Targeting of class II major histocompatibility complex molecules to endocytic compartments is mediated by their association with the invariant chain (Ii). Although the identity of certain sorting signals located in Ii's cytoplasmic tail is known, proteins that interact with Ii's cytoplasmic tail in living cells remain to be identified. Synthesis of a biotinylated trimeric Ii cytoplasmic tail allowed the retrieval of two proteins that interact with this domain. We identify one of them as the 70-kDa heat-shock cognate protein (hsc70), the uncoating ATPase of clathrin-coated vesicles, and the other as its mitochondrial homologue, the glucose-regulated protein grp75. Expression of Ii in COS cells results in the formation of large endocytic compartments. We observe extensive colocalization of hsc70 with Ii in these macrosomes. Expression of a dominant-negative (K71M) green fluorescent protein-tagged version of hsc70 counteracted the ability of Ii to modify the endocytic pathway, demonstrating an interaction in vivo of Ii with hsc70 as part of the machinery responsible for macrosome formation.
The atypical two-electron oxidation of thioanisole and its p-methyl, p-methoxy, and p-nitro analogues by horseradish peroxidase, contrary to earlier reports, stereoselectively produces the (8) sulfoxides in 60-
1.0 A bstract Vaccines help reduce new infections, but interventions that can prevent the disease from transitioning to a severe stage are rather limited. Dysregulated IFN kinetics are mostly exploited by pathogenic viruses, including SARS-CoV-2. The clinical benefits of systemically infused IFN are, unfortunately, mired by undesired side effects. To address this situation, we engineered a T cell to synthesize interferons (IFNs) as antiviral proteins upon recognizing the virus envelop protein of SARS-CoV-2, i.e., anti-SARS T-cell Biofactory . The T-cell Biofactory, capable of regulating the IFN expression with spatiotemporal resolution within the infected tissues, can mitigate these concerns. In this work, we determined the prophylactic and therapeutic effects of the type-I and type-III IFNs produced from the T-cell Biofactory against SARS-CoV-2 infection in host cells and investigated the expression profiles of ensuing IFN-stimulated genes (ISGs). To enable the translation of T-cell Biofactory as an effective antiviral countermeasure, we also investigated an irradiation dose that renders the T-cell Biofactory non-proliferative and thus non-oncogenic. The ongoing public health crisis motivated us to direct the T-cell Biofactory technology to target SARS-CoV-2. The T-cell Biofactory, based on T cells engineered with chimeric antigen receptors (CAR T cells), is a platform technology that can be rapidly re-engineered and become available for targeting any new pathogen.
Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.
Formation of the ferryl (FeIV=O) porphyrin radical cation known as Compound I in the reaction of horseradish peroxidase (HRP) with H2O2 is catalyzed by His-42, a residue that facilitates the binding of H2O2 to the iron and subsequent rupture of the dioxygen bond. An H42A mutation was shown earlier to decrease the rate of Compound I formation by a factor of approximately 10(6) and of guaiacol oxidation by a factor of approximately 10(4). In contrast, an F41A mutation has little effect on peroxidative catalysis (Newmyer, S. L., and Ortiz de Montellano, P. R. (1995) J. Biol. Chem. 270, 19430-19438). We report here construction, expression, and characterization of the F41H/H42A double mutant. The pH profile for guaiacol oxidation by this double mutant has a broad maximum at approximately pH 6.3. Addition of H2O2 produces a Compound I species (lambdamax = 406 nm) that is reduced by 1 eq of K4Fe(CN)6 to the ferric state (lambdamax = 407 nm) without the detectable formation of Compound II. A fraction of the heme chromophore is lost in the process. The rate of Compound I formation for the F41H/H42A double mutant is 3.0 x 10(4) M-1 s-1. This is to be compared with 0.9 x 10(7) M-1 s-1 for wild-type HRP and 19 M-1 s-1 for the H42A mutant. The kcat values for guaiacol oxidation by wild-type, H42A, and F41H/H42A HRP are 300, 0.015, and 1.8 s-1. The corresponding kcat values for ABTS oxidation are 4900, 0.41, and 100 s-1, respectively. These results show that a histidine at position 41 substitutes, albeit imperfectly, for His-42 in peroxidative turnover of the enzyme. The F41H/H42A double mutant has peroxidative properties intermediate between those of the native enzyme and the H42A mutant. The F41H/H42A double mutant, however, is a considerably better thioanisole sulfoxidation and styrene epoxidation catalyst than native or H42A HRP. The surrogate catalytic residue introduced by the F41H mutation thus partially compensates for the H42A substitution used to increase access to the ferryl oxygen.
The tumor microenvironment (TME), embedded within the dense fibrous extracellular matrix (ECM) common to many solid tumors, significantly contributes to drug resistance and immunosuppression, thereby reducing the effectiveness of antitumor agents. Increasing the therapeutic dosage in the systemic circulation often fails to improve efficacy and typically results in cachexia and morbidity. Therefore, the challenge lies in developing a technology that can selectively break down the ECM—where the TME resides—without affecting normal tissues.
Methods
To address this challenge, we have engineered CD4 T cells to synthesize calibrated amounts of ECM-degrading enzymes after engaging the antigen-presenting tumor cells. This will mitigate the undesired systemic effect of directly infusing such ECM-degrading enzymes and focusing their effect only within the solid TME.
Results
Primary T cells were engineered to express ECM-degrading enzymes upon engaging Folate Receptor-alpha (FRα) as the target tumor antigen. Enzyme production by these T cells, triggered by FRα-presenting cells, was quantified using ELISA, showing a correlation with stimulation duration, target cell numbers, and the number of engineered T cells. A synthetic fluorogenic substrate confirmed enzyme activity after 48 hours of stimulation. The enzymes produced by these T cells effectively degraded ECM coatings and were inhibited by an enzyme-specific inhibitor. A high concentration of degraded ECM components was observed when ECM-enriched tumor explants derived from subcutaneous and peritoneal mouse tumors were treated with enzyme-enriched cell culture supernatant from engineered primary T cells that express the ECM-degrading enzymes, compared to those that did not. To streamline clinical translation, we optimized our engineered primary T cells, achieving a 100-fold expansion in 14 days and a 9-fold increase in the expression of the desired protein compared to prior iterations.
Conclusions
The rationale for this technology in treating solid tumors is that it limits the effect of ECM-degrading enzymes with spatiotemporal resolution to within the TME. Thus, degraded ECM will be more easily infiltrated by the innate immune system and many antitumor agents that are currently in the market. It will also allow efflux of the tumor metabolites potentially reducing the immunosuppressive nature of the TME.
Acknowledgements
Research reported in this publication was supported in part by the National Institute of Biomedical Imaging and Bioengineering (DP2EB024245: NIH Director's New Innovator Award Program (https://commonfund.nih.gov/newinnovator); and the National Cancer Institute (R21CA236640, R33CA247739) of the National Institutes of Health (NIH).
Ethics Approval
The in vivo validation of our T-cell based delivery system was performed in mice at SRI International in accordance with the guidelines from the Institutional Animal Care and Use Committee (Approval # 22001).
The NK-92MI, a fast-growing cytolytic cell line with a track record of exerting clinical efficacy, is transformed into a vector for synthesizing calibrated amounts of desired engineered proteins at our disease site, that is, NK-cell Biofactory. This provides an allogeneic option to the previously published T-cell-based living vector that is limited by high manufacturing cost and product variability. The modularity of this pathway, which combines a "target" receptor with an "effector" function, enables reprogramming of the NK-cell Biofactory to target diseases with specific molecular biomarkers, such as cancer, viral infections, or auto-immune disorders, and overcome barriers that may affect the advancement of NK-cell therapies.
The CD4 T cell, when engineered with a chimeric antigen receptor (CAR) containing specific intracellular domains, has been transformed into a zero-order drug-delivery platform. This introduces the capability of prolonged, disease-specific engineered protein biologics production, at the disease site. Experimental findings demonstrate that CD4 T cells offer a solution when modified with a CAR that includes 4-1BB but excludes CD28 intracellular domain. In this configuration, they achieve ~3X transduction efficiency of CD8 T cells, ~2X expansion rates, generating ~5X more biologic, and exhibit minimal cytolytic activity. Cumulatively, this addresses two main hurdles in the translation of cell-based drug delivery: scaling the production of engineered T cell ex vivo and generating sufficient biologics in vivo. When programmed to induce IFNβ upon engaging the target antigen, the CD4 T cells outperforms CD8 T cells, effectively suppressing cancer cell growth in vitro and in vivo. In summary, this platform enables precise targeting of disease sites with engineered protein-based therapeutics while minimizing healthy tissue exposure. Leveraging CD4 T cells' persistence could enhance disease management by reducing drug administration frequency, addressing critical challenges in cell-based therapy.
Solution 1H NMR has been used to assign a major portion of the heme environment and the substrate-binding pocket of resting state horseradish peroxidase, HRP, despite the high-spin iron(III) paramagnetism, and a quantitative interpretive basis of the hyperfine shifts is established. The effective assignment protocol included 2D NMR over a wide range of temperatures to locate residues shifted by paramagnetism, relaxation analysis, and use of dipolar shifts predicted from the crystal structure by an axial paramagnetic susceptibility tensor normal to the heme. The most effective use of the dipolar shifts, however, is in the form of their temperature gradients, rather than by their direct estimation as the difference of observed and diamagnetic shifts. The extensive assignments allowed the quantitative determination of the axial magnetic anisotropy, Δχax = −2.50 × 10-8 m3/mol, oriented essentially normal to the heme. The value of Δχax together with the confirmed T-2 dependence allow an estimate of the zero-field splitting constant D = 15.3 cm-1, which is consistent with pentacoordination of HRP. The solution structure was generally indistinguishable from that in the crystal (Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nature Structural Biology 1997, 4, 1032−1038) except for Phe68 of the substrate-binding pocket, which was found turned into the pocket as found in the crystal only upon substrate binding (Henriksen, A.; Schuller, D. J.; Meno, K.; Welinder, K. G.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8054−8060). The reorientation of several rings in the aromatic cluster adjacent to the proximal His170 is found to be slow on the NMR time scale, confirming a dense, closely packed, and dynamically stable proximal side up to 55 °C. Similar assignments on the H42A-HRP mutant reveal conserved orientations for the majority of residues, and only a very small decrease in Δχax or D, which dictates that five-coordination is retained in the mutant. The two residues adjacent to residue 42, Ile53 and Leu138, reorient slightly in the mutant H42A protein. It is concluded that effective and very informative 1H NMR studies of the effect of either substrate binding or mutation can be carried out on resting state heme peroxidases.
