-seq analysis revealed that the PDT group had increased levels of CD8+ activated T cells and CD8+ cytotoxic T cells, but decreased levels of exhausted CD8+ T cells. PDT treatment also enhanced the infiltration of CD8+ T cells into tumors and increased the production of key effector molecules, including granzyme B and perforin 1. These findings provide insight into immune-therapeutic modulation for CRC patients and highlight the potential of PDT in overcoming immune evasion and enhancing antitumor immunity.

Increased Response to Immune Checkpoint Inhibitors with Dietary Methionine Restriction in a Colorectal Cancer Model.

Dietary methionine restriction (MR), defined as a reduction of methionine intake by around 80%, has been shown to reproducibly decrease tumor growth and synergize with cancer therapies. In this study, we combined DMR with immune checkpoint inhibitors (ICIs) in a model of colon adenocarcinoma. In vitro, we observed that MR increased the expression of MHC-I and PD-L1 in both mouse and human colorectal cancer cells. We also saw an increase in the gene expression of STING, a known inducer of type I interferon signaling. Inhibition of the cGAS-STING pathway, pharmacologically or with siRNA, blunted the increase in MHC-I and PD-L1 surface and gene expression following MR. This indicated that the cGAS-STING pathway, and interferon in general, played a role in the immune response to MR. We then combined dietary MR with ICIs targeting CTLA-4 and PD-1 in an MC38 colorectal cancer tumor model developed in immunocompetent C57BL/6 mice. The combination treatment was five times more effective at reducing the tumor size than ICIs alone in male mice. We noted sex differences in the response to dietary MR, with males showing a greater response than females. Finally, we observed an increase in membrane staining for the PD-L1 protein in MC38 tumors from animals who were fed an MR diet. MHC-I was highly expressed in all tumors and showed no expression difference when comparing tumors from control and MR-treated mice. These results indicated that MR increased PD-L1 expression both in vitro and in vivo and improved the response to ICIs in mice.

Cordycepin synergizes with CTLA-4 blockade to remodel the tumor microenvironment for enhanced cancer immunotherapy

The strategy of using immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, leading to remarkable clinical outcomes. However, certain cancer types and patient demographics continue to face unique challenges. As a result, it is vital to investigate combination therapies that involve ICIs to boost therapeutic efficacy. Cordycepin, an adenosine derivative composed of adenine and pentose, holds immense promise for treating inflammation and cancer. Our recent research has demonstrated that the combined treatment of cordycepin and the anti-CD47 antibody significantly curtails tumor growth and extends the lifespan of tumor-bearing mice. In the current study, we showed that the combination of cordycepin and CTLA-4 blockade had a profound impact on suppressing tumor growth. We utilized the MC38 and CT26 tumor models to evaluate the therapeutic effect of cordycepin, CTLA-4 blockade, and their combined approach. Flow cytometry results unveiled that cordycepin, when combined with CTLA-4 blockade, considerably augmented the presence of tumor-infiltrating CD8+T cells and diminished the population of Foxp3+Tregs within the tumor microenvironment (TME). Additionally, we employed single-cell analysis to examine the TME’s reconfiguration upon the combined treatment of anti-CTLA-4 and cordycepin. We observed a significant impact on inhibiting tumor growth and substantially extended survival in tumor-bearing mice. Our data also demonstrated an increased proportion of effector CD8+T cells in the combined treatment group compared to all other groups, while exhausted CD8+T cells diminished in the combined group compared to the anti-CTLA-4 treatment alone. In conclusion, our findings supported the idea that combining cordycepin and CTLA-4 blockade could modify the effector and exhaustion status of CD8+T cells, thereby bolstering CD8+T-cell-mediated anti-tumor immunity in the TME. Collectively, our current study successfully established a combination therapeutic strategy utilizing cordycepin and CTLA-4 blockade. This strategy demonstrated a significant synergistic effect against cancer, highlighting its importance in cancer treatment.

Preparation of an Ultrahigh-DAR PDL1 monoclonal antibody-polymeric-SN38 conjugate for precise colon cancer therapy

Antibody-drug conjugates (ADCs) are the most potent active tumor-targeting agents used clinically. However, the preparation of ADCs with high drug-to-antibody ratios (DARs) remains a major challenge. Herein, a Fab-nondestructive SN38-loaded antibody-polymeric-drug conjugate (APDC), aPDL1-NPLG-SN38, was prepared that had a DAR as high as 72 for the first time, by increased numbers of payload binding sites via the carboxyl groups of poly (l-glutamic acid) (PLG). The bonding of Fc-III-4C peptide with PLG-graft-mPEG/SN38 (Fc-NPLG-SN38) was achieved using a click reaction between azide and DBCO groups. The aPDL1-NPLG-SN38 conjugate was then synthesized by the high-affinity interaction between the Fc-III-4C peptide in Fc-NPLG-SN38 and the crystallizable fragment (Fc) of PDL1 monoclonal antibody (aPDL1). This approach avoided the potential deleterious effects on the Fab structure of the monoclonal antibody. The aqueous environment used in its preparation helped maintain monoclonal antibody recognition capability. Through the specific recognition by aPDL1 of PDL1 that is highly expressed on MC38 tumors, the accumulation of aPDL1-NPLG-SN38 in the tumors was 2.8-fold greater than achieved with IgG-NPLG-SN38 that had no active tumor-targeting capability. aPDL1-NPLG-SN38 exhibited excellent therapeutic properties in both medium-sized and large MC38 tumor animal models. The present study provides the details of a novel preparation strategy for SN38-loaded ADCs having a high DAR.

Hyaluronic acid-bilirubin nanomedicine-based combination chemoimmunotherapy

Despite significant advances in immune checkpoint blockade (ICB), immunosuppression mediated by tumor-associated myeloid cells (TAMCs) poses a major barrier to cancer immunotherapy. In addition, while immunogenic cell death (ICD) provides a viable approach to inducing anti-tumor immune response, it remains unknown how to effectively trigger ICD while addressing immunosuppressive TAMCs. Here, we show that SC144, a gp130 inhibitor that blocks the IL-6/gp130/STAT3 pathway, induces ICD of tumor cells and polarizes macrophages to M1-phenotype in vitro. However, as SC144 also induces killing of CD8+ T-cells, we sought to deliver SC144 selectively to tumor cells and TAMCs. Toward this goal, we have developed hyaluronic acid-bilirubin nanoparticles (HABN) that accumulate in CD44hi tumor cells and TAMCs. Systemic administration of SC144 loaded in HABN (SC144@HABN) induces apoptosis and ICD of tumor cells, increases the ratio of M1-like to M2-like macrophages, and decreases the frequency of myeloid-derived suppressor cells and CD4+ regulatory T-cells, while promoting anti-tumor CD8+ T-cells. Moreover, SC144@HABN combined with anti-PD-L1 ICB efficiently eliminates MC38 tumors and ICB-resistant 4T1 tumors. Overall, our work demonstrates a therapeutic strategy based on coordinated ICD induction and TAMC modulation and highlights the potential of combination chemoimmunotherapy.

Ncoa2 Promotes CD8+ T cell-Mediated Antitumor Immunity by Stimulating T-cell Activation via Upregulation of PGC-1α Critical for Mitochondrial Function.

Nuclear receptor coactivator 2 (Ncoa2) is a member of the Ncoa family of coactivators, and we previously showed that Ncoa2 regulates the differentiation of induced regulatory T cells. However, it remains unknown if Ncoa2 plays a role in CD8+ T-cell function. Here, we show that Ncoa2 promotes CD8+ T cell-mediated immune responses against tumors by stimulating T-cell activation via upregulating PGC-1α expression to enhance mitochondrial function. Mice deficient in Ncoa2 in T cells (Ncoa2fl/fl/CD4Cre) displayed defective immune responses against implanted MC38 tumors, which associated with significantly reduced tumor-infiltrating CD8+ T cells and decreased IFNγ production. Consistently, CD8+ T cells from Ncoa2fl/fl/CD4Cre mice failed to reject tumors after adoptive transfer into Rag1-/- mice. Further, in response to TCR stimulation, Ncoa2fl/fl/CD4Cre CD8+ T cells failed to increase mitochondrial mass, showed impaired oxidative phosphorylation, and had lower expression of PGC-1α, a master regulator of mitochondrial biogenesis and function. Mechanically, T-cell activation-induced phosphorylation of CREB triggered the recruitment of Ncoa2 to bind to enhancers, thus, stimulating PGC-1α expression. Forced expression of PGC-1α in Ncoa2fl/fl/CD4Cre CD8+ T cells restored mitochondrial function, T-cell activation, IFNγ production, and antitumor immunity. This work informs the development of Ncoa2-based therapies that modulate CD8+ T cell-mediated antitumor immune responses.

Inhibition of tumor intrinsic BANF1 activates antitumor immune responses via cGAS-STING and enhances the efficacy of PD-1 blockade

BackgroundBANF1 is well known as a natural opponent of cyclic GMP-AMP synthase (cGAS) activity on genomic self-DNA. However, the roles of BANF1 in tumor immunity remain unclear. Here, we investigate...

Novel neoantigen peptides and tumor regression in the MC38 colorectal cancer model.

28 Background: Neoantigen vaccine is an emerging modality to treat cancer by unleashing cytotoxic T cells against tumor-specific mutant antigens. Neoantigens are de-novo changes in tumor cells and are hence more immunogenic and less susceptible to off-target toxicities. In building our own pipeline for neoantigen discovery, we used the mouse colon adenocarcinoma cell line MC38 as a validation model. Methods: We sequenced MC38 cells by whole exome and RNA sequencing to shortlist variants. Candidates were confirmed by sanger sequencing, then analyzed for binding affinity to MHC class I H2-Kb and H2-Db. We selected top 10 epitopes for facile modification to maximize immunogenicity by taking into account peptide characteristics, such as hydrophobicity. In-vivo immunogenicity was tested by subcutaneous injections of pooled peptides in C57BL/6J mice. Amongst the 10 peptides tested, we picked five that elicited greatest IFNγ secretion in re-stimulated splenocytes. Predicted binding affinity did not correlate with in-vivo immunogenicity. We inoculated MC38 cells into the left flank of C57BL/6J mice and started treatment four days later. Mice were injected twice weekly with PBS (group 1), CpG only (group 2), anti-PDL1 only (group 3), Peptide pool + CpG (group 4) and Peptide pool + CpG + anti-PDL1 (group 5) (n=8 per group). CpG ODN 1826 is a TLR9 agonist and acts as an adjuvant, while anti-PDL1 is an immune checkpoint inhibitor. We monitored tumor size, animal weights and food intake every 2-3 days. Results: In control group 1, tumor size exponentially increased by 36 folds on the 18th day of treatment. Groups 2, 3, 4 and 5 saw 16, 13, 8 and 5-fold increases in tumor size respectively, 21 days post-treatment. No tumors were found in 3/7 mice in group 5 upon sacrifice, suggesting tumor regression. Tumor control in groups 4 and 5 corroborated with significant IFNγ secretion from neoantigen re-stimulated splenocytes. We observed 38-45 fold expansion in the number of CD8+ KLRG1+ short-lived effector cells and significant increase in PD-1+ TIM3+ exhausted T cells in group 4 and 5 relative to control. Reps1 and Adpgk neoantigen peptides are widely reported and used in MC38 syngenic model. However, they were not included because both were undetected by sequencing. Cell line authentication by STR profiling confirmed that our cells matched that of MC38 from Kerafast. Conclusions: Using an in-house pipeline, we derived a set of novel neoantigen peptides that robustly controlled tumor growth in the MC38 tumor challenge model. In-silico binding predictions did not correlate with in-vivo patterns of IFNγ secretion. In-vivo tests for IFNγ induction after peptide vaccination is a better measure for potential in tumor control. Upregulation of T-cell exhaustion markers after peptide vaccination explains the improved response with anti-PDL1. Reps1 and Adpgk mutations were absent in our cells, which calls caution to researchers conducting similar research.

BAT6026, a novel anti-OX40 antibody with enhanced antibody dependent cellular cytotoxicity effect for cancer immunotherapy.

OX40 (CD134), a member of the TNF receptor superfamily, is a widely studied costimulatory immune checkpoint. Several OX40 agonistic antibodies are in the clinical stage for cancer treatment, among which PF-04518600 is the leader and currently in phase II trial. It has been recognized that one potential mode of action for anti-OX40 antibodies is the deletion of intratumoral Tregs. Thus, a novel human anti-OX40 antibody, BAT6026, was generated with enhanced antibody dependent cellular cytotoxicity (ADCC) via fucose deletion to strengthen its Treg depletion activity. This characteristic of BAT6026 differentiates it from other previously reported anti-OX40 antibodies in the field of tumor therapy. The affinity of BT6026 to OX40 was 0.28nM, approximately 8 times stronger than that of PF-04518600. BAT6026 effectively competed for the binding of ligand OX40L to OX40, whereas PF-04518600 only partially competed. Moreover, compared to PF-04518600, BAT6026 activated T cells more effectively when clustered by FcγRs engagement and stimulated SEB-pretreated PBMCs to secrete IL-2 cytokines in vitro. In addition, BAT6026 demonstrated stronger anti-tumor activity than PF-04518600 in an OX40-humanized mouse MC38 tumor model. BAT6026 also showed a significantly synergistic effect on tumor inhibition when combined treatment with PD-1 antibody. Analysis of tumor-infiltrating T cells revealed that BAT6026 treatment significantly reduced Treg cells and increased CD8+ T cells in tumor. Preclinical safety assessment in non-human primates demonstrated a good safety profile for BAT6026. Together these data warrant further development of BAT6026 into clinical trials for patients with cancer.

Targeted co-delivery of resiquimod and a SIRPα variant by liposomes to activate macrophage immune responses for tumor immunotherapy

Tumor-associated macrophages (TAMs) are the major immune cells infiltrating the tumor microenvironment (TME) and typically exhibit an immunosuppressive M2-like phenotype, which facilitates tumor growth and promotes resistance to immunotherapy. Additionally, tumor cells tend to express high levels of CD47, a “don't eat me” signal, that obstructs macrophage phagocytosis. Consequently, re-educating TAMs in combination with CD47 blockage is promising to trigger intense macrophage immune responses against tumors. As a toll-like receptor 7/8 agonist, resiquimod (R848) possesses the capacity to re-educate TAMs from M2 type to M1 type. We found that intratumoral administration of R848 synergistically improved the antitumor immunotherapeutic effect of CV1 protein (a SIRPα variant with high antagonism to CD47). However, the poor bioavailability and potential toxicity of this combo strategy remain a challenge. Here, a TAMs-targeted liposome (named: R-LS/M/CV1) co-delivering R848 and CV1 protein was constructed via decorating mannose on the liposomal surface. R-LS/M/CV1 exhibited high abilities of targeting, re-education and pro-phagocytosis of tumor cells to M2 macrophages in vitro. Intratumoral administration of R-LS/M/CV1 remarkedly eliminated tumor burden in the MC38 tumor model via repolarization of TAMs to M1 type, pro-phagocytosis of TAMs against tumors, and recruitment of tumor-infiltrating T cells. More encouragingly, due to the double targeting to TAMs and tumor cells of mannose and CV1 protein, R-LS/M/CV1 effectively accumulated at the tumor site, thereby not only remarkedly inhibiting tumors, but also exerting no hematological and histopathological toxicity when administered systemically. Our integrated strategy based on re-educating TAMs and CD47 blockade provides a promising approach to trigger macrophage immune responses against tumors for immunotherapy.

TARGETING TREG CELLS BY TNFR2 ANTIBODY INDUCES TUMOR REGRESSION IN VIVO

Abstract Background Tumor necrosis factor receptor 2 (TNFR2) is considered an appealing target due to its low-level expression on immune cells, but it could be upregulated on regulatory T cells (Tregs) in the tumor microenvironment which plays key roles in Treg proliferation and function. It has been demonstrated that Tregs with high TNFR2 expression were the most suppressive subsets among all Treg populations in the tumor. While early studies showed that TNFR2 co-stimulates naïve T cells, it has been revealed later that TNFR2 also limits CD8+ T-cell-mediated viral clearance and anti-tumor immunity by inducing rapid contraction of CD8+ T cells. Consistent with these findings, several publications reported that anti-TNFR2 antibody treatment exhibited robust antitumor efficacy. In short, TNFR2 signaling is critical in regulating immune response in different diseases. In the current study, the anti-human TNFR2 antibody SBT-1901 was developed and the anti-tumor activity was evaluated. Materials and methods SBT-1901 was generated through hybridoma and humanization technologies. The binding affinity and specificity were tested by ELISA, FACS, and OCTET. The function of SBT-1901 was tested in TNFR2-Fas overexpressing Ramos cells and in Treg proliferation assay. The in vivo anti-tumor activity and pharmacokinetics of SBT-1901 were evaluated in the human TNFR2 transgenic mouse model (Biocytogen) bearing MC38 tumor. Results SBT-1901 binds to the extracellular domain of human and cynomolgus TNFR2 with the affinity of single digit-nanomolar. It competes with TNFα for binding to TNFR2 receptor and inhibits TNFα-induced TNFR2-Fas overexpressing RAMOS cell death. SBT-1901 also blocks TNFα induced Treg proliferation in human PBMC. In addition, SBT-1901 significantly inhibits MC38 tumor growth in a dose-dependent manner as a monotherapy and enhances anti-tumor efficacy of mPD-1 antibody in a combination study in human TNFR2 transgenic mouse model. SBT-1901 is currently under preclinical development. Conclusions Our studies show that SBT-1901 exhibits a very potent anti-tumor efficacy in vivo as a single agent or the combination. Therefore, it is highly valuable to further develop SBT-1901 for human cancer treatment.

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

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

Tumor cell-intrinsic c-Myb upregulation stimulates antitumor immunity in a murine colorectal cancer model.

The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. While no differences in proliferation, apoptosis, and angiogenesis of tumors were evident tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAMs). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding 2-microglobulin and IFNβ, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.

Intratumoral injection of IL-12-encoding mRNA targeted to CSF1R and PD-L1 exerts potent anti-tumor effects without substantial systemic exposure

IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in the clinic. Currently, the intratumoral injection of IL-12-encoding mRNA or DNA to avoid such side effects is being evaluated in clinical trials. In this study, we aimed to improve this strategy by further favoring IL-12 tethering to the tumor. We generated invitro transcribed mRNAs encoding murine single-chain IL-12 fused to diabodies binding to CSF1R and/or PD-L1. These targeted molecules are expressed in the tumor microenvironment, especially on myeloid cells. The binding capacity of chimeric constructs and the bioactivity of IL-12 were demonstrated invitro and invivo. Doses as low as 0.5μg IL-12-encoding mRNA achieved potent antitumor effects in subcutaneously injected B16-OVA and MC38 tumors. Treatment delivery was associated with increases in IL-12p70 and IFN-γ levels in circulation. Fusion of IL-12 to the diabodies exerted comparable efficacy against bilateral tumor models. However, it achieved tethering to myeloid cells infiltrating the tumor, resulting in nearly undetectable systemic levels of IL-12 and IFN-γ. Overall, tethering IL-12 to intratumoral myeloid cells in the mRNA-transferred tumors achieves similar efficacy while reducing the dangerous systemic bioavailability of IL-12.

Intratumoral IL15 Improves Efficacy of Near-Infrared Photoimmunotherapy.

IL15 is a potent inducer of differentiation and proliferation of CD8+ T and natural killer (NK) cells, making it a promising candidate for cancer immunotherapy. However, limited efficacy of systemic monotherapy utilizing intravenous IL15 suggests the needs for alternative routes of administration or combination treatment with other therapies. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective anticancer treatment that elicits a massive release of tumor antigens and immunogenic signals. Here, we investigated whether intratumoral IL15 can enhance the effectiveness of cancer cell-targeted NIR-PIT using syngeneic murine tumor models. Intratumoral injection of IL15 was more effective than intraperitoneal IL15 in vivo in suppressing tumor growth and inducing intratumoral immune responses. When the efficacy of CD44-targeted NIR-PIT was compared in vivo between IL15-secreting MC38 (hIL15-MC38) and parental MC38 tumors, the hIL15-MC38/NIR-PIT group showed the best tumor growth inhibition and survival. In addition, the hIL15-MC38/NIR-PIT group showed significant dendritic cell maturation and significant increases in the number and Granzyme B expression of tumor-infiltrating CD8+ T, NK, and natural killer T cells compared with the treated parental line. Furthermore, intratumoral IL15 injection combined with CD44-targeted NIR-PIT showed significant tumor control in MC38 and Pan02-luc tumor models. In bilateral tumor models, CD44-targeted NIR-PIT in hIL15-MC38 tumors significantly suppressed the growth of untreated MC38 tumors, suggesting abscopal effects. Mice that achieved complete response after the combination therapy completely rejected later tumor rechallenge. In conclusion, local IL15 administration synergistically improves the efficacy of cancer cell-targeted NIR-PIT probably by inducing stronger anticancer immunity, indicating its potential as an anticancer treatment strategy.

Ultrasound-responsive low-dose doxorubicin liposomes trigger mitochondrial DNA release and activate cGAS-STING-mediated antitumour immunity

DNA derived from chemotherapeutics-killed tumor cells is one of the most important damage-associated molecular patterns that can activate the cGAS-STING (cyclic GMP-AMP synthase—stimulator of interferon genes) pathway in antigen-presenting cells (APCs) and promote antitumor immunity. However, conventional chemotherapy displays limited tumor cell killing and ineffective transfer of stable tumor DNA to APCs. Here we show that liposomes loaded with an optimized ratio of indocyanine green and doxorubicin, denoted as LID, efficiently generate reactive oxygen species upon exposure to ultrasound. LID plus ultrasound enhance the nuclear delivery of doxorubicin, induce tumor mitochondrial DNA oxidation, and promote oxidized tumor mitochondrial DNA transfer to APCs for effective activation of cGAS-STING signaling. Depleting tumor mitochondrial DNA or knocking out STING in APCs compromises the activation of APCs. Furthermore, systemic injection of LID plus ultrasound over the tumor lead to targeted cytotoxicity and STING activation, eliciting potent antitumor T cell immunity, which upon the combination with immune checkpoint blockade leads to regression of bilateral MC38, CT26, and orthotopic 4T1 tumors in female mice. Our study sheds light on the importance of oxidized tumor mitochondrial DNA in STING-mediated antitumor immunity and may inspire the development of more effective strategies for cancer immunotherapy.

Characterization of 405B8H3(D-E), a newly engineered high affinity chimeric LAG-3 antibody with potent antitumor activity.

Lymphocyte activation gene-3 (LAG-3) is a type I transmembrane protein with structural similarities to CD4. Overexpression of LAG-3 enables cancer cells to escape immune surveillance, while its blockade reinvigorates exhausted T cells and strengthens anti-infection immunity. Blockade of LAG-3 may have antitumor effects. Here, we generated a novel anti-LAG-3 chimeric antibody, 405B8H3(D-E), through hybridoma technology from monoclonal antibodies produced in mice. The heavy-chain variable region of the selected mouse antibody was grafted onto a human IgG4 scaffold, while a modified light-chain variable region was coupled to the human kappa light-chain constant region. 405B8H3(D-E) could effectively bind LAG-3-expressing HEK293 cells. Moreover, it could bind cynomolgus monkey (cyno) LAG-3 expressed on HEK293 cells with a higher affinity than the reference anti-LAG-3 antibody BMS-986016. Furthermore, 405B8H3(D-E) promoted interleukin-2 secretion and was able to block the interactions of LAG-3 with liver sinusoidal endothelial cell lectin and major histocompatibility complex II molecules. Finally, 405B8H3(D-E) combined with anti-mPD-1-antibody showed effective therapeutic potential in the MC38 tumor mouse model. Therefore, 405B8H3(D-E) is likely to be a promising candidate therapeutic antibody for immunotherapy.

Nuanced role for dendritic cell intrinsic IRE1 RNase in the regulation of antitumor adaptive immunity.

In cancer, activation of the IRE1/XBP1s axis of the unfolded protein response (UPR) promotes immunosuppression and tumor growth, by acting in cancer cells and tumor infiltrating immune cells. However, the role of IRE1/XBP1s in dendritic cells (DCs) in tumors, particularly in conventional type 1 DCs (cDC1s) which are cellular targets in immunotherapy, has not been fully elucidated. Here, we studied the role of IRE1/XBP1s in subcutaneous B16/B78 melanoma and MC38 tumors by generating loss-of-function models of IRE1 and/or XBP1s in DCs or in cDC1s. Data show that concomitant deletion of the RNase domain of IRE1 and XBP1s in DCs and cDC1s does not influence the kinetics of B16/B78 and MC38 tumor growth or the effector profile of tumor infiltrating T cells. A modest effect is observed in mice bearing single deletion of XBP1s in DCs, which showed slight acceleration of melanoma tumor growth and dysfunctional T cell responses, however, this effect was not recapitulated in animals lacking XBP1 only in cDC1s. Thus, evidence presented here argues against a general pro-tumorigenic role of the IRE1/XBP1s pathway in tumor associated DC subsets.