Tumor immune contexture is a determinant of anti-CD19 CAR T cell efficacy in large B cell lymphoma.

Axicabtagene ciloleucel (axi-cel) is an anti-CD19 chimeric antigen receptor (CAR) T cell therapy approved for relapsed/refractory large B cell lymphoma (LBCL) and has treatment with similar efficacy across conventional LBCL subtypes. Toward patient stratification, we assessed whether tumor immune contexture influenced clinical outcomes after axi-cel. We evaluated the tumor microenvironment (TME) of 135 pre-treatment and post-treatment tumor biopsies taken from 51 patients in the ZUMA-1 phase 2 trial. We uncovered dynamic patterns that occurred within 2 weeks after axi-cel. The biological associations among Immunoscore (quantification of tumor-infiltrating T cell density), Immunosign 21 (expression of pre-defined immune gene panel) and cell subsets were validated in three independent LBCL datasets. In the ZUMA-1 trial samples, clinical response and overall survival were associated with pre-treatment immune contexture as characterized by Immunoscore and Immunosign 21. Circulating CAR T cell levels were associated with post-treatment TME T cell exhaustion. TME enriched for chemokines (CCL5 and CCL22), γ-chain receptor cytokines (IL-15, IL-7 and IL-21) and interferon-regulated molecules were associated with T cell infiltration and markers of activity. Finally, high density of regulatory T cells in pre-treatment TME associated with reduced axi-cel-related neurologic toxicity. These findings advance the understanding of LBCL TME characteristics associated with clinical responses to anti-CD19 CAR T cell therapy and could foster biomarker development and treatment optimization for patients with LBCL.

Axicabtagene ciloleucel in relapsed or refractory indolent non-Hodgkin lymphoma (ZUMA-5): a single-arm, multicentre, phase 2 trial.

BACKGROUND: Most patients with advanced-stage indolent non-Hodgkin lymphoma have multiple relapses. We assessed axicabtagene ciloleucel autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in relapsed or refractory indolent non-Hodgkin lymphoma. METHODS: ZUMA-5 is a single-arm, multicentre, phase 2 trial being conducted at 15 medical cancer centres in the USA and two medical cancer centres in France. Patients were eligible if they were aged 18 years or older, with histologically confirmed indolent non-Hodgkin lymphoma (follicular lymphoma or marginal zone lymphoma), had relapsed or refractory disease, previously had two or more lines of therapy (including an anti-CD20 monoclonal antibody with an alkylating agent), and an Eastern Cooperative Oncology Group performance score of 0 or 1. Patients underwent leukapheresis and received conditioning chemotherapy (cyclophosphamide at 500 mg/m2 per day and fludarabine at 30 mg/m2 per day on days -5, -4, and -3) followed by a single infusion of axicabtagene ciloleucel (2 × 106 CAR T cells per kg) on day 0. The primary endpoint was overall response rate (complete response and partial response) assessed by an independent review committee per Lugano classification. The primary activity analysis was done after at least 80 treated patients with follicular lymphoma had been followed up for at least 12 months after the first response assessment at week 4 after infusion. The primary analyses were done in the per-protocol population (ie, eligible patients with follicular lymphoma who had 12 months of follow-up after the first response assessment and eligible patients with marginal zone lymphoma who had at least 4 weeks of follow-up after infusion of axicabtagene ciloleucel). Safety analyses were done in patients who received an infusion of axicabtagene ciloleucel. This study is registered with ClinicalTrials.gov, NCT03105336, and is closed to accrual. FINDINGS: Between June 20, 2017, and July 16, 2020, 153 patients were enrolled and underwent leukapheresis, and axicabtagene ciloleucel was successfully manufactured for all enrolled patients. As of data cutoff (Sept 14, 2020), 148 patients had received an infusion of axicabtagene ciloleucel (124 [84%] who had follicular lymphoma and 24 [16%] who had marginal zone lymphoma). The median follow-up for the primary analysis was 17·5 months (IQR 14·1-22·6). Among patients who were eligible for the primary analysis (n=104, of whom 84 had follicular lymphoma and 20 had marginal zone lymphoma), 96 (92%; 95% CI 85-97) had an overall response and 77 (74%) had a complete response. The most common grade 3 or worse adverse events were cytopenias (104 [70%] of 148 patients) and infections (26 [18%]). Grade 3 or worse cytokine release syndrome occurred in ten (7%) patients and grade 3 or 4 neurological events occurred in 28 (19%) patients. Serious adverse events (any grade) occurred in 74 (50%) patients. Deaths due to adverse events occurred in four (3%) patients, one of which was deemed to be treatment-related (multisystem organ failure). INTERPRETATION: Axicabtagene ciloleucel showed high rates of durable responses and had a manageable safety profile in patients with relapsed or refractory indolent non-Hodgkin lymphoma. FUNDING: Kite, a Gilead Company.

Earlier corticosteroid use for adverse event management in patients receiving axicabtagene ciloleucel for large B-cell lymphoma.

Axicabtagene ciloleucel (axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for relapsed or refractory large B-cell lymphoma (R/R LBCL). To reduce axi-cel-related toxicity, several exploratory safety management cohorts were added to ZUMA-1 (NCT02348216), the pivotal phase 1/2 study of axi-cel in refractory LBCL. Cohort 4 evaluated the rates and severity of cytokine release syndrome (CRS) and neurologic events (NEs) with earlier corticosteroid and tocilizumab use. Primary endpoints were incidence and severity of CRS and NEs. Patients received 2 × 106 anti-CD19 CAR T cells/kg after conditioning chemotherapy. Forty-one patients received axi-cel. Incidences of any-grade CRS and NEs were 93% and 61%, respectively (grade ≥ 3, 2% and 17%). There was no grade 4 or 5 CRS or NE. Despite earlier dosing, the cumulative cortisone-equivalent corticosteroid dose in patients requiring corticosteroid therapy was lower than that reported in the pivotal ZUMA-1 cohorts. With a median follow-up of 14·8 months, objective and complete response rates were 73% and 51%, respectively, and 51% of treated patients were in ongoing response. Earlier and measured use of corticosteroids and/or tocilizumab has the potential to reduce the incidence of grade ≥ 3 CRS and NEs in patients with R/R LBCL receiving axi-cel.

Enrichment of circulating tumor-derived extracellular vesicles from human plasma.

Extracellular vesicles (EVs) are gaining considerable traction within the liquid biopsy arena, as carriers of information from cells in distant sites that may not be accessible for biopsy. Therefore, there is a need to develop methods to enrich for specific EV subtypes, based on their cells of origin. Here we describe the development of an automated method to enrich tumor-derived EVs from plasma using the CellSearch technology compared to Total EVs isolated using differential ultracentrifugation (DUC). We use a modified CellSearch protocol to enrich EpCAM+ EVs from the plasma of patients with non-small cell lung carcinoma (NSCLC) and triple negative breast cancer (TNBC). As a test case, we examined PD-L1, an immune checkpoint ligand known to be expressed in some tumor tissues, to demonstrate enrichment for EpCAM+ EVs. For this purpose, we developed two custom immunoassays utilizing the Simoa HD-1 analyzer (Quanterix) to detect PD-L1 in EVs and interrogate specific EV populations from human plasma. PD-L1 was present in Total EVs from the plasma of healthy individuals and cancer patients, since it is also expressed on several immune cells. However, EpCAM+ EVs were only detectable from the plasma of cancer patients, suggesting these are tumor-derived EVs. As low as 250 µL of plasma could be used to reliably detect PD-L1 from patient-derived EpCAM+ EVs. In summary, this report demonstrates the development of a robust tumor-derived EV enrichment method from human blood. Furthermore, this proof-of-concept study is extendable to other known cancer-specific proteins expressed on EVs exuded from tumors.

LGR5 in breast cancer and ductal carcinoma in situ: a diagnostic and prognostic biomarker and a therapeutic target.

BACKGROUND: Novel biomarkers are required to discern between breast tumors that should be targeted for treatment from those that would never become clinically apparent and/or life threatening for patients. Moreover, therapeutics that specifically target breast cancer (BC) cells with tumor-initiating capacity to prevent recurrence are an unmet need. We investigated the clinical importance of LGR5 in BC and ductal carcinoma in situ (DCIS) to explore LGR5 as a biomarker and a therapeutic target. METHODS: We stained BC (n = 401) and DCIS (n = 119) tissue microarrays with an antibody against LGR5. We examined an LGR5 knockdown ER- cell line that was orthotopically transplanted and used for in vitro colony assays. We also determined the tumor-initiating role of Lgr5 in lineage-tracing experiments. Lastly, we transplanted ER- patient-derived xenografts into mice that were subsequently treated with a LGR5 antibody drug conjugate (anti-LGR5-ADC). RESULTS: LGR5 expression correlated with small tumor size, lower grade, lymph node negativity, and ER-positivity. ER+ patients with LGR5high tumors rarely had recurrence, while high-grade ER- patients with LGR5high expression recurred and died due to BC more often. Intriguingly, all the DCIS patients who later died of BC had LGR5-positive tumors. Colony assays and xenograft experiments substantiated a role for LGR5 in ER- tumor initiation and subsequent growth, which was further validated by lineage-tracing experiments in ER- /triple-negative BC mouse models. Importantly, by utilizing LGR5high patient-derived xenografts, we showed that anti-LGR5-ADC should be considered as a therapeutic for high-grade ER- BC. CONCLUSION: LGR5 has distinct roles in ER- vs. ER+ BC with potential clinical applicability as a biomarker to identify patients in need of therapy and could serve as a therapeutic target for high-grade ER- BC.

MMP9 modulates the metastatic cascade and immune landscape for breast cancer anti-metastatic therapy.

Metastasis, the main cause of cancer-related death, has traditionally been viewed as a late-occurring process during cancer progression. Using the MMTV-PyMT luminal B breast cancer model, we demonstrate that the lung metastatic niche is established early during tumorigenesis. We found that matrix metalloproteinase 9 (MMP9) is an important component of the metastatic niche early in tumorigenesis and promotes circulating tumor cells to colonize the lungs. Blocking active MMP9, using a monoclonal antibody specific to the active form of gelatinases, inhibited endogenous and experimental lung metastases in the MMTV-PyMT model. Mechanistically, inhibiting MMP9 attenuated migration, invasion, and colony formation and promoted CD8+ T cell infiltration and activation. Interestingly, primary tumor burden was unaffected, suggesting that inhibiting active MMP9 is primarily effective during the early metastatic cascade. These findings suggest that the early metastatic circuit can be disrupted by inhibiting active MMP9 and warrant further studies of MMP9-targeted anti-metastatic breast cancer therapy.

CBP/p300 Drives the Differentiation of Regulatory T Cells through Transcriptional and Non-Transcriptional Mechanisms.

Regulatory T cells (Treg) are immunosuppressive and negatively impact response to cancer immunotherapies. CREB-binding protein (CBP) and p300 are closely related acetyltransferases and transcriptional coactivators. Here, we evaluate the mechanisms by which CBP/p300 regulate Treg differentiation and the consequences of CBP/p300 loss-of-function mutations in follicular lymphoma. Transcriptional and epigenetic profiling identified a cascade of transcription factors essential for Treg differentiation. Mass spectrometry analysis showed that CBP/p300 acetylates prostacyclin synthase, which regulates Treg differentiation by altering proinflammatory cytokine secretion by T and B cells. Reduced Treg presence in tissues harboring CBP/p300 loss-of-function mutations was observed in follicular lymphoma. Our findings provide novel insights into the regulation of Treg differentiation by CBP/p300, with potential clinical implications on alteration of the immune landscape. SIGNIFICANCE: This study provides insights into the dynamic role of CBP/p300 in the differentiation of Tregs, with potential clinical implications in the alteration of the immune landscape in follicular lymphoma.

Single cell-produced and in vitro-assembled anti-FcRH5/CD3 T-cell dependent bispecific antibodies have similar in vitro and in vivo properties.

Bispecific antibody production using single host cells has been a new advancement in the antibody engineering field. We previously showed comparable in vitro biological activity and in vivo mouse pharmacokinetics (PK) for two novel single cell variants (v10 and v11) and one traditional dual cell in vitro-assembled anti-human epidermal growth factor receptor 2/CD3 T-cell dependent bispecific (TDB) antibodies. Here, we extended our previous work to assess single cell-produced bispecific variants of a novel TDB against FcRH5, a B-cell lineage marker expressed on multiple myeloma (MM) tumor cells. An in vitro-assembled anti- FcRH5/CD3 TDB antibody was previously developed as a potential treatment option for MM. Two bispecific antibody variants (designs v10 and v11) for manufacturing anti-FcRH5/CD3 TDB in single cells were compared to in vitro-assembled TDB in a dual-cell process to understand whether differences in antibody design and production led to any major differences in their in vitro biological activity, in vivo mouse PK, and PK/pharmacodynamics (PD) or immunogenicity in cynomolgus monkeys (cynos). The binding, in vitro potencies, in vitro pharmacological activities and in vivo PK in mice and cynos of these single cell TDBs were comparable to those of the in vitro-assembled TDB. In addition, the single cell and in vitro-assembled TDBs exhibited robust PD activity and comparable immunogenicity in cynos. Overall, these studies demonstrate that single cell-produced and in vitro-assembled anti-FcRH5/CD3 T-cell dependent bispecific antibodies have similar in vitro and in vivo properties, and support further development of single-cell production method for anti-FcRH5/CD3 TDBs and other single-cell bispecifics.

New Horizons in Advocacy Engaged Physical Sciences and Oncology Research.

To address cancer as a multifaceted adaptive system, the increasing momentum for cross-disciplinary connectivity between cancer biologists, physical scientists, mathematicians, chemists, biomedical engineers, computer scientists, clinicians, and advocates is fueling the emergence of new scientific frontiers, principles, and opportunities within physical sciences and oncology. In parallel to highlighting the advances, challenges, and acceptance of advocates as credible contributors, we offer recommendations for addressing real world hurdles in advancing equitable partnerships among advocacy stakeholders.

Overcoming Barriers of Age to Enhance Efficacy of Cancer Immunotherapy: The Clout of the Extracellular Matrix.

There is a growing list of cancer immunotherapeutics approved for use in a population with an increasing number of aged individuals. Cancer immunotherapy (CIT) mediates tumor destruction by activating anti-tumor immune responses that have been silenced through the oncogenic process. However, in an aging individual, immune deregulation is positively correlated with age. In this context, it is vital to examine the age-related changes in the tumor microenvironment (TME) and specifically, those directly affecting critical players to ensure CIT efficacy. Effector T cells, regulatory T cells, myeloid-derived suppressor cells, tumor-associated macrophages, and tumor-associated neutrophils play important roles in promoting or inhibiting the inflammatory response, while cancer-associated fibroblasts are key mediators of the extracellular matrix (ECM). Immune checkpoint inhibitors function optimally in inflamed tumors heavily invaded by CD4 and CD8 T cells. However, immunosenescence curtails the effector T cell response within the TME and causes ECM deregulation, creating a biophysical barrier impeding both effective drug delivery and pro-inflammatory responses. The ability of the chimeric antigen receptor T (CAR-T) cell to artificially induce an adaptive immune response can be modified to degrade essential components of the ECM and alleviate the age-related changes to the TME. This review will focus on the age-related alterations in ECM and immune-stroma interactions within the TME. We will discuss strategies to overcome the barriers of immunosenescence and matrix deregulation to ameliorate the efficacy of CIT in aged subjects.

Cancer Immunotherapy Getting Brainy: Visualizing the Distinctive CNS Metastatic Niche to Illuminate Therapeutic Resistance.

The advent of cancer immunotherapy (CIT) and its success in treating primary and metastatic cancer may offer substantially improved outcomes for patients. Despite recent advancements, many malignancies remain resistant to CIT, among which are brain metastases, a particularly virulent disease with no apparent cure. The immunologically unique niche of the brain has prompted compelling new questions in immuno-oncology such as the effects of tissue-specific differences in immune response, heterogeneity between primary tumors and distant metastases, and the role of spatiotemporal dynamics in shaping an effective anti-tumor immune response. Current methods to examine the immunobiology of metastases in the brain are constrained by tissue processing methods that limit spatial data collection, omit dynamic information, and cannot recapitulate the heterogeneity of the tumor microenvironment. In the current review, we describe how high-resolution, live imaging tools, particularly intravital microscopy (IVM), are instrumental in answering these questions. IVM of pre-clinical cancer models enables short- and long-term observations of critical immunobiology and metastatic growth phenomena to potentially generate revolutionary insights into the spatiotemporal dynamics of brain metastasis, interactions of CIT with immune elements therein, and influence of chemo- and radiotherapy. We describe the utility of IVM to study brain metastasis in mice by tracking the migration and growth of fluorescently-labeled cells, including cancer cells and immune subsets, while monitoring the physical environment within optical windows using imaging dyes and other signal generation mechanisms to illuminate angiogenesis, hypoxia, and/or CIT drug expression within the metastatic niche. Our review summarizes the current knowledge regarding brain metastases and the immune milieu, presents the current status of CIT and its prospects in targeting brain metastases to circumvent therapeutic resistance, and proposes avenues to utilize IVM to study CIT drug delivery and therapeutic efficacy in preclinical models that will ultimately facilitate novel drug discovery and innovative combination therapies.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Adaptive Immune Regulation of Mammary Postnatal Organogenesis.

Postnatal organogenesis occurs in an immune competent environment and is tightly controlled by interplay between positive and negative regulators. Innate immune cells have beneficial roles in postnatal tissue remodeling, but roles for the adaptive immune system are currently unexplored. Here we show that adaptive immune responses participate in the normal postnatal development of a non-lymphoid epithelial tissue. Since the mammary gland (MG) is the only organ developing predominantly after birth, we utilized it as a powerful system to study adaptive immune regulation of organogenesis. We found that antigen-mediated interactions between mammary antigen-presenting cells and interferon-γ (IFNγ)-producing CD4+ T helper 1 cells participate in MG postnatal organogenesis as negative regulators, locally orchestrating epithelial rearrangement. IFNγ then affects luminal lineage differentiation. This function of adaptive immune responses, regulating normal development, changes the paradigm for studying players of postnatal organogenesis and provides insights into immune surveillance and cancer transformation.

The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells?

Cancer stem cells (CSCs) are tumor cells that have the principal properties of self-renewal, clonal tumor initiation capacity, and clonal long-term repopulation potential. CSCs reside in niches, which are anatomically distinct regions within the tumor microenvironment. These niches maintain the principle properties of CSCs, preserve their phenotypic plasticity, protect them from the immune system, and facilitate their metastatic potential. In this perspective, we focus on the CSC niche and discuss its contribution to tumor initiation and progression. Since CSCs survive many commonly employed cancer therapies, we examine the prospects of targeting the niche components as preferable therapeutic targets.

Blastocyst implantation failure relates to impaired translational machinery gene expression.

Oocyte quality is a well-established determinant of embryonic fate. However, the molecular participants and biological markers that affect and may predict adequate embryonic development are largely elusive. Our aim was to identify the components of the oocyte molecular machinery that part take in the production of a healthy embryo. For this purpose, we used an animal model, generated by us previously, the oocytes of which do not express Cx43 (Cx43(del/del)). In these mice, oogenesis appears normal, fertilisation does occur, early embryonic development is successful but implantation fails. We used magnetic resonance imaging analysis combined with histological examination to characterise the embryonic developmental incompetence. Reciprocal embryo transfer confirmed that the blastocyst evolved from the Cx43(del/del) oocyte is responsible for the implantation disorder. In order to unveil the genes, the impaired expression of which brings about the development of defective embryos, we carried out a genomic screening of both the oocytes and the resulting blastocysts. This microarray analysis revealed a low expression of Egr1, Rpl21 and Eif4a1 in Cx43(del/del) oocytes and downregulation of Rpl15 and Eif4g2 in the resulting blastocysts. We propose that global deficiencies in genes related to the expression of ribosomal proteins and translation initiation factors in apparently normal oocytes bring about accumulation of defects, which significantly compromise their developmental capacity. The blastocysts resulting from such oocytes, which grow within a confined space until implantation, may be unable to generate enough biological mass to allow their expansion. This information could be implicated to diagnosis and treatment of infertility, particularly to IVF.

Matrix metalloproteinase-9 deficiency phenocopies features of preeclampsia and intrauterine growth restriction.

The pregnancy complication preeclampsia (PE), which occurs in approximately 3% to 8% of human pregnancies, is characterized by placental pathologies that can lead to significant fetal and maternal morbidity and mortality. Currently, the only known cure is delivery of the placenta. As the etiology of PE remains unknown, it is vital to find models to study this common syndrome. Here we show that matrix metalloproteinase-9 (MMP9) deficiency causes physiological and placental abnormalities in mice, which mimic features of PE. As with the severe cases of this syndrome, which commence early in gestation, MMP9-null mouse embryos exhibit deficiencies in trophoblast differentiation and invasion shortly after implantation, along with intrauterine growth restriction or embryonic death. Reciprocal embryo transfer experiments demonstrated that embryonic MMP9 is a major contributor to normal implantation, but maternal MMP9 also plays a role in embryonic trophoblast development. Pregnant MMP9-null mice bearing null embryos exhibited clinical features of PE as VEGF dysregulation and proteinuria accompanied by preexisting elevated blood pressure and kidney pathology. Thus, our data show that fetal and maternal MMP9 play a role in the development of PE and establish the MMP9-null mice as a much-needed model to study the clinical course of this syndrome.

Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis.

Mammary epithelial stem cells are vital to tissue expansion and remodeling during various phases of postnatal mammary development. Basal mammary epithelial cells are enriched in Wnt-responsive cells and can reconstitute cleared mammary fat pads upon transplantation into mice. Lgr5 is a Wnt-regulated target gene and was identified as a major stem cell marker in the small intestine, colon, stomach, and hair follicle, as well as in kidney nephrons. Here, we demonstrate the outstanding regenerative potential of a rare population of Lgr5-expressing (Lgr5(+)) mammary epithelial cells (MECs). We found that Lgr5(+) cells reside within the basal population, are superior to other basal cells in regenerating functional mammary glands (MGs), are exceptionally efficient in reconstituting MGs from single cells, and exhibit regenerative capacity in serial transplantations. Loss-of-function and depletion experiments of Lgr5(+) cells from transplanted MECs or from pubertal MGs revealed that these cells are not only sufficient but also necessary for postnatal mammary organogenesis.

Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.

Little is known about the dynamics of cancer cell death in response to therapy in the tumor microenvironment. Intravital microscopy of chemotherapy-treated mouse mammary carcinomas allowed us to follow drug distribution, cell death, and tumor-stroma interactions. We observed associations between vascular leakage and response to doxorubicin, including improved response in matrix metalloproteinase-9 null mice that had increased vascular leakage. Furthermore, we observed CCR2-dependent infiltration of myeloid cells after treatment and that Ccr2 null host mice responded better to treatment with doxorubicin or cisplatin. These data show that the microenvironment contributes critically to drug response via regulation of vascular permeability and innate immune cell infiltration. Thus, live imaging can be used to gain insights into drug responses in situ.