Emerging monoclonal antibody therapies in the treatment of metastatic urothelial carcinoma.

INTRODUCTION: The treatment landscape for advanced-stage, unresectable or metastatic urothelial carcinoma (mUC) has shifted dramatically over a short period of time, with new therapeutic agents available for clinical use. However, despite these recent advances in the field, mUC continues to be a disease with significant morbidity and mortality and remains generally incurable. While platinum-based therapy remains the backbone of therapy, many patients are ineligible for chemotherapy or have failed initial chemotherapy treatment. In post-platinum treated patients, immunotherapy and antibody drug conjugates have provided incremental advances, but agents with better therapeutic index guided by precision medicine are needed. AREAS COVERED: This article covers the available monoclonal antibody therapies in mUC excluding immunotherapy and antibody drug conjugates. Included are a review of data utilizing monoclonal antibodies targeting VEG-F, HER-2, FGFR, and KIR-2 in the setting of mUC. A literature search from 6/2022- 9/2022 was performed utilizing PubMed with key terms including urothelial carcinoma, monoclonal antibody, VEG-F, HER-2, FGFR. EXPERT OPINION: Often used in combination with immunotherapy or other therapeutic agents, monoclonal antibody therapies have exhibited efficacy in mUC in early trials. Upcoming clinical trials will further explore their full clinical utility in treating mUC patients.

Memory Precursors and Short-Lived Effector T cell Subsets Have Different Sensitivities to TGFß.

After exposure to an antigen, CD8 T cells reach a decision point about their fate: to become either short-lived effector cells (SLECs) or memory progenitor effector cells (MPECs). SLECs are specialized in providing an immediate effector function but have a shorter lifespan and lower proliferative capacity compared to MPECs. Upon encountering the cognate antigen during an infection, CD8 T cells rapidly expand and then contract to a level that is maintained for the memory phase after the peak of the response. Studies have shown that the contraction phase is mediated by TGFß and selectively targets SLECs, while sparing MPECs. The aim of this study is to investigate how the CD8 T cell precursor stage determines TGFß sensitivity. Our results demonstrate that MPECs and SLECs have differential responses to TGFß, with SLECs being more sensitive to TGFß than MPECs. This difference in sensitivity is associated with the levels of TGFßRI and RGS3, and the SLEC-related transcriptional activator T-bet binding to the TGFßRI promoter may provide a molecular basis for increased TGFß sensitivity in SLECs.

Barriers and Opportunities for CAR T-Cell Targeting of Solid Tumors.

CAR T-cell therapy has transformed the treatment of hematological malignancies of the B cell lineage. However, the quest to fulfil the same promise for solid tumors is still in its infancy. This review summarizes some of the challenges that the field is trying to overcome for effective treatment of human carcinomas, including tumor heterogeneity, the paucity of truly tumor-specific targets, immunosuppression and metabolic restrictions at solid tumor beds, and defective T-cell trafficking. All these barriers are being currently investigated and, in some cases, targeted, by multiple independent groups. With clinical interventions against multiple human malignancies and different platforms under accelerated clinical development, the next few years will see an array of cellular therapies, including CAR T-cells, progressively becoming routine interventions to eliminate currently incurable diseases, as it happened with some hematological malignancies.

CD8(+) T cells sabotage their own memory potential through IFN-γ-dependent modification of the IL-12/IL-15 receptor α axis on dendritic cells.

CD8(+) T cell responses have been shown to be regulated by dendritic cells (DCs) and CD4(+) T cells, leading to the tenet that CD8(+) T cells play a passive role in their own differentiation. In contrast, by using a DNA vaccination model, to separate the events of vaccination from those of CD8(+) T cell priming, we demonstrate that CD8(+) T cells, themselves, actively limit their own memory potential through CD8(+) T cell-derived IFN-γ-dependent modification of the IL-12/IL-15Rα axis on DCs. Such CD8(+) T cell-driven cytokine alterations result in increased T-bet and decreased Bcl-2 expression, and thus decreased memory progenitor formation. These results identify an unrecognized role for CD8(+) T cells in the regulation of their own effector differentiation fate and a previously uncharacterized relationship between the balance of inflammation and memory formation.

NKG2D receptor signaling shapes T cell thymic education.

The role of natural killer group 2D (NKG2D) in peripheral T cells as a costimulatory receptor is well established. However, its contribution to T cell thymic education and functional imprint is unknown. Here, we report significant changes in development, receptor signaling, transcriptional program, and function in T cells from mice lacking NKG2D signaling. In C57BL/6 (B6) and OT-I mice, we found that NKG2D deficiency results in Vß chain usage changes and stagnation of the double-positive stage in thymic T cell development. We found that the expression of CD5 and CD45 in thymocytes from NKG2D deficient mice were reduced, indicating a direct influence of NKG2D on the strength of T cell receptor (TCR) signaling during the developmental stage of T cells. Depicting the functional consequences of NKG2D, peripheral OT-I NKG2D-deficient cells were unresponsive to ovalbumin peptide stimulation. Paradoxically, while αCD3/CD28 agonist antibodies led to phenotypic T cell activation, their ability to produce cytokines remained severely compromised. We found that OT-I NKG2D-deficient cells activate STAT5 in response to interleukin-15 but were unable to phosphorylate ERK or S6 upon TCR engagement, underpinning a defect in TCR signaling. Finally, we showed that NKG2D is expressed in mouse and human thymic T cells at the double-negative stage, suggesting an evolutionarily conserved function during T cell development. The data presented in this study indicate that NKG2D impacts thymic T cell development at a fundamental level by reducing the TCR threshold and affecting the functional imprint of the thymic progeny. In summary, understanding the impact of NKG2D on thymic T cell development and TCR signaling contributes to our knowledge of immune system regulation, immune dysregulation, and the design of immunotherapies.

Immune Assessment Today: Optimizing and Standardizing Efforts to Monitor Immune Responses in Cancer and Beyond.

As part of a symposium, current and former directors of Immune Monitoring cores and investigative oncologists presented insights into the past, present and future of immune assessment. Dr. Gnjatic presented a classification of immune monitoring technologies ranging from universally applicable to experimental protocols, while emphasizing the need for assay harmonization. Dr. Obeng discussed physiologic differences among CD8 T cells that align with anti-tumor responses. Dr. Lyerly presented the Soldano Ferrone lecture, commemorating the passionate tumor immunologist who inspired many, and covered a timeline of monitoring technology development and its importance to immuno-oncology. Dr. Sonabend presented recent achievements in glioblastoma treatment, accentuating the range of monitoring techniques that allowed him to refine patient selection for clinical trials. Dr. Guevara-Patiño focused on hypoxia within the tumor environment and stressed that T cell viability is not to be confused with functionality. Dr. Butterfield accentuated monitoring of dendritic cell metabolic (dys)function as a determinant for tumor vaccine success. Lectures were interspersed with select abstract presentations. To summarize the concepts, Dr. Maecker from Stanford led an informative forum discussion, pointing towards the future of immune monitoring. Immune monitoring continues to be a guiding light towards effective immunotherapeutic strategies.

Stimulation of the glucocorticoid-induced TNF receptor family-related receptor on CD8 T cells induces protective and high-avidity T cell responses to tumor-specific antigens.

Treatment of tumor-bearing mice with a stimulatory Ab to glucocorticoid-induced TNFR family-related receptor (GITR) has previously been shown to elicit protective T cell responses against poorly immunogenic tumors. However, the role of GITR stimulation on CD8 T cells and the nature of tumor rejection Ags have yet to be determined. In this study, we show that a stimulatory mAb to GITR (clone DTA-1) acts directly on CD8 T cells, but not on CD4(+)CD25(+) regulatory T (T(reg)) cells, in B16 tumor-bearing mice to induce concomitant immunity against secondary B16 tumors, as well as protective memory following surgical excision of the primary tumor. Melanoma growth itself induced GITR expression on tumor-specific CD8 T cells, providing a mechanism whereby these cells may respond to stimulatory anti-GITR. Unexpectedly, in contrast to T(reg) cell depletion therapy with anti-CD4, GITR stimulation induced very weak CD8 T cell responses to melanocyte differentiation Ags expressed by the tumor, and did not induce autoimmune vitiligo. Accordingly, GITR-stimulated hosts that were primed with B16 melanoma rejected B16, but not the unrelated JBRH melanoma, indicating that tumor rejection Ags are tumor-specific rather than shared. In support of this, we show that GITR stimulation induces CD8 T cell responses to a tumor-specific Ag, and that these responses are of higher functional avidity compared with those induced by T(reg) cell depletion. We conclude that stimulation of GITR on effector CD8 T cells results in high-avidity T cell responses to tumor-specific Ags, thereby inducing potent antitumor immunity in the absence of autoimmunity.

Development of tumor-infiltrating CD8+ T cell memory precursor effector cells and antimelanoma memory responses are the result of vaccination and TGF-ß blockade during the perioperative period of tumor resection.

A main goal of cancer immunology research is the formation of Ag-specific memory T cell immunity capable of activation upon tumor re-encounter. The requirements necessary to overcome the inhibitory signals present in the tumor microenvironment and form such memory T cell responses are unknown. In contrast to previous studies targeting tumors expressing highly immunogenic model Ags, we demonstrate that alleviating tumor-induced suppression along with vaccination against authentic Ags during the perioperative period provides long-lasting protection against a highly suppressive and poorly immunogenic melanoma. In this study, we employed DNA vaccination with an immunologically optimized mouse melanoma-shared Ag, Trp1ee/ng, combined with systemic TGF-ß blockade during the perioperative period of primary tumor resection, to confer protection against B16 melanoma, and against JBRH, an independently derived melanoma unrelated to B16. Importantly, we demonstrate that correlative to memory responses, perioperative immunotherapy increases the formation of tumor-infiltrating and tumor-reactive CD8(+) T cells expressing low levels of the transcription factor T-bet, defined as memory precursor effector cells. We show that conditions for an immunologically fertile environment are met when TGF-ß blockade and vaccination are applied during the perioperative period of primary tumor resection. These findings address limitations of current CD8(+) T cell immunotherapies against cancer by generating effective CD8(+) T cell memory recall responses.

Autoimmunity and tumor immunity induced by immune responses to mutations in self.

Little is known about the consequences of immune recognition of mutated gene products, despite their potential relevance to autoimmunity and tumor immunity. To identify mutations that induce immunity, here we have developed a systematic approach in which combinatorial DNA libraries encoding large numbers of random mutations in two syngeneic tyrosinase-related proteins are used to immunize black mice. We show that the libraries of mutated DNA induce autoimmune hypopigmentation and tumor immunity through cross-recognition of nonmutated gene products. Truncations are present in all immunogenic clones and are sufficient to elicit immunity to self, triggering recognition of normally silent epitopes. Immunity is further enhanced by specific amino acid substitutions that promote T helper cell responses. Thus, presentation of a vast repertoire of antigen variants to the immune system can enhance the generation of adaptive immune responses to self.

NKG2D signaling shifts the balance of CD8 T cells from single cytokine- to polycytokine-producing effector cells.

CD8 T cells play a critical role in immunity against intracellular pathogens and cancer. A primary objective of T cell-based vaccine strategies is the induction of durable and effective immune responses. Achieving this goal involves more than simply boosting the numbers of responding T cells. Of particular interest is the induction of CD8 T cells with polycytokine capability, specifically with the ability of CD8 T cells to co-produce IFNγ, TNFα and IL-2. The presence of these polycytokine-producing CD8 T cells correlates strongly with protection against foreign pathogens and cancer. Therefore, approaches capable of inducing such polyfunctional responses are needed. NKG2D engagement on CD8 T cells has been shown to result in increased effector response. However, the manner in which NKG2D engagement results in improved CD8 T cell effector response is unclear. Here we demonstrate in vitro and in vivo that NKG2D engagement by its natural ligand, Rae-1ε, shifts the balance from single cytokine to polycytokine (IL-2, IFNγ, and TFNα) production. These data define a previously unrecognized process in which NKG2D costimulation on CD8 T cells results in improved effector responses.

Antibody-Drug Conjugates in the Treatment of Urothelial Cancer.

Antibody-drug conjugates (ADCs) have transformed the treatment landscape in oncology and become an essential therapeutic modality. In urothelial carcinoma (UC), the two ADCs that have been especially successful in clinical practice are enfortumab vedotin and sacituzumab govitecan. These drugs are currently approved as monotherapy for later lines of treatment in locally advanced or metastatic UC and have had a significant impact for patients with limited treatment options. Combinational trials, as well as additional ADCs, are currently being investigated in the treatment of UC for subsequent lines of therapy as overall survival rates remain dismal.

Bispecific CD33/CD123 targeted chimeric antigen receptor T cells for the treatment of acute myeloid leukemia.

CD33 and CD123 are expressed on the surface of human acute myeloid leukemia blasts and other noncancerous tissues such as hematopoietic stem cells. On-target off-tumor toxicities may limit chimeric antigen receptor T cell therapies that target both CD33 and CD123. To overcome this limitation, we developed bispecific human CD33/CD123 chimeric antigen receptor (CAR) T cells with an "AND" logic gate. We produced novel CD33 and CD123 scFvs from monoclonal antibodies that bound CD33 and CD123 and activated T cells. Screening of CD33 and CD123 CAR T cells for cytotoxicity, cytokine production, and proliferation was performed, and we selected scFvs for CD33/CD123 bispecific CARs. The bispecific CARs split 4-1BB co-stimulation on one scFv and CD3ζ on the other. In vitro testing of cytokine secretion and cytotoxicity resulted in selecting bispecific CAR 1 construct for in vivo analysis. The CD33/CD123 bispecific CAR T cells were able to control acute myeloid leukemia (AML) in a xenograft AML mouse model similar to monospecific CD33 and CD123 CAR T cells while showing no on-target off-tumor effects. Based on our findings, human CD33/CD123 bispecific CAR T cells are a promising cell-based approach to prevent AML and support clinical investigation.

EGFR Inhibition by Cetuximab Modulates Hypoxia and IFN Response Genes in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) has one of the most hypoxic and immunosuppressive tumor microenvironments (TME) among solid tumors. However, there is no proven therapeutic strategy to remodel the TME to be less hypoxic and proinflammatory. In this study, we classified tumors according to a Hypoxia-Immune signature, characterized the immune cells in each subgroup, and analyzed the signaling pathways to identify a potential therapeutic target that can remodel the TME. We confirmed that hypoxic tumors had significantly higher numbers of immunosuppressive cells, as evidenced by a lower ratio of CD8+ T cells to FOXP3+ regulatory T cells, compared with nonhypoxic tumors. Patients with hypoxic tumors had worse outcomes after treatment with pembrolizumab or nivolumab, anti-programmed cell death-1 inhibitors. Our expression analysis also indicated that hypoxic tumors predominantly increased the expression of the EGFR and TGFß pathway genes. Cetuximab, an anti-EGFR inhibitor, decreased the expression of hypoxia signature genes, suggesting that it may alleviate the effects of hypoxia and remodel the TME to become more proinflammatory. Our study provides a rationale for treatment strategies combining EGFR-targeted agents and immunotherapy in the management of hypoxic HNSCC. Significance: While the hypoxic and immunosuppressive TME of HNSCC has been well described, comprehensive evaluation of the immune cell components and signaling pathways contributing to immunotherapy resistance has been poorly characterized. We further identified additional molecular determinants and potential therapeutic targets of the hypoxic TME to fully leverage currently available targeted therapies that can be administered with immunotherapy.

Combination oncolytic virus, radiation therapy, and immune checkpoint inhibitor treatment in anti-PD-1-refractory cancer.

BACKGROUND: Immunotherapies are becoming front-line treatments for many advanced cancers, and combinations of two or more therapies are beginning to be investigated. Based on their individual antitumor capabilities, we sought to determine whether combination oncolytic virus (OV) and radiation therapy (RT) may improve cancer outcomes. METHODS: To investigate the activity of this combination therapy, we used in vitro mouse and human cancer cell lines as well as a mouse model of skin cancer. After initial results, we further included immune checkpoint blockade, whose addition constituted a triple combination immunotherapy. RESULTS: Our findings demonstrate that OV and RT reduce tumor growth via conversion of immunologically 'cold' tumors to 'hot', via a CD8+ T cell-dependent and IL-1α-dependent mechanism that is associated with increased PD-1/PD-L1 expression, and the triple combination of OV, RT, and PD-1 checkpoint inhibition impedes tumor growth and prolongs survival. Further, we describe the response of a PD-1-refractory patient with cutaneous squamous cell carcinoma who received the triple combination of OV, RT, and immune checkpoint inhibitor (ICI), and went on to experience unexpected, prolonged control and survival. He remains off-treatment and is without evidence of progression for >44 months since study entry. CONCLUSIONS: Effective systemic antitumor immune response is rarely elicited by a single therapy. In a skin cancer mouse model, we demonstrate improved outcomes with combination OV, RT, and ICI treatment, which is associated with mechanisms involving augmented CD8+ T cell infiltration and IL-1α expression. We report tumor reduction and prolonged survival of a patient with skin cancer treated with combination OV, RT, and ICI. Overall, our data provide strong rationale for combining OV, RT, and ICI for treatment of patients with ICI-refractory skin and potentially other cancers.

Gut Microbial Shifts Indicate Melanoma Presence and Bacterial Interactions in a Murine Model.

Through a multitude of studies, the gut microbiota has been recognized as a significant influencer of both homeostasis and pathophysiology. Certain microbial taxa can even affect treatments such as cancer immunotherapies, including the immune checkpoint blockade. These taxa can impact such processes both individually as well as collectively through mechanisms from quorum sensing to metabolite production. Due to this overarching presence of the gut microbiota in many physiological processes distal to the GI tract, we hypothesized that mice bearing tumors at extraintestinal sites would display a distinct intestinal microbial signature from non-tumor-bearing mice, and that such a signature would involve taxa that collectively shift with tumor presence. Microbial OTUs were determined from 16S rRNA genes isolated from the fecal samples of C57BL/6 mice challenged with either B16-F10 melanoma cells or PBS control and analyzed using QIIME. Relative proportions of bacteria were determined for each mouse and, using machine-learning approaches, significantly altered taxa and co-occurrence patterns between tumor- and non-tumor-bearing mice were found. Mice with a tumor had elevated proportions of Ruminococcaceae, Peptococcaceae.g_rc4.4, and Christensenellaceae, as well as significant information gains and ReliefF weights for Bacteroidales.f__S24.7, Ruminococcaceae, Clostridiales, and Erysipelotrichaceae. Bacteroidales.f__S24.7, Ruminococcaceae, and Clostridiales were also implicated through shifting co-occurrences and PCA values. Using these seven taxa as a melanoma signature, a neural network reached an 80% tumor detection accuracy in a 10-fold stratified random sampling validation. These results indicated gut microbial proportions as a biosensor for tumor detection, and that shifting co-occurrences could be used to reveal relevant taxa.

T cell repertoire in peripheral blood as a potential biomarker for predicting response to concurrent cetuximab and nivolumab in head and neck squamous cell carcinoma.

BACKGROUND: T cell receptor (TCR) signaling profile is a fundamental property that underpins both adaptive and innate immunity in the host. Despite its potential clinical relevance, the TCR repertoire in peripheral blood has not been thoroughly explored for its value as an immunotherapy efficacy biomarker in head and neck squamous cell carcinoma (HNSCC). The purpose of the present study is to characterize and compare the TCR repertoire in peripheral blood mononuclear cells (PBMC) from patients with HNSCC treated with the combination of cetuximab and nivolumab. METHODS: We used the immunoSEQ assay to sequence the TCR beta (TCR-B) chain repertoire from serially obtained PBMC at baseline and during the treatments from a total of 41 patients who received the combination (NCT03370276). Key TCR repertoire metrics, including diversity and clonality, were calculated and compared between patients with different therapy responses and clinical characteristics (eg, human papillomavirus (HPV) status and smoking history). Patient survival outcomes were compared according to patient groups stratified by the TCR-B clonotyping. To confirm the observed patterns in TCR spectrum, samples from patients who achieved complete response (CR) and partial response (PR) were further profiled with the immunoSEQ deep resolution assay. RESULTS: Our data indicated that the patients who achieved CR and PR had an increased TCR sequence diversity in their baseline samples, this tendency being more pronounced in HPV-negative patients or those with a smoking history. Notably, the CR/PR group had the lowest proportion of patients with oligoclonal TCR clones (2 out of 8 patients), followed by the stable disease group (9 out of 20 patients) and lastly the progressive disease group (7 out of 10 patients). An overall trend toward favorable patient survival was also observed in the polyclonal group. Finally, we reported the shared TCR clones across patients within the same response group, as well as the shared clones by aligning immunoSEQ reads with TCR data retrieved from The Cancer Genome Atlas- head and neck squamous cell carcinoma (TCGA-HNSC) cohort. CONCLUSIONS: Our data suggest that, despite the great clinical heterogeneity of HNSCC and the limited responders in the present cohort, the peripheral TCR repertoires from pretreatment PBMC may be developed as biomarkers for the benefit of immunotherapy in HNSCC.

Priming with very low-affinity peptide ligands gives rise to CD8(+) T-cell effectors with enhanced function but with greater susceptibility to transforming growth factor (TGF)ß-mediated suppression.

While the effects of TCR affinity and TGFß on CD8(+) T-cell function have been studied individually, the manner in which TCR affinity dictates susceptibility to TGFß-mediated suppression remains unknown. To address this issue, we utilized OVA altered peptide ligands (APLs) of different affinities in the OT-I model. We demonstrate that while decreased TCR ligand affinity initially results in weakened responses, such interactions prime the resultant effector cells to respond more strongly to cognate antigen upon secondary exposure. Despite this, responses by CD8(+) T cells primed with lower-affinity TCR ligands are more effectively regulated by TGFß. Susceptibility to TGFß-mediated suppression is associated with downregulation of RGS3, a recently recognized negative regulator of TGFß signaling, but not expression of TGFß receptors I/II. These results suggest a novel tolerance mechanism whereby CD8(+) T cells are discriminately regulated by TGFß according to the affinity of the ligand on which they were initially primed. In addition, because of the major role played by TGFß in tumor-induced immune suppression, these results identify the affinity of the priming ligand as a primary concern in CD8(+) T-cell-mediated cancer immunotherapeutic strategies.

CD8 co-receptor promotes susceptibility of CD8+ T cells to transforming growth factor-ß (TGF-ß)-mediated suppression.

CD8+ T cell function depends on a finely orchestrated balance of activation/suppression signals. While the stimulatory role of the CD8 co-receptor and pleiotropic capabilities of TGF-ß have been studied individually, the influence of CD8 co-receptor on TGF-ß function in CD8+ T cells is unknown. Here, we show that while CD8 enhances T cell activation, it also enhances susceptibility to TGF-ß-mediated immune suppression. Using Jurkat cells expressing a full-length, truncated or no αßCD8 molecule, we demonstrate that cells expressing full-length αßCD8 were highly susceptible, αßCD8-truncated cells were partially susceptible, and CD8-deficient cells were completely resistant to suppression by TGF-ß. Additionally, we determined that inhibition of Lck rendered mouse CD8+ T cells highly resistant to TGF-ß suppression. Resistance was not associated with TGF-ß receptor expression but did correlate with decreased Smad3 and increased Smad7 levels. These findings highlight a previously unrecognized third role for CD8 co-receptor which appears to prepare activated CD8+ T cells for response to TGF-ß. Based on the important role which TGF-ß-mediated suppression plays in tumor immunology, these findings unveil necessary considerations in formulation of CD8+ T cell-related cancer immunotherapy strategies.

The Role of the NKG2D in Vitiligo.

Vitiligo is an acquired multifactorial disease that affects melanocytes and results in skin depigmentation. In this review, we examine the role of cells stress and self-reactive T cells responses. Given the canonical and non-canonical functions of NKG2D, such as authenticating stressed target and enhance TCR signaling, we examine how melanocyte stress leads to the expression of ligands that are recognized by the activating receptor NKG2D, and how its signaling results in the turning of T cells against self (melanocyte suicide by proxy). We also discuss how this initiation phase is followed by T cell perpetuation, as NKG2D signaling results in self-sustained long-lasting T cells, with improved cytolytic properties.

Corrigendum: The Role of NKG2D in Vitiligo.

[This corrects the article DOI: 10.3389/fimmu.2021.624131.].