A model for cancer chemo-immunotherapy treatment schedules

Differential Allele-Specific Expression Uncovers Breast Cancer Genes Dysregulated by Cis Noncoding Mutations.

CAR-based cell therapies have revolutionized cancer treatment, however, applications beyond targeting lineage antigens are challenging due to expression of targeted antigens in healthy cells posing a risk for on-target off-tissue toxicities. This presents an opportunity to leverage synthetic biological logic gated gene circuits, such as NOT gate, to expand cancer targets for CAR-based cell therapies. We have constructed a first-in-class NOT gate in CAR-NK cells to protect healthy cells from CAR-mediated cytotoxicity. An inhibitory CAR (iCAR) recognizes a safety antigen expressed on healthy cells and suppresses activating CAR (aCAR) functions, significantly reducing NK cell activity. Multiple iCARs with intracellular co-inhibitory domains containing immunoreceptor tyrosine-based inhibitory motifs have shown to suppress over 50% of aCAR-mediated killing (p<0.05) and significantly reduce TNFa secretion (p<0.0005) in an antigen-specific manner. Here we describe a robust cancer and safety antigen pairing discovery method for the development of NOT gated CAR-NK therapies. A bioinformatics pipeline uses transcriptomics data to discover and prioritize tumor and healthy tissue antigens. We have identified genes differentially expressed in healthy vs tumor tissue and selected leads based on antigens' co-expression in healthy tissue, subcellular localization, antigen topology (presence of extracellular domain(s)), and antibody availability. Such antigen pairs have been validated in primary tissue samples. In AML, targeting the critical leukemic stem cell (LSC) population via antigens such as FLT3 leads to hematopoietic toxicity due to expression in healthy hematopoietic stem cells (HSCs). Comparative bioinformatic analysis between AML and healthy human bone marrow mononuclear cell (BMMC) samples identified 10 surface antigens differentially expressed between HSCs and AML cells that could be used as NOT gate targets to protect HSCs from CAR-mediated toxicity. We further validated one of the top candidate targets, EMCN, by flow cytometry and confirmed significant differential protein expression between HSCs and LSCs. Similarly, in CEA+ tumors, significant on-target off-tissue toxicities occur in healthy epithelium resulting in colitis and lung damage. We prioritized 3 healthy tissue antigens preferentially expressed in intestinal and lung epithelial cells compared to cancer cells: VSIG2, CPM and SLC26A2. IHC analysis confirmed that these targets are expressed at higher levels in the healthy tissues compared to CEA+ tumor, making them attractive candidates to use in a NOT gate circuit to improve the therapeutic window of CEA CAR-NK cells. Using a bioinformatics discovery and validation pipeline coupled with NOT logic gated CAR-NK cells, we can selectively target tumor antigens, while protecting healthy tissues, to create cell therapies with greater efficacy, precision and control. Citation Format: Alba Gonzalez, Assen Roguev, Nicholas W. Frankel, Brian S. Garrison, Derrick Lee, Marcus Gainer, Alyssa Mullenix, Russell M. Gordley, Kathryn A. Loving, Jenny Chien, Gary Lee. Development of logic-gated CAR-NK cells to reduce target-mediated healthy tissue toxicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB028.

Reading the Tea Leaves: Anticarcinogenic Properties of (-)-Epigallocatechin-3-Gallate

Reading the Tea Leaves: Anticarcinogenic Properties of (-)-Epigallocatechin-3-Gallate

This paper discusses possible mechanisms for the selective effect of weakly ionized nonequilibrium plasma and currents in electrolytes on healthy and cancerous cells in physiological saline in a Petri dish. The interaction with the plasma source leads to a change in osmotic pressure, which affects the electro-mechanical properties of cell membranes in healthy and cancerous cells in different ways. The currents arising in the electrolyte charge the membranes of healthy and cancerous cells to a different potential difference due to the different values of the membranes’ dielectric constants. We hypothesized: 1. The dielectric permeability of cancer cell membranes is lower than that of healthy cells, as is the capacity of a unit of the membrane surface, and therefore, the additional potential difference acquired by the membrane through charging with currents induced in the intercellular electrolyte is greater in cancer cells. This can lead to electroporation of cancer cell membranes, resulting in their apoptosis, but does not affect healthy cells. 2. It is known from the literature that the equilibrium potential differences on the membrane (resting potential) of cancer and healthy cells are noticeably different. Therefore, a change in the potential difference on the membrane due to currents in the extracellular fluid can affect the permeability and transport properties of the membranes. It can also be a reason for the selective effect of the nonequilibrium plasma interaction with healthy and cancerous cells in physiological saline.

An operation in itself is a kind of trauma and may lead to immunosuppression followed by a bounce back. Not many studies exist that describe dynamics of the distribution of peripheral blood (PB) immune cells during the perioperative period. Considering this scarcity, we aggregated the data on the dynamics of immune cells in patients with digestive system resections during the perioperative period and the relationship with short- and long-term prognoses. By the systematic retrieval of documents, we collected perioperative period data on white blood cells (WBC), lymphocytes, neutrophil–lymphocyte ratio (NLR), CD4+ T cells, CD8+ T cells, helper T cells (Th), B cells, natural killer cells (NK), dendritic cells (DCs), regulatory T cells (Tregs), regulatory B cells (Bregs), and Myeloid derived suppressor cells (MDSC). The frequency and distribution of these immune cells and the relationship with the patient’s prognosis were summarized. A total of 1916 patients’ data were included. Compared with before surgery, WBC, lymphocytes, CD4+ cells, CD8+ T cells, MDSC, and NK cells decreased after surgery, and then returned to preoperative levels. After operation DCs increased, then gradually recovered to the preoperative level. No significant changes were found in B cell levels during the perioperative period. Compared with the preoperative time-point, Tregs and Bregs both increased postoperatively. Only high levels of the preoperative and/or postoperative NLR were found to be related to the patient’s prognosis. In summary, the surgery itself can cause changes in peripheral blood immune cells, which might change the immunogenicity. Therefore, the immunosuppression caused by the surgical trauma should be minimized. In oncological patients this might even influence long-term results.

S100P-Binding Protein, S100PBP, Mediates Adhesion through Regulation of Cathepsin Z in Pancreatic Cancer Cells

Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176- control cells, their response proved to be target-specific. Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity.

Aberrant O-glycosylation is a hallmark of cancer and can be used to improve druggability of targets for cancer immunotherapy. Tumors express cell surface proteins decorated with truncated O-glycans like Thomsen-Friedenreich (TF), Thomsen-nouveau (Tn) or sialylated Tn (sTn) antigens, which are virtually absent from normal tissues. Development of therapeutic mAbs against these hapten glycotopes is often hampered by weak affinities and low specificity, whereas peptide epitope targeting mAbs usually cannot distinguish between normal and tumor tissue. Both drawbacks can be circumvented by addressing combined protein/carbohydrate epitopes with mAbs that enable high affinity binding and high tumor specificity at the same time. We generated mAb GT-008 recognizing the glycoprotein CD24 with fundamental role in tumorigenesis and tumor progression, which is expressed on solid tumors, but also on healthy immune cells and tissues. GT-008 binds CD24 only in presence of a tumor-associated glycosylation thereby leading to reduced binding of the protein expressed on healthy cells.GT-008 was generated by immunization and single B cell cloning using different on- and off-target glycoforms for selection and screening. Binding and tumor selectivity were analyzed by ELISA, flow cytometry and immunohistochemistry on healthy and tumor tissues. Functionality was investigated by in vitro internalization and cytotoxicity assays with tumor and healthy cells. Compared to a conventional glycosylation-independent control antibody, GT-008 binds the protein target only in presence of TF O-glycosylation, but not the non-glycosylated protein, other glycoproteins carrying TF nor a synthetic TF O-glycoconjugate. GT-008 differentiates against the sialylated glycoform present on normal tissues. This leads to reduced binding to healthy human immune cells and tissues compared to the control mAb, whereas binding to cancer tissues from different indications (e.g. endometrium, breast, ovarian, prostate, a.o.) was retained. Additionally, its glycosylation dependent binding translates into tumor-selective internalization of GT-008 in vitro while sparing out CD24-positive immune cells. GT-008 has the potential to overcome limitations of conventional protein-targeting mAbs currently in clinical development by circumventing immune cell depletion and on-target/off-tumor toxicities associated with target expression on healthy cells. With its improved therapeutic window due to the strong differentiation between healthy and tumor tissue, GT-008 is exceptionally suitable for different therapeutic modalities, such as drug delivery (ADC, RIT) or cellular therapies (CARs). An in vitro and in vivo proof-of-concept study as therapeutic radioimmunoconjugate has been initiated. Citation Format: Johanna Gellert, Andreas Franz, Manon Weis, Evelyn Hartung, Stephanie Gurka, Antje Danielczyk, Lisa Weiß, Patrik Kehler. Improving druggability of a promising tumor target by the novel glycosylation-dependent mAb GT-008 for treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3429.

The Tumor Microenvironment: A Druggable Target for Metastatic Disease?

Event Abstract Back to Event Dendritic pro-drug for local and selective treatment of locally advanced breast cancer Nuria Oliva1, Mariana Atilano1, 2*, João Conde1, 3*, Elazer R. Edelman1, 4* and Natalie Artzi1, 5* 1 MIT, IMES, United States 2 IQS, Chemical Engineering, Spain 3 Queen Mary University of London, School of Engineering and Materials Science, United Kingdom 4 BWH, Harvard Medical School, Cardiovascular Division, United States 5 BWH, Harvard Medical School, Department of Medicine, United States Introduction: Systemic neoadjuvant therapy has been established as the preferred therapeutic approach for locally advanced breast cancer, downstaging the disease and preventing mastectomy. However, complications of systemic chemotherapy are devastating. Local therapy would prevent high concentrations of circulating drug and reduce off-target tissue retention. Yet, the means to attain ideal release kinetics and selective uptake remain elusive. We have developed a novel class of biocompatible and biodegradable adhesive materials based on dendrimers and dextrans[1] that can coat the tumor and locally release drugs in a controlled manner. In this work, I have developed and optimized a dendritic pro-drug capable of discerning between healthy and cancer cells. It selectively enters EGFR-overexpressing breast cancer cells through receptor-mediated endocytosis (RME) and releases doxorubicin inside the cells. They will be added to our adhesive hydrogel for local and sustained delivery. Materials and Methods: PAMAM dendrimer generation 5 (Dendritech) was conjugated to EGF-mimicking peptides[2] (Biopolymer Lab, MIT) and also to doxorubicin (Cayman) through a pH-sensitive linker[3]. Cancer cells (MDA-MB-468, ATCC) and healthy mammary epithelial cells (HMEpC, ATCC) were cultured in their recommended media. Cells were treated with 10 uM tagged-dendrimer solutions and uptake was assessed by FACS. Doxorubicin release from the dendrimer was monitored through UV-VIS spectroscopy in PBS (pH 7.4) and acetate buffer (pH 5.5). Cancer and healthy cells were incubated with 10 uM dendritic pro-drug for 48 hours to study cytotoxicity. Results and Discussion: Fluorescence microscopy showed indiscriminate uptake of naked dendrimer independent of cell type (Fig. 1a-b), while dendrimer-peptide uptake was higher in EGFR+ cancer cells than in EGFR- healthy cells (Fig. 1d-e). Blocking of the receptor using an antibody caused abrogation of dendrimer-peptide uptake, but not naked dendrimer (Fig 1c and f). These results were corroborated by FACS (not shown). Taken together, these data prove that our dendrimer conjugates are being uptaken by RME through EGFR, as opposed to diffusion-driven uptake observed for naked dendrimer. Doxorubicin was conjugated to dendrimer-peptide through a cis-aconityl pH-sensitive linker to form the dendritic pro-drug. Incubation of the dendritic pro-drug in PBS (pH 7.4) showed no statistically significant doxorubicin release over 12 hours, while 45% of the drug was released in acetate buffer (pH 5.5) in the first 3 hours (not shown), thus corroborating pH-triggered release. The dendritic pro-drug showed 86% cytotoxicity after 48 hours in cancer cells, while no toxicity was observed in healthy cells (Fig. 1g-k). Conclusions: We have demonstrated that we can successfully develop a dendritic pro-drug that selectively treats EGFR-overexpressing tumors while minimizing side effects in healthy cells surrounding the tumor. The dendritic pro-drug will be incorporated to our adhesive hydrogel and release kinetics and in vivo efficacy will be assessed. Generalization of this platform with peptides targeting other commonly overexpressed growth factor receptors in cancer (FGF2R, VEGFR or PDGFR) will expand the targeting capabilities of our delivery system. We aim to develop a delivery platform capable of treating tumors in a local and selective manner. Marie Curie International Outgoing Fellowship and Funding (FP7-PEOPLE-2013-IOF, Project 626386); Dr. Dong Soo Yun for cryo-TEM assistance at the Peterson Nanotechnology Materials Core Facility; KI MIT Biopolymers Lab; Dr. Glenn Paradis for FACS assistance with Cancer Center Support (FACS core); KI Genomics Core/ MIT BioMicro Center; Timothy E. Cheng for assistance with computation of methods

Significance High platelet counts and advanced stage of ovarian cancer go hand-in-hand in promoting each other in a feed-forward loop that results in blood coagulation and chemotherapy resistance. This results in a high incidence of death due to thrombosis in ovarian cancer patients, and especially in patients with the ovarian clear cell carcinoma histologic type. Objective and Hypothesis We sought to develop an experimental model of the positive interactions between platelets and cancer cells and test the hypothesis that interference with platelet clotting will inhibit this interaction. Approach The effects of platelets on spheroid formation by cancer or healthy epithelial cells were evaluated using a magnetic 3D cancer spheroids assay. The ES2 and MESOV cell lines, which represent clear cell carcinoma and high grade serous histologies, respectively were used for the ovarian cancer cells. Primary human fallopian tube secretory epithelial cell cultures were used to represent healthy cells. The spheroids were imaged and measured using the Optronix GelCount colony counter and evaluated using metabolic viability (MTT) and protein concentration (SRB) assays. The shear-free platelet aggregation assay was performed in the presence of healthy or cancer cells or their conditioned media. Furthermore, we evaluated possible interruption of this feed-forward loop using antiplatelet agents: aspirin—a cyclooxygenase (COX)-1 and -2 inhibitor, celecoxib—a selective COX-2 inhibitor, clopidogrel—an ADP binding inhibitor, dipyridamole—an ADP uptake inhibitor, eptifibatide—a platelet's GP IIb/IIIa inhibitor, and prostacyclin—a platelet aggregation inhibitor. Results Incubation of platelets with cancer spheroids as they are forming decreased the size and density of the spheres in less than 15 minutes of exposure. The MTT assay indicated that condensed spheres were just as live and viable as the spheres that formed in the absence of platelets. Incubation of cancer cells or cancer cells’ conditioned media with platelets caused clumping of platelets in a cancer cells number-dependent manner. Healthy cells’ conditioned media did not cause platelets aggregation. Pre-treating platelets with up to a 1 mM of aspirin, clopidogrel, dipyridamole, and prostacyclin did not prevent cancer cell-induced aggregation, unlike celecoxib, which prevented aggregation at high concentrations, and eptifibatide, which was able to prevent aggregation at low concentrations as 0.1 µM. Conclusions The positive interaction between platelets and cancer cells can be mimicked in co-culture conditions. This interaction appears to involve platelets GPIIb/IIIa binding.

Cancer and healthy cells have distinct distributions of molecular properties and thus respond differently to drugs. Cancer drugs ideally kill cancer cells while limiting harm to healthy cells. However, the inherent variance among cells in both cancer and healthy cell populations increases the difficulty of selective drug action. Here we formalize a classification framework based on the idea that an ideal cancer drug should maximally discriminate between cancer and healthy cells. More specifically, this discrimination should be performed on the basis of measurable cell markers. We divide the problem into three parts which we explore with examples. First, molecular markers should discriminate cancer cells from healthy cells at the single-cell level. Second, the effects of drugs should be statistically predicted by these molecular markers. Third, drugs should be optimized for classification performance. We find that expression levels of a handful of genes suffice to discriminate well between individual cells in cancer and healthy tissue. We also find that gene expression predicts the efficacy of some cancer drugs, suggesting that these cancer drugs act as suboptimal classifiers using gene profiles. Finally, we formulate a framework that defines an optimal drug, and predicts drug cocktails that may target cancer more accurately than the individual drugs alone. Conceptualizing cancer drugs as solving a discrimination problem in the high-dimensional space of molecular markers promises to inform the design of new cancer drugs and drug cocktails.

Current cancer treatments damage healthy cells and tissues, causing short-term and long-term side effects. New treatments are desired that show greater selectivity toward cancer cells and evade the common mechanisms of multidrug resistance. Membranolytic anticancer peptides (mACPs) hold promise against cancer and multidrug resistance. Amphipathicity, hydrophobicity, and net charge of mACPs participate in their respective interactions with cell membranes and their overall inhibition of cancer cells. To support the design of cell-line selective mACPs, we investigated the relationships that amino acid composition, physicochemical properties, sequence motifs, and sequence homology could have with their potency and selectivity towards several healthy and cancer cell lines. Sequence length and net charge are known to affect the selectivity of mACPs between cancer and healthy cell lines. Our study reveals that increasing the net charge or flexibility (i. e., small and aliphatic residues) influences their selectivity between cancer cell lines with comparable lipid compositions.

Communication between cells is a vital process that governs cellular organization and coordination in an organism. Thus, multiple mechanisms of cell communication have evolved to respond to all the needs of an organism. The modes of cell-cell communication range from the ones that require direct contact between cells, such as cytoplasmic bridges (CBs) and gap junctions, to others that may take place over long distances throughout the organism, such as hormone signaling. Precise integration of data acquired from all these different modes of communication maintains tissue homeostasis and allows optimal adaptation of an organism to various stresses. Cellular senescence represents one of the outcomes of cellular response to stress.1 Senescent cells execute essential functions in different physiological and pathophysiological conditions. They are present in pre-malignant lesions, sites of tissue damage, aging tissues and even during embryonic development. In all these places senescent cells communicate with cells in their surroundings and modulate the function of these cells.2-5 The effect of senescent cells on nearby cells is commonly attributed to the secretion of cytokines, chemokines and matrix metalloproteinases.4,6 We have recently demonstrated that in addition to secretion, senescent cells affect neighboring cells by direct intercellular protein transfer (IPT).2 Proteins from senescent cells are directly transferred to recipient neighboring cells, such as immune and cancer cells, triggering activation of signaling pathways in these cells, ultimately leading to changes in cellular behavior. We have detected IPT from senescent cells and demonstrated that these cells form CBs with other cells, including immune cells, cancer and non-cancer epithelial cells.2 CBs, which in some conditions are called tunneling nanotubes, are open on both edges and allow transfer of cytoplasmic content to neighboring cells. Interestingly, we also identified mitochondria and lysosomes in CBs, implying that organelles might be transferred between the cells. Transfer of these organelles was indeed identified in several other systems. Since dysfunctional mitochondria are thought to play a role in cellular senescence, it can be speculated that transfer of the dysfunctional mitochondria to healthy neighboring cells may induce senescence in these cells. Transfer of cytoplasmic content or whole organelles might serve therefore, for communication of cellular stress. The functional consequences of cell communication of senescent cells through IPT are not completely understood and could be cell type dependent. For instance, we have reported that IPT facilitates elimination of senescent cells by NK cells since transfer of proteins to NK cells correlated with NK cell activation and cytotoxicity. Remarkably, inhibition of CDC42 in senescent cells resulted in decreased IPT and impaired elimination of the senescent cells by NK cells.2 In addition to interaction with NK cells senescent cells also communicate with other components of the innate and adaptive immune systems.2,5,6 Thus, it is possible that senescent cells transfer proteins to other types of immune cells and alter their behavior. To fulfill their function immune cells respond to cytokines and chemokines using receptor-ligand interactions to activate cellular responses (Fig. 1). IPT can serve as an additional mechanism regulating activity of immune cells following contact mediated interaction with resident cells and thereby fine-tune the immune response. Figure 1. Senescent cells communicate with NK cells by multiple mechanisms. Senescent cells influence NK cells by (i) senescent associated secretory phenotype (SASP); (ii) specific receptor ligand interactions; (iii) cytoplasmic bridges. These diverse mechanisms ... Senescent cells can impact tumorigenesis, mainly by secretion of pro-inflammatory cytokines.3,5 Our data demonstrate that senescent cells form CBs with cancer cells, thereby implementing IPT in interaction of senescent and cancer cells. Since the interaction of these cells is long lasting, in contrast to the interaction with NK cells, higher amount and number of proteins could potentially be transferred. The functional impact of IPT from senescent cells to cancer cells is unknown, as it could potentially promote or restrain cancer cells. Senescent cells could transfer cell cycle inhibitors (i.e. p16, p15 and p21) to cancer cells, thereby inhibiting their proliferation. Conversely, cancer cells may also receive organelles and proteins to support their rapid proliferation. In addition to pathological conditions, senescent cells modulate physiological processes, including embryonic development.4 During embryonic development human placental syncytiotrophoblast exhibit features and molecular markers of cellular senescence.7 The syncytiotrophoblast is a multinucleate epithelium which supports fetal growth by creating an interface between maternal and fetal circulation. The syncytiotrophoblast cells communicate with subjacent layer of mono-nucleated cytotrophoblasts to promote division and fusion of cytotrophoblasts into the syncytiotrophoblast. These cells also communicate with immune cells to control immune tolerance at the maternal-fetal interface. Therefore, senescent syncytiotrophoblast cells might also form CBs to facilitate these non-cell autonomous effects which are essential for embryonic development. Overall, IPT from senescent cells represents a pivotal mode of cellular communication with possible physiological outcomes on cancer progression, tissue repair, embryonic development, aging and immune modulation.