Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-01-27
2002-06-25
Caputa, Anthony C. (Department: 1642)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S021800, C424S114000, C424S184100, C424S184100, C424S193100, C424S278100, C530S350000, C530S351000
Reexamination Certificate
active
06410509
ABSTRACT:
The present invention relates to the use of LAG-3 and CD4, and in a more general way, the use of MHC class II ligands or MHC class II-like ligands as adjuvants for vaccines, in order to boost an antigen specific immune response, as well as the use of LAG-3 as a therapeutical agent in cancer immunotherapy.
It is now recognized that the proteins encoded by MHC Class II region are involved in many aspects of immune recognition, including the interaction between different lymphoid cells such as lymphocytes and antigen presenting cells. Different observations have also shown that other mechanisms which do not take place via CD4 participate in the effector function of T helper lymphocytes.
The lymphocyte activation gene 3 (LAG-3) expressed in human CD4
+
and CD8
+
activated T-cells as well as in activated NK cells encodes a 503 amino-acids (aa) type I membrane protein with four extracellular immunoglobulin superfamily (IgSF) domains (1) and is a ligand for MHC class II molecules (2). Analysis of this sequence revealed notable patches of identity with stretches of aminoacids sequences found at the corresponding positions in CD4, although the overall aminoacids sequence homology with human CD4 is barely above background level (approximately 20% sequence identity). There are also some internal sequence homologies in the LAG-3 molecule between domains 1 (D1) and 3 (D3) as well as between domains 2 (D2) and 4 (D4) suggesting that LAG-3 has evolved like CD4 by gene duplication from a preexisting 2 IgSF structure (1). In addition, LAG-3 and CD4 genes are located in a very close proximity on the distal part of the short arm of chromosome 12 (3). LAG-3 and CD4 can therefore be regarded as evolutionary “first cousins” within the IgSF (2).
Like CD4, hLAG-3 is composed of lg like ectodomains with a WxC signature motif in domains 2 and 4; however a difference with CD4 is the presence of an extraloop sequence in domain 1 (recognized by the 17B4mAb) 30 and an intracytoplasmic proline rich motif (EP repeats) in human LAG-3 (hLAG-3). Recently, murine lymphocyte activation gene 3 (mLAG-3) was cloned and approximately 70% of homology was found with hLAG-3, with the same proline rich motif in the intracytoplasmic tail.
Antigen specific stimulation of CD4
+
T-cell clones in the presence of anti-LAG-3 mAb leads to extended proliferation and cytokines production (5). It has been suggested a regulatory role of hLAG-3 on CD4
+
T lymphocyte activated, by cross-linking MHC class II molecules expressed on T-cells with LAG-3 lg fusion proteins (6). LAG-3 MHC class II interaction inhibits signals through MHC class II molecules expressed on CD4
+
T-cells (decrease of proliferation and cytokines production), suggesting that both LAG-3 and MHC class II are effector molecules for the down-regulation of T helper cell mediated immune responses. The hLAG-3 lg fusion protein was found to bind xenogenic MHC class II molecules (murine and monkey). In addition, the mLAG-3 has been proposed to transduce a positive signal in effector cells, since transgenic mice with a LAG-3 null mutation have a defect in the NK cell compartment (7).
Mouse tumor cell lines engineered to express membrane (B7.1, B7.2. CD95L, . . .) or secreted molecules (IL-2, IL-12,. . .) are often used to investigate immune responses or antitumor effects. This approach implies that many tumor cells are potentially antigenic (9), and become immunogenic when they express molecules. Experimental mouse tumors are classified as intrinsically immunogenic when, after a single injection into syngenic mice as nonreplicating cell vaccines, they elicit a protective immune response against a subsequent lethal challenge. Tumors that do not retain this residual immunogenicity are defined as poorly immunogenic or nonimmunogenic.
Antitumor immune responses are mediated primarily by T-cells (12). Recent studies have implicated a deficit in efficient antigen presentation and T-cell priming as being problematic for the practical implementation of an ideal tumor vaccine. Indeed, it has been demonstrated that transfecting tumor cells with genes coding for various cytokines, such as IL-2, IL-4, IL-12 or GM-CSF or genes coding for co-stimulatory molecules such as B7 not only led to primary rejection of the modified cells but often elicited protective immunity against subsequent challenge with unmodified tumor cells (13).
Professional antigen presenting cells (APCs) are capable of taking up, processing and presenting antigen to T-cells in the context of co-stimulatory signals required for T-cell activation, leading to optimal antigen presentation. In particular, it is well established that MHC class II
+
dendritic cells (DCs) play a crucial role in processing and presenting antigens to the immune system. The inventors hypothesized that tumor immunogenicity would be increased if tumor could be modified to directly trigger host APCs such as macrophages and dendritic cells. Indeed, it has been reported that cross-linking of MHC class II molecules specifically expressed by such cells, using mAb or superantigens, transduces signals resulting in TNF&agr; and IL-12 production (14, 15). They had previously reported that Lymphocyte Activation Gene-3 (LAG-3), which is embedded in the CD4 locus (1, 16), encodes a protein that binds human and murine MHC class II molecules with higher affinities than CD4 (17, 6).
The inventors of the instant application have investigated whether hLAG-3, human CD4 (hCD4) and mLAG-3 expression on three MHC class II-mouse tumors (the poorly immunogenic sarcoma MCA 205 and the nonimmunogenic TSIA+RENCA adenocarcinoma) can mediate an immune response so as to reject mouse tumor and can induce systemic immunity.
As a result, they have discovered that human or murine LAG-3, whether expressed as membrane proteins in solid tumor cell lines or inoculated into mice as a soluble protein induced a potent immunity against highly malignant murine tumors. The immunity was T-cell dependent and antigen-specific.
They have further investigated the role of CD4 and found that human CD4 (hCD4) also induced a systemic antitumor response.
The induced immunity has been found to be T-cell mediated, since the same antitumor response was obtained with Nude mice lacking T-lymphocytes.
The antitumor effect was still found when using different tumor cell lines exhibiting different intrinsic immunogenicity as well as different strains of mice expressing different MHC genes.
Furthermore, the hLAG-3 and hCD4 induced effects were observed when tumor cell lines expressing hLAG-3 or hCD4 were injected at a distant site from the initial inoculation site of the wild-type tumor cell lines.
Furthermore, systemic administration of soluble hLAG-3 directly induces an inhibition of in vivo tumor growth.
All the aforementioned results demonstrate that LAG-3 and CD4 are able to elicit an antigen specific T-cell mediated immune response and may be useful as a tool in immunotherapy, in order to prevent the occurrence of a cancer among populations at risk or more generally in any immunotherapy involving an antigen-specific T-cell mediated immune response, and that LAG-3 is further useful as a tool for inhibiting in vivo tumor growth.
The inventors have further demonstrated that soluble LAG-3 when administered together with an antigen against which an immune response is sought, was able to work as an adjuvant for a vaccine.
This role can be explained by an improved presentation of the antigen by professional APCs (dendritic cells and macrophages) located underneath the skin and triggered via MHC class II.
Accordingly, since induction of a CD8
+
T-cell immunity is involved in viral (e.g. AIDS, hepatitis and herpes) and intra-cellular parasitic and bacterial (e.g. leprosy tuberculosis) infections and cancer, LAG-3 will be particularly useful for therapeutic vaccination against the pathogen agents involved in these diseases as well as in cancer treatment.
According to one of its aspects, the present invention relates to the use of a MHC class II ligand or a MHC class
Browdy and Neimark
Canella Karen A.
Caputa Anthony C.
Institut Gustave-Roussy
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