Neo-intline: integrated pipeline enables neoantigen design through the in-silico presentation of T-cell epitope.

Neoantigen vaccines are one of the most effective immunotherapies for personalized tumour treatment. The current immunogen design of neoantigen vaccines is usually based on whole-genome sequencing (WGS) and bioinformatics prediction that focuses on the prediction of binding affinity between peptide and MHC molecules, ignoring other peptide-presenting related steps. This may result in a gap between high prediction accuracy and relatively low clinical effectiveness. In this study, we designed an integrated in-silico pipeline, Neo-intline, which started from the SNPs and indels of the tumour samples to simulate the presentation process of peptides in-vivo through an integrated calculation model. Validation on the benchmark dataset of TESLA and clinically validated neoantigens illustrated that neo-intline could outperform current state-of-the-art tools on both sample level and melanoma level. Furthermore, by taking the mouse melanoma model as an example, we verified the effectiveness of 20 neoantigens, including 10 MHC-I and 10 MHC-II peptides. The in-vitro and in-vivo experiments showed that both peptides predicted by Neo-intline could recruit corresponding CD4+ T cells and CD8+ T cells to induce a T-cell-mediated cellular immune response. Moreover, although the therapeutic effect of neoantigen vaccines alone is not sufficient, combinations with other specific therapies, such as broad-spectrum immune-enhanced adjuvants of granulocyte-macrophage colony-stimulating factor (GM-CSF) and polyinosinic-polycytidylic acid (poly(I:C)), or immune checkpoint inhibitors, such as PD-1/PD-L1 antibodies, can illustrate significant anticancer effects on melanoma. Neo-intline can be used as a benchmark process for the design and screening of immunogenic targets for neoantigen vaccines.

Effect of BAFF blockade on the B cell receptor repertoire and transcriptome in a mouse model of systemic lupus erythematosus.

Introduction: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. Anti-B-cell-activating factor (BAFF) therapy effectively depletes B cells and reduces SLE disease activity. This research aimed to evaluate the effect of BAFF blockade on B cell receptor (BCR) repertoire and gene expression. Methods: Through next-generation sequencing, we analyzed gene expression and BCR repertoire in MRL/lpr mice that received long-term anti-BAFF therapy. Based on gene expression profiles, we predicted the relative proportion of immune cells using ImmuCellAI-mouse, validating our predictions via flow cytometry and FluoroSpot. Results: The loss of BCR repertoire diversity and richness, along with increased clonality and differential frequency distribution of the immunoglobulin heavy chain variable (IGHV) segment gene usage, were observed in BAFF-blockade mice. Meanwhile, the distribution of complementarity-determining region 3 (CDR3) length and CDR3 amino acid usage remained unaffected. BAFF blockade resulted in extensive changes in gene expression, particularly that of genes related to B cells and immunoglobulins. Besides, the tumor necrosis factor (TNF)-α responses and interferon (IFN)-α/γ were downregulated, consistent with the decrease in IFN-γ and TNF-α serum levels following anti-BAFF therapy. In addition, BAFF blockade significantly reduced B cell subpopulations and plasmacytoid dendritic cells, and caused the depletion of antibody-secreting cells. Discussion: Our comparative BCR repertoire and transcriptome analyses of MRL/lpr mice subjected to BAFF blockade provide innovative insights into the molecular pathophysiology of SLE.

Targeting CD89 on tumor-associated macrophages overcomes resistance to immune checkpoint blockade.

BACKGROUND: Despite the survival benefits observed with immune checkpoint blockade (ICB) treatment-programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1), many patients with cancer have not benefited from these agents because of impaired antigen presentation and other resistance mechanisms. To overcome resistance to checkpoint therapy, we designed bispecific antibodies (BsAbs) targeting CD89 and tumor antigens. We demonstrated their immunomodulatory efficacy as a separate treatment strategy or combined with immune checkpoint inhibitors. METHODS: We have previously generated a heterodimeric one-arm IgG1 Fc-based bispecific antibody. For animal efficacy studies, murine tumors in a humanized transgenic mice model were used to determine the effects of CD89-bispecific antibodies on antigen presentation and immune cell recruitment. The efficacy of the CD89 bispecific antibody against tumors resistant to pembrolizumab was evaluated in double-transgenic mice. RESULTS: BsAbs targeting CD89 on tumor-associated macrophages (TAMs) increased the ratio of M1:M2 and activated the antigen presentation, leading to increased cytotoxic T cell-mediated tumor regression. CD89-BsAbs also potentiated a combinational antitumor effect with PD-1/PD-L1 inhibitors and overcame the ICB resistance by augmenting cytotoxic T-cell infiltration and reshaping tumor immune microenvironment. In an hCD89/hPD-1 double transgenic mouse model engrafted with pembrolizumab-resistant B16-HER2 tumor cells, the HER2-CD89 bispecific antibody potently inhibited tumor growth. CONCLUSIONS: CD89 BsAbs targeting tumor antigens and TAMs controlled tumor growth in animal models by improving antigen presentation and T-cell infiltration. Our results suggest a general strategy for overcoming resistance to ICB.

Reversing PD-1 Resistance in B16F10 Cells and Recovering Tumour Immunity Using a COX2 Inhibitor.

Immunotherapy is an effective method for tumour treatment. Anti-programmed cell death protein 1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) monoclonal antibodies play a significant role in immunotherapy of most tumours; however, some patients develop drug resistance to PD-1/PD-L1 therapy. Cyclooxygenase-2 (COX2) is expressed in various solid tumours, and prostaglandin E2 (PGE2) drives the development of malignant tumours. We developed a drug-resistant B16F10 (B16F10-R) tumour mouse model through four rounds of selection in vivo. Subsequently, we investigated changes in PD-L1 expression and lymphocyte infiltration in B16F10-NR and B16F10-R tumours. Additionally, we explored the role of COX2 in acquired resistance to pembrolizumab, an anti-PD-1 treatment. Immune cell infiltration was significantly decreased in resistant tumours compared to B16F10-NR tumours; however, ptgs2 gene expression was significantly elevated in resistant tumours. Aspirin or celecoxib combined with pembrolizumab can effectively reverse tumour drug resistance. In addition, ptgs2 knockout or the use of the EP4 inhibitor E7046 abrogated drug resistance to anti-PD-1 treatment in B16F10-R tumour cells. Our study showed that inhibition of the COX2/PGE2/EP4 axis could increase the number of immune cells infiltrating the tumour microenvironment and recover drug-resistant tumour sensitivity to pembrolizumab. Thus, we highlight COX2 inhibition as a promising therapeutic target for drug-resistant tumours for future consideration.

A Novel Anti-B7-H3 × Anti-CD3 Bispecific Antibody with Potent Antitumor Activity.

B7-H3 plays an important role in tumor apoptosis, proliferation, adhesion, angiogenesis, invasion, migration, and evasion of immune surveillance. It is overexpressed in various human solid tumor tissues. In patients, B7-H3 overexpression correlates with advanced stages, poor clinical outcomes, and resistance to therapy. The roles of B7-H3 in tumor progression make it a potential candidate for targeted therapy. Here, we generated a mouse anti-human B7-H3 antibody and demonstrated its binding activity via Tongji University Suzhou Instituteprotein-based and cell-based assays. We then developed a novel format anti-B7-H3 × anti-CD3 bispecific antibody based on the antibody-binding fragment of the anti-B7-H3 antibody and single-chain variable fragment structure of anti-CD3 antibody (OKT3) and demonstrated that this bispecific antibody mediated potent cytotoxic activities against various B7-H3-positive tumor cell lines in vitro by improving T cell activation and proliferation. This bispecific antibody also demonstrated potent antitumor activity in humanized mice xenograft models. These results revealed that the novel anti-B7-H3 × anti-CD3 bispecific antibody has the potential to be employed in treatment of B7-H3-positive solid tumors.

A HER2 target antibody drug conjugate combined with anti-PD-(L)1 treatment eliminates hHER2+ tumors in hPD-1 transgenic mouse model and contributes immune memory formation.

PURPOSE: Disitamab vedotin (RC48) is an HER2-directed antibody-drug conjugate, emerging as an effective strategy for cancer therapy, which not only enhances antitumor immunity in previous animal models but also improves clinical outcomes for patients such as with gastric cancer, urothelium carcinoma, and HER2 low-expressing breast cancer. Here, we explore the combination therapeutic efficacy of this novel HER2-targeting ADC with immune checkpoint inhibitors in a human HER2-expressing syngeneic breast cancer model. METHODS: The human HER2+ cancer cell line is constructed by stable transfection and individual clones were isolated by single-cell sorting. Flow cytometry was performed to determine its binding activity. Cytotoxic effect was determined using an MTT assay with the supplement of RC48. Human PD-1 transgenic mice were used to analyze the in vivo antitumor effects of the ADC and its combination therapy with PD-1/PD-L1 antibody. RESULTS: The combination of RC48 and PD-1/PD-L1 immune checkpoint inhibition significantly enhanced tumor suppression and antitumor immunity. Tumor rejection in the synergistic groups was accompanied by massive T cell infiltration and immune marker activation. Furthermore, the combination therapy promoted immunological memory formation in the tumor eradication animals, protecting them from tumor rechallenge. CONCLUSION: A novel HER2-targeting ADC combined with immune checkpoint inhibitors can achieve remarkable effects in mice and elicit long-lasting immune protection in a hHER2+ murine breast cancer model. This study provides insights into the efficacy of RC48 therapeutic activity and a rationale for potential therapeutic combination strategies with immunotherapy.

Adaptive resistance in tumors to anti-PD-1 therapy through re-immunosuppression by upregulation of GPNMB expression.

Acquired resistance to the antitumor activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in various types of cancers has increasingly been observed during treatment. To gain insight into the molecular mechanism underlying anti-PD-1 therapy resistance, we developed a mouse MC38 colon adenocarcinoma cell line that was made resistant to anti-PD-1 treatment through repeated in vivo selection. We compared the transcriptomic profiles of anti-PD-1 therapy-resistant and -sensitive tumors using RNA sequencing analysis. The immunosuppressive molecule transmembrane glycoprotein NMB (GPNMB) was significantly upregulated in resistant tumor cells, as determined using quantitative real-time polymerase chain reaction and immunofluorescence analyses. Furthermore, deletion of GPNMB in resistant cells successfully restored sensitivity to anti-PD-1 treatment in vivo. Collectively, our results indicate that tumors may develop resistance to anti-PD-1 therapy by upregulating their expression of the immunosuppressive molecule GPNMB. Furthermore, GPNMB is a potential, targetable biomarker for monitoring adaptive resistance to therapeutic PD-1 blockade, and identification of this immunosuppressive molecule may be a breakthrough for new therapies.

Simultaneous exposure to FcγR and FcαR on monocytes and macrophages enhances antitumor activity in vivo.

Therapeutic antibodies are effective for tumor immunotherapy and exhibit prominent clinical effects. All approved antibody therapeutics utilize IgG as the molecular format. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a key mechanism for tumor cell killing by antibodies. For IgG antibodies, ADCC depends on FcγR-expressing cells, such as natural killer (NK) cells. However, in patients with a high tumor burden, antibody therapeutics may lose efficacy owing to exhaustion of FcγR-expressing effector cells as well as the inhibitory effects of certain FcγRs on effector cells. To achieve more potent effector functions, we engineered an anti-CD20 antibody to contain both IgG Fc and IgA Fc domains. These engineered antibodies interacted with both IgG and IgA Fc receptors (FcγR and FcαR) and recruited a broader range of effector cells, including monocytes, macrophages, neutrophils, and NK cells, thereby enhancing antibody-dependent cellular phagocytosis. Using transgenic mice expressing the FcαRI (CD89) in macrophages, we demonstrated that recombinant antibodies bearing the chimeric IgG and IgA Fc exhibited potent in vivo antitumor activity. Additionally, in a short-term peritoneal model using CD20-transfected LLC target cells, the in vivo cytotoxic activity of hybrid recombinant antibodies was mediated by macrophages with significant reduction in the absence of FcαRI. Our findings supported targeting of FcαRI on monocytes and macrophages for improved tumor immunotherapy.

CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy.

Since tumors are often infiltrated by macrophages, it would be advantageous to turn these types of cells into cytotoxic effector cells. Here, we have designed a novel bispecific antibody (BsAb) that targets both tumor antigen (CD20) and the FcαRI receptor (CD89). This antibody could be used to lyse tumors by connecting tumor cells to CD89-expressing immune effector cells such as macrophages and neutrophils. Previously there were very limited attempts to exploit FcαRI-expressing cells as effector cells for tumor cell-killing, largely due to the lack of an appropriate in vivo model, since mice do not express a human CD89 homolog. In this study, we used a transgenic mouse strain with specific expression of CD89 on macrophages and monocytes. In this transgenic mouse model, the CD89 bispecific antibody showed significant anti-tumor activities, demonstrating that bispecific antibodies can redirect macrophages, including M2 macrophages, to mediate additional effector function in the tumor microenvironment. This approach realized the full potential of the innate immune system and could be applied to other tumor-associated antigens especially the solid tumors, thus has potential to translate into clinical benefits in human cancers.