Evaluation of Cellular Targeting by Fab' vs Full-Length Antibodies in Antibody-Nanoparticle Conjugates (ANCs) Using CD4 T-cells.

Targeted delivery of chemotherapeutic drugs can improve their therapeutic efficiency by localizing their toxic effects at the diseased site. This is often achieved either by direct conjugation of drugs to antibodies targeting overexpressed receptors on cancer cells (antibody-drug conjugates/ADCs) or by conjugating antibodies to nanoparticles bearing drugs (antibody-nanoparticle conjugates/ANCs). Here, we report a platform for utilizing hinge cysteines on antigen-binding fragment (Fab') of an anti-CD4 antibody for site-specific conjugation to nanoparticles giving rise to anti-CD4 Fab'-nanoparticle conjugates (Fab'-NCs). We demonstrate a convenient route for obtaining functional anti-CD4 Fab' from full-length antibody and examine the targeted delivery efficiencies of anti-CD4 Fab'-NCs vs ANCs for selective delivery to CD4high mT-ALL cells. Our results indicate that higher avidity of full-length anti-CD4 antibody, i.e., protein alone translated to higher binding ability to CD4high mT-ALL cells in comparison with anti-CD4 Fab' alone. However, the targeted delivery efficiency of anti-CD4 Fab'-NCs was comparable to ANCs indicating that the avidity of Fab' is restored in a nanoparticle-conjugate format. Fab'-NCs are equally capable of achieving targeted drug delivery to CD4high T-cells as ANCs and are a versatile alternative to ANCs by offering site-selective modification strategy while retaining their advantages.

Protein Kinase C Theta Modulates PCMT1 through hnRNPL to Regulate FOXP3 Stability in Regulatory T Cells.

T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype.

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells.

Regulatory T cells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory T cells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the T cell-specific kinase, protein kinase C theta, which promotes T cell effector functions and represses regulatory T cell differentiation, augments regulatory T cell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 T cells. When differentiated ex vivo into induced regulatory T cells, treated cells expressed elevated levels of the regulatory T cell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory T cells. The in vitro suppressive capacity of these induced regulatory T cells was 10-fold greater than that of T cells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory T cells were superior to non-treated T cells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory T cells as a cell-based therapy for immune modulation.

Protein-Antibody Conjugates (PACs): A Plug-and-Play Strategy for Covalent Conjugation and Targeted Intracellular Delivery of Pristine Proteins.

We report here on protein-antibody conjugates (PACs) that are used for antibody-directed delivery of protein therapeutics to specific cells. PACs have the potential to judiciously combine the merits of two prolific therapeutic approaches-biologics and antibody-drug conjugates. We utilize spherical polymer brushes to construct PACs using the combination of two simple and efficient functionally orthogonal click chemistries. In addition to the synthesis and characterization of these nanoparticles, we demonstrate that PACs are indeed capable of specifically targeting cells based on the presence of target antigen on the cell surface to deliver proteins. The potentially broad adaptability of PACs opens up new opportunities for targeted biologics in therapeutics and sensing.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells.

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular ß-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

Targeting CD4+ Cells with Anti-CD4 Conjugated Mertansine-Loaded Nanogels.

CD4+ T lymphocytes play an important role in controlling many malignancies. The modulation of CD4+ T cells through immunomodulatory or cytotoxic drugs could change the course of disease progression for disorders such as autoimmunity, immunodeficiency, and cancer. Here, we demonstrate that anti-CD4 conjugated polymeric nanogels can deliver a small molecule cargo to primary CD4+ T cells and a CD4high T cell lymphoma. The antibody conjugation not only increased the uptake efficiency of the nanogel (NG) by CD4+ T cells but also decreased the non-specific uptake of the NG by CD4- lymphocytes. For T lymphoma cell lines, the mertansine-loaded conjugate displayed a dose-dependent cell growth inhibition at 17 ng/mL antibody concentration. On the other hand, antibody-drug conjugate (ADC)-type formulation of the anti-CD4 reached similar levels of cell growth inhibition only at the significantly higher concentration of 1.8 µg/mL. NG and antibody conjugates have the advantage of carrying a large payload to a defined target in a more efficient manner as it needs far less antibody to achieve a similar outcome.

Alpha-1 Antitrypsin-Expressing Mesenchymal Stromal Cells Confer a Long-Term Survival Benefit in a Mouse Model of Lethal GvHD.

Acute graft-versus-host disease is a frequent complication associated with allogeneic hematopoietic stem cell transplantation. Patients that become refractory to initial steroid treatment have a poor prognosis. apceth-201 consists of human allogeneic mesenchymal stromal cells, engineered by lentiviral transduction to express the protease inhibitor alpha-1 antitrypsin, to augment the anti-inflammatory potential of the mesenchymal stromal cells. We show that apceth-201 mesenchymal stromal cells efficiently suppress T cell proliferation and polarize macrophages to an anti-inflammatory M2 type, in vitro. To assess the in vivo efficacy of apceth-201, it was tested in two different mouse models of acute graft-versus-host disease. Control animals in a humanized model succumbed quickly to disease, whereas median survival was doubled in apceth-201-treated animals. The product was also tested in a graft-versus-host disease model system that closely mimics haploidentical hematopoietic stem cell transplantation, an approach that is now being evaluated for use in the clinic. Control animals succumbed quickly to disease, whereas treatment with apceth-201 resulted in long-term survival of 57% of the animals. Within 25 days after the second injection, clinical scores returned to baseline in responding animals, indicating complete resolution of graft-versus-host disease. These promising data have led to planning of a phase I study using apceth-201.

Notch and T Cell Function - A Complex Tale.

Notch drives critical decisions in a multitude of developmental decisions in many invertebrate and vertebrate organisms including flies, worms, fish, mice and humans. Therefore, it is not surprising that Notch family members also play a key role in cell fate choices in the vertebrate immune system. This review highlights the critical function of Notch in the development of mature T lymphocytes from hematopoietic precursors and describes the role of Notch in mature T cell activation, proliferation and differentiation.

Notch signalling during peripheral T-cell activation and differentiation.

For many years, researchers have focused on the contribution of Notch signalling to lymphoid development. Only recently have investigators begun to ask what role, if any, Notch has during the activation and differentiation of naive CD4(+) T cells in the periphery. As interest in this issue grows, it is becoming increasingly clear that the main role of Notch signalling, to regulate cell-fate decisions, might also be influential in peripheral T cells.

Impact of Notch1 Deletion in Macrophages on Proinflammatory Cytokine Production and the Outcome of Experimental Autoimmune Encephalomyelitis.

Notch signaling is involved in regulating TLR-mediated responses in activated macrophages. In this study, we investigated the impact of Notch signaling in macrophages in an experimental autoimmune encephalomyelitis (EAE) model. To examine the impact of deficiency in Notch signaling in activated macrophages in EAE, an adoptive transfer of activated macrophages derived from Notch1(fl/fl) × Mx1cre(+/-) (Notch1 knockout [N1KO]) or CSL/Rbp-jκ(fl/fl) × Mx1cre(+/-) (CSL/RBP-Jκ KO) mice was performed prior to induction of EAE. Mice receiving activated N1KO macrophages showed decreased severity of EAE compared with mice receiving wild-type or CSL/RBP-Jκ KO macrophages. In vitro restimulation of splenocytes by myelin oligodendrocyte glycoprotein 35-55 peptide from these mice revealed that cells from mice receiving N1KO macrophages produced significantly less IL-17 compared with the control mice, whereas IFN-γ production was similar in both groups. We found that activated N1KO, but not CSL/RBP-Jκ KO, macrophages produced less IL-6 and had lower CD80 expression compared with wild-type and did not exhibit any defect in IL-12p40/70 production, whereas activated macrophages from CSL/RBP-Jκ KO mice phenocopied γ-secretase inhibitor treatment for reduced IL-12p40/70 production. Furthermore, the nuclear translocation of the NF-κB subunit c-Rel was compromised in γ-secretase inhibitor-treated and CSL/RBP-Jκ KO but not N1KO macrophages. These results suggest that Notch1 and CSL/RBP-Jκ in macrophages may affect the severity of EAE differently, possibly through modulating IL-6 and CD80 expression, which is involved in the Th17 but not Th1 response.

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.

Regulation of oxidative stress by methylation-controlled J protein controls macrophage responses to inflammatory insults.

Mitochondria contribute to macrophage immune function through the generation of reactive oxygen species, a byproduct of the mitochondrial respiratory chain. MCJ (also known as DnaJC15) is a mitochondrial inner membrane protein identified as an endogenous inhibitor of respiratory chain complex I. Here we show that MCJ is essential for the production of tumor necrosis factor by macrophages in response to a variety of Toll-like receptor ligands and bacteria, without affecting their phagocytic activity. Loss of MCJ in macrophages results in increased mitochondrial respiration and elevated basal levels of reactive oxygen species that cause activation of the JNK/c-Jun pathway, lead to the upregulation of the TACE (also known as ADAM17) inhibitor TIMP-3, and lead to the inhibition of tumor necrosis factor shedding from the plasma membrane. Consequently, MCJ-deficient mice are resistant to the development of fulminant liver injury upon lipopolysaccharide administration. Thus, attenuation of the mitochondrial respiratory chain by MCJ in macrophages exquisitely regulates the response of macrophages to infectious insults.

γ-Secretase inhibitors and modulators.

γ-Secretase is a fascinating, multi-subunit, intramembrane cleaving protease that is now being considered as a therapeutic target for a number of diseases. Potent, orally bioavailable γ-secretase inhibitors (GSIs) have been developed and tested in humans with Alzheimer's disease (AD) and cancer. Preclinical studies also suggest the therapeutic potential for GSIs in other disease conditions. However, due to inherent mechanism based-toxicity of non-selective inhibition of γ-secretase, clinical development of GSIs will require empirical testing with careful evaluation of benefit versus risk. In addition to GSIs, compounds referred to as γ-secretase modulators (GSMs) remain in development as AD therapeutics. GSMs do not inhibit γ-secretase, but modulate γ-secretase processivity and thereby shift the profile of the secreted amyloid ß peptides (Aß) peptides produced. Although GSMs are thought to have an inherently safe mechanism of action, their effects on substrates other than the amyloid ß protein precursor (APP) have not been extensively investigated. Herein, we will review the current state of development of GSIs and GSMs and explore pertinent biological and pharmacological questions pertaining to the use of these agents for select indications. This article is part of a Special Issue entitled: Intramembrane Proteases.

Importance of sequence specific hydrophobicity in synthetic protein transduction domain mimics.

A new series of synthetic protein transduction domain mimics (PTDMs) was designed to analyze the importance of guanidine and phenyl group segregation along the backbone on their membrane interaction and cellular internalization abilities. ROMP was utilized to synthesize three polymers: nonsegregated homopolymers, intermediately segregated gradient copolymers, and strongly segregated block copolymers. In order to understand the role of functional group segregation on activity, it was important to design monomers that enabled these three different polymer topologies, or constitutional macromolecular isomers, to be prepared with identical chemical compositions. The structure-activity relationships were evaluated by both a biophysical assay, using dye-loaded vesicles, and by in vitro cellular uptake studies of fluorescently labeled chains. The results showed that functional group segregation impacts activity. In general, the nonsegregated homopolymer was the most active in both assays but also showed larger, ill-defined aggregates compared to either the gradient or block copolymers. It was also the most cytotoxic of the three isomers. As a result, the gradient copolymer with intermediate segregation optimizes activity and solubility with low cytotoxicity. This study gives new design guidelines for the development of PTDMs.

Novel protein transduction domain mimics as nonviral delivery vectors for siRNA targeting NOTCH1 in primary human T cells.

RNA interference technology has recently been highlighted as a powerful research method as well as a potential therapeutic treatment for several diseases. However, the delivery of small interfering RNA (siRNA) into T cell lines and primary blood cells is exceedingly challenging, as they are resistant to transfection by conventional reagents. As a result, there is an unmet need for nonviral, efficient, and easily prepared carriers for siRNA delivery into hard-to-transfect cell types. Here, we report a novel system based on protein transduction domain mimics (PTDMs), generated by ring opening metathesis polymerization, for intracellular delivery of siRNA molecules. PTDM-based siRNA delivery induced efficient NOTCH1 knockdown in Jurkat T cells and human peripheral blood mononuclear cells without any measured toxicity. Furthermore, delivering siRNA to NOTCH1 in human peripheral blood cells modulated cell proliferation and differentiation of T cells into T(H)1 cells.

Exopolysaccharide-Treated Dendritic Cells Effectively Ameliorate Acute Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is a primary and often lethal complication of allogenic hematopoietic stem cell transplantation (HSCT). Prophylactic regimens for GVHD are given as standard pretransplantation therapy; however, up to 50% of these patients develop acute GVHD (aGVHD) and require additional immunosuppressive intervention. Using a mouse GVHD model, we previously showed that injecting mice with exopolysaccharide (EPS) from Bacillus subtilis prior to GVHD induction significantly increased 80-day survival after transplantation of complete allogeneic major histocompatibility complex-mismatched cells. To ask whether EPS might also inhibit GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of A2neg human peripheral blood mononuclear cells (PBMCs). Because we could not inject human donors with EPS, we transferred EPS-pretreated dendritic cells (DCs) to inhibit aGVHD. We derived these DCs from CD34+ human cord blood cells, treated them with EPS, and then injected them together with PBMCs into the NSG-HLA-A2 mice. We found that all mice that received untreated DCs were dead by day 35, whereas 25% of mice receiving EPS-treated DCs (EPS-DCs) survived. This DC cell therapy could be readily translatable to humans, because we can generate large numbers of human EPS-DCs and use them as an "off the shelf" treatment for patients undergoing HSCT.

Antibody responses in blood and saliva post COVID-19 bivalent booster do not reveal an Omicron BA.4/BA.5- specific response.

Introduction: Current SARS-CoV-2 strains continue to mutate and attempt to evade the antibody response elicited by previous exposures and vaccinations. In September of 2022, the first updated SARS-CoV-2 vaccines, designed to create immune responses specific for the variants circulating in 2022, were approved. These new vaccines, known commonly as the bivalent boost(er), include mRNA that encodes both the original Wuhan-Hu-1 spike protein as well as the spike protein specific to the Omicron BA.4 and BA.5 variants. Methods: We recruited volunteers from University of Massachusetts student, faculty and staff members to provide samples of blood and saliva at four different time points, including pre-boost and three times post boost and analyzed samples for antibody production as well as neutralization of virus. Results: Our data provide a comprehensive analysis of the antibody response following a single dose of the bivalent boost over a 6-month period and support previous findings that the response induced after the bivalent boost does not create a strong BA.4/BA.5-specific antibody response. Conclusion: We found no evidence of a specific anti-BA.4/BA.5 response developing over time, including in a sub-population of individuals who become infected after a single dose of the bivalent booster. Additionally, we present data that support the use of saliva samples as a reliable alternative to blood for antibody detection against specific SARS-CoV-2 antigens.

Canonical and non-canonical Notch signaling in CD4⁺ T cells.

For T cells to become fully activated, they must integrate a myriad of signals, both extrinsic and intrinsic. External stimuli accrued through various cell surface receptors are transduced and amplified through a coordinated circuitry of signaling cascades that ultimately result in the transcription of new genes. Along the way, extracellular and intracellular signaling components function to impart a fully activated state. Evidence is accumulating to show that the Notch family of cell surface receptors, long known to function as transcriptional regulators through their interactions with the canonical nuclear binding protein CSL/RBP-J, may also be playing an as-yet-unappreciated role in T cell activation by virtue of its signaling via non-canonical as well as nonnuclear mechanisms. In this review we will discuss these and other better-known means by which Notch signaling influences T cell responses.

Notch signaling regulates mouse and human Th17 differentiation.

Th17 cells are known to play a critical role in adaptive immune responses to several important extracellular pathogens. Additionally, Th17 cells are implicated in the pathogenesis of several autoimmune and inflammatory disorders as well as in cancer. Therefore, it is essential to understand the mechanisms that regulate Th17 differentiation. Notch signaling is known to be important at several stages of T cell development and differentiation. In this study, we report that Notch1 is activated in both mouse and human in vitro-polarized Th17 cells and that blockade of Notch signaling significantly downregulates the production of Th17-associated cytokines, suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies, as well as chromatin immunoprecipitation, that IL-17 and retinoic acid-related orphan receptor γt are direct transcriptional targets of Notch signaling in Th17 cells. Finally, in vivo inhibition of Notch signaling reduced IL-17 production and Th17-mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Thus, this study highlights the importance of Notch signaling in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.