Combination immunotherapy of chlorogenic acid liposomes modified with sialic acid and PD-1 blockers effectively enhances the anti-tumor immune response and therapeutic effects

Melanoma is one of the most common malignant tumors. The anti-PD-1 antibody is used for the treatment of metastatic melanoma. Treatment success is only 35–40% and a range of immune-related adverse reactions can occur. Combination of anti-PD1 antibody therapy with other oncology therapies has been attempted. Herein, we assessed whether chlorogenic acid liposomes modified with sialic acid (CA-SAL) combined with anti-PD1 antibody treatment was efficacious as immunotherapy for melanoma. CA-SAL liposomes were prepared and characterized. In a mouse model of B16F10 tumor, mice were treated with an anti-PD1 antibody, CA-SAL, or combination of CA-SAL + anti-PD1 antibody, and compared with no treatment controls. The tumor inhibition rate, tumor-associated macrophages (TAMs) phenotype, T-cell activity, and safety were investigated. We observed a significant decrease in the proportion of M2-TAMs and CD4⁺Fop3⁺ T cells, while there was a significant increase in the proportion of M1-TAMs and CD8⁺ T cells, and in the activity of T cells, and thus in the tumor inhibition rate. No significant toxicity was observed in major organs. CA-SAL and anti-PD1 Ab combination therapy presented synergistic anti-tumor activity, which enhanced the efficacy of the PD-1 checkpoint blocker in a mouse model of melanoma. In summary, combination immunotherapy of CA-SAL and anti-PD1 Ab has broad prospects in improving the therapeutic effect of melanoma, and may provide a new strategy for clinical treatment.     

Gemcitabine and checkpoint blockade exhibit synergistic anti-tumor effects in a model of murine lung carcinoma

Lung cancer is leading cause of cancer death in the world. Chemotherapy is currently one of the standard treatments for lung cancer. Gemcitabine is a pyrimidine nucleoside drug which has been approved by FDA to treat lung cancer. However, acquired resistance inevitable develops after Gemcitabine treatment, limiting clinical efficacy. Lewis lung carcinoma (LLC) cells were treated with Gemcitabine and cell apoptosis and programmed cell death-ligand 1 (PD-L1) expression were analyzed by flow cytometry. LLC mouse model was established to analysis the proportion and programmed cell death-1 (PD-1) expression of CD8 + T cells. Anti-tumor effect by treating with Gemcitabine and anti-PD-1 antibody was measured through in vivo LLC mouse model. Gemcitabine treatment induces tumor cell apoptosis and PD-L1 expression. Further study showed that Gemcitabine treatment also increases CD8⁺ and CD4⁺ T cells proportion, PD-1 and PD-L1 expression in LLC mouse model. Combination therapy with Gemcitabine and αPD-1 not only has strong anti-tumor effect, but also could inhibit postsurgical recurrence of LLC. Our findings demonstrated that the combination therapy of Gemcitabine and αPD-1 is an effective therapeutic strategy for lung cancer.     

A novel cyclic peptide targeting LAG-3 for cancer immunotherapy by activating antigen-specific CD8+ T cell responses

PD-1 and CTLA-4 antibodies offer great hope for cancer immunotherapy. However, many patients are incapable of responding to PD-1 and CTLA-4 blockade and show low response rates due to insufficient immune activation. The combination of checkpoint blockers has been proposed to increase the response rates. Besides, antibody drugs have disadvantages such as inclined to cause immune-related adverse events and infiltration problems. In this study, we developed a cyclic peptide C25 by using Ph.D.-C7C phage display technology targeting LAG-3. As a result, C25 showed a relative high affinity with human LAG-3 protein and could effectively interfere the binding between LAG-3 and HLA-DR (MHC-II). Additionally, C25 could significantly stimulate CD8⁺ T cell activation in human PBMCs. The results also demonstrated that C25 could inhibit tumor growth of CT26, B16 and B16-OVA bearing mice, and the infiltration of CD8⁺ T cells was significantly increased while FOXP3⁺ Tregs significantly decreased in the tumor site. Furthermore, the secretion of IFN-γ by CD8⁺ T cells in spleen, draining lymph nodes and especially in the tumors was promoted. Simultaneously, we exploited T cells depletion models to study the anti-tumor mechanisms for C25 peptide, and the results combined with MTT assay confirmed that C25 exerted anti-tumor effects via CD8⁺ T cells but not direct killing. In conclusion, cyclic peptide C25 provides a rationale for targeting the immune checkpoint, by blockade of LAG-3/HLA-DR interaction in order to enhance anti-tumor immunity, and C25 may provide an alternative for cancer immunotherapy besides antibody drugs.     

Abrogation of HnRNP L enhances anti-PD-1 therapy efficacy via diminishing PD-L1 and promoting CD8+ T cell-mediated ferroptosis in castration-resistant prostate cancer

Owing to incurable castration-resistant prostate cancer (CRPC) ultimately developing after treating with androgen deprivation therapy (ADT), it is vital to devise new therapeutic strategies to treat CRPC. Treatments that target programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for human cancers with clinical benefit. However, many patients, especially prostate cancer, fail to respond to anti-PD-1/PD-L1 treatment, so it is an urgent need to seek a support strategy for improving the traditional PD-1/PD-L1 targeting immunotherapy. In the present study, analyzing the data from our prostate cancer tissue microarray, we found that PD-L1 expression was positively correlated with the expression of heterogeneous nuclear ribonucleoprotein L (HnRNP L). Hence, we further investigated the potential role of HnRNP L on the PD-L1 expression, the sensitivity of cancer cells to T-cell killing and the synergistic effect with anti-PD-1 therapy in CRPC. Indeed, HnRNP L knockdown effectively decreased PD-L1 expression and recovered the sensitivity of cancer cells to T-cell killing in vitro and in vivo, on the contrary, HnRNP L overexpression led to the opposite effect in CRPC cells. In addition, consistent with the previous study, we revealed that ferroptosis played a critical role in T-cell-induced cancer cell death, and HnRNP L promoted the cancer immune escape partly through targeting YY1/PD-L1 axis and inhibiting ferroptosis in CRPC cells. Furthermore, HnRNP L knockdown enhanced antitumor immunity by recruiting infiltrating CD8⁺ T cells and synergized with anti-PD-1 therapy in CRPC tumors. This study provided biological evidence that HnRNP L knockdown might be a novel therapeutic agent in PD-L1/PD-1 blockade strategy that enhanced anti-tumor immune response in CRPC.     

Apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression

Increasing studies confirm that anti-angiogenesis can increase the effectiveness of immunotherapy. In this study, we found that an angiogenesis inhibitor apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression. Apatinib treatment upregulated PD-L1 expression in various colon cancer cells both at the mRNA and protein levels. Further, apatinib-treated cancer cells hampered activation and IFN-γ secretion of T cells in the co-culture system, which was reversed by the anti-PD-1 antibody. Based on this, the combination of apatinib with anti-PD-1 on colon cancer growth in mice was examined. The combination treatment showed more significant inhibition on the growth of transplanted tumors in mice than single-drug treatment. Overall, our study here showed the enhancement of anti-PD-1 antitumor efficacy in a syngeneic mouse model (CT-26 cells in Balb/c) by the angiogenesis inhibitor apatinib via upregulating PD-L1 expression as well as angiogenesis inhibition, which may provide a rationale for the combination of apatinib and anti-PD-1 antibody for colorectal cancer treatment in the clinic.     

PD-1 blockade augments humoral immunity through ICOS-mediated CD4+ T cell instruction

Successful applications of PD-1/PD-L1 blockade in multiple cancers highlight the efficacy of immunotherapy mediated by enhancing CD8⁺ T cell immunity both in mouse and human. How PD-1 blockade affects humoral immunity remains unclear. Herein we demonstrated that treatment of anti-PD-1 antibody led to the increase in both total IgG and OVA-specific IgG in OVA-immunized mice. However, no effect was observed on Ab affinity maturation. Accumulation of germinal center (GC) and memory B cells was observed in the spleens together with elevated percentages of plasma cells in the spleens and bone marrow. More interestingly, dramatic infiltration of CD4⁺ T cells was apparent in GCs after PD-1 blockade with a significant increase in the expression of ICOS. When CD4⁺ T cells and B cells from OVA-immunized mice were co-cultured with neutralizing anti-PD-1 Ab in vitro, PD-1 blockade recapitulated the up-regulation of ICOS expression on CD4⁺ T cells with the activation of ERK signaling. Suppression of ERK activation not only reduced ICOS expression on CD4⁺ T cells but also attenuated IgG production upon PD-1 blockade. Taken together, PD-1 blockade enhances humoral immunity. This process partially relies on more accumulation of CD4⁺ T cells in GCs with the up-regulation of ICOS expression and the promotion of B cell terminal differentiation. The regulatory pattern of PD-1 blockade illustrated here provides a new mechanism of how immune checkpoint molecules regulating humoral immune responses.     

Remodeling “cold” tumor immune microenvironment via epigenetic-based therapy using targeted liposomes with in situ formed albumin corona

There is a close connection between epigenetic regulation, cancer metabolism, and immunology. The combination of epigenetic therapy and immunotherapy provides a promising avenue for cancer management. As an epigenetic regulator of histone acetylation, panobinostat can induce histone acetylation and inhibit tumor cell proliferation, as well as regulate aerobic glycolysis and reprogram intratumoral immune cells. JQ1 is a BRD4 inhibitor that can suppress PD-L1 expression. Herein, we proposed a chemo-free, epigenetic-based combination therapy of panobinostat/JQ1 for metastatic colorectal cancer. A novel targeted binary-drug liposome was developed based on lactoferrin-mediated binding with the LRP-1 receptor. It was found that the tumor-targeted delivery was further enhanced by in situ formation of albumin corona. The lactoferrin modification and endogenous albumin adsorption contribute a dual-targeting effect on the receptors of both LRP-1 and SPARC that were overexpressed in tumor cells and immune cells (e.g., tumor-associated macrophages). The targeted liposomal therapy was effective to suppress the crosstalk between tumor metabolism and immune evasion via glycolysis inhibition and immune normalization. Consequently, lactic acid production was reduced and angiogenesis inhibited; TAM switched to an anti-tumor phenotype, and the anti-tumor function of the effector CD8⁺ T cells was reinforced. The strategy provides a potential method for remodeling the tumor immune microenvironment (TIME).     

Remodeling “cold” tumor immune microenvironment via epigenetic-based therapy using targeted liposomes with in situ formed albumin corona

There is a close connection between epigenetic regulation, cancer metabolism, and immunology. The combination of epigenetic therapy and immunotherapy provides a promising avenue for cancer management. As an epigenetic regulator of histone acetylation, panobinostat can induce histone acetylation and inhibit tumor cell proliferation, as well as regulate aerobic glycolysis and reprogram intratumoral immune cells. JQ1 is a BRD4 inhibitor that can suppress PD-L1 expression. Herein, we proposed a chemo-free, epigenetic-based combination therapy of panobinostat/JQ1 for metastatic colorectal cancer. A novel targeted binary-drug liposome was developed based on lactoferrin-mediated binding with the LRP-1 receptor. It was found that the tumor-targeted delivery was further enhanced by in situ formation of albumin corona. The lactoferrin modification and endogenous albumin adsorption contribute a dual-targeting effect on the receptors of both LRP-1 and SPARC that were overexpressed in tumor cells and immune cells (e.g., tumor-associated macrophages). The targeted liposomal therapy was effective to suppress the crosstalk between tumor metabolism and immune evasion via glycolysis inhibition and immune normalization. Consequently, lactic acid production was reduced and angiogenesis inhibited; TAM switched to an anti-tumor phenotype, and the anti-tumor function of the effector CD8⁺ T cells was reinforced. The strategy provides a potential method for remodeling the tumor immune microenvironment (TIME).KEY WORDS: Tumor immune microenvironment, Tumor-associated macrophage, Epigenetic therapy, Immune checkpoint, Angiogenesis, Panobinostat, JQ1, Liposome     

Modulating the tumor immune microenvironment with sunitinib malate supports the rationale for combined treatment with immunotherapy

Small molecule inhibitors have proven useful in the treatment of a variety of tumors, but they are often limited by unsustainable benefits and confer resistance quickly. Immunotherapy can result in durable clinical responses, but activity only occurs in a minority of patients. The unfavorable tumor microenvironment (TME) is an important factor limiting immunotherapy. An appropriate understanding of how small molecule inhibitors modulate the TME may optimize the combination of targeted treatment and immunotherapy in managing tumors. In this study, we found that transient treatment with sunitinib malate inhibited the disorganized extension of tumor vessels, pericytes and collagen IV but increased the relative ratio of pericyte-wrapping blood vessels with alleviated hypoxia in tumors, which resulted from tumor vascular normalization. Sunitinib malate increased infiltration of CD8⁺ T cells and decreased regulatory T cells (Tregs), accompanied by inhibited expression of TGF-β1 and IL-10 and increased CCL-28, IFN-γ and IL-12, but no significant inhibition of myeloid-derived suppressor cells (MDSCs) was observed. In addition, sunitinib malate increased the levels of PD-1 and PD-L1 in TME, combined with anti-PD-1 therapy showed a significant reduction in tumor burden compared with either monotherapy, suggesting that anti-PD-1 therapy is reasonable after sunitinib malate treatment.     

Andrographolide sulfonate ameliorates experimental colitis in mice by inhibiting Th1/Th17 response

Inflammatory bowel disease (IBD) is a chronic, relapsing and remitting condition of inflammation involves overproduction of pro-inflammatory cytokines and excessive functions of inflammatory cells. However, current treatments for IBD may have potential adverse effects including steroid dependence, infections and lymphoma. Therefore new therapies for the treatment of IBD are desperately needed. In the present study, we aimed to examine the effect of andrographolide sulfonate, a water-soluble form of andrographolide (trade name: Xi-Yan-Ping Injection), on murine experimental colitis induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). Andrographolide sulfonate was administrated through intraperitoneal injection to mice with TNBS-induced colitis. TNBS-induced body weight loss, myeloperoxidase activity, shortening of the colon and colonic inflammation were significantly ameliorated by andrographolide sulfonate. Both the mRNA and protein levels of pro-inflammatory cytokines were reduced by andrographolide sulfonate administration. Moreover, andrographolide sulfonate markedly suppressed the activation of p38 mitogen-activated protein kinase as well as p65 subunit of nuclear factor-κB (NF-κB). Furthermore, CD4⁺ T cell infiltration as well as the differentiation of Th1 (CD4⁺IFN-γ⁺) and Th17 (CD4⁺IL17A⁺) subset were inhibited by andrographolide sulfonate. In summary, these results suggest that andrographolide sulfonate ameliorated TNBS-induced colitis in mice through inhibiting Th1/Th17 response. Our study shows that water-soluble andrographolide sulfonate may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders.     

Improving cancer immunotherapy via co-delivering checkpoint blockade and thrombospondin-1 downregulator

The use of checkpoint-blockade antibodies is still restricted in several malignancies due to the modest efficacy,despite considerable success in anti-tumor immunotherapy.The poor response of cancer cells to immune destruction is an essential contributor to the failure of checkpoint therapy.We hypothesized that combining checkpoint therapy with natural-product chemosensitizer could enhance immune response.Herein,a targeted diterpenoid derivative was integrated with the checkpoint blockade(anti-CT...     

l-arginine and docetaxel synergistically enhance anti-tumor immunity by modifying the immune status of tumor-bearing mice

l-arginine (l-Arg) supplementation has been reported to enhance the function of immune cells, including dendritic cells (DCs) and T lymphocytes, in cancer models thereby countering the suppressive effects of myeloid-derived suppressor cells (MDSCs). The balance of the active immune cells is one factor that determines the progression of cancers in vivo. Docetaxel (DTX), an immunomodulatory chemotherapeutic agent, is now widely used in several types of malignancies including breast cancer. We hypothesized that the combination of DTX and l-Arg would elicit a more robust antitumor response than either molecule alone. To test this hypothesis we utilized BALB/c mice inoculated with 4T1 mammary carcinoma cells. DTX and l-Arg synergistically limited tumor growth in vivo and moderately increased the life span of tumor bearing mice. The anti-tumor effects were associated with the proliferation of splenic CD8⁺ CTL and CD4⁺ Th1 effector cells, as well as increased serum levels of interferon gamma. More importantly, DTX + l-Arg effectively increased anti-tumor immunity within the tumor microenvironment. Furthermore, the combined therapy increased the number of myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, potent activators of the T cell response, and enhanced expression of the maturation markers CD86 and MHC II (required for antigen presentation). The combination therapy also reduced the proliferation of MDSCs. These data suggest that DTX + l-Arg may be a novel therapeutic strategy for breast cancer patients.     

Bispecific Antibody-Bound T Cells as a Novel Anticancer Immunotherapy

Chimeric antigen receptor T (CAR-T) cell therapy is one of the promising anticancer treatments. It shows a high overall response rate with complete response to blood cancer. However, there is a limitation to solid tumor treatment. Additionally, this currently approved therapy exhibits side effects such as cytokine release syndrome and neurotoxicity. Alternatively, bispecific antibody is an innovative therapeutic tool that simultaneously engages specific immune cells to disease-related target cells. Since programmed death ligand 1 (PD-L1) is an immune checkpoint molecule highly expressed in some cancer cells, in the current study, we generated αCD3xαPD-L1 bispecific antibody (BiTE) which can engage T cells to PD-L1⁺ cancer cells. We observed that the BiTE-bound OT-1 T cells effectively killed cancer cells in vitro and in vivo. They substantially increased the recruitment of effector memory CD8⁺ T cells having CD8⁺CD44⁺CD62L^low phenotype in tumor. Interestingly, we also observed that BiTE-bound polyclonal T cells showed highly efficacious tumor killing activity in vivo in comparison with the direct intravenous treatment of bispecific antibody, suggesting that PD-L1-directed migration and engagement of activated T cells might increase cancer cell killing. Additionally, BiTE-bound CAR-T cells which targets human Her-2/neu exhibited enhanced killing effect on Her-2-expressing cancer cells in vivo, suggesting that this could be a novel therapeutic regimen. Collectively, our results suggested that engaging activated T cells with cancer cells using αCD3xαPD-L1 BiTE could be an innovative next generation anticancer therapy which exerts simultaneous inhibitory functions on PD-L1 as well as increasing the infiltration of activated T cells having effector memory phenotype in tumor site.     

Enhanced chemo-immunotherapy against melanoma by inhibition of cholesterol esterification in CD8+ T cells

Cholesterol facilitated the formation of T cell receptor on cytotoxic CD8⁺ T lymphocytes (CTLs). However, the activation of CD8⁺ T cells always resulted in the upregulation of acetyl-CoA acetyltransferase-1 (ACAT-1) and enhanced the esterification of cholesterol. To relieve the suppression on CD8⁺ T cells, an ACAT-1 inhibitor avasimibe was combined with chemo-immunotherapy. Paclitaxel and immunoadjuvant αGC were co-encapsulated in liposomes modified with pH sensitive TH peptide (PTX/αGC-TH-Lip). After intravenous injections, the combination of avasimibe significantly elevated the free cholesterol level and relieved the inhibition of CD8⁺ T cells caused by PTX/αGC-TH-Lip, leading to enhanced CTL responses and anti-tumor effects of PTX/αGC-TH-Lip in B16F10 melanoma xenograft and lung metastasis models. The adoptive immunotherapy further confirmed the enhanced anti-tumor immune responses of the combined strategy. The combination of avasimibe and PTX/αGC-TH-Lip was proven as a feasible approach to enhance the antitumor effects of chemo-immunotherapy by relieving the inhibition of CD8⁺ T cells.     

Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1highCD11b+ cells elimination in hepatocellular carcinoma

Cryoablation (CRA) and microwave ablation (MWA) are two main local treatments for hepatocellular carcinoma (HCC). However, which one is more curative and suitable for combining with immunotherapy is still controversial. Herein, CRA induced higher tumoral PD-L1 expression and more T cells infiltration, but less PD-L1^highCD11b⁺ myeloid cells infiltration than MWA in HCC. Furthermore, CRA had better curative effect than MWA for anti-PD-L1 combination therapy in mouse models. Mechanistically, anti-PD-L1 antibody facilitated infiltration of CD8⁺ T cells by enhancing the secretion of CXCL9 from cDC1 cells after CRA therapy. On the other hand, anti-PD-L1 antibody promoted the infiltration of NK cells to eliminate PD-L1^highCD11b⁺ myeloid cells by antibody-dependent cell-mediated cytotoxicity (ADCC) effect after CRA therapy. Both aspects relieved the immunosuppressive microenvironment after CRA therapy. Notably, the wild-type PD-L1 Avelumab (Bavencio), compared to the mutant PD-L1 atezolizumab (Tecentriq), was better at inducing the ADCC effect to target PD-L1^highCD11b⁺ myeloid cells. Collectively, our study uncovered the novel insights that CRA showed superior curative effect than MWA in combining with anti-PD-L1 antibody by strengthening CTL/NK cell immune responses, which provided a strong rationale for combining CRA and PD-L1 blockade in the clinical treatment for HCC.     

PD0325901, an ERK inhibitor,enhances the efficacy of PD-1 inhibitor in non-small cell lung carcinoma

ERK pathway regulated the programmed death ligand-1 (PD-L1) expression which was linked to the response of programmed death-1 (PD-1)/PD-L1 blockade therapy. So it is deducible that ERK inhibitor could enhance the efficacy of PD-1 inhibitor in cancer immunotherapy. In this study, PD0325901, an oral potent ERK inhibitor, strongly enhanced the efficacy of PD-1 antibody in vitro and in vivo models in non-small cell lung carcinoma (NSCLC) cells. Mechanistically, PD0325901 or shRNA-ERK1/2 significantly downregulated the PD-L1 expression in NSCLC cells and increased the CD3⁺ T cells infiltration and functions in tumor tissue. There was a positive correlation between the p-ERK1/2 expression and PD-L1 expression in patients with NSCLC. And the patients with low p-ERK1/2 expression were observed a high response rate of PD-1/PD-L1 blockage therapy. Our results demonstrate that PD0325901, an ERK inhibitor, can enhance the efficacy of PD-1 blockage against NSCLC in vitro and in vivo models. And the combination of ERK inhibitor such as PD0325901 and PD-1/PD-L1 blockage is a promising regimen and encouraged to be further confirmed in the treatment of patients with NSCLC.     

Inhibition of human cervical carcinoma growth by cytokine-induced killer cells in nude mouse xenograft model

Cervical cancer is a major cause of cancer mortality in women worldwide and is an important public health problem for adult women in developing countries. Despite aggressive treatment with surgery and chemoradiation, the outcomes for cervical cancer patients remain poor. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against human cervical cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The resulting populations of CIK cells comprised 95% CD3⁺, 3% CD3⁻CD56⁺, 35% CD3⁺CD56⁺, 11% CD4⁺, < 1% CD4⁺CD56⁺, 80% CD8⁺, and 25% CD8⁺CD56⁺. At an effector-target cell ratio of 100:1, CIK cells destroyed 56% of KB-3-1 human cervical cancer cells, as measured by the ⁵¹Cr-release assay. In addition, CIK cells at doses of 3 and 10 million cells per mouse inhibited 34% and 57% of KB-3-1 tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for cervical cancer patients.     

Naloxone can improve the anti-tumor immunity by reducing the CD4CD25Foxp3 regulatory T cells in BALB/c mice

New strategies that stimulate cell-mediated immunity (CMI) against tumors and inhibit regulatory T cells are needed to improve the outcome of cancer immunotherapy. The aim of this study was to enhance the anti-tumor immunity of gp96 vaccine through naloxone administration. Therefore, we used BALB/c mouse model of fibrosarcoma tumor and analyzed the tumor size, splenocyte proliferation, spleen and tumor-infiltrated lymphocytes. Tumor and spleen CD4⁺CD25⁺Foxp3⁺ regulatory T lymphocytes, cytotoxic activity of the splenocytes, IFN-γ and IL-4 secretion were assessed to describe the anti-tumor immune response. Our findings showed that co-administration of gp96 and naloxone has resulted in a significant reduction in CD4⁺CD25⁺Foxp3⁺ regulatory T cells in the spleen. The results indicated that on days 27 and 32 the tumors in the gp96 + Nal group grew significantly slower. Moreover, co-administration of gp96 and naloxone significantly increased the intra-tumor CD8⁺ T cells and cytotoxic activity. In addition the results indicated a significant increase in the proliferation of splenocytes and IFN-γ production. Our results demonstrate that naloxone is an effective immunoadjuvant in cancer immunotherapy.     

VEGFR2-targeted antibody fused with IFNαmut regulates the tumor microenvironment of colorectal cancer and exhibits potent anti-tumor and anti-metastasis activity

Although interferon α (IFNα) and anti-angiogenesis antibodies have shown appropriate clinical benefit in the treatment of malignant cancer, they are deficient in clinical applications. Previously, we described an anti-vascular endothelial growth factor receptor 2 (VEGFR2)-IFNα fusion protein named JZA01, which showed increased in vivo half-life and reduced side effects compared with IFNα, and it was more effective than the anti-VEGFR2 antibody against tumors. However, the affinity of the IFNα component of the fusion protein for its receptor-IFNAR1 was decreased. To address this problem, an IFNα-mutant fused with anti-VEGFR2 was designed to produce anti-VEGFR2-IFNαmut, which was used to target VEGFR2 with enhanced anti-tumor and anti-metastasis efficacy. Anti-VEGFR2-IFNαmut specifically inhibited proliferation of tumor cells and promoted apoptosis. In addition, anti-VEGFR2-IFNαmut inhibited migration of colorectal cancer cells and invasion by regulating the PI3K–AKT–GSK3β–snail signal pathway. Anti-VEGFR2-IFNαmut showed superior anti-tumor efficacy with improved tumor microenvironment (TME) by enhancing dendritic cell maturation, dendritic cell activity, and increasing tumor-infiltrating CD8⁺ T cells. Thus, this study provides a novel approach for the treatment of metastatic colorectal cancer, and this design may become a new approach to cancer immunotherapy.KEY WORDS: Anti-VEGFR2, IFNαmut, Tumor microenvironment, Colorectal cancer, Liver metastasis, Cancer immunotherapy