A LAG-3-Specific Agonist Antibody for the Treatment of T Cell-Induced Autoimmune Diseases.

T cells chronically stimulated with the same peptide tend to express exhaustion markers such as PD-1 or LAG-3. Deficiencies in the PD-1 and LAG-3 pathways have been linked to the development of autoimmune diseases. IMP761 is a LAG-3-specific humanized agonist Ab with immunosuppressive properties both in vitro and in vivo in an Ag-specific delayed-type hypersensitivity (DTH) model in the cynomolgus macaque (Macaca fascicularis). IMP761 inhibits TCR-mediated NFAT activation and Ag-induced human T cell proliferation and activation. In the DTH model, assessment of T cell infiltration and gene expression profile at the DTH biopsy site corresponds to immunosuppression of an Ag-induced T cell response. IMP761 is the first LAG-3-specific agonist product candidate, acting upstream on activated T cells, the root cause of self-Ag-specific T cell-induced autoimmune diseases.

Paclitaxel plus Eftilagimod Alpha, a Soluble LAG-3 Protein, in Metastatic, HR+ Breast Cancer: Results from AIPAC, a Randomized, Placebo Controlled Phase IIb Trial.

PURPOSE: Eftilagimod alpha (efti), a soluble lymphocyte activation gene (LAG-3) protein and MHC class II agonist, enhances innate and adaptive immunity. Active Immunotherapy PAClitaxel (AIPAC) evaluated safety and efficacy of efti plus paclitaxel in patients with predominantly endocrine-resistant, hormone receptor-positive, HER2-negative metastatic breast cancer (ET-resistant HR+ HER2- MBC). PATIENTS AND METHODS: Women with HR+ HER2- MBC were randomized 1:1 to weekly intravenous paclitaxel (80 mg/m2) and subcutaneous efti (30 mg) or placebo every 2 weeks for six 4-week cycles, then monthly subcutaneous efti (30 mg) or placebo maintenance. Primary endpoint was progression-free survival (PFS) by blinded independent central review. Secondary endpoints included overall survival (OS), safety/tolerability, pharmacokinetics/pharmacodynamics, and quality of life. Exploratory endpoints included cellular biomarkers. RESULTS: 114 patients received efti and 112 patients received placebo. Median age was 60 years (91.6% visceral disease, 84.1% ET-resistant, 44.2% with previous CDK4/6 inhibitor treatment). Median PFS at 7.3 months was similar for efti and placebo. Median OS was not significantly improved for efti (20.4 vs. 17.5 months; HR, 0.88; P = 0.197) but became significant for predefined exploratory subgroups. EORTC QLQC30-B23 global health status was sustained for efti but deteriorated for placebo. Efti increased absolute lymphocyte, monocyte and secondary target cell (CD4, CD8) counts, plasma IFNγ and CXCL10 levels. CONCLUSIONS: Although the primary endpoint, PFS, was not met, AIPAC confirmed expected pharmacodynamic effects and demonstrated excellent safety profile for efti. OS was not significantly improved globally (2.9-month difference), but was significantly improved in exploratory biomarker subgroups, warranting further studies to clarify efti's role in patients with ET-resistant HER2- MBC.

MHC class II engagement by its ligand LAG-3 (CD223) contributes to melanoma resistance to apoptosis.

Melanoma is the most aggressive skin cancer in humans that often expresses MHC class II (MHC II) molecules, which could make these tumors eliminable by the immune system. However, this MHC II expression has been associated with poor prognosis, and there is a lack of immune-mediated eradication. The lymphocyte activation gene-3 (LAG-3) is a natural ligand for MHC II that is substantially expressed on melanoma-infiltrating T cells including those endowed with potent immune-suppressive activity. Based on our previous data showing the signaling capacity of MHC II in melanoma cells, we hypothesized that LAG-3 could contribute to melanoma survival through its MHC II signaling capacity in melanoma cells. In this study, we demonstrate that both soluble LAG-3 and LAG-3-transfected cells can protect MHC II-positive melanoma cells, but not MHC II-negative cells, from FAS-mediated and drug-induced apoptosis. Interaction of LAG-3 with MHC II expressed on melanoma cells upregulates both MAPK/Erk and PI3K/Akt pathways, albeit with different kinetics. Inhibition studies using specific inhibitors of both pathways provided evidence of their involvement in the LAG-3-induced protection from apoptosis. Altogether, our data suggest that the LAG-3-MHC II interaction could be viewed as a bidirectional immune escape pathway in melanoma, with direct consequences shared by both melanoma and immune cells. In the future, compounds that efficiently hinder LAG-3-MHC II interaction might be used as an adjuvant to current therapy for MHC II-positive melanoma.

Immunophenotyping of Peripheral Blood Mononuclear Cells in Septic Shock Patients With High-Dimensional Flow Cytometry Analysis Reveals Two Subgroups With Differential Responses to Immunostimulant Drugs.

Sepsis is associated with a dysregulated inflammatory response to infection. Despite the activation of inflammation, an immune suppression is often observed, predisposing patients to secondary infections. Therapies directed at restoration of immunity may be considered but should be guided by the immune status of the patients. In this paper, we described the use of a high-dimensional flow cytometry (HDCyto) panel to assess the immunophenotype of patients with sepsis. We then isolated peripheral blood mononuclear cells (PBMCs) from patients with septic shock and mimicked a secondary infection by stimulating PBMCs for 4 h in vitro with lipopolysaccharide (LPS) with or without prior exposure to either IFN-γ, or LAG-3Ig. We evaluated the response by means of flow cytometry and high-resolution clustering cum differential analysis and compared the results to PBMCs from healthy donors. We observed a heterogeneous immune response in septic patients and identified two major subgroups: one characterized by hypo-responsiveness (Hypo) and another one by hyper-responsiveness (Hyper). Hypo and Hyper groups showed significant differences in the production of cytokines/chemokine and surface human leukocyte antigen-DR (HLA-DR) expression in response to LPS stimulation, which were observed across all cell types. When pre-treated with either interferon gamma (IFN-γ) or lymphocyte-activation gene 3 (LAG)-3 recombinant fusion protein (LAG-3Ig) prior to LPS stimulation, cells from the Hypo group were shown to be more responsive to both immunostimulants than cells from the Hyper group. Our results demonstrate the importance of patient stratification based on their immune status prior to any immune therapies. Once sufficiently scaled, this approach may be useful for prescribing the right immune therapy for the right patient at the right time, the key to the success of any therapy.

First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity.

BACKGROUND: IMP321 is a recombinant soluble LAG-3Ig fusion protein that binds to MHC class II with high avidity and mediates APC and then antigen-experienced memory CD8+ T cell activation. We report clinical and biological results of a phase I/II in patients with metastatic breast carcinoma (MBC) receiving first-line paclitaxel weekly, 3 weeks out of 4. METHODS: MBC patients were administered one dose of IMP321 s.c. every two weeks for a total of 24 weeks (12 injections). The repeated single doses were administered the day after chemotherapy at D2 and D16 of the 28-day cycles of paclitaxel (80 mg/m2 at D1, D8 and D15, for 6 cycles). Blood samples were taken 13 days after the sixth and the twelfth IMP321 injections to determine sustained APC, NK and memory CD8 T cell responses. RESULTS: Thirty MBC patients received IMP321 in three cohorts (doses: 0.25, 1.25 and 6.25 mg). IMP321 induced both a sustained increase in the number and activation of APC (monocytes and dendritic cells) and an increase in the percentage of NK and long-lived cytotoxic effector-memory CD8 T cells. Clinical benefit was observed for 90% of patients with only 3 progressors at 6 months. Also, the objective tumor response rate of 50% compared favorably to the 25% rate reported in the historical control group. CONCLUSIONS: The absence of toxicity and the demonstration of activity strongly support the future development of this agent for clinical use in combined first-line regimens. TRIAL REGISTRATION: ClinicalTrials.gov NCT00349934.

Eftilagimod alpha, a soluble lymphocyte activation gene-3 (LAG-3) protein plus pembrolizumab in patients with metastatic melanoma.

BACKGROUND: To evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of eftilagimod alpha (efti), a soluble lymphocyte activation gene-3 protein, in combination with the programmed cell death-1 (PD-1) antagonist pembrolizumab. METHODS: The study was divided into two parts; parts A and B, where part A was the dose escalation part and part B was an extension part of the study. Patients with metastatic melanoma were treated with efti plus the standard dose of pembrolizumab. Blood samples were assayed to determine plasma pharmacokinetic parameters, detect efti antibody formation and determine long-lived CD8 T cell responses and associated pharmacodynamic parameters. RESULTS: Twenty-four patients with melanoma received pembrolizumab and bi-weekly subcutaneous (s.c.) injections of efti at doses 1 mg, 6 mg or 30 mg/injection for up to 6 months (part A) or 30 mg/injection for up 12 months (part B). No dose-limiting toxicities were reported and the main adverse event for efti was injection site reactions. Sustained systemic exposure to the product was obtained in all patients following s.c. injections of 30 mg dose. Treatment induced an increase in activated CD8 and CD4 T cell counts, and in some of the soluble biomarkers, particularly interferon (IFN)-γ, a Th1 signature cytokine. An overall response rate (ORR) of 33% was observed in patients partly with pembrolizumab-refractory of part A and ORR of 50% was observed in patients with PD-1 naïve of part B. CONCLUSIONS: Efti was well tolerated in combination with pembrolizumab with encouraging antitumor activity. This warrants further clinical studies of this new combination therapy combining an antigen-presenting cell activator with an immune checkpoint inhibitor.

IMP321 (sLAG-3), an immunopotentiator for T cell responses against a HBsAg antigen in healthy adults: a single blind randomised controlled phase I study.

BACKGROUND: LAG-3 (CD223) is a natural high affinity ligand for MHC class II. The soluble form (sLAG-3) induces maturation of monocyte-derived dendritic cells in vitro and is used as a potent Th1-like immune enhancer with many antigens in animal models. To extend this observation to human, a proof of concept study was conducted with a clinical-grade sLAG-3, termed IMP321, coinjected with alum-non-absorbed recombinant hepatitis B surface antigen. METHODS: In a randomised, single blind controlled phase I dose escalation study, 48 seronegative healthy volunteers aged 18-55 years were vaccinated at 0, 4 and 8 weeks by subcutaneous injection with 10 microg HBsAg mixed with saline (control) or with IMP321 at one of four doses (3, 10, 30 and 100 microg). To evaluate the efficacy of this three injections over 2 months immunization protocol, an additional control group was injected with the commercial vaccine Engerix-B. RESULTS: IMP321 was very well tolerated. Indeed, a lower incidence of adverse events was reported from the HBsAg plus IMP321 groups than from the Engerix-B group. HBsAg-specific antibody responses (anti-HBs) appeared sooner and were higher at 8 and 12 weeks in IMP321 recipients compared to HBsAg control subjects. More importantly, increased numbers of responders to HBsAg were found in IMP321 recipients compared HBsAg group, as revealed by higher post-vaccination frequencies of CD4 Th1 or CD8 Tc1 antigen specific T cells. IMP321 induced CD4 Th1 antigen-specific T cells in some of these naïve individuals after only one injection, especially in the 10 and 30 microg dose groups. CONCLUSION: IMP321 as an adjuvant to HBsAg was well-tolerated and enhanced T cell response vaccine immunogenicity (i.e. induced both CD4 Th1 and CD8 Tc1 antigen-specific T cells). This latter property has allowed the development of IMP321 as an immunopotentiator for therapeutic vaccines.

A post-ubiquitination role for MDM2 and hHR23A in the p53 degradation pathway.

Abrogation of ubiquitin/proteasome-dependent turnover of p53 is critical for its activation. UbL-UBA proteins, including human homolog of Rad23 (hHR23) proteins, may regulate proteasomal degradation of substrates such as p53, due to their ability to interact with both ubiquitinated substrates and the proteasome. siRNA-mediated depletion of hHR23A or hHR23B in human cell lines accelerated p53 degradation. In contrast, overexpression of hHR23 proteins led to the accumulation of ubiquitinated p53, and purified hHR23 proteins also blocked p53 proteasome degradation in vitro. An hHR23-MDM2 complex was identified, suggesting that MDM2 and hHR23 cooperate in the regulation of p53 proteasome degradation. Consistent with this hypothesis, an MDM2 mutant that demonstrated increased binding in vivo to hHR23A was able to ubiquitinate, but not degrade p53. Moreover, the defective phenotype of this MDM2 mutant was rescued by siRNA knockdown of hHR23A. Our data indicate that MDM2 acts at a step in the p53 degradation pathway after ubiquitination, to counteract hHR23 inhibition of p53 turnover. Moreover, our data suggest the possibility that ubiquitin ligase/UbL-UBA protein complexes, as exemplified by the MDM2/hHR23 complex, may serve a general role in regulating substrate degradation by the proteasome.

A phase I pharmacokinetic and biological correlative study of IMP321, a novel MHC class II agonist, in patients with advanced renal cell carcinoma.

PURPOSE: To evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of IMP321, a recombinant soluble LAG-3Ig fusion protein which agonizes MHC class II-driven dendritic cell activation. EXPERIMENTAL DESIGN: Patients with advanced renal cell carcinoma were treated with escalating doses of IMP321 s.c. Blood samples were assayed to determine plasma pharmacokinetic parameters, detect human anti-IMP321 antibody formation, and determine long-lived CD8 T cell responses. RESULTS: Twenty-one advanced renal cell carcinoma patients received 119 injections of IMP321 at doses ranging from 0.050 to 30 mg/injection s.c. biweekly for 6 injections. No clinically significant adverse events were observed. Good systemic exposure to the product was obtained following s.c. injections of doses above 6 mg. IMP321 induced both sustained CD8 T-cell activation and an increase in the percentage of long-lived effector-memory CD8 T cells in all patients at doses above 6 mg. Tumor growth was reduced and progression-free survival was better in those patients receiving higher doses (>6 mg) of IMP321: 7 of 8 evaluable patients treated at the higher doses experienced stable disease at 3 months compared with only 3 of 11 in the lower dose group (P = 0.015). CONCLUSION: The absence of toxicity and the demonstration of activity at doses above 6 mg warrant further disease-directed studies of IMP321 in combined regimens (e.g., chemoimmunotherapy).

IMP321 (sLAG-3) safety and T cell response potentiation using an influenza vaccine as a model antigen: a single-blind phase I study.

sLAG-3 (IMP321), a natural high affinity ligand for MHC class II, was tested for safety, tolerability and its ability to increase Th-1-type T cell responses to a commercial trivalent split influenza vaccine (Agrippal) in a phase I single-blinded, randomized, controlled clinical trial. Twenty healthy volunteers were first injected with increasing doses of IMP321 alone (safety for first-in-man use). Then 40 volunteers were recruited into 4 consecutive cohorts of 10 subjects, who were randomly assigned to receive the flu vaccine plus 3, 10, 30 or 100 microg IMP321 or the flu vaccine plus saline control. All vaccine formulations were found to be generally well tolerated with similar frequency and intensity of adverse reaction in groups receiving IMP321 as in controls. Post-vaccination humoral immune responses, as determined 29 and 57 days later by assay of hemagglutinin inhibition activity were similar for both IMP321 and control groups. In contrast, the addition of 10, 30 or 100 microg IMP321 to the flu vaccine resulted in higher levels of Th1-type (IFN-gamma, TNF-alpha or IL-2) flu-specific CD4 T cells in PBMC recovered at D29 and D57 and tested in a short-term ex vivo restimulation assay (6-colour FACS analysis after intra-cellular staining of cytokines). In summary, IMP321 as an adjuvant to a model antigen (Agrippal) was well-tolerated and may enhance T cell response vaccine immunogenicity.

A soluble form of lymphocyte activation gene-3 (IMP321) induces activation of a large range of human effector cytotoxic cells.

The principal antitumor immune response is mediated through the activation of type 1 cytotoxic (Tc1) CD8 T cells, NK cells, and monocytes/macrophages. In this study, we investigated the potency of a clinical-grade soluble form of lymphocyte activation gene-3 protein (IMP321), a physiological high-affinity MHC class II binder, at inducing in PBMCs an appropriate cytotoxic-type response in short-term ex vivo assays. We found that IMP321 binds to a minority (<10%) of MHC class II + cells in PBMCs, including all myeloid dendritic cells, and a small fraction of monocytes. Four hours after addition of IMP321 to PBMCs, these myeloid cells produce TNF-alpha and CCL4 as determined by intracellular staining. At 18 h, 1% of CD8+ T cells and 3.7% NK cells produce Tc1 cytokines such as IFN-gamma and/or TNF-alpha (mean values from 60 blood donors). Similar induction was observed in metastatic cancer patient PBMCs, but the values were lower for the NK cell subset. Early APC activation by IMP321 is needed for this Tc1-type activation because pure sorted CD8+ T cells could not be activated by IMP321. Only Ag-experienced, fully differentiated granzyme+ CD8 T cells (effector and effector memory but not naive or central memory T cells) are induced by IMP321 to full Tc1 activation. In contrast to IMP321, TLR1-9 agonists induce IL-10 and are therefore unable to induce this Tc1 IFN-gamma+ response. Thus, IMP321 has many properties that confirm its potential to be a new class of immunopotentiator in cancer patients.

A soluble LAG-3 protein as an immunopotentiator for therapeutic vaccines: Preclinical evaluation of IMP321.

A soluble LAG-3 (CD223) molecule (sLAG-3) is a natural high affinity ligand for MHC class II. It is known to induce maturation of monocyte-derived dendritic cells in vitro and is used as a vaccine adjuvant to induce CD4 Th1 and CD8 T cell responses in vivo. The work presented herein was performed to study the biological activity of a preclinical grade human sLAG-3 protein, called IMP321: (i) binding on murine APC, (ii) induction of dendritic cell maturation, (iii) safety of repeated injections in mice. The results show that IMP321 binds MHC class II(+) murine APC, induces the maturation of dendritic cells and is well tolerated either injected alone or as an adjuvant with strongly immunogenic antigens.

Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis.

Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.

Polyubiquitination of p53 by a ubiquitin ligase activity of p300.

Rapid turnover of the tumor suppressor protein p53 requires the MDM2 ubiquitin ligase, and both interact with p300-CREB-binding protein transcriptional coactivator proteins. p53 is stabilized by the binding of p300 to the oncoprotein E1A, suggesting that p300 regulates p53 degradation. Purified p300 exhibited intrinsic ubiquitin ligase activity that was inhibited by E1A. In vitro, p300 with MDM2 catalyzed p53 polyubiquitination, whereas MDM2 catalyzed p53 monoubiquitination. E1A expression caused a decrease in polyubiquitinated but not monoubiquitinated p53 in cells. Thus, generation of the polyubiquitinated forms of p53 that are targeted for proteasome degradation requires the intrinsic ubiquitin ligase activities of MDM2 and p300.

Yin Yang 1 is a negative regulator of p53.

Yin Yang 1 (YY1) is a transcription factor that plays an essential role in development. However, the full spectrum of YY1's functions and mechanism of action remains unclear. We find that YY1 ablation results in p53 accumulation due to a reduction of p53 ubiquitination in vivo. Conversely, YY1 overexpression stimulates p53 ubiquitination and degradation. Significantly, recombinant YY1 is sufficient to induce Hdm2-mediated p53 polyubiquitination in vitro, suggesting that this function of YY1 is independent of its transcriptional activity. We identify direct physical interactions of YY1 with Hdm2 and p53 and show that the basis for YY1-regulating p53 ubiquitination is its ability to facilitate Hdm2-p53 interaction. Importantly, the tumor suppressor p14ARF compromises the Hdm2-YY1 interaction, which is important for YY1 regulation of p53. Taken together, these findings identify YY1 as a potential cofactor for Hdm2 in the regulation of p53 homeostasis and suggest a possible role for YY1 in tumorigenesis.