Metabolic Response to Stress Differentiates Heterogeneous Cancer Cells with Varying Metastatic Potential

Intratumoral heterogeneity is ubiquitously present within primary tumors and contributes to intractable behaviors such as metastasis and mutability spatiotemporally. Mounting evidence has shown that heterogeneous cell populations can adversely affect cell metabolism and metastatic potential. The cell’s only fluorescent molecules within the electron transport chain, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH), can allow the quantitation of cell metabolism. We demonstrate the use of the optical redox ratio (FAD/(NADH+FAD)) to determine the metabolic behaviors of a heterogeneous panel of cells with varying metastatic programs at normal conditions and following acute hypoxia. At normal conditions, we reveal an attenuation in the optical redox ratio as metastatic potential decreases, not including the non-metastatic cell line. We reveal that reoxygenating the clonogenic cells after hypoxia enabled further differences in the optical redox ratio for the highly metastatic (increased by 43 ± 9%), semi-metastatic (increased by 33 ± 4%), and non-metastatic (decreased by 14 ± 7%) cell lines. This work coalesces two potential strategies for cancer treatment: 1) the optical redox ratio to assess cell metabolic features and therapy-induced changes 2) the method of inducing a “stress” test to identify further differences in heterogeneous cell populations

Nanoparticle T cell Engagers as a Modular Platform for Cancer Immunotherapy

Immunotherapy has advanced significantly in recent years due to its promising clinical outcomes in a variety of malignancies and holds great promise in becoming the “cure” for cancer. Cancer immunotherapy is the treatment that stimulates a person’s own immune system to recognize, target, and eliminate cancer cells. As the field progresses with emerging and novel strategies, the ability to manipulate the immune system while mitigating toxicities is the goal for clinical translation. To control for both efficacy and safety, biomaterials have been incorporated into immunotherapies to achieve tissue- and/or cell-specific immunomodulation, overcome immunosuppression, and address tumor microenvironment heterogeneity.T cell-based immunotherapy, such as chimeric antigen receptor (CAR)-T cells, has shown promising clinical outcomes in many cancers. CAR-T cells are autologous T cells that have been virally transfected to express an engineered CAR construct, containing a synthesized fragment that targets the desired surface antigen on the target cell. However, this therapy has significant limitations such as toxicity, the long-term safety profile of the viral vector, the need to perform quality control testing frequently throughout the production of CAR-T cells, the high costs associated with extensive labor and expensive facility equipment, complex production, and the inability to target multiple tumor antigens with one CAR-T cell. In addition to CAR-T cells, T cell-based therapy can be pursued with T cell engagers (TCEs). TCEs consists of two single chain variable fragments which are connected by a protein linker. One of the domains recognizes a tumor-associated surface antigen, while the other recognizes the T cell using the CD3 receptor. TCEs demonstrate high potency and efficacy against tumor cells and exploit the use of endogenous T cells, circumventing the limitation of genetically engineering extracted patient T cells to express CARs. The disadvantages of TCEs, however, include toxicity, laborious and tedious production, short pharmacokinetics (PK), and the inability to target multiple cancer surface markers Moreover, both CART and TCE therapies confer the development of antigen-less clones, causing tumor escape and relapse in multi-clonal diseases; and inability to induce T cell persistent activation, ultimately causing T cell exhaustion. We developed nanoparticle-based bispecific T cell engagers (nanoBTCEs), which are liposomes decorated with anti-CD3 monoclonal antibodies targeting T cells, and monoclonal antibodies targeting one cancer antigen. NanoBTCEs 1) have a long half-life of about 60 hours, which enables once-a-week administration instead of continuous infusion; 2) induce T cell activation in the presence of Waldenstrom Macroglobulinemia (WM) and multiple myeloma (MM) cells; and 3) induce T cell-mediated cancer cell lysis of WM and MM cells. Due to the nanoparticulate nature of nanoBTCEs, we solved the PK problem, enabled simple and cheap production, and created an off-the-shelf platform for cancer immunotherapy. For multi-clonal diseases such as MM, we also developed nanoparticle-based multispecific T cell engagers (nanoMuTEs), which are liposomes decorated with anti-CD3 monoclonal antibodies targeting T cells, and monoclonal antibodies targeting more than one cancer antigen. NanoMuTEs targeting multiple cancer antigens showed greater efficacy in MM cells in vitro and in vivo, compared to nanoBTCEs targeting only one cancer antigen. Unlike nanoBTCEs, treatment with nanoMuTEs didn’t cause downregulation (or loss) of a single antigen and prevented the development of antigen-less tumor escape. Our nanoparticle-based immuno-engaging technology provides a solution for the major limitations of current immunotherapy technologies. In addition, a major disadvantage TCEs have is that their T cell activation and persistence is weaker than CAR-T cells, which is why CAR-T cells have a greater anti-tumor response compared to TCEs. Methods to activate T cells include the use of lectins, such as phytohemagglutinin (PHA) which is commonly only used for research purposes ex vivo, but not in vivo. PHA binds to glycoproteins on the T cell receptor and stimulates T cells more significantly compared to other forms of T cell activators such as phorbol 12-myristate 13- acetate, ionomycin, and concanavalin A. Yet, PHA has not been used to activate T cells in vivo, for immunotherapy, due to its biological instability and toxicity. The instability stems from its protein-nature, which causes its degradation and short bioavailability profile in the blood while toxicity can cause death due to agglutination of red and white blood cells. Therefore, to take full advantage of PHA as an immune activator, an approach of circumventing the limitations of PHA while also preserving function is needed. We report the encapsulation of PHA in a liposome which increased the in vivo stability, reduce toxicity, and activated T cells in vitro and in vivo, and induced killing of tumor cells in vitro and in vivo. The liposomal PHA is a new form of pan- cancer immunotherapy which acts regardless of tumor antigens and thus does not induce antigen- less tumor escape while also circumventing current obstacles of T cell exhaustion. In conclusion, our nanoTCE platform uses nanoparticles to create a relatively simple, reproducible, and off-the-shelf solution to overcome the major limitations of current immunotherapy techniques such as TCEs and CAR-T cells. The nanoTCE targets each antigen with the high specificity of monoclonal antibodies which enables the creation of a more robust immunotherapy technology to take advantage of the immune system for an effective response. Our system enables the customization of the nanoTCE as an immunotherapy with the use of existing monoclonal antibodies for the targeting of any desired cancer or immune cell antigen. This simple, customizable, specific, translational, and efficacious nanoTCE platform provides the flexibility to engage any immune cell for the treatment of the cancer of interest and can be used for personalized medicine based on the cancer antigens presented by the patient’s tumor

Liposomal phytohemagglutinin: In vivo T-cell activator as a novel pan-cancer immunotherapy

Immunotherapy is an attractive approach for treating cancer. T-cell engagers (TCEs) are a type of immunotherapy that are highly efficacious; however, they are challenged by weak T-cell activation and short persistence. Therefore, alternative solutions to induce greater activation and persistence of T cells during TCE immunotherapy is needed. Methods to activate T cells include the use of lectins, such as phytohemagglutinin (PHA). PHA has not been used to activate T cells in vivo, for immunotherapy, due to its biological instability and toxicity. An approach to overcome the limitations of PHA while also preserving its function is needed. In this study, we report a liposomal PHA which increased PHA stability, reduced toxicity and performed as an immunotherapeutic that is able to activate T cells for the use in future cancer immunotherapies to circumvent current obstacles in immunosuppression and T-cell exhaustion

Nanoparticle T cell engagers for the treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common type of leukemia and has a 5-year survival rate of 25%. The standard-of-care for AML has not changed in the past few decades. Promising immunotherapy options are being developed for the treatment of AML; yet, these regimens require highly laborious and sophisticated techniques. We create nanoTCEs using liposomes conjugated to monoclonal antibodies to enable specific binding. We also recreate the bone marrow niche using our 3D culture system and use immunocompromised mice to enable use of human AML and T cells with nanoTCEs. We show that CD33 is ubiquitously present on AML cells. The CD33 nanoTCEs bind preferentially to AML cells compared to Isotype. We show that nanoTCEs effectively activate T cells and induce AML killin

Nanoparticle T-cell engagers as a modular platform for cancer immunotherapy

T-cell-based immunotherapy, such as CAR-T cells and bispecific T-cell engagers (BiTEs), has shown promising clinical outcomes in many cancers; however, these therapies have significant limitations, such as poor pharmacokinetics and the ability to target only one antigen on the cancer cells. In multiclonal diseases, these therapies confer the development of antigen-less clones, causing tumor escape and relapse. In this study, we developed nanoparticle-based bispecific T-cell engagers (nanoBiTEs), which are liposomes decorated with anti-CD3 monoclonal antibodies (mAbs) targeting T cells, and mAbs targeting the cancer antigen. We also developed a nanoparticle that targets multiple cancer antigens by conjugating multiple mAbs against multiple cancer antigens for T-cell engagement (nanoMuTEs). NanoBiTEs and nanoMuTEs have a long half-life of about 60 h, which enables once-a-week administration instead of continuous infusion, while maintaining efficacy in vitro and in vivo. NanoMuTEs targeting multiple cancer antigens showed greater efficacy in myeloma cells in vitro and in vivo, compared to nanoBiTEs targeting only one cancer antigen. Unlike nanoBiTEs, treatment with nanoMuTEs did not cause downregulation (or loss) of a single antigen, and prevented the development of antigen-less tumor escape. Our nanoparticle-based immuno-engaging technology provides a solution for the major limitations of current immunotherapy technologies

Targeting CD47 as a novel immunotherapy for multiple myeloma

Multiple myeloma (MM) remains to be incurable despite recent therapeutic advances. CD47, an immune checkpoint known as the don\u27t eat me signal, is highly expressed on the surface of various cancers, allowing cancer cells to send inhibitory signals to macrophages and impede phagocytosis and immune response. In this study, we hypothesized that blocking the don\u27t eat me signaling using an anti-CD47 monoclonal antibody will induce killing of MM cells. We report that CD47 expression was directly correlated with stage of the disease, from normal to MGUS to MM. Moreover, MM cells had remarkably higher CD47 expression than other cell populations in the bone marrow. These findings indicate that CD47 is specifically expressed on MM and can be used as a potential therapeutic target. Further, blocking of CD47 using an anti-CD47 antibody induced immediate activation of macrophages, which resulted in induction of phagocytosis and killing of MM cells in the 3D-tissue engineered bone marrow model, as early as 4 hours. These results suggest that macrophage checkpoint immunotherapy by blocking the CD47 don\u27t eat me signal is a novel and promising strategy for the treatment of MM, providing a basis for additional studies to validate these effects in vivo and in patients

Bispecific T Cell Engagers for the Treatment of Multiple Myeloma: Achievements and Challenges

MM is the second most common hematological malignancy and represents approximately 20% of deaths from hematopoietic cancers. The advent of novel agents has changed the therapeutic landscape of MM treatment; however, MM remains incurable. T cell-based immunotherapy such as BTCEs is a promising modality for the treatment of MM. This review article discusses the advancements and future directions of BTCE treatments for MM

Bispecific T Cell Engagers for the Treatment of Multiple Myeloma: Achievements and Challenges

MM is the second most common hematological malignancy and represents approximately 20% of deaths from hematopoietic cancers. The advent of novel agents has changed the therapeutic landscape of MM treatment; however, MM remains incurable. T cell-based immunotherapy such as BTCEs is a promising modality for the treatment of MM. This review article discusses the advancements and future directions of BTCE treatments for MM