Background
ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to ...inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 1.
Methods
Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8
+
tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data.
Results
ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (
K
D
) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound
89
Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing
89
Zr immuno-PET reagents.
Conclusion
89
Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.
With the advent of checkpoint inhibitors, there is increasing need to study the dynamics of CD8+ T-cells in the tumor microenviroment. In this article, we describe a semi-automated method to quantify ...and interrogate spatial relationships between T-cells and collagenous stroma in human and mouse tissue samples. The assay combines CD8 immunohistochemistry with modified Masson’s trichrome. Slides are scanned and digital images are analyzed using an adjustable MATLAB algorithm, allowing for high-throughput quantification of cytotoxic T-cells and collagen. This method provides a flexible tool for unbiased quantification of T-cells and their interactions with tumor cells and tumor microenvironment in tissue samples.
Immune contexture of paediatric cancers Thakur, Meghna Das; Franz, Carl J.; Brennan, Laura ...
European journal of cancer (1990),
07/2022, Letnik:
170
Journal Article
Recenzirano
The clinical development of immune checkpoint–targeted immunotherapies has been disappointing so far in paediatric solid tumours. However, as opposed to adults, very little is known about the immune ...contexture of paediatric malignancies.
We investigated by gene expression and immunohistochemistry (IHC) the immune microenvironment of five major paediatric cancers: Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), medulloblastoma (MB) and neuroblastoma (NB; 20 cases each; n = 100 samples total), and correlated them with overall survival.
NB and RMS tumours had high immune cell gene expression values and high T-cell counts but were low for antigen processing cell (APC) genes. OS and ES tumours showed low levels of T-cells but the highest levels of APC genes. OS had the highest levels of macrophages (CSF1R, CD163 and CD68), whereas ES had the lowest. MB appeared as immune deserts. Tregs (FOXP3 staining) were higher in both RMS and OS. Most tumours scored negative for PD-L1 in tumour and immune cells, with only 11 of 100 samples positive for PD-L1 staining. PD-L1 and OX40 levels were generally low across all five indications. Interestingly, NB had comparable levels of CD8 by IHC and by gene expression to adult tumours. However, by gene expression, these tumours were low for T-cell cytotoxic molecules GZMB, GZMA and PRF1. Surprisingly, the lower the level of tumour infiltrative CD8 T-cells, the better the prognosis was in NB, RMS and ES. Gene expression analyses showed that MYCN-amplified NB have higher amounts of immune suppressive cells such as macrophages, myeloid-derived suppressor cells and Tregs, whereas the non-MYCN-amplified tumours were more infiltrated and had higher expression levels of Teff.
Our results describe the quality and quantity of immune cells across five major paediatric cancers and provide some key features differentiating these tumours from adult tumour types. These findings explain why anti-PD(L)1 might not have had single agent success in paediatric cancers. These results provides the rationale for the development of biologically stratified and personalised immunotherapy strategies in children with relapsing/refractory cancers.
•The immune contexture of paediatric tumours remains unknown compared with adult cancers.•No PD-L1 expression was found on cancer and immune cells of 5 major paediatric cancers.•CD163, CSF1R and CD68 myeloid cells were abundant in all tumours except Ewing sarcoma.•Some tumours have high levels of infiltrating T-cells but lack efficient activation.•Biologically stratified immunotherapy strategies are needed for paediatric oncology.
Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and ...somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire. Therefore, in this report, we used a novel inducible AID-knockout mouse model and discovered that deleting AID in adult mice caused spontaneous germinal center formation. Deep sequencing of the IgH repertoire revealed that Ab diversification begins early in life and evolves over time. Our data suggest that activated B cells form germinal centers at steady state and facilitate continuous diversification of the B cell repertoire. In support, we identified shared B cell lineages that were class switched and showed age-dependent rates of mutation. Our data provide novel context to the genesis of the B cell repertoire that may benefit the understanding of autoimmunity and the strength of an immune response to infection.
Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain ...containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient
antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.
Abstract
Background: Highly multiplexed spatial analysis of tumor tissue is necessary to improve understanding of anti-tumor immunity. Moreover, spatial interrogation of protein and nucleic acid ...targets simultaneously can better characterize immune cell subsets and targets such as cytokines that are difficult to detect by immunohistochemistry (IHC). NanoString's Digital Spatial Profıling (DSP) technology can detect and quantify proteins and RNA at highly multiplexed levels. We compared DSP to flow cytometry (FCM), IHC and in-situ hybridization (ISH) to assess its sensitivity and specificity. Ongoing studies will assess the prevalence and distribution of immune cell subsets, cytokines and tumor cells in 10 non-small cell lung cancer (NSCLC) cases.
Methods: 20 FFPE NSCLC cases and 1 peripheral blood mononuclear cell (PBMC) pellet were assayed on the DSP platform. Regions of interest (300 um x 400 um) or single cell masks were selected from H&E or fluorescence-stained sections. Serial sections were then probed with a cocktail of primary antibodies conjugated to DNA oligos via a UV-cleavable linker for 29 protein and probes for 39 RNA targets. Oligos from the ROIs were then released via UV-mediated linker cleavage and quantitated on the nCounter® Analysis System. Repeat analysis was performed on 10 cases to assess reproducibility between serial sections. A subset of cases was stained for CD3, CD4, CD8, CD45, PD-1, and PD-L1 by IHC and counted by image analysis. FCM was performed on the PBMC sample. Counts from both methods were compared to nCounter counts.
Results: Comparison of DSP to FCM in PBMC showed good correlation (R2=0.65). Correlation between DSP counts and IHC was robust for CD3 (R2=0.72), CD4 (R2=0.80), and CD45 (R2=0.90). 21 of 29 protein markers showed good/excellent reproducibility (R2 >0.70) on separate DSP runs. 7 of 29 showed fair/good reproducibility across tissue sections (R2=0.50-0.70). RNA analysis of NSCLC samples by both DSP and ISH is ongoing. Analysis of both PBMC and NSCLC samples suggests that target abundance and section-to-section variability may explain differences in performance and reproducibility between sections. For example, low abundance in NSCLC tissue and section-to-section variability was observed for CD19 and CD56 with corresponding lower correlation between replicates (R2=0.68 and R2=0.52, respectively).
Conclusion: DSP shows high section to section reproducibility and fair/good correlation with FCM and IHC. We find performance can be affected by differences in antibody clones or lots and/or section to section variability, particularly for low abundance cell types. Multiplexed RNA analysis of NSCLC samples is ongoing and will document the sensitivity and specificity of DSP compared to traditional ISH and the potential utility of DSP in assessing cytokines in the tumor microenvironment.
Citation Format: James Ziai, Patrick Caplazi, Jérémie Decalf, Yan Liang, Patricia de Almeida, Daniel Zollinger, Alison Van Schoiack, Joseph Beechem, Jane Grogan, Matthew Albert. Highly multiplexed analysis of immune cell subsets in non-small cell lung cancer: validation of protein and RNA analysis by the Nanostring Digital Spatial Profiling (DSP) platform abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2089.
NUT midline carcinomas (NMC) are a rare, recently described class of poorly-differentiated tumors that exhibit rapid onset and highly aggressive clinicopathologic behavior. These tumors are defined ...by rearrangement of the nuclear protein in testis (NUT) gene on chromosome 15q14, most commonly in a balanced translocation with the BRD4 gene on chromosome 19p13.1, resulting in the characteristic BRD4-NUT fusion gene and protein which blocks epithelial differentiation through chromatin binding. NMC frequently involve midline structures of adolescents and young adults and affect the head and neck region in 50% of cases. To our knowledge, only one case has been previously reported involving a salivary gland. Here, we present a case of a NMC of the salivary gland in an adolescent male presenting with an intermittently painful left submandibular mass of 3 months duration.
Although immune checkpoint inhibitors (ICIs) are established as effective cancer therapies, overcoming therapeutic resistance remains a critical challenge. Here we identify interleukin 6 (IL-6) as a ...correlate of poor response to atezolizumab (anti-PD-L1) in large clinical trials of advanced kidney, breast, and bladder cancers. In pre-clinical models, combined blockade of PD-L1 and the IL-6 receptor (IL6R) causes synergistic regression of large established tumors and substantially improves anti-tumor CD8+ cytotoxic T lymphocyte (CTL) responses compared with anti-PD-L1 alone. Circulating CTLs from cancer patients with high plasma IL-6 display a repressed functional profile based on single-cell RNA sequencing, and IL-6-STAT3 signaling inhibits classical cytotoxic differentiation of CTLs in vitro. In tumor-bearing mice, CTL-specific IL6R deficiency is sufficient to improve anti-PD-L1 activity. Thus, based on both clinical and experimental evidence, agents targeting IL-6 signaling are plausible partners for combination with ICIs in cancer patients.
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•High IL-6 associates with poor atezolizumab efficacy in patients with advanced cancer•IL6R- and PD-L1-blocking antibodies combine synergistically to treat murine tumors•IL-6-STAT3 signaling blocks cytotoxic effector differentiation of CD8+ T cells•Cell-intrinsic IL-6 signaling in CD8+ T cells limits pre-clinical anti-PD-L1 activity
Identifying clinically actionable drivers of therapeutic resistance is a major objective for cancer immunotherapy. Here, Huseni et al. identify IL-6 as a correlate of poor clinical response to atezolizumab (anti-PD-L1) therapy and demonstrate that IL-6 impairs anti-PD-L1 efficacy by restricting the anti-tumor functions of cytotoxic T cells.
Abstract FDA approval for chimeric antigen receptor (CAR) T cell therapies targeting hematological cancers has unleashed the potential of utilizing live T cells as therapeutics. However, CAR-mediated ...targeting of solid tumors is currently limited by a paucity of suitable tumor antigens that are both specific and highly expressed on the tumor cell surface. In this report we explore the possibility of harnessing tumor-specific expression of endogenous retroviruses (ERVs) as a universal target for CAR T cell therapy. We found that gp70, the envelope protein from a murine ERV, is highly expressed on the surface of tumor cell lines derived from a diverse array of murine tumor types. We utilized the B16 melanoma model as a representative poorly inflamed tumor that does not respond to existing immunotherapies to examine efficacy of gp70 CAR T cells. T cells engineered to express a gp70 CAR suppress tumor growth in vivo and recognize B16 tumor cells with improved sensitivity compared to T cells expressing two previously described dominant tumor antigens (gp100 or P15e) due to low expression of MHC-I on B16 tumors. Co-incubation of B16 tumor cells with gp70 CAR T cells results in upregulation of MHC-I on the tumor cell surface suggesting that in addition to killing tumor cells directly, gp70 CAR T cells could facilitate recognition of the tumor by endogenous T cells through local release T cell derived IFNγ. We postulate that utilization of CAR T cells specific for cell surface ERV envelope proteins offers a promising strategy that could be harnessed to achieve local release of T cell derived payloads specifically within the tumor microenvironment. Citation Format: Sophie M. Lehar, Srirupa Roy, Lisa Liao, Haiyin Chen, Yongmei Chen, Mayra Cruz Tleugabulova, Eric Janezic, Azadeh Madjidi, Tamaki Jones, Vincent Javinal, James Ziai, Ira Mellman, Jill Schartner, Andrey Shaw. Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB340.
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