Viviana P. Lutzky

Suppression of Metastases Using a New Lymphocyte Checkpoint Target for Cancer Immunotherapy

UNLABELLED CD96 has recently been shown as a negative regulator of mouse natural killer (NK)-cell activity, with Cd96(-/-)mice displaying hyperresponsive NK cells upon immune challenge. In this study, we have demonstrated that blocking CD96 with a monoclonal antibody inhibited experimental metastases in three different tumor models. The antimetastatic activity of anti-CD96 was dependent on NK cells, CD226 (DNAM-1), and IFNγ, but independent of activating Fc receptors. Anti-CD96 was more effective in combination with anti-CTLA-4, anti-PD-1, or doxorubicin chemotherapy. Blocking CD96 in Tigit(-/-)mice significantly reduced experimental and spontaneous metastases compared with its activity in wild-type mice. Co-blockade of CD96 and PD-1 potently inhibited lung metastases, with the combination increasing local NK-cell IFNγ production and infiltration. Overall, these data demonstrate that blocking CD96 is a new and complementary immunotherapeutic strategy to reduce tumor metastases. SIGNIFICANCE This article illustrates the antimetastatic activity and mechanism of action of an anti-CD96 antibody that inhibits the CD96-CD155 interaction and stimulates NK-cell function. Targeting host CD96 is shown to complement surgery and conventional immune checkpoint blockade.

Novel Approach to the Formulation of an Epstein-Barr Virus Antigen-Based Nasopharyngeal Carcinoma Vaccine

ABSTRACT Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.

Cytotoxic T Cell Adoptive Immunotherapy as a Treatment for Nasopharyngeal Carcinoma

ABSTRACT Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). We assess the safety and tolerability of adoptive transfer of autologous cytotoxic T lymphocytes (CTLs) specific for the EBV latent membrane protein (LMP) in a patient with recurrent NPC. After infusion, the majority of pulmonary lesions were no longer evident, although the primary tumor did not regress.

Optimization of LMP-specific CTL expansion for potential adoptive immunotherapy in NPC patients

Nasopharyngeal carcinoma (NPC) is Epstein–Barr virus (EBV) positive in all undifferentiated cases, expressing the latency II phenotype of latent membrane proteins (LMPs) 1 and 2, in addition to EBV nuclear antigen (EBNA) 1. Several studies have attempted to treat NPC with EBV‐specific cytotoxic T lymphocyte (CTL) with a partial response. To improve this therapy, there is a need to expand CTL targeted to the latency II antigens of EBV, rather than the immunodominant EBV nuclear antigens 3–6 peptides typically expanded by lymphoblastoid cells. In order to maximize the expansion of LMP‐specific CTL in vitro for use in adoptive immunotherapy of nasopharyngeal carcinoma patients, we used lymphoblastoid cell lines coated with synthetic peptides corresponding to CTL determinants from the LMP proteins. We investigated several issues pertaining to the expansion of an immunologically weak CTL response, including peptide and interleukin‐2 concentration, and screening assays for selecting the optimal peptide for use in expansion of LMP‐specific CTL. Although screening of ex vivo peripheral blood mononuclear cells did not prove to be useful in the selection of an LMP peptide for use in CTL cultures, the peptide and interleukin‐2 concentrations were critical for the maximum expansion of CTL. Therefore, it is imperative that stimulation protocols are optimized for the expansion of LMP‐specific CTL.

Biomarkers for Cancers of the Head and Neck

Head and neck cancer is a broad term used to describe malignancies that arise in the nasal and oral cavities, pharynx and larynx, as well as the paranasal sinuses. Head and neck squamous cell carcinoma (HNSCC) affects the squamous epithelium of the oral cavity, tongue and oropharynx, excluding the nasopharynx. Recent advances in molecular technology, including gene expression and proteomic profiling appear to offer the potential for the development of specific biomarkers including diagnostic tools which may act as an aid to guide therapy for this malignancy. The other human head and neck cancer included in this review, nasopharyngeal carcinoma (NPC) is a malignancy derived from the undifferentiated epithelium of the nasopharyngeal cavity, and is considered here as a separate entity because its strong association with Epstein-Barr virus (EBV) presents the opportunity for the development of virus related and unrelated biomarkers. In particular, IgA antibodies to EBV and high levels of EBV DNA in serum samples of NPC patients have been recorded. This review aims to summarize some current and also potential new biomarkers that could be used for screening, diagnosis, monitoring and prognostic prediction for cancers of the head and neck, including NPC and HNSCC.

CD96 targeted antibodies need not block CD96-CD155 interactions to promote NK cell anti-metastatic activity

ABSTRACT CD96 is a transmembrane glycoprotein Ig superfamily receptor, expressed on various T cell subsets and NK cells, that interacts with nectin and nectin-like proteins, including CD155/polio virus receptor (PVR). Here, we have compared three rat anti-mouse CD96 mAbs, including two that block CD96-CD155 (3.3 and 6A6) and one that does not block CD96-CD155 (8B10). Using flow cytometry, we demonstrated that both mAbs 3.3 and 6A6 bind to the first Ig domain of mouse CD96 and compete with CD155 binding, while mAb 8B10 binds to the second Ig domain and does not block CD155. While Fc isotype was irrelevant concerning the anti-metastatic activity of 3.3 mAb, in four different experimental metastases models and one spontaneous metastasis model, the relative order of anti-metastatic potency was 6A6 > 3.3 > 8B10. The metastatic burden control of all of the anti-CD96 clones was highly dependent on NK cells and IFN-γ. Consistent with its inability to block CD96-CD155 interactions, 8B10 retained anti-metastatic activity in CD155-deficient mice, whereas 3.3 and 6A6 lost potency in CD155-deficient mice. Furthermore, 8B10 retained most of its anti-metastatic activity in IL-12p35-deficient mice whereas the activity of 3.3 and 6A6 were partially lost. All three mAbs were inactive in CD226-deficient mice. Altogether, these data demonstrate anti-CD96 need not block CD96-CD155 interactions (ie. immune checkpoint blockade) to promote NK cell anti-metastatic activity.

Deficiency of host CD96 and PD-1 or TIGIT enhances tumor immunity without significantly compromising immune homeostasis

ABSTRACT Multiple non-redundant immunosuppressive pathways co-exist in the tumor microenvironment and their co-targeting can increase clinical responses. Indeed, concurrent blockade of CTLA-4 and PD-1 in patients with advanced melanoma increased clinical responses over monotherapy alone although the frequency and severity of immune related adverse events (irAEs) also increased. Nevertheless, a substantial number of patients still display an innate resistance phenotype and are unresponsive to current approved immunotherapies even when utilized in combination. In this study, we generated Pdcd1−/−CD96−/− and Tigit−/−CD96−/− mice to investigate how loss of CD96 in combination with PD-1 or TIGIT impacts on immune homeostasis and hence the potential of inducing immune related toxicities following co-targeting of these pairs of receptors. The ability of Pdcd1−/−CD96−/− and Tigit−/−CD96−/− mice to suppress primary tumor growth was also assessed using the MC38 colon carcinoma and SM1WT1 BRAF-mutated melanoma tumor models. Both Pdcd1−/−CD96−/− or Tigit−/−CD96−/− mice displayed no overt perturbations in immune homeostasis over what was previously reported with Pdcd1−/− or Tigit−/− mice even when aged for 22 months. Interestingly, increased suppression of subcutaneous tumor growth and complete responses was seen in Pdcd1−/−CD96−/− mice compared to Pdcd1−/− or CD96−/− mice depending upon the tumor model. In contrast, in these models, growth suppression in Tigit−/−CD96−/− were similar to Tigit−/− or CD96−/− . This enhanced anti-tumor efficacy of Pdcd1−/−CD96−/− appeared to be due to favorable changes in the ratio of CD8+ T cells to T regulatory cells or CD11b+GR-1hi myeloid cells in the tumor microenvironment. Co-targeting CD96 and PD-1 may increase anti-tumor immunity over targeting PD-1 alone and potentially not induce serious immune-related toxicities and thus appears a promising strategy for clinical development.

The immune checkpoint CD96 defines a distinct lymphocyte phenotype and is highly expressed on tumor-infiltrating T cells

CD96 has recently been shown to be a potent immune checkpoint molecule in mice, but a similar role in humans is not known. In this study, we provide a detailed map of CD96 expression across human lymphocyte lineages, the kinetics of CD96 regulation on T‐cell activation and co‐expression with other conventional and emerging immune checkpoint molecules. We show that CD96 is predominantly expressed by T cells and has a unique lymphocyte expression profile. CD96high T cells exhibited distinct effector functions on activation. Of note, CD96 expression was highly correlated with T‐cell markers in primary and metastatic human tumors and was elevated on antigen‐experienced T cells and tumor‐infiltrating lymphocytes. Collectively, these data demonstrate that CD96 may be a promising immune checkpoint to enhance T‐cell function against human cancer and infectious disease.

Anomalies in T Cell Function Are Associated With Individuals at Risk of Mycobacterium abscessus Complex Infection

The increasing global incidence and prevalence of non-tuberculous mycobacteria (NTM) infection is of growing concern. New evidence of person-to-person transmission of multidrug-resistant NTM adds to the global concern. The reason why certain individuals are at risk of NTM infections is unknown. Using high definition flow cytometry, we studied the immune profiles of two groups that are at risk of Mycobacterium abscessus complex infection and matched controls. The first group was cystic fibrosis (CF) patients and the second group was elderly individuals. CF individuals with active M. abscessus complex infection or a history of M. abscessus complex infection exhibited a unique surface T cell phenotype with a marked global deficiency in TNFα production during mitogen stimulation. Importantly, immune-based signatures were identified that appeared to predict at baseline the subset of CF individuals who were at risk of M. abscessus complex infection. In contrast, elderly individuals with M. abscessus complex infection exhibited a separate T cell phenotype underlined by the presence of exhaustion markers and dysregulation in type 1 cytokine release during mitogen stimulation. Collectively, these data suggest an association between T cell signatures and individuals at risk of M. abscessus complex infection, however, validation of these immune anomalies as robust biomarkers will require analysis on larger patient cohorts.