Altered BMP2/4 Signaling in Stem Cells and Their Niche: Different Cancers but Similar Mechanisms, the Example of Myeloid Leukemia and Breast Cancer

Understanding mechanisms of cancer development is mandatory for disease prevention and management. In healthy tissue, the microenvironment or niche governs stem cell fate by regulating the availability of soluble molecules, cell-cell contacts, cell-matrix interactions, and physical constraints. Gaining insight into the biology of the stem cell microenvironment is of utmost importance, since it plays a role at all stages of tumorigenesis, from (stem) cell transformation to tumor escape. In this context, BMPs (Bone Morphogenetic Proteins), are key mediators of stem cell regulation in both embryonic and adult organs such as hematopoietic, neural and epithelial tissues. BMPs directly regulate the niche and stem cells residing within. Among them, BMP2 and BMP4 emerged as master regulators of normal and tumorigenic processes. Recently, a number of studies unraveled important mechanisms that sustain cell transformation related to dysregulations of the BMP pathway in stem cells and their niche (including exposure to pollutants such as bisphenols). Furthermore, a direct link between BMP2/BMP4 binding to BMP type 1 receptors and the emergence and expansion of cancer stem cells was unveiled. In addition, a chronic exposure of normal stem cells to abnormal BMP signals contributes to the emergence of cancer stem cells, or to disease progression independently of the initial transforming event. In this review, we will illustrate how the regulation of stem cells and their microenvironment becomes dysfunctional in cancer via the hijacking of BMP signaling with main examples in myeloid leukemia and breast cancers.

TCR signal strength defines distinct mechanisms of T cell dysfunction and cancer evasion

T cell receptor (TCR) signal strength is a key determinant of T cell responses. We developed a cancer mouse model in which tumor-specific CD8 T cells (TST cells) encounter tumor antigens with varying TCR signal strength. High-signal-strength interactions caused TST cells to up-regulate inhibitory receptors (IRs), lose effector function, and establish a dysfunction-associated molecular program. TST cells undergoing low-signal-strength interactions also up-regulated IRs, including PD1, but retained a cell-intrinsic functional state. Surprisingly, neither high- nor low-signal-strength interactions led to tumor control in vivo, revealing two distinct mechanisms by which PD1hi TST cells permit tumor escape; high signal strength drives dysfunction, while low signal strength results in functional inertness, where the signal strength is too low to mediate effective cancer cell killing by functional TST cells. CRISPR-Cas9–mediated fine-tuning of signal strength to an intermediate range improved anti-tumor activity in vivo. Our study defines the role of TCR signal strength in TST cell function, with important implications for T cell–based cancer immunotherapies.

Low-affinity CAR T cells exhibit reduced trogocytosis, preventing fratricide and antigen-negative tumor escape while preserving anti-tumor activity

ABSTRACTChimeric antigen receptor (CAR) T cells using the high-affinity CD19 binding domain FMC63 are an effective treatment for patients with relapsed and aggressive B cell lymphoma. However, antigen loss and poor CAR T cell persistence remain common causes for relapse in these patients. Using primary patient samples, we now show that FMC63-based CAR T cells confer rapid antigen loss in all major tumor types currently approved for treatment with CD19 CAR T cells via trogocytosis, the stripping of antigen from tumor cells by CAR T cells. We show that CAR T cell-mediated trogocytosis can be dramatically reduced across a wide range of B cell malignancies by replacing FMC63 with a low affinity CD19 antibody. This reduction in trogocytosis does not alter the direct anti-tumor activity of CD19 CAR T cells but prevents the emergence of antigen-negative tumor cells and significantly increases CAR T cell viability by reducing fratricide of CD19 CAR T cells following trogocytosis.TEASERA reduction in CAR affinity does not affect tumor killing but prolongs T cell persistence and prevents antigen-negative tumor escape.

Prediction of anti-CD25 and 5-FU treatments efficacy for pancreatic cancer using a mathematical model

Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with rising incidence and with 5-years overall survival of less than 8%. PDAC creates an immune-suppressive tumor microenvironment to escape immune-mediated eradication. Regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSC) are critical components of the immune-suppressive tumor microenvironment. Shifting from tumor escape or tolerance to elimination is the major challenge in the treatment of PDAC. Results In a mathematical model, we combine distinct treatment modalities for PDAC, including 5-FU chemotherapy and anti- CD25 immunotherapy to improve clinical outcome and therapeutic efficacy. To address and optimize 5-FU and anti- CD25 treatment (to suppress MDSCs and Tregs, respectively) schedule in-silico and simultaneously unravel the processes driving therapeutic responses, we designed an in vivo calibrated mathematical model of tumor-immune system (TIS) interactions. We designed a user-friendly graphical user interface (GUI) unit which is configurable for treatment timings to implement an in-silico clinical trial to test different timings of both 5-FU and anti- CD25 therapies. By optimizing combination regimens, we improved treatment efficacy. In-silico assessment of 5-FU and anti- CD25 combination therapy for PDAC significantly showed better treatment outcomes when compared to 5-FU and anti- CD25 therapies separately. Due to imprecise, missing, or incomplete experimental data, the kinetic parameters of the TIS model are uncertain that this can be captured by the fuzzy theorem. We have predicted the uncertainty band of cell/cytokines dynamics based on the parametric uncertainty, and we have shown the effect of the treatments on the displacement of the uncertainty band of the cells/cytokines. We performed global sensitivity analysis methods to identify the most influential kinetic parameters and simulate the effect of the perturbation on kinetic parameters on the dynamics of cells/cytokines. Conclusion Our findings outline a rational approach to therapy optimization with meaningful consequences for how we effectively design treatment schedules (timing) to maximize their success, and how we treat PDAC with combined 5-FU and anti- CD25 therapies. Our data revealed that a synergistic combinatorial regimen targeting the Tregs and MDSCs in both crisp and fuzzy settings of model parameters can lead to tumor eradication.

793 Targeting engineered interleukin-2 (IL-2) to antigen specific T cells via novel biologic platforms

BackgroundA key challenge with IL-2 immunotherapy for cancers is lack of selectivity for anti-tumor immune cells and safety liabilities related to indiscriminate activation of immune cells. The CUE-100 series of Immuno-STATs (ISTs) are designed to selectively activate tumor-specific T cells while avoiding IL-2 toxicities due to systemic activation. CUE-100 series ISTs are rationally engineered Fc fusion proteins comprised of bivalent tumor-peptide-HLA (pHLA) complexes and four affinity-attenuated IL-2 molecules to preferentially engage and activate tumor-specific T cells directly in the patient. Emerging clinical data from our lead candidate CUE-101, which targets HPV-specific T cells in 2L+ R/M HNSCCC, provides PoC for the approach and builds confidence for broad applications in numerous cancers. Building on the CUE-100 series framework, our Neo-STAT (NST) platform contains HLA molecules manufactured with an “empty” peptide-binding pocket, into which diverse tumor-peptides can be chemically conjugated, hence addressing tumor heterogeneity in a cost- and time-efficient manner. Our RDI-STAT (Re-Directed Immuno-STAT) platform further expands the CUE-100 series by redirecting the pre-existing protective viral-specific T cell repertoire to target tumor cells via scFv moieties. RDI-STATs are designed to circumvent potential tumor escape mechanisms linked to HLA loss or defects in antigen-presenting pathways. We present here preclinical data supporting the mechanism of action of these platforms to enhance anti-tumor immune responses.MethodsNSTs were engineered with “empty” HLA-A*0201, into which relevant antigenic peptides were conjugated, and assessed for capacity to expand T cells. RDI-STATs were engineered with TAA-specific scFv and viral-specific pHLA complexes, and assessed for their capacity to induce redirected killing of tumor cells while avoiding systemic activation of all T cells.ResultsThe NST platform demonstrated that different T cell epitopes can be efficiently conjugated into the HLA-binding pocket, and that these molecules activate and expand antigen specific T cells in vitro. RDI-STATs were able to expand anti-viral T cell repertoires and drive anti-viral T cell redirected killing of TAA-expressing cells. In contrast to pan anti-CD3 bispecific molecules, RDI-STATs demonstrated significantly lower induction of pro-inflammatory cytokines.ConclusionsThe IST, NST, and RDI-STAT platforms provide novel opportunities for selective targeting of IL-2 to tumor-relevant T cells while avoiding global immune activation and cytokine release. The scalability and versatility of NSTs highlight the potential to target multiple TAA T cell responses, while RDI-STATs highlight a novel means to harness antiviral immunity against cancer, especially in cases where the tumor may escape immune detection due to loss of HLA.

Epitope spreading driven by the joint action of CART cells and pharmacological STING stimulation counteracts tumor escape via antigen-loss variants

BackgroundTarget antigen (Ag) loss has emerged as a major cause of relapse after chimeric antigen receptor T (CART)-cell therapy. We reasoned that the combination of CART cells, with the consequent tumor debulking and release of Ags, together with an immunomodulatory agent, such as the stimulator of interferon gene ligand (STING-L) 2′3′-cyclic GMP-AMP (2′3′-cGAMP), may facilitate the activation of an endogenous response to secondary tumor Ags able to counteract this tumor escape mechanism.MethodsMice bearing B16-derived tumors expressing prostate-specific membrane Ag or gp75 were treated systemically with cognate CART cells followed by intratumoral injections of 2′3′-cGAMP. We studied the target Ag inmunoediting by CART cells and the effect of the CART/STING-L combination on the control of STING-L-treated and STING-L-non-treated tumors and on the endogenous antitumor T-cell response. The role of Batf3-dependent dendritic cells (DCs), stimulator of interferon gene (STING) signaling and perforin (Perf)-mediated killing in the efficacy of the combination were analyzed.ResultsUsing an immune-competent solid tumor model, we showed that CART cells led to the emergence of tumor cells that lose the target Ag, recreating the cancer immunoediting effect of CART-cell therapy. In this setting, the CART/STING-L combination, but not the monotherapy with CART cells or STING-L, restrained tumor progression and enhanced overall survival, showing abscopal effects on distal STING-L-non-treated tumors. Interestingly, a secondary immune response against non-chimeric antigen receptor-targeted Ags (epitope spreading), as determined by major histocompatibility complex-I-tetramer staining, was fostered and its intensity correlated with the efficacy of the combination. This was consistent with the oligoclonal expansion of host T cells, as revealed by in-depth T-cell receptor repertoire analysis. Moreover, only in the combination group did the activation of endogenous T cells translate into a systemic antitumor response. Importantly, the epitope spreading and the antitumor effects of the combination were fully dependent on host STING signaling and Batf3-dependent DCs, and were partially dependent on Perf release by CART cells. Interestingly, the efficacy of the CART/STING-L treatment also depended on STING signaling in CART cells.ConclusionsOur data show that 2′3′-cGAMP is a suitable adjuvant to combine with CART-cell therapy, allowing the induction of an endogenous T-cell response that prevents the outgrowth of Ag-loss tumor variants.

Upregulation of CD22 by Chidamide promotes CAR T cells functionality

Treatment failure or relapse due to tumor escape caused by reduction in target antigen expression has become a challenge in the field of CART therapy. Target antigen density is closely related to the effectiveness of CART therapy, and reduced or lost target antigen expression limits the efficacy of CART therapy and hinders the durability of CAR T cells. Epigenetic drugs can regulate histones for molecular modifications to regulate the transcriptional, translational and post-translational modification processes of target agents, and we demonstrated for the first time the role in regulating CD22 expression and its effect on the efficacy of CD22 CART. In this paper, we found that Chidamide promoted the expression of CD22 on the surface of B-cell tumor cells in vitro and in vivo, and enhanced the function of CD22 CART. As for mechanisms, we demonstrated that Chidamide did not affect CD22 mRNA transcription, but significantly increased the expression of total CD22 protein, indicating that Chidamide may upregulate cell surface CD22 expression by affecting the distribution of CD22 protein. In summary, our results suggest that Chidamide may enhance the efficacy of CD22 CART by inhibiting histone deacetylases to regulate post-transcriptional modifications that affect protein distribution to increase the expression of CD22 on the cell surface.

10.04 A library of novel cancer testis specific T-cell receptors for T-cell receptor gene therapy

BackgroundThe positive clinical effect of T-cell receptor(TCR) gene therapy on tumor regression has previously been demonstrated by NY-ESO-1 TCR-gene therapy. To seriously increase the number of cancer patients that can be treated with TCR-gene therapy we aim to identify a novel set of high-affinity Cancer Testis (CT) specific TCRs targeting different CT-antigens in a variety of prevalent HLA-class I alleles.Materials and MethodsIn this study, we selected by bioinformatic tools the most promising CT-genes to target, and from these genes we identified by HLA-peptidomics the naturally processed and presented HLA-class I peptides. With these peptides HLA-tetramers were generated, and by MACS enrichment and single cell sorting CT-specific CD8+ T-cell clones were selected from the allo-HLA repertoire of healthy donors. By performing several different functional assays the high function avidity CT-clones with a safe recognition pattern were selected. To evaluate the potential for clinical application in TCR-gene therapy, TCRs were sequenced, and transferred into peripheral blood derived CD8+ T cells.ResultsIn total we identified, 7 novel CT-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6 and MAGE-A9 expressing tumors cells in the context of HLA-A1, -A2, -A3, -B7, -C7 and -B35.ConclusionsWith this set of 7 novel CT-specific TCRs we expand the arsenal of tumor specific TCRs. With this expanding library of TCRs it would be possible to select in future for each cancer patient, based on HLA typing and gene expression, a useful TCR to generate a personalized TCR-gene therapy products. In addition, patients could be treated with multiple TCRs to enhance the efficacy and increase the durability of clinical responses by reducing the likelihood of tumor escape.Disclosure InformationM.A.J. de Rooij: None. D.M. Steen: None. D. Remst: None. A. Wouters: None. M.G.D. Kester: None. R.S. Hagedoorn: None. P.A. van Veelen: None. E.M.E. Verdegaal: None. J.H.F. Falkenburg: None. M.H.M. Heemskerk: None.

High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood–brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.