Helene Margaret Finney

Activation of resting human primary T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain.

Chimeric receptors that include CD28 signaling in series with TCRζ in the same receptor have been demonstrated to activate prestimulated human primary T cells more efficiently than a receptor providing TCRζ signaling alone. We examined whether this type of receptor can also activate resting human primary T cells, and whether molecules other than CD28 could be included in a single chimeric receptor in series with TCRζ to mediate the activation of resting human primary T cells. Human CD33-specific chimeric receptors were generated with CD28, inducible costimulator, CD134, or CD137 signaling regions in series with TCRζ signaling region and transfected by electroporation into resting human primary T cells. Their ability to mediate Ag-specific activation was analyzed in comparison with a receptor providing TCRζ signaling alone. Inclusion of any of the costimulatory signaling regions in series with TCRζ enhanced the level of specific Ag-induced IL-2, IFN-γ, TNF-α, and GM-CSF cytokine production and enabled resting primary T cells to survive and proliferate in response to Ag in the absence of any exogenous factors. Inclusion of CD28, inducible costimulator, or CD134 enhanced TCRζ-mediated, Ag-specific target cell lysis. Chimeric receptors providing B7 and TNFR family costimulatory signals in series with TCRζ in the same receptor can confer self-sufficient clonal expansion and enhanced effector function to resting human T cells. This type of chimeric receptor may now be used to discover the most potent combination of costimulatory signals that will improve current immunotherapeutic strategies.

Functional skewing of the global CD8 T cell population in chronic hepatitis B virus infection

The inflamed liver in chronic hepatitis B virus (HBV) infection (CHB) is characterized by a large influx of non–virus-specific CD8 T cells. Little is known about the functional capacity of these lymphocytes, which could provide insights into mechanisms of failure of viral control and liver damage in this setting. We compared the effector function of total circulating and intrahepatic CD8 T cells in CHB patients and healthy donors. We demonstrated that CD8 T cells from CHB patients, regardless of their antigen specificity, were impaired in their ability to produce interleukin-2 and proliferate upon TCR-dependent stimulation. In contrast, these CD8 T cells had preserved production of the proinflammatory cytokines interferon-γ and tumor necrosis factor-α. This aberrant functional profile was partially attributable to down-regulation of the proximal T cell receptor signaling molecule CD3ζ, and could be corrected in vitro by transfection of CD3ζ or replenishment of the amino acid arginine required for its expression. We provide evidence for depletion of arginine in the inflamed hepatic microenvironment as a potential mechanism for these defects in global CD8 T cell signaling and function. These data imply that polarized CD8 T cells within the HBV-infected liver may impede proliferative antiviral effector function, while contributing to the proinflammatory cytokine environment.

The Loss of Telomerase Activity in Highly Differentiated CD8+CD28−CD27− T Cells Is Associated with Decreased Akt (Ser473) Phosphorylation

The enzyme telomerase is essential for maintaining the replicative capacity of memory T cells. Although CD28 costimulatory signals can up-regulate telomerase activity, human CD8+ T cells lose CD28 expression after repeated activation. Nevertheless, telomerase is still inducible in CD8+CD28− T cells. To identify alternative costimulatory pathways that may be involved, we introduced chimeric receptors containing the signaling domains of CD28, CD27, CD137, CD134, and ICOS in series with the CD3 zeta (ζ) chain into primary human CD8+ T cells. Although CD3 ζ-chain signals alone were ineffective, triggering of all the other constructs induced proliferation and telomerase activity. However, not all CD8+CD28− T cells could up-regulate this enzyme. The further fractionation of CD8+CD28− T cells into CD8+CD28− CD27+ and CD8+CD28−CD27− subsets showed that the latter had significantly shorter telomeres and extremely poor telomerase activity. The restoration of CD28 signaling in CD8+CD28−CD27− T cells could not reverse the low telomerase activity that was not due to decreased expression of human telomerase reverse transcriptase, the enzyme catalytic subunit. Instead, the defect was associated with decreased phosphorylation of the kinase Akt, that phosphorylates human telomerase reverse transcriptase to induce telomerase activity. Furthermore, the defective Akt phosphorylation in these cells was specific for the Ser473 but not the Thr308 phosphorylation site of this molecule. Telomerase down-regulation in highly differentiated CD8+CD28−CD27− T cells marks their inexorable progress toward a replicative end stage after activation. This limits the ability of memory CD8+ T cells to be maintained by continuous proliferation in vivo.

Cytokine Induced Killer cells for cell therapy of acute myeloid leukemia: improvement of their immune activity by expression of CD33-specific chimeric receptors

Background Cytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine-induced killer cells with chimeric receptors specific for the CD33 myeloid antigen. Design and Methods SFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour 51chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate (3H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34+ hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation. Results Cytokine-induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34+ progenitor cells, residual clonogenic activity was preserved. Conclusions Our results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.

In vitro comparison of three different chimeric receptor-modified effector T-cell populations for leukemia cell therapy.

The identification of the optimal T-cell effector subtype is a crucial issue for adoptive cell therapy with chimeric receptor-modified T cells. The ideal T cell population must be able to home toward tumor site, exert prolonged antitumoral activity, and display minimal toxicity against normal tissues. Therefore, we characterized the in vitro antitumoral properties of three effector T-cell populations: Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs), cytokine-induced killer (CIK) cells, and &ggr;9&dgr;2 T (GDT) cells, after transduction with a chimeric receptor specific for the CD33 antigen, broadly expressed on acute myeloid leukemia cells. EBV-CTLs, CIK, and GDT cells were generated and transduced with high efficiency with a retroviral vector coding for an anti-CD33-&zgr; chimeric receptor without alterations of their native phenotype. Anti-CD33-&zgr; chimeric receptor-redirected T cells displayed analogous in vitro chemotactic activity toward CXCL12. In addition, anti-CD33-&zgr; chimeric receptor-expressing EBV-CTLs, CIK, and GDT cells showed potent and similar cytotoxicity against several CD33+ leukemic targets both in short-term 4-hours-51 Chromium-release assays (mean killing vs primary leukemic cells at effector:target ratio of 5:1; 50%, 61%, and 50% for EBV-CTLs, CIK, and GDT cells, respectively) and in long-term assays, where they were cocultured with leukemic cells for 6 days on stromal mesenchymal cells (mean survival of primary leukemic cells at effector:target ratio of 1:100; 18%, 16%, and 29% for EBV-CTLs, CIK, and GDT cells, respectively). Moreover, all effector cells acquired consistent capability to proliferate in vitro after contact with CD33+cells and to release high and comparable levels of immunostimulatory cytokines, while secreting similar low amount of immunoregulatory cytokines as the unmanipulated counterpart. Our results indicate that expression of an anti-CD33-&zgr; chimeric receptor potently and similarly increase the antileukemic functions of different effector T-cell subtypes, underlying the impossibility to identify a more potent T-cell population through in vitro analysis, and consistently with recent observations that have emerged from clinical trials with chimeric receptor-modified T cells, suggesting the need to perform such type of studies in the human setting.

Creation of a gated antibody as a conditionally functional synthetic protein

The ability to conditionally direct antibodies is a potentially powerful application for Synthetic Biology in Medicine. Here we show that control of antibody binding through site-specific, chemical phosphorylation of a recognition domain creates a ‘gated’ antibody (Ab). This displays a crude Boolean logic where binding is induced in an enzyme-AND-antigen dependent manner. This ‘AND-Ab’ is therefore active only in the presence of two biomarker inputs: the simultaneous expression of a (cell surface) antigen and secreted enzyme to generate function in vitro, on cells and in mammalian tissue. Such gated Abs, either alone or in combination, could allow the application of logic strategies to enhance precision in biological interrogation, modulation and in therapy.

Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration.

ABSTRACT Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).