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Open Access Publications from the University of California

Scholarly Works (29 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2017)
The ability of immune cells to survey tissues and sense pathologic insults and deviations makes them a unique platform for interfacing with the body and disease. With the rapid advancement of synthetic biology, we can now engineer and equip immune cells with new sensors and controllable therapeutic response programs to sense and treat diseases that our natural immune system cannot normally handle. Here we review the current state of engineered immune cell therapeutics and their unique capabilities compared to small molecules and biologics. We then discuss how engineered immune cells are being designed to combat cancer, focusing on how new synthetic biology tools are providing potential ways to overcome the major roadblocks for treatment. Finally, we give a long-term vision for the use of synthetic biology to engineer immune cells as a general sensor-response platform to precisely detect disease, to remodel disease microenvironments, and to treat a potentially wide range of challenging diseases.
    Cover page: Synthetic Immunology: Hacking Immune Cells to Expand Their Therapeutic Capabilities
    • Thesis
    • Peer Reviewed
    UC San Francisco Electronic Theses and Dissertations (2019)

    The immune system is central to the development and resolution of complex diseases that still challenge modern therapeutic medicine such as autoimmunity and cancer. Recent blunt immune system manipulations, such as systemic monoclonal antibodies, have shown great promise in subsets of these conditions. But more controlled therapeutic manipulation of immune cell function must involve the ultimate source of that functionality, the genome itself. First, we manipulated immune cell genomes by extending high efficiency gene knockout technologies to primary human and mouse T, B, and NK cells using CRISPR/Cas9 RNP electroporation. Yet only so much therapeutic potential can be expected from removing endogenous genetic instructions. The ability to target large amounts of new genetic material to specific sites in immune cell genomes offers a potentially infinite number of therapeutic modifications. Second, we thus present non-viral genome targeting, a simple high efficiency method for the targeted integration of large (>3 kB) sequences of DNA into diverse primary human immune cell types. We applied non-viral genome targeting in three ways: the correction of a monogenic autoimmune mutation in patient cells; the replacement of T cell’s endogenous TCR with a new specificity, such as for a common melanoma antigen; and the integration of synthetic DNA sequences into endogenous genetic loci for a new class of therapeutic cell modifications, termed Genetically Engineered Endogenous Proteins. Finally, we extended non-viral genome targeting to develop a robust targeted pooled knockin method, allowing for libraries of large DNA modifications integrated into endogenous genomic sites to be rapidly screened for their functional effects on primary human T cells. Overall, human immune cell specificity and function can now be engineered without costly viral vectors or random integrations through non- viral genome targeting, opening the next chapter in immune cell therapies for cancer and autoimmunity. And beyond its therapeutic potential, large scale modifications of the endogenous genome will enable new avenues of biology exploring the evolutionary choices, trade offs, and paths untaken, that comprise our collective genome.

      Cover page: Non-viral engineering of immune cell specificity and function
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2022)
      IL-12 is an essential cytokine involved in the generation of memory or memory-like NK cells. Mouse cytomegalovirus infection triggers NK receptor-induced, ligand-specific IL-12-dependent NK cell expansion, yet specific IL-12 stimulation ex vivo leading to NK cell proliferation and expansion is not established. Here, we show that IL-12 alone can sustain human primary NK cell survival without providing IL-2 or IL-15 but was insufficient to promote human NK cell proliferation. IL-12 signaling analysis revealed STAT5 phosphorylation and weak mTOR activation, which was enhanced by activating NK receptor upregulation and crosslinking leading to STAT5-dependent, rapamycin-sensitive, or TGFβ-sensitive NK cell IL-12-dependent expansion, independently of IL-12 receptor upregulation. Prolonged IL-2 culture did not impair IL-12-dependent ligand-specific NK cell expansion. These findings demonstrate that activating NK receptor stimulation promotes differential IL-12 signaling, leading to human NK cell expansion, and suggest adopting strategies to provide IL-12 signaling in vivo for ligand-specific IL-2-primed NK cell-based therapies.
        Cover page: Differential IL-12 signaling induces human natural killer cell activating receptor-mediated ligand-specific expansion
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2015)
        There is rapidly growing interest in learning how to engineer immune cells, such as T lymphocytes, because of the potential of these engineered cells to be used for therapeutic applications such as the recognition and killing of cancer cells. At the same time, our knowhow and capability to logically engineer cellular behavior is growing rapidly with the development of synthetic biology. Here we describe how synthetic biology approaches are being used to rationally alter the behavior of T cells to optimize them for therapeutic functions. We also describe future developments that will be important in order to construct safe and precise T cell therapeutics.
          Cover page: Synthetic biology approaches to engineer T cells
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2015)
          Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches.
            Cover page: Early Signaling in Primary T Cells Activated by Antigen Presenting Cells Is Associated with a Deep and Transient Lamellal Actin Network.
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2015)
            There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed "ON-switch" CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control.
              Cover page: Remote control of therapeutic T cells through a small molecule–gated chimeric receptor
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2016)
              The Notch protein is one of the most mechanistically direct transmembrane receptors-the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.
                Cover page: Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2016)
                T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.
                  Cover page: Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2017)
                  Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 [PD-1]) have both opened new avenues for cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies.
                    Cover page: CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2023)
                    T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.
                      Cover page: Hyperstabilization of T cell microvilli contacts by chimeric antigen receptors
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