The PTPN2/PTPN1 inhibitor ABBV-CLS-484 unleashes potent anti-tumour immunity.

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

Reversion analysis reveals the in vivo immunogenicity of a poorly MHC I-binding cancer neoepitope.

High-affinity MHC I-peptide interactions are considered essential for immunogenicity. However, some neo-epitopes with low affinity for MHC I have been reported to elicit CD8 T cell dependent tumor rejection in immunization-challenge studies. Here we show in a mouse model that a neo-epitope that poorly binds to MHC I is able to enhance the immunogenicity of a tumor in the absence of immunization. Fibrosarcoma cells with a naturally occurring mutation are edited to their wild type counterpart; the mutation is then re-introduced in order to obtain a cell line that is genetically identical to the wild type except for the neo-epitope-encoding mutation. Upon transplantation into syngeneic mice, all three cell lines form tumors that are infiltrated with activated T cells. However, lymphocytes from the two tumors that harbor the mutation show significantly stronger transcriptional signatures of cytotoxicity and TCR engagement, and induce greater breadth of TCR reactivity than those of the wild type tumors. Structural modeling of the neo-epitope peptide/MHC I pairs suggests increased hydrophobicity of the neo-epitope surface, consistent with higher TCR reactivity. These results confirm the in vivo immunogenicity of low affinity or 'non-binding' epitopes that do not follow the canonical concept of MHC I-peptide recognition.

Strong Antitumor Activity of Bevacizumab and Aflibercept in Neuroendocrine Carcinomas: In-Depth Preclinical Study.

BACKGROUND: Neuroendocrine carcinoma (NEC) is a rare and very aggressive tumor. It has been greatly understudied, and very little is known about optimal treatment strategy for patients with this disease. The purpose of this study was to evaluate in vivo whether anti-vascular endothelial growth factor (VEGF) drugs could be a therapeutic alternative for these tumors with a poor prognosis. METHODS: We have developed 2 xenograft models using either human cell line derived from lung (H460) or from colon (COLO320) NEC to assess the effect of 2 antiangiogenic drugs, aflibercept and bevacizumab, on tumor growth and their pathological characteristics. Additionally, tumors were subjected to immunohistochemistry staining and proteins were measured with Western blot and ELISA. RESULTS: Both aflibercept and bevacizumab showed significant antitumor activity (p < 0.001). In the H460 model, aflibercept resulted in 94% tumor growth inhibition (TGI) and bevacizumab treatment resulted in 72.2% TGI. Similarly, in the COLO320 model, aflibercept and bevacizumab resulted in 89.3 and 84% TGI, respectively. Moreover, antitumor activity occurs early after treatment initiation. Using Tumor Control Index score, which address the kinetics of tumor growth in a way comparable to the methods used in human clinical studies, we confirmed that both drugs inhibit significantly tumor growth. When tumor stabilization was evaluated, aflibercept shows higher ability to stabilize NEC tumors than bevacizumab. CONCLUSION: Results derived from this study strongly support anti-VEGF therapies, especially aflibercept, as a novel therapeutic option in NECs. Further studies are necessary, but our observations encourage the evaluation of antiangiogenics in clinical trials combined with standard chemotherapy.

Mass spectrometry driven exploration reveals nuances of neoepitope-driven tumor rejection.

Neoepitopes are the only truly tumor-specific antigens. Although potential neoepitopes can be readily identified using genomics, the neoepitopes that mediate tumor rejection constitute a small minority, and there is little consensus on how to identify them. Here, for the first time, we use a combination of genomics, unbiased discovery MS immunopeptidomics and targeted MS to directly identify neoepitopes that elicit actual tumor rejection in mice. We report that MS-identified neoepitopes are an astonishingly rich source of tumor rejection mediating neoepitopes. MS has also demonstrated unambiguously the presentation by MHC I, of confirmed tumor rejection neoepitopes which bind weakly to MHC I; this was done using DCs exogenously loaded with long peptides containing the weakly binding neoepitopes. Such weakly MHC I-binding neoepitopes are routinely excluded from analysis, and our demonstration of their presentation, and their activity in tumor rejection, reveals a broader universe of tumor-rejection neoepitopes than presently imagined. Modeling studies show that a mutation in the active neoepitope alters its conformation such that its T cell receptor-facing surface is significantly altered, increasing its exposed hydrophobicity. No such changes are observed in the inactive neoepitope. These results broaden our understanding of antigen presentation and help prioritize neoepitopes for personalized cancer immunotherapy.

CD11c+ MHCIIlo GM-CSF-bone marrow-derived dendritic cells act as antigen donor cells and as antigen presenting cells in neoepitope-elicited tumor immunity against a mouse fibrosarcoma.

Dendritic cells play a critical role in initiating T-cell responses. In spite of this recognition, they have not been used widely as adjuvants, nor is the mechanism of their adjuvanticity fully understood. Here, using a mutated neoepitope of a mouse fibrosarcoma as the antigen, and tumor rejection as the end point, we show that dendritic cells but not macrophages possess superior adjuvanticity. Several types of dendritic cells, such as bone marrow-derived dendritic cells (GM-CSF cultured or FLT3-ligand induced) or monocyte-derived ones, are powerful adjuvants, although GM-CSF-cultured cells show the highest activity. Among these, the CD11c+ MHCIIlo sub-set, distinguishable by a distinct transcriptional profile including a higher expression of heat shock protein receptors CD91 and LOX1, mannose receptors and TLRs, is significantly superior to the CD11c+ MHCIIhi sub-set. Finally, dendritic cells exert their adjuvanticity by acting as both antigen donor cells (i.e., antigen reservoirs) as well as antigen presenting cells.

Bacterial ghost of avian pathogenic E. coli (APEC) serotype O78:K80 as a homologous vaccine against avian colibacillosis.

Avian Colibacillosis is among the major causes of economic loss in the poultry industry worldwide, with a more vivid impact on developing countries. The involvement of several bacteria has made it challenging to develop effective vaccines for this disease, particularly because it is notoriously difficult to make a vaccine that contains all the contributing pathogenic bacteria. Here, we report the design and fabrication of a bacterial ghost (BG) of E. coli O78:K80, which is among the major bacterial serotypes responsible for this disease. The generated ghost is then exploited as a homologous vaccine against Avian Colibacillosis. We demonstrate that hole formation in the cell wall of E. coli O78:K80 can happen properly in optical densities as high as 0.8 compared to the 0.3-0.4 standard for bacteria like E. coli TOP10. This is especially advantageous for mass production of this ghost which is a vital factor in development of any BG-based vaccine. Compared to E. coli TOP10, we faced a great challenge in transforming the wild type bacteria with the E-lysis plasmid which was probably due to higher thickness of the cell wall in O78:K80. This, however, was addressed by treating the cell wall with a different combination of ions.The vaccine was administered to Ross 308 broiler chickens via injection as well as through their respiratory system at a dose of 1010 BGs, repeated 3 times at weekly intervals. Chickens were then challenged with the wild type O78:K80 at a dose of 1011 bacteria together with Infectious Bronchitis H120 vaccine (as immunosuppressant) one week after the last immunization. Air sac lesions were significantly reduced in BG vaccinated groups in comparison with the control group. The levels of IFNγ, IgA and IgY were measured in the serum of immunized chickens as an indication of immune response and were compared with those of the chickens vaccinated with killed bacteria. The results show that O78:K80 BG can be used as an efficient homologous vaccine against Colibacillosis disease in poultry. We expect our findings can serve as the starting point for designing more sophisticated vaccines that contain all three major pathogenic bacteria involved in avian Colibacillosis.

Tumor Control Index as a new tool to assess tumor growth in experimental animals.

Measurement of tumor diameters, tumor volumes, or area under the curve has been traditionally used to quantitate and compare tumor growth curves in immune competent as well as immune-compromised mice and rats. Here, using tumor growth data from a large number of mice challenged with live tumor cells, we describe the use of a new composite parameter, Tumor Control Index (TCI) as an alternative method to do the same. This index, comprised of three distinct values, the Tumor Inhibition Score, Tumor Rejection Score, and Tumor Stability Score, provides a complete picture of nearly every aspect of tumor growth in large numbers of animals, can be deduced automatically from tumor diameter or volume data, and can be used to compare several groups of animals in different experiments. This automatically derivable index also corresponds neatly to the use of complete and partial responses and tumor stability data generated in human tumors, and can be used to assess the efficacy of interventions to be used in clinical studies.

Neoepitopes as cancer immunotherapy targets: key challenges and opportunities.

Over the last half century, it has become well established that cancers can elicit a host immune response that can target them with high specificity. Only within the last decade, with the advances in high-throughput gene sequencing and bioinformatics approaches, are we now on the forefront of harnessing the host's immune system to treat cancer. Recently, some strides have been taken toward understanding effective tumor-specific MHC I restricted epitopes or neoepitopes. However, many fundamental questions still remain to be addressed before this therapy can live up to its full clinical potential. In this review, we discuss the major hurdles that lie ahead and the work being done to address them.