Molecular characterization of HLA class II binding to the LAG-3 T cell co-inhibitory receptor.

Immune checkpoint inhibitors (antibodies that block the T cell co-inhibitory receptors PD-1/PD-L1 or CTLA-4) have revolutionized the treatment of some forms of cancer. Importantly, combination approaches using drugs that target both pathways have been shown to boost the efficacy of such treatments. Subsequently, several other T cell inhibitory receptors have been identified for the development of novel immune checkpoint inhibitors. Included in this list is the co-inhibitory receptor lymphocyte activation gene-3 (LAG-3), which is upregulated on T cells extracted from tumor sites that have suppressive or exhausted phenotypes. However, the molecular rules that govern the function of LAG-3 are still not understood. Using surface plasmon resonance combined with a novel bead-based assay (AlphaScreenTM ), we demonstrate that LAG-3 can directly and specifically interact with intact human leukocyte antigen class II (HLA-II) heterodimers. Unlike the homologue CD4, which has an immeasurably weak affinity using these biophysical approaches, LAG-3 binds with low micromolar affinity. We further validated the interaction at the cell surface by staining LAG-3+ cells with pHLA-II-multimers. These data provide new insights into the mechanism by which LAG-3 initiates T cell inhibition.

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.

AIPAC: a Phase IIb study of eftilagimod alpha (IMP321 or LAG-3Ig) added to weekly paclitaxel in patients with metastatic breast cancer.

Eftilagimod alpha (IMP321), a soluble dimeric recombinant form of LAG-3, is a first-in-class antigen presenting cell activator under clinical development. By stimulating dendritic cells through MHC class II molecules, IMP321 was proven to induce sustained immune responses. Combining active immunotherapy with a standard cytotoxic chemotherapy regimen represents a promising novel strategy that might lead to therapeutic improvements in metastatic breast cancer. Here, we describe the rationale and design of AIPAC (NCT02614833), a double-blind, randomized, multicenter Phase IIb study evaluating IMP321 plus paclitaxel as a first-line chemotherapy compared with paclitaxel plus placebo in hormone receptor-positive metastatic breast cancer patients. The primary end point is progression-free survival and key secondary objectives include overall survival, safety, quality of life and objective response rate.

MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial.

BACKGROUND: Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear. METHODS: We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion. RESULTS: After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7⁻ CD45RA⁺/⁻) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1⁻CD160⁻TIM3⁻LAG3⁻2B4⁺/⁻). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group. CONCLUSIONS: Vaccination with IMP321 as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs induced more robust and durable cellular antitumor immune responses, supporting further development of IMP321 as an adjuvant for future immunotherapeutic strategies.

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.

LAG-3 expression defines a subset of CD4(+)CD25(high)Foxp3(+) regulatory T cells that are expanded at tumor sites.

Human natural regulatory CD4(+) T cells comprise 5-10% of peripheral CD4(+)T cells. They constitutively express the IL-2Ralpha-chain (CD25) and the nuclear transcription Foxp3. These cells are heterogeneous and contain discrete subsets with distinct phenotypes and functions. Studies in mice report that LAG-3 has a complex role in T cell homeostasis and is expressed in CD4(+)CD25(+) T regulatory cells. In this study, we explored the expression of LAG-3 in human CD4(+) T cells and found that LAG-3 identifies a discrete subset of CD4(+)CD25(high)Foxp3(+) T cells. This CD4(+)CD25(high)Foxp3(+)LAG-3(+) population is preferentially expanded in the PBMCs of patients with cancer, in lymphocytes of tumor-invaded lymph nodes and in lymphocytes infiltrating visceral metastasis. Ex vivo analysis showed that CD4(+)CD25(high)Foxp3(+)LAG-3(+) T cells are functionally active cells that release the immunosuppressive cytokines IL-10 and TGF-beta1, but not IL-2. An in vitro suppression assay using CD4(+)CD25(high)LAG-3(+) T cells sorted from in vitro expanded CD4(+)CD25(high) regulatory T cells showed that this subset of cells is endowed with potent suppressor activity that requires cell-to-cell contact. Our data show that LAG-3 defines an active CD4(+)CD25(high)Foxp3(+) regulatory T cell subset whose frequency is enhanced in the PBMCs of patients with cancer and is expanded at tumor sites.

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.

Lymphocyte activation gene-3 fusion protein increases the potency of a granulocyte macrophage colony-stimulating factor-secreting tumor cell immunotherapy.

PURPOSE: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy, which is known to stimulate a potent and long-lasting antigen-specific immune response in combination with lymphocyte activation gene-3 fusion protein (LAG-3Ig), which has been shown to act as an adjuvant for priming T helper type 1 and cytotoxic T-cell responses. EXPERIMENTAL DESIGN: Survival and immune monitoring studies were done in the B16 melanoma model. GM-CSF-secreting tumor cell immunotherapy was administered as a single s.c. injection and LAG-3Ig was administered s.c. at the immunotherapy site. RESULTS: The studies reported here show that combining LAG-3Ig with GM-CSF-secreting tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with increased numbers of systemic IFN gamma-secreting CD8+ T cells and a significantly increased infiltration of activated effector CD8+ T cells into the tumor. Moreover, an increase in antigen-specific IgG1 humoral responses was detected in serum of animals injected with the combination therapy compared with animals injected with either therapy alone. CONCLUSION: LAG-3Ig combined with a GM-CSF-secreting tumor cell immunotherapy stimulated both cellular and humoral antitumor immune responses that correlated with prolonged survival in tumor-bearing animals.

Soluble human LAG-3 molecule amplifies the in vitro generation of type 1 tumor-specific immunity.

The adjuvant activities of the human lymphocyte activation gene-3 (LAG-3) molecule have been evaluated in a human setting by investigating the ability of a soluble recombinant human LAG-3 protein (hLAG-3Ig) to enhance the in vitro induction of viral- and tumor-specific CTLs. We found that soluble human LAG-3 significantly sustained the generation and expansion of influenza matrix protein Melan-A/MART-1 and survivin-specific CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMC) of both cancer patients and healthy donors, showing its ability to boost CD8+ T-cell memory response or to prime naive T cells in vitro. The peptide-specific T cells generated in the presence of hLAG-3Ig were endowed with cytotoxic activity and enhanced release of type 1 cytotoxic T (Tc1) cytokines and were able to recognize tumor cells expressing their nominal antigen. Phenotype and cytokine/chemokines produced by antigen-presenting cells (APC) of PBMCs exposed in vitro for 2 days to peptide and hLAG-3Ig indicate that the LAG-3-mediated adjuvant effect may depend on a direct activation of circulating APCs. Our data revealed the activity of hLAG-3Ig in inducing tumor-associated, antigen-specific CD8+ T-cell responses in a human setting and strongly support the conclusion that this recombinant protein is a potential candidate adjuvant for cancer vaccines.

Rescuing CD4+CD25+ regulatory T-cell functions in rheumatoid arthritis by cytokine-targeted monoclonal antibody therapy.

CD4+CD25+ regulatory T cells (Tregs) play a crucial role in controlling the development of autoimmune diseases such as rheumatoid arthritis (RA). However, despite an increased number of Tregs, the persistence of inflammation in the rheumatoid joints suggests that Tregs are unable to suppress ongoing disease, perhaps due to an inhibition of their functions by pro-inflammatory cytokines. Treatment of RA patients with anti-TNF-alpha monoclonal antibodies such as infliximab and adalimumab has been found to induce and restore the functions of Tregs. Thus, manipulation of the pro-inflammatory environment in the inflamed synovia via neutralization of inflammatory cytokines by monoclonal antibodies could represent a novel therapeutic strategy for restoring the suppressive functions of Tregs and induction and/or expansion of Tregs in order to reinforce tolerance mechanisms.

Depletion of LAG-3 positive cells in cardiac allograft reveals their role in rejection and tolerance.

BACKGROUND: Lymphocyte-activated gene-3 (LAG-3, CD223) is upregulated during the early stages of T-cell activation and could be the target of cytotoxic antibodies for induction therapy in transplantation. METHODS: Fully vascularized heterotopic allogeneic heart transplantation was performed in rats across a full major histocompatibility complex-mismatch barrier (LEW.1W into LEW.1A). Recipients received two injections (day 0 and 3) of cytotoxic antibodies directed to the extra-loop of LAG-3 immunoglobulin (Ig)-like N-terminal domain or control antibodies. RESULTS: LAG-3 mRNA transcripts accumulated in cardiac allografts undergoing rejection, but not in peripheral lymphoid organs. Administration of anti-LAG-3 antibodies on the day of transplantation did not modify alloreactivity of T lymphocytes from the spleen and did not change the alloantibody response. However, it inhibited graft infiltration by effector mononuclear cells, reduced intragraft levels of interferon-gamma mRNA and prolonged allograft survival from 6 days in controls to a median of 27 days. Anti-LAG-3 antibodies were also active in prolonging survival when administered in a delayed manner, after rejection onset. LAG-3 being also expressed by activated regulatory T (Treg) cells, we tested the effect of anti-LAG-3 antibodies on graft acceptance after donor blood transfusions, a Treg-dependent tolerance induction model. We found that tolerance induction was prevented by anti-LAG-3 antibodies. CONCLUSIONS: Targeting LAG-3-positive cells with cytotoxic antibodies is immunosuppressive in transplantation by depleting effectors T cells and therefore may represent a treatment for rejection episodes focused only on pathogenic cells. However, it might not be compatible with tolerance-induction strategies.

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.

New Cancer Immunotherapy Agents in Development: a report from an associated program of the 31stAnnual Meeting of the Society for Immunotherapy of Cancer, 2016.

This report is a summary of 'New Cancer Immunotherapy Agents in Development' program, which took place in association with the 31st Annual Meeting of the Society for Immunotherapy of Cancer (SITC), on November 9, 2016 in National Harbor, Maryland. Presenters gave brief overviews of emerging clinical and pre-clinical immune-based agents and combinations, before participating in an extended panel discussion with multidisciplinary leaders, including members of the FDA, leading academic institutions and industrial drug developers, to consider topics relevant to the future of cancer immunotherapy.

A soluble lymphocyte activation gene-3 (sLAG-3) protein as a prognostic factor in human breast cancer expressing estrogen or progesterone receptors.

In contrast to the long-held belief that breast cancer is a weakly immunogenic tumor, accumulating evidence indicates an immune infiltrate is an invariable finding in breast cancers, raising hopes that immunotherapy for breast cancers may succeed in targeted patients, specifically those with either regional or minimal residual disease. However, no immunologically related prognostic factor has yet been established that may help to define subsets of patients who are more prone to respond to immunotherapy. High levels of soluble LAG-3 protein (sLAG-3) in sera has previously been shown to be associated, as a Th1 marker, to resistance to tuberculosis in large series of patients. We therefore hypothesized that, if cell-mediated immune mechanisms are indeed important for improved prognosis, high levels of sLAG-3 might be correlated with improved survival in some subsets of breast cancer patients. Studying a cohort of 246 patient's sera collected in 1994 at time of first diagnosis, we found that both disease-free and overall survival rates were greater in patients with estrogen or progesterone receptor positive tumor cells who had detectable levels of sLAG-3 at diagnosis versus patients with undetectable sLAG-3 levels. These results indicate that sLAG-3 may be a valuable marker for prognosis in some subsets of breast cancers and, more importantly, that cell-mediated mechanisms such as Th1 responses do have an impact on survival, a pre-requisite before the setting-up of immunotherapy protocols as a form of adjuvant therapy for breast cancer.

LAG-3 enables DNA vaccination to persistently prevent mammary carcinogenesis in HER-2/neu transgenic BALB/c mice.

Within 33 weeks of life, all 10 mammary glands of virgin BALB/c mice transgenic for the transforming rat HER-2/neu oncogene under the mammary tumor virus promoter (BALB-neuT mice) progress from atypical hyperplasia to invasive palpable carcinoma. Repeated DNA vaccination with plasmids coding for the extracellular and transmembrane domain of the protein product of rat HER-2/neu (r-p185(neu)) delayed tumor onset and reduced tumor multiplicity, but this protection eventually declined, and few mice were tumor free at 1 year of age. Association of plasmid vaccination with administration of soluble mouse LAG-3 (lymphocyte activation gene-3/CD223) generated by fusing the extracellular domain of murine LAG-3 to a murine IgG2a Fc portion (mLAG-3Ig) elicited a stronger and sustained protection that kept 70% of 1-year-old mice tumor free. Moreover, this combined vaccination, which was performed when multiple in situ carcinomas were already evident, extended disease-free survival and reduced carcinoma multiplicity. Inhibition of carcinogenesis was associated with markedly reduced epithelial cell proliferation and r-p185(neu) expression, whereas the few remaining hyperplastic foci were heavily infiltrated by reactive leukocytes. A stronger and enduring r-p185(neu)-specific cytotoxicity, a sustained release of IFN-gamma and interleukin 4, and a marked expansion of both CD8(+)/CD11b(+)/CD28(+) effector and CD8(+)/CD11b(+)/CD28(-) memory effector T-cell populations were induced in immunized mice. This combined vaccination also elicited a quicker and higher antibody response to r-p185(neu), as well as an early antibody isotype switch. These data suggest that the appropriate costimulation provided by mLAG-3Ig enables DNA vaccination to establish an effective protection, probably by enhancing cross-presentation of the DNA coded antigen.

Vaccination with LAG-3Ig (IMP321) and Peptides Induces Specific CD4 and CD8 T-Cell Responses in Metastatic Melanoma Patients--Report of a Phase I/IIa Clinical Trial.

PURPOSE: Cancer vaccines aim to generate and maintain antitumor immune responses. We designed a phase I/IIa clinical trial to test a vaccine formulation composed of Montanide ISA-51 (Incomplete Freund's Adjuvant), LAG-3Ig (IMP321, a non-Toll like Receptor agonist with adjuvant properties), and five synthetic peptides derived from tumor-associated antigens (four short 9/10-mers targeting CD8 T-cells, and one longer 15-mer targeting CD4 T-cells). Primary endpoints were safety and T-cell responses. EXPERIMENTAL DESIGN: Sixteen metastatic melanoma patients received serial vaccinations. Up to nine injections were subcutaneously administered in three cycles, each with three vaccinations every 3 weeks, with 6 to 14 weeks interval between cycles. Blood samples were collected at baseline, 1-week after the third, sixth and ninth vaccination, and 6 months after the last vaccination. Circulating T-cells were monitored by tetramer staining directly ex vivo, and by combinatorial tetramer and cytokine staining on in vitro stimulated cells. RESULTS: Side effects were mild to moderate, comparable to vaccines with Montanide alone. Specific CD8 T-cell responses to at least one peptide formulated in the vaccine preparation were found in 13 of 16 patients. However, two of the four short peptides of the vaccine formulation did not elicit CD8 T-cell responses. Specific CD4 T-cell responses were found in all 16 patients. CONCLUSIONS: We conclude that vaccination with IMP321 is a promising and safe strategy for inducing sustained immune responses, encouraging further development for cancer vaccines as components of combination therapies.

Orchestration and Prognostic Significance of Immune Checkpoints in the Microenvironment of Primary and Metastatic Renal Cell Cancer.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) has shown durable responses to checkpoint blockade therapies. However, important gaps persist in the understanding of its immune microenvironment. This study aims to investigate the expression and prognostic significance of immune checkpoints in primary and metastatic ccRCC, in relation with mature dendritic cells (DC) and T-cell densities. EXPERIMENTAL DESIGN: We investigated the infiltration and the localization of CD8(+) T cells and mature DC, and the expression of immune checkpoints (PD-1, LAG-3, PD-L1, and PD-L2) in relation with prognosis, in 135 primary ccRCC tumors and 51 ccRCC lung metastases. RNA expression data for 496 primary ccRCC samples were used as confirmatory cohort. RESULTS: We identify two groups of tumors with extensive CD8(+) T-cell infiltrates. One group, characterized by high expression of immune checkpoints in the absence of fully functional mature DC, is associated with increased risk of disease progression. The second group, characterized by low expression of immune checkpoints and localization of mature DC in peritumoral immune aggregates (tertiary lymphoid structures), is associated with good prognosis. CONCLUSIONS: The expression of the immune checkpoints and the localization of DC in the tumor microenvironment modulate the clinical impact of CD8(+) T cells in ccRCC.