Abstract:
The present disclosure relates to the use of a recombinant LAG-3 or derivatives thereof in order to boost a monocyte-mediated immune response, in particular to elicit an increase in the number of monocytes in blood. This finds use in the development of novel therapeutic agents for the treatment of an infectious disease or cancer.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a National Phase Entry of International Application No. PCT/IB2008/002653, filed on Oct. 3, 2008, which claims priority to European Application EP 07291214.0, filed on Oct. 5, 2007, both of which are incorporated by reference herein. 
     
    
     BACKGROUND 
       [0002]    1. Technical Field 
         [0003]    The present invention relates to the use of recombinant LAG-3 or derivatives thereof in order to boost a monocyte-mediated immune response. It enables an increase in monocyte numbers in blood. It finds many applications in particular in the development of novel therapeutic agents in cancer immunotherapy. 
         [0004]    2. State of the Art 
         [0005]    In the description which follows, the references between brackets [ ] refer to the attached reference list. The lymphocyte activation gene 3 (hlag-3) expressed in human CD4 +  and CD8 +  activated T cells as well as in activated NK cells encodes a 503 amino acids type I membrane protein (LAG-3) with four extracellular immunoglobulin superfamily (IgSF) domains [1]. A murine lymphocyte activation gene 3 (mlag-3) was cloned and approximatively 70% of homology was found with hlag-3, with the same proline rich motif in the intracytoplasmic tail. 
         [0006]    LAG-3 (CD223), described as being a natural high affinity ligand for MHC class II, is known to induce maturation of monocyte-derived dendritic cells in vitro, and is used as an immunotherapy adjuvant to induce CD4 T helper type 1 responses and CD8 T cell responses in vivo [2]. Further information regarding LAG-3 and its use as an immunostimulant may be found in TRIEBEL et al. [1], TRIEBEL et al. [3], and HUARD et al. [4]. Some forms of soluble LAG-3 can bind to MHC class II molecules and can induce dendritic cells to mature and migrate to secondary lymphoid organs where they can prime naïve CD4-helper and CD8-cytotoxic T cells leading to tumour rejection [5]. More recently a recombinant soluble human LAG-3lg fusion protein (IMP321) was shown to activate a large range of effector cells in both innate and acquired immune responses, for example inducing monocytes-macrophages to secrete cytokines/chemokines [6]. 
         [0007]    Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts. They constitute between three to eight percent of the leukocytes in the blood. Monocytes circulate in the bloodstream for about 24 hours (half-life of 8 hours) and then typically move into tissues throughout the body. In the tissues, monocytes mature into macrophages, epithelioid cells or antigen-presenting cells (APCs, for example dendritic cells). Monocytes are responsible for phagocytosis (ingestion) of foreign substances in the body. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the pathogen directly via pattern-recognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody, termed antibody-dependent cell-mediated cytotoxicity (ADCC). Due to their secretion and phagocytosis properties, monocytes-macrophages occur in aspecific and specific immune response. 
         [0008]    The study of membrane markers allows the identification of monocyte populations, mature or not, dystrophic or not. The molecules present on monocyte membranes, mature or not, are almost always non specific but correspond to the following activities:
       receptor for the Fc fragment of IgG (CD16, CD32, CD64),   receptor for the Fc fragment of IgE (CD23),   receptor for complement fractions (CD11b, CD21/CD35),   leukocyte adhesion proteins (CD11a, CD11c),   protein facilitating binding to LPS of Gram—bacteria (CD14),   protein with tyrosine phosphatase activity (CD45).       
 
       SUMMARY 
       [0015]    The authors of the present invention have now discovered, entirely unexpectedly, that human LAG-3 or derivatives thereof when inoculated into patients with highly malignant tumors, for example patients with metastatic breast cancer (MBC) or metastatic renal clear-cell carcinoma (MRCC), induced a potent immunity which is monocyte dependent. Said induced immunity manifests itself by a significant increase in blood monocyte counts. 
         [0016]    This result was achieved by means of plural administration of LAG-3 or derivatives thereof to patients receiving immunotherapy or chemo-immunotherapy. This result is rather surprising since binding to, and activation of, monocytes is not expected to be followed by monocyte expansion. Indeed monocytes are end-of-differenciation hematopoietic cells and can not proliferate. They can stay in the blood as monocytes or differentiate toward either macrophages or dendritic cells under the influence of different cytokines, until they die. Thus it is believed, without limitation to the following theory, that the mechanism of action involved may be a proliferative signal directed to hematopoietic precursor cells (before the promonocyte stage) residing in the bone marrow, or an increase in the half-life or residence time of mature circulating monocytes. 
         [0017]    Therefore the present invention relates to the use of a recombinant LAG-3 protein or derivative thereof that elicits monocyte mediated immune response, for the manufacture of a medicament inducing an increase in monocyte numbers for the treatment of an infectious disease or cancer. By “derivatives of LAG-3”, in the sense of the present invention, is meant mutants, variants and fragments of LAG-3 provided that they maintain the ability of LAG-3 to bind MHC class II molecules. 
         [0018]    Thus, the following forms of LAG-3 may be used:
       the whole LAG-3 protein,   a soluble polypeptide fragment thereof consisting of at least one of the four immunoglobulin extracellular domains, namely the soluble part of LAG-3 comprised of the extracellular region stretching from the amino acid 23 to the amino acid 448 o the LAG-3 sequence disclosed in French patent Application FR 90 00 126,   a fragment of LAG-3 consisting of substantially all of the first and second domains,   a fragment of LAG-3 consisting of substantially all of the first and second domains or all of the four domains, such as defined in WO 95/30750,   a mutant form of soluble LAG-3 or a fragment thereof comprising the D1 and D2 extracellular domains and consisting of:
           a substitution of an amino acid at one of the following positions:   position 73 where ARG is substituted with GLU,   position 75 where ARG is substituted with ALA or GLU,   position 76 where ARG is substituted with GLU,   or a combination of two or more of those substitutions,   a substitution of an amino acid at one of the following positions:   position 30 where ASP is substituted with ALA,   position 56 where HIS is substituted with ALA,   position 77 where TYR is substituted with PHE,   position 88 where ARG is substituted with ALA,   position 103 where ARG is substituted with ALA,   position 109 where ASP is substituted with GLU,   position 115 where ARG is substituted with ALA,   or a deletion of the region comprised between the position 54 and the position 66,   or a combination of two or more of those substitutions.   
               
 
         [0039]    Those mutants are described by HUARD et al. (Proc. Natl. Acad. Sci. USA, 11 : 5744-5749, 1997).
       a physiological variant of LAG-3 comprised of the soluble 52 kDa protein containing D1, D2 and D3.   a recombinant soluble human LAG-3lg fusion protein (IMP321), a 200-kDa dimer produced in Chinese hamster ovary cells transfected with a plasmid encoding for the extracellular domain of hLAG-3 fused to the human IgG1 Fc.       
 
         [0042]    By “medicament”, in the sense of the present invention, is meant an effective plurality of doses of a recombinant LAG-3 protein or derivative thereof. By “effective plurality of doses of a recombinant LAG-3 protein or derivative thereof”, in the sense of the present invention, is meant a formulation that allows administration of one dose of a recombinant LAG-3 protein or derivative thereof every one to several weeks for at least 12 weeks, and preferably for at least 24 weeks, separated by 13-day±2 days administration-free intervals. Advantageously, the administration is an every-two-week schedule. By “one dose of a recombinant LAG-3 protein or derivative thereof”, in the sense of the present invention, is meant a formulation that allows one administration in the range of 0.25-30 mg, preferably 1-6.25 mg, more preferably 6-30 mg, and for example about 1.25 mg of recombinant LAG-3 protein or derivative thereof to a patient in need thereof having a body mass index (weight/height 2 ) in the range of 18-30 kg/m 2 . 
         [0043]    The recombinant LAG-3 or derivatives thereof are administered in a free form, for example in a soluble form by inoculating them systemically, for example as a subcutaneous, intramuscular or intravenous injection, preferably as a subcutaneous injection. Said recombinant LAG-3 or derivatives thereof may also be formulated so as to allow administration with a compound having anti-cancer or anti-infectious disease immunotherapeutical or chemotherapeutical properties. By “administration with a compound”, in the sense of the present invention, is meant an administration of a recombinant LAG-3 or derivative thereof before, with, or subsequent to, the administration of said compound. By “compound having anti-cancer or anti-infectious disease chemotherapeutical properties”, in the sense of the present invention, is meant for example a chemotherapy agent selected from the group consisting of taxanes (paclitaxel, docetaxel), gemcitabine and anthracyclines (doxorubicine) or an anti-viral agent such as ribavirin. 
         [0044]    In a particular embodiment of the invention, recombinant LAG-3 protein or derivative thereof is administered to patients after the first administration of the cytotoxic compound having anti-cancer or anti-infectious disease chemotherapeutical properties. Advantageously, recombinant LAG-3 protein or derivative thereof is administered to patients is administered 12 to 96 hours after the administration of the cytotoxic compound having anti-cancer or anti-infectious disease chemotherapeutical properties. In another embodiment, recombinant LAG-3 protein or derivative thereof is administered to patients is administered one or two days after the first administration of the compound having anti-cancer or anti-infectious disease chemotherapeutical properties. In another particular embodiment of the invention, recombinant LAG-3 protein or derivative and the cytotoxic compound having anti-cancer or anti-infectious disease chemotherapeutical properties are administered simultaneously, separately or sequentially. 
         [0045]    Advantageously, in this particular embodiment of the invention, recombinant LAG-3 protein or derivative thereof is administered at least six times, for example seven times, ten times, twelve times or more. Advantageously, in this particular embodiment of the invention, recombinant LAG-3 protein or derivative thereof is administered on an every-two-week schedule. Advantageously, recombinant LAG-3 protein or derivative thereof is administered at a dose comprised between 0.25 to 30 mg, eventually at a dose comprised between 6 to 30 mg, eventually at a dose comprised between 8 to 25 mg, eventually between 12 and 20 mg. By “compound having anti-cancer or anti-infectious disease immunotherapeutical properties”, in the sense of the present invention, is also meant for example a compound selected from the group consisting of therapeutic antibodies killing tumour cells through ADCC (antibody-dependent cell cytotoxicity), and mixtures thereof, and preferably from the group consisting of rituximab, cetuximab, edrecolomab, and trastuzumab. 
         [0046]    In a particular embodiment of the invention, recombinant LAG-3 protein or derivative thereof and therapeutic antibodies are administered to patients simultaneously, separately or sequentially. Advantageously, in a particular embodiment of the invention, recombinant LAG-3 protein or derivative thereof is administered to patients the same day as therapeutic antibodies. 
         [0047]    The present invention also relates to kit-of-parts, i.e. a combined preparation, containing recombinant LAG-3 protein or derivative thereof and a therapeutic antibody for simultaneous, separate or sequential use. Advantageously, the kit-of-parts contains recombinant LAG-3 protein or derivative thereof and a therapeutic antibody selected from the group consisting of rituximab, cetuximab, edrecolomab, and trastuzumab. Preferentially, the kit-of-part of the invention contain recombinant LAG-3 protein or derivative thereof and rituximab. 
         [0048]    In the kit-of-parts of the invention, recombinant LAG-3 protein or derivative thereof and a therapeutic antibody form a functional unity, because of a synergistic cytotoxic effect between the two components. This effect is a new joint effect, because the two components administered alone does not have the same effect as when administered as a combined preparation. The present invention also relates to kit-of-parts, i.e. a combined preparation, containing recombinant LAG-3 protein or derivative thereof and a compound having anti-cancer or anti-infectious disease chemotherapeutical properties for simultaneous, separate or sequential use. Advantageously, the kit-of-parts contains recombinant LAG-3 protein or derivative thereof and a compound having anti-cancer or anti-infectious disease chemotherapeutical properties selected from the group consisting of taxanes (paclitaxel, docetaxel), gemcitabine and anthracyclines (doxorubicine). 
         [0049]    The present invention also relates to a method for treating a pathological condition involving a monocyte mediated immune response, comprising administering the medicament as above defined to a patient in need thereof. By “pathological condition involving a monocyte mediated immune response”, in the sense of the present invention, is meant viral infectious diseases, parasitic infectious diseases, bacterial infectious diseases, and cancer. Other advantages may also appear to one skilled in the art from the non-limitative examples given below, and illustrated by the enclosed figures. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0050]      FIG. 1  represents fluorescence-activated cell sorting (FACS) analysis of monocytes (i.e. CD14 + CD45 + cells) in PBMCs from metastatic breast carcinoma patients. 
           [0051]      FIG. 2  represents fluorescence-activated cell sorting (FACS) analysis of monocytes (i.e. CD14 + CD45 +  cells) in fresh whole blood from metastatic breast carcinoma patients. 
           [0052]      FIG. 3  represents fluorescence-activated cell sorting (FACS) analysis of monocytes (i.e. CD14 + CD45 +  cells) in fresh whole blood from metastatic renal cell cancer patients. 
           [0053]      FIG. 4  represents fluorescence-activated cell sorting (FAGS) analysis of monocytes (i.e. CD14 + CD45 +  cells) in fresh whole blood from metastatic breast carcinoma patients. 
           [0054]      FIG. 5  represents the pharmacokinetic profiles of IMP321 measured by ELISA in the plasma of metastatic renal cell cancer patients. 
           [0055]      FIG. 6  represents the flow cytometry analysis of PBMC cultured in different conditions with rituximab and/or IMP321. 
       
    
    
     DETAILED DESCRIPTION 
     Example 1  
     Monocytes Increase in Metastatic Breast Cancer (MBC) Patients Using Low IMP321 Dose 
       [0056]    Five MBC patients, receiving chemotherapy known to induce tumour cell apoptosis, each received one subcutaneous IMP321 dose of 0.25 mg 1-2 days after chemotherapy every other week, for 24 weeks, separated by 14-day administration-free intervals. Blood samples were collected in heparinated lithium tubes (Vacutainer; BD Biosciences) from each patient, 14 days after the last IMP321 injection (i.e. looking at lasting immunomodulatory effects of the product), at 3 months (Day 85) and 6 months (Day 170). PBMCs were isolated on Ficoll-Paque gradient (Pharmacia) using LeucoSep tubes (Greiner Bio-One), and used immediately. The increase in number of monocytes was analysed by fluorescence-activated cell sorting (FACS) in said fresh PBMC samples (because monocytes are sensitive to freezing), and compared with the monocyte counts carried out on fresh PBMC samples collected before IMP321 administration (Day 1). 
         [0057]    The results are represented in  FIG. 1 . The results showed a 2.5-fold (at 3 months, Day 85) and a 3.5-fold (at 6 months, Day 170) mean increase of monocyte counts at this low IMP321 dose clinical protocol. In order to confirm the above results, a more direct and probably more accurate approach was carried out, which was to quantify directly ex-vivo the number of monocytes in whole blood (i.e. without prior purification of PBMCs on Ficoll gradient) by first measuring the exact volume of blood to be analyzed with diluted fluorescent beads and then counting the number of CD14 +  cells (i.e. monocytes) in the gated CD45 +  (leukocytes) cells present in this whole blood volume. 
         [0058]    The results are represented in  FIG. 2 . The results showed a 4.4-fold mean increase at Day 170 (2.8-fold at Day 85) when IMP321 was given at low dose (0.25 mg) for a long period of time, 6 months, with 12 injections, showing strong and direct stimulation of the targeted MHC class II +  monocyte-like hematopoietic cells. 
       Example 2 
     Monocytes Increase in Metastatic Renal Clear-cell Carcinoma (MRCC) Patients Using High IMP321 Dose 
       [0059]    Three MRCC patients each received one subcutaneous IMP321 dose of 6.25 mg every other week, for 12 weeks, separated by 14-day administration-free intervals. Blood samples were collected as described above from each patient, 14 days after the last IMP321 injection (i.e. looking at lasting immunomodulatory effects of the product), at 2 months (Day 57) and 3 months (Day 85), and used immediately. The expansion of CD14 +  CD45 +  cells was analysed by FACS in fresh blood samples (because monocytes are sensitive to freezing), and compared with the monocyte counts carried out on fresh blood samples collected before IMP321 administration (Day 1). 
         [0060]    The results are represented in  FIG. 3 . The results showed a 2-fold (at 3 months, Day 85) mean increase of monocyte counts with this high IMP321 dose clinical protocol where patients received only 6 injections. 
       Example 3 
     Monocytes Increase in Metastatic Breast Carcinoma Patients Receiving Paclitaxel and IMP321 Doses 
       [0061]    Patients receiving as a first line chemotherapy for metastatic breast carcinoma 6 cycles of paclitaxel (80 mg/m 2  given i.v.) on days 1, 8, and 15 of a 28 day cycle, received 1-30 mg s.c. (sub-cutaneous) IMP321 on days 2 and 16 of each 28-day cycle. Alternatively, IMP321 was administered at days 3 or 17. Accordingly, each patient received a standard 6-month course of weekly paclitaxel with 12 s.c. injections of IMP321, each injection being given one to two days after paclitaxel administration on an every-two-week schedule. The increase in absolute monocyte counts per microliter of fresh blood was analysed by fluorescence-activated cell sorting (FACS), 14 days after the last injection, at 3 months (Day 85) and 6 months (Day 170) compared to day 1. 
         [0062]    The results obtained in patients injected with a low dose IMP321 (1.25 mg) are represented in  FIG. 4 . These data showed that doses of 1.25 mg in most if not all patients ( FIG. 4 ) induce an expansion of the monocyte subset pool in the blood. It is predicted that the optimal dose regimen for IMP321 will be between 6 and 30 mg/injection. These doses have been shown to be safe and give an acceptable systemic exposure based on the results of pharmacokinetics data obtained in metastatic renal cell cancer patients ( FIG. 5 ). A blood concentration of IMP321 superior to 1 ng/ml for at least 24 hours after s.c. injection could be obtained in patients injected by IMP321 doses of more than 6 mg ( FIG. 5 ). 
       Example 4 
     Treatment of Advanced Pancreas Cancer Patients Receiving Gemcitabine and IMP321 Doses 
       [0063]    Patients, receiving as a first line chemotherapy for advanced pancreas cancer (or patients not eligible for surgical removal of the tumor) 6 cycles of standard gemcitabine (1 gm/m 2  given i.v. over 30 min) on days 1, 8, and 15 of a 28 day cycle, receive in addition 6 to 30 mg s.c. IMP321 on days 2 and 16 of each 28-day cycle. Alternatively, IMP321 is administered at days 3 or 17. Accordingly, each patient receives a standard 6-month course of gemcitabine with 12 s.c. injections of IMP321, each injection being given one to two days after gemcitabine administration on an every-two-week schedule. The number of monocytes is analysed by fluorescence-activated cell sorting (FACS) as in example 1. 
       Example 5 
     Induction of Increased Rituximab-mediated ADCC by Low Doses IMP321 
       [0064]    PBMCs are first incubated for 40 hours with IL-2 (100 U/ml), with or without IMP321 (at the concentrations 0 μg/m, 0.03 μg/ml or 0.1 μg/ml). PBMCs are then incubated with increasing concentrations of rituximab (0, 0.5 and 5 μg/ml) in the presence of target cells (i.e. human CD20 +  Raji B cells). Raji cells were first labeled with CFSE (carboxy-fluorescein succinimidyl ester), incubated in medium with rituximab at 0, 0.5 or 5 μg/ml and cocultured with effector cells at an effector-target ratio of 25:1 for 6 hours at 37° C. The cells were then incubated with 7-AAD (7-amino-actinomycin-D) for 15 min on ice and analyzed by flow cytometry to determine the percentage of dead CFSE +  7-AAD +  Raji target cells (i.e. % of cytotoxicity). 
         [0065]    The results are presented in  FIG. 6 . Increasing the concentration of rituximab increased the percentage of cytotoxicity, clearly showing a dose-dependent ADCC activity. When 0.03 or 0.1 μg/ml IMP321 is added, the percentage of cytotoxicity greatly increased. For instance, a 30% cytotoxicity is observed with 0.5 μg/ml rituximab in the presence of 0.1 μg/ml IMP321 which is superior to the 25% cytotoxicity value obtained with 5 μg/ml rituximab in the absence of IMP321. Thus, adding 0.1 μg/ml IMP321 potentializes 10-15 fold the activity of rituximab because a superior cytotoxicity is obtained with 10 time less antibody when a low dose IMP321 (0.1 μg/ml) is added. These data show the synergistic effect between rituximab and IMP321. 
       REFERENCE LIST  
       [0000]    
       
         [1] TRIEBEL et al., J. Exp. Med., 171 : 1393-1405, 1990; 
         [2] BRIGNONE et al., J. Immune Based Ther Immunotherapies, 5: 5, 2007; 
         [3] TRIEBEL et al., Trends Immunol., 24 : 619-622, 2003; 
         [4] HUARD et al., Proc. Natl. Acad. Sci. USA, 94 : 5744-5749, 1997; 
         [5] PRIGENT et al., Eur. J. Immunol., 29 : 3867-3876, 1999; and 
         [6] BRIGNONE et al., J. Immunol., 179: 4202-4211, 2007.