Patent Application
20210317210
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 3, 2021, is named LB64510-1 D1C1 US SEQ LIST and is 21.3 KB in size.
The present invention is in the field of immunotherapy. More specifically, it relates to the treatment or prevention of organ transplant rejection or for treating autoimmune disease. The invention relates to molecule binding to LAG-3 protein and causing depletion of LAG-3+ activated T cells. More specifically, it relates to cytotoxic LAG-3-specific monoclonal antibody or fragment thereof.
Lymphocyte activation gene-3 (LAG-3, CD223) is upregulated during the early stages of T-cell activation. The present invention is based on the analysis of the effects of cytotoxic antibodies against LAG-3 in acute cardiac allograft rejection (in vivo animal studies) and in in vitro experiments where selected LAG-3 monoclonal antibodies are efficient at low doses (<0.1 μg/ml) at depleting LAG-3+ activated effector T cells.
Selectively depleting activated T lymphocytes might represent an immunosuppressive induction treatment able to result in the development of regulatory cells supporting a long-term survival of allogeneic organs in mice and primates (1). This has actually been demonstrated with anti-CD40L antibodies that deplete in vivo activated T cells through a Fc-dependent mechanism (2). However, anti-CD40L antibodies also target activated platelets in humans and affect the stability of arterial thrombi (3). Therefore the development of monoclonal antibodies to other molecules specific for T-cell activation has catalyzed attempts to achieve immunosuppression. One such molecule is LAG-3, which engages Class II on dendritic cells (DC) with a high affinity, enabling DC to become activated (4-6). The LAG-3 protein is expressed in vivo in activated CD4+ and CD8+ lymphocytes residing in inflamed secondary lymphoid organs or tissues but not in spleen, thymus or blood (7). In addition, LAG-3 can function as a negative regulator of activated human CD4 and CD8 T cells by inhibiting early events in primary activation (8).
The invention provides a molecule binding to LAG-3 protein and causing depletion of LAG-3+ activated T cells. Said depletion can be measured by changes in peripheral blood lymphocyte numbers, in a tissue or an organ.
In a preferred embodiment the molecule binding to LAG-3 protein is a cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof causing depletion of LAG-3+ activated T cells, said antibody comprising an Fc fragment from the human IgGI or IgM (or mouse IgG2a) subclass and an Fab fragment which binds LAG-3 protein, said antibody exhibiting a complement-dependent cytotoxicity (CDC) and/or an antibody dependent cell cytotoxicity activity (ADCC).
In one embodiment, the anti-LAG-3 monoclonal antibody is IMP731 or a biologically active fragment thereof.
Another aspect of the invention is a method of depleting LAG-3+ activated T cells in a mammal, the method comprising administering monoclonal antibody IMP731 or a biologically active fragment thereof to said mammal.
Another embodiment of the invention is a pharmaceutical composition comprising monoclonal antibody IMP731 or a biologically active fragment thereof.
Another embodiment of the invention is a method of treating diseases associated with LAG-3+ activated T cells in a subject, the method comprising administering an effective amount of a pharmaceutical composition comprising monoclonal antibody IMP731 or a biologically active fragment thereof to said subject. Such disease may be an auto-immune disease. Exemplary auto-immune diseases include autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, mixed essential cryoglobulinemia, systemic lupus erythematosus, insulin-dependent diabetes mellitus, rheumatoid arthritis, Grave's disease, Hashimoto's thyroiditis, myasthenia gravis, psoriasis and multiple sclerosis.
The present invention further provides a method for treating or preventing organ transplant rejection or for treating auto-immune disease. Said method comprises the administration, to a mammalian subject, of a therapeutically effective amount of a cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof. In one embodiment, the monoclonal antibody is IMP731.
Another embodiment of the invention is directed to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the kappa region of IMP731 or a biologically active fragment thereof.
Another embodiment is directed to a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence encoding the kappa region of IMP731 or a biologically active fragment thereof.
Another embodiment is directed to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the gamma region of IMP731 or a biologically active fragment thereof.
Another embodiment is directed to a polypeptide encoded by nucleic acid molecule comprising a nucleic acid sequence encoding the gamma region of IMP731 or a biologically active fragment thereof.
Another embodiment is directed to an antibody that binds LAG-3 protein and depletes LAG-3+ activated T cells and comprises a light chain variable region comprising the 3 CDRs in
Another embodiment of the invention is directed to a method of identifying a molecule that binds LAG-3 protein and may deplete LAG-3+ activated T cells, the method comprising providing an assay in which a candidate molecule and IMP371 compete for binding on an LAG-3 protein, wherein when said candidate blocks IMP371 from binding said LAG-3 protein, said candidate is identified as a molecule that binds LAG-3 protein and may deplete LAG-3+ activated T cells.
Another embodiment of the invention is directed to a method of assessing a candidate molecule for cytotoxicity on LAG-3+ activated T cells, the method comprising incubating T-cells in a CMV peptide containing medium with the candidate molecule and under the same conditions, incubating T-cells in a CMV peptide containing medium without the candidate molecule and comparing the percentage of T cells expressing an activation marker with the candidate molecule against the percentage of T cells expressing an activation markers without the candidate molecule, wherein a reduction in the percentage of activated T-cells with the candidate molecule indicates the cytotoxicity of the candidate molecule on LAG-3+ activated T cells.
A) hLAG-3-transfected CHO were dissociated from culture plastic using Versene buffer containing cation-chelating agent, incubated with indicated concentrations of A9H12 or
17B4 mAbs for 30 min at 40 C, washed and then incubated with a FITC-conjugated goat anti-mouse IgG+M (H+L) secondary antibody (5 μg/ml, Coulter Inc.) for 30 minutes at 4° C. After washing, cells were analysed by flow cytometry using a FACSCanto™ (BD Biosciences) and means of fluorescence intensity were plotted as a function of antibody concentration.
B) PBMCs from a healthy volunteer were stimulated for 2 days with SEB (1 μg/ml, Sigma Aldrich) to induce the expression of LAG-3 on T cells. PBMCs were stained as above. Data represent a weighted percentage, calculated as the percentage of LAG-3+ cells in PBMCs×mean of fluorescence intensity of the LAG-3+ cells, plotted as a function of antibody concentration.
A) hLAG-3-transfected and wild type CHO cells were labelled with FITC-conjugated anti-LAG-3 mAb (17B4) and the expression of LAG-3 on cell surface was analysed by flow cytometry using a FACSCanto™. The histogram plots represent the mean fluorescence intensity of wt CHO (gray) and LAG-3+ CHO (dark).
B) hLAG-3-transfected and wt CHO cells were washed in complete medium (MEM supplemented with 10% heat inactivated Foetal Calf Serum, FCS) and incubated with 0.1 μg/ml of A9H12 LAG-3 mAb or mlgG2a isotype-control mAb (Southern Biotechnologies) in complete medium for 30 min at 4° C. Cells were then washed and incubated in complete medium (-Complement) or in MEM supplemented with 10% of freshly resuspended rabbit serum (Cerdalane Inc.) (+Complement) for 1 hour at 37° C. After washing, cells were stained with 7-AAD (Coulter Inc.) for 15 minutes at room temperature and immediately analysed by flow cytometry to determine the percentage of 7-AAD-positive cells corresponding to dead cells. Data represent the percentage of dead cells in each condition on hLAG-3-transfected and wt CHO cells as indicated.
C) LAG-3+ CHO cells were incubated with indicated concentrations of A9H112 LAG-3 mAb for 30 min at 4° C. and then incubated with MEM supplemented with 25% rabbit serum for 1 hour at 37° C. After washing, cells were stained with 7-AAD (Coulter Inc.) and analysed by flow cytometry. The percentage of specific cytotoxicity is calculated according to the following formula
(Sample Death−Spontaneous Death)×100
(Maximal Death−Spontaneous Death)
where Sample Death is the percentage of 7-AAD-positive cells in each condition, Spontaneous Death, the percentage of 7-AAD-positive cells without mAb and Maximal Death, the percentage of 7-AAD-positive cells with 10 μg/ml mAb.
D) LAG-3+ CHO cells were incubated with 0.1 μg/ml A9H12, 17B4 or 31 G11 LAG-3 mAb or with their corresponding isotype controls (IgG2a, IgG1 or IgM, respectively) for 30 min at 4° C. and then incubated with MEM supplemented with 25% rabbit serum for 1 hour at 37° C. Specific cytotoxicity was determined as above with a Maximal Death corresponding to 10 μg/ml A9H12 (left panel) and 0.1 μg/ml A9H112 (right panel).
E) PBMCs were stimulated with SEB (1 μg/ml) to induce LAG-3 expression on T cells and then used as target cells in the CDC assay in the presence of A9H112 or 31 G11 LAG-3 mAb or their isotype controls. After staining cells with fluorochrome-conjugated CD3, CD4, CD8, CD25 and 17B4, the percentage of 7-AAD-positive cells was analysed on the indicated T cell subpopulations. Data represent the percentage of dead cells in each population (with spontaneous death in the absence of mAb being subtracted).
A) Effector cells (PBMCs) were stimulated with IL-2 (100 IU/ml, BD Biosciences) for 1 day. Target cells (hLAG-3-transfected CHO cells) were labelled with CFSE (a fluorescent vital dye) and incubated with 1 μg/ml A9H12, mlgG2a, 17B4 or mlgGI for 20 min at room temperature. Effector cells and target cells were then mixed at indicated E:T ratios (E:T, Effector:Target) and incubated for 16 hours at 37° C. Non-adherent and adherent cells were harvested using Versene reagent, stained with 7-AAD and immediately analysed by flow cytometry to determine the percentage of 7-AAD-positive cells in the CFSE-positive population. Data represent the percentage of dead cells, with the non-specific cell death in the presence of the isotype control being subtracted.
B) CFSE-labelled wild-type or LAG-3+ CHO target cells were incubated with indicated concentrations of A9H12 or mlgG2a and IL-2-stimulated PBMCs were added at a 50:1 E:T ratio and incubated for 16 hours at 37° C. Cell death was analysed as above and data represent the percentage of dead cells in CFSE-positive cells in the presence of A9H12 or its isotype-matched IgG2a control mAb.
Male DBA/1 mice (n=22) were injected i.d. with bovine type II collagen (200 μg) emulsified in CFA containing 250 lug M. tuberculosis.
A) hLAG-3-transfected CHO were dissociated from culture plastic using VERSENE™ buffer containing cation-chelating agent, incubated with indicated concentrations of IMP731 Ab or its isotype control hIgG1 (Chemicon) for 30 min at 4° C., washed and then incubated with a FITC-conjugated F(ab)′2 goat anti-human IgG1 secondary antibody (5 μg/ml, SBA) for 30 minutes at 4° C. After washing, cells were analysed by flow cytometry using a FACSCanto™ (BD Biosciences) and the means of fluorescence intensity were plotted as a function of antibody concentration.
B) PBMCs from a healthy volunteer were stimulated for 2 days with SEB (1 μg/ml, Sigma Aldrich) to induce the expression of LAG-3 on T cells. PBMCs were stained as above. Data represent a weighted percentage, calculated as the percentage of LAG-3+ cells in PBMCs×mean of fluorescence intensity of the LAG-3+ cells, plotted as a function of antibody concentration.
hLAG-3-transfected CHO cells were incubated with 1 μg/ml of IMP731 Ab or hlgGI isotype-control mAb (Chemicon) in complete medium (MEM supplemented with 10% heat inactivated Foetal Calf Serum, FCS) for 30 min at 4° C. Cells were then washed and incubated in complete medium (without Complement) or in MEM supplemented with 25% of freshly resuspended rabbit serum (Cerdalane Inc.) (with Complement) for 1 hour at 37° C. After washing, cells were stained with 7-AAD (BD Biosciences) for 15 minutes at room temperature and immediately analysed by flow cytometry to determine the percentage of 7-AAD-positive cells corresponding to dead cells. Data represent the percentage of dead cells in each condition as indicated.
A) Effector cells (PBMCs) were stimulated with IL-2 (100 IU/ml, BD Biosciences) for 1 day. Target cells (hLAG-3-transfected CHO cells) were labelled with CFSE (a fluorescent vital dye) and incubated with 1 μg/ml IMP731 or hlgGI for 10 min at room temperature. Effector cells and target cells were then mixed at indicated E:T ratios (E:T, Effector:Target) and incubated for 16 hours at 37° C. Cells were stained with 7-AAD and immediately analysed by flow cytometry to determine the percentage of 7-AAD-positive cells in the CFSE-positive population. Data represent the percentage of dead cells.
B) CFSE-labelled LAG-3+ CHO target cells were incubated with indicated concentrations of IMP731 or hlgGI and IL-2-stimulated PBMCs were added at a 50:1 E:T ratio and incubated for 16 hours at 37° C. Cell death was analysed as above in CFSE-positive population. The percentage of specific cytotoxicity, calculated according to the following formula
(Sample Death−Spontaneous Death)×100
(Maximal Death−Spontaneous Death)
where Sample Death is the percentage of 7-AAD-positive cells in each condition, Spontaneous Death, the percentage of 7-AAD-positive cells without Ab and Maximal Death, the percentage of 7-AAD-positive cells with 1 μg/ml IMP731
C) Effector cells (PBMCs) were stimulated with IL-2 (100 IU/ml, BD Biosciences) for 1 day. Target cells (hLAG-3+ CHO cells or hLAG-3− CHO cells) were labelled with CFSE (a fluorescent vital dye) and incubated with 1 μg/ml IMP731 or hlgGI for 10 min at room temperature. Effector cells and target cells were then mixed at indicated E:T ratios (E:T, Effector:Target) and incubated for 16 hours at 37° C. Cells were stained with 7-AAD and immediately analysed by flow cytometry to determine the percentage of 7-AAD-positive cells in the CFSE-positive population. Data represent the percentage of dead cells.
The present invention provides molecules binding to LAG-3 protein and causing depletion of LAG-3+ activated T cells. Said depletion can be measured by changes in peripheral blood lymphocyte numbers, a tissue or an organ.
The present invention relates preferably to human LAG-3 protein (hLAG-3 also named hereafter LAG-3). In a preferred embodiment the molecule binding to LAG-3 protein is a cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof causing depletion of LAG-3+ activated T cells, said antibody comprising an Fc fragment from the human IgGI or IgM (or mouse IgG2a) subclass and an Fab fragment which binds LAG-3 protein, said antibody exhibiting a complement-dependent cytotoxicity (CDC) and/or an antibody dependent-cell cytotoxicity activity (ADCC).
Lymphocyte activation gene-3 (LAG-3, CD223) is up-regulated during the early stages of T-cell activation. The present invention is based on the analysis of the effects of cytotoxic antibodies against LAG-3 in acute cardiac allograft rejection and in rheumatoid arthritis (in vivo animal studies) and in in vitro experiments where selected LAG-3 monoclonal antibodies are efficient at low doses (<0.1 μg/ml) at depleting LAG-3+ activated effector T cells.
Fully vascularized heterotopic allogeneic heart transplantation was performed in rats across a full-MHC mismatch barrier (LEW.1W into LEW.1A). Recipients received two injections (day 0 and 3) of antibodies directed to the extraloop domain of LAG-3 or control antibodies. Graft survival, histology, mRNA transcripts and alloreactivity of lymphocytes were tested.
It was first noted that LAG-3 mRNA molecules accumulate in cardiac allografts undergoing rejection, but not in peripheral lymphoid organs. Administration of anti-LAG-3 antibodies inhibited graft infiltration by effectors mononuclear cells and prolonged allograft survival from 6 days in control antibodies-treated recipients to a median of 27 days.
It was found that cells expressing LAG-3 infiltrate rejected heart allografts and that targeting LAG-3 using cytotoxic antibodies as induction monotherapy delays acute rejection by reducing graft infiltration by T cells and monocytes.
Experiments showing that short courses of CD40L antibody therapy could achieve long-term graft survival in mice and primates have been initially interpreted as an effect of costimulation blockade. However, Monk et al. (2) showed that much of the efficacy of anti-CD40L therapy derives not from costimulation blockade, but from destruction of activated T cells. The outcome is a selective purging of potentially aggressive T cells that have experienced antigen.
Collagen-induced arthritis (CIA) is a well-described animal model for rheumatoid arthritis. Collagen-induced arthritis is an autoimmune disease inducible in rats, mice and primates by immunization with heterologous type II collagen. The resulting joint pathology resembles rheumatoid arthritis with synovial proliferation, cell infiltration, cartilage erosion and bone resorption in the most severe cases (12).
Using particular immunization protocols, early studies have established a hierarchy of responsiveness to CIA linked to the H-2 haplotype, with H-2q (e. g. DBA/1 mice) being the most and H-2b (e.g. C57BL/6 mice) amongst the least responsive strains. However, some studies have shown that responsiveness to CIA may be less restricted by the MHC class II than previously thought and may be just as dependent on immunization conditions (13). The variety of type II collagen (CII) from different species and the preparation of complete Freund's adjuvant (CFA) with different concentrations of Mycobacterium tuberculosis were important parameters for arthritis development. Inglis et al. have shown that chicken, but not bovine, CII was capable of inducing disease in C57BL/6 mice, with an incidence of 50% to 75%. This is in contrast to DBA/1 mice, in which bovine, mouse and chicken CII all induced disease, with an incidence of 80% to 100%. The phenotype of arthritis was milder in C57BL/6 mice than in DBA/1 mice, with less swelling and a more gradual increase in clinical score (14). Moreover, male mice are frequently preferred for CIA studies, as the incidence of arthritis is somewhat higher in male than in female mice.
In mice, CIA is induced by an i.d. injection of type II collagen (CII) in the presence of CFA, usually followed by an i.p. boost injection of CII, without adjuvant, 21 days later. However, there are reported variations for almost every aspect of the immunization procedure and even in the highly susceptible DBA/1 strain the time of onset, severity and incidence of CIA can be variable (13, 15).
Therapeutic antibodies for the treatment of auto-immune diseases have already been described, like the TNFa mAbs in rheumatoid arthritis. By definition, LAG-3 (Lymphocyte Activation Gene-3) is a marker for recently activated effector T cells. Depleting these effector LAG-3+ T cells will lead to targeted immunosuppression (i.e. only activated T cells are suppressed, not all T cells as with corticoids or cyclosporin). This very specific immunosuppression should lead to higher therapeutic indices compared to classical immunosuppressive agents or to therapeutic antibodies (e.g. HUMIRA™, REMICADE™) or soluble receptors (e.g. ENBREL™) blocking TNFa. Thus, LAG-3 is a promising target available for a therapeutic depleting mAb approach to eliminate auto-reactive activated effector T cells.
Molecules that bind to LAG-3 protein and cause depletion of LAG-3+ activated T cells, according to the present invention, include antibodies (mono or polyclonal, preferably monoclonal) and fragment thereof, peptides and organic small molecules.
Cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof according to the present invention causes depletion of more than 30% preferably more than 50% of LAG-3+ activated T cells.
Cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof according to the invention comprises antibodies with a murine IgG2a or a human IgGI Fc region giving strong CDC or ADCC properties.
Cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof according to the present invention exhibits (i) more than 50% of maximal CDC activity at a mAb concentration of less than 0.1 μg/ml and/or (ii) more than 50% of maximal ADCC activity at a mAb concentration of less than 0.1 μg/ml.
Cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof, which suppresses a DTH reaction in a mammal after a single dose of 0.1 mg/kg.
Molecules binding to LAG-3 protein and more particularly cytotoxic anti-LAG-3 monoclonal antibody, causing depletion of LAG-3+ activated T cells and antibody, can be produced by methods well known to those skilled in the art.
Antibodies generated against CD223 polypeptides can be obtained by administering, in particular by direct injection, CD223 polypeptides to an animal, preferably a non-human. The antibody so obtained will then bind the CD223 polypeptides itself. In this manner, even a sequence encoding only a fragment of the CD223 polypeptide can be used to generate antibodies binding the whole native CD223 polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (9), the trioma technique, the human B-cell hybridoma technique (10).
Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be readily used to produce single chain antibodies to CD223 polypeptides. Also, transgenic mice may be used to express humanized antibodies to immunogenic CD223 polypeptides.
An antibody of the present invention includes intact antibodies, an antibody fragment, a humanized antibody, a conjugate, a fusion protein or a bi-specific antibody that contains a VH-VL pair where the CDRs form the antigen binding site. The invention also includes antibodies in which the Complementarity Determining Regions (CDRs) of one of the antibodies of the invention, such as those set forth in
For instance, a first monoclonal antibody according to the present invention, called A9H12, is produced by the hybridoma deposited at the CNCM on Apr. 27, 2007 under the access number CNCM I-3755.
A second monoclonal antibody according to the present invention, called 31 G11, is produced by the hybridoma deposited at the CNCM on Apr. 27, 2007 under the access number CNCM I-3756.
A third monoclonal antibody according to the present invention, called IMP731, is produced by chimerization of monoclonal antibody A9H112 with human IgG1 Fc region, as described and characterized in Examples 4, 5 and 6.
The antibodies of the present invention include those that have the same CDRs as the above antibodies (A9H12, 31 G11 or IMP731) but have differences due to glycosylation patterns or post translational modifications.
The invention is also directed to the use of a cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof for the manufacture of a medicament for treating or preventing organ transplant rejection or for treating autoimmune disease.
Biologically active fragments of the monoclonal antibodies of the present invention exhibit some or all of the biological activities of the monoclonal antibody. For example, biologically active fragments of IMP731 exhibit the same high binding affinity as IMP731, as assessed in a standard assay such as a BIACORE™ analysis. Biological activity may also be assessed by other in vitro or in vivo assays, which are known to the skilled artisan and which are used to assess and characterize the monoclonal antibodies of the invention. One such assay assesses DTH reaction in baboons. Another in vitro assay is the ADCC bioassay described in Example 5 and
The present invention also relates to an antibody other than IMP731 which comprises the kappa or gamma regions of IMP731 and which has the same biological activity as IMP731. In another embodiment, the invention relates to antibodies or fragments thereof that compete with IMP731 in binding LAG-3 protein and in causing depletion of LAG-3+ activated T cells.
The nucleic acid sequences encoding the kappa and gamma regions of IMP 731 are set forth in
The present invention further provides a method for treating or preventing organ transplant rejection or for treating autoimmune disease. Said method comprises the administration to a mammalian subject a therapeutically effective amount of a cytotoxic anti-LAG-3 monoclonal antibody or fragment thereof.
Organ transplant rejection refers to the graft of an organ in an allogenic host. It may be useful for treating organisms suffering from conditions resulting in an abnormally high T-cell population or deleterious T-cell activity, for example graft rejection mediated by host T-cells, graft vs. host disease and T-cell mediated autoimmune and inflammatory diseases such as rheumatoid arthritis, type 1 diabetes, muscular sclerosis, etc. The methods of the invention may be applied to any organism which contains T-cells that express CD223. This includes, but is not limited to, any mammal and particularly includes humans and mice.
Auto-immune diseases are diseases in which the subject's own immune system reacts against the subject cells. Auto-immune disease which are amenable to treatments according to the present invention include autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, mixed essential cryoglobulinemia, systemic lupus erythematosus, insulin-dependent diabetes mellitus, rheumatoid arthritis, Grave's disease, Hashimoto's thyroiditis, myasthenia gravis, psoriasis and multiple sclerosis.
A method for depleting LAG-3+ activated T cells from a sample from a patient according to the present invention comprises reacting the sample with an antibody composition comprising an antibody described above.
A pharmaceutical composition according to the present invention comprises from 0.1 to 300 mg per dose, preferably from 0.5 to 30 mg per dose, and more preferably from 5 to 10 mg per dose, of a cytotoxic monoclonal antibody described above and one or more pharmaceutically acceptable carriers, adjuvants and/or diluents for administration to a mammal. In another embodiment, the pharmaceutical composition according to the invention comprises from 0.01 to 30 mg per kg, preferably from 0.1 to 3 mg per kg, and more preferably from 0.5 to 1 mg per kg of the cytotoxic monoclonal antibody described above. One of skill in the art would understand that the pharmaceutical compositions of the invention could be adjusted to contain amounts of the cytotoxic monoclonal antibody that would vary depending upon the health, age, weight, and condition of the subject being treated.
The pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form according to methods well known to the skilled artisan. Suitable carriers, for instance, vehicles, adjuvants, excipients and diluents are well known to the skilled artisan. Suitable formulations include formulations for oral, aerosol, parenteral, subcutaneous, transdermal, transmucosal, intestinal, intramedullary, intravenous, intranasal, intraocular, intravenous and interperitoneal administration or injection are exemplary and are not intended to be limiting. Techniques for formulating the pharmaceuticals of the present invention may be found in Remington's Pharmaceutical Sciences 18th ed. Mack Publishing Co., Easton, Pa. (1990).
The pharmaceutical compositions of the invention may contain the cytotoxic monoclonal antibody of the invention in combination with one or more other therapeutic agents.
The following examples further illustrate the invention but should not be construed as limiting the scope of the invention.
Material and Methods
Animals and Transplantations
Eight- to 12-week-old male Lewis. IW (LEW. 1W, haplotype RTu) and Lewis. IA (LEW. 1A, haplotype RT1a) congeneic rats (Centre d'Elevage Janvier, Le Genest-Saint-Isle, France), differed in their entire MHC region. Heterotopic LEW.IW heart transplantation was performed as previously described (11). Graft survival was evaluated by palpation through the abdominal wall.
Anti-LAG-3 Antibodies
A synthetic peptide corresponding to the extraloop domain of the rat LAG-3 protein (NCBI accession nb DQ438937; peptide DQPASIPALDLLQGMPSTRRHPPHR) (SEQ ID NO. 1) was linked to ovalbumin and used to immunise two rabbits. Pre-immune and immune sera, collected on day 63 after the 4th immunisation, were assayed by ELISA on immunogen and peptide and by flow cytometry on Con-A activated rat spleen cells. Pre-immune sera were negative in both assays. Pooled immune sera presented a titer (dilution for 50% signal) of 1/60000 by ELISA and of 1/1000 by FRCS, and presented a specificity for activated T cells.
Complement-Dependent Cytotoxicity Assay
Complement-mediated antibody-dependent cytotoxicity was tested using rabbit sera against Lewis 1A T cells in a 51Cr release assay. A total of 2×106 Lewis 1 A T cells were labelled with 30 μCi of 51Cr for 90 min at 37° C. in RPMI (GIBCO) with 10% FCS. After three washes, T cells were distributed in 96 V-bottomed plates and incubated with rabbit complement and serial dilutions of heat-inactivated rabbit serum. After 4 h at 37° C., 51Cr release was measured in the supernatants using a scintillation counter. Specific cytotoxicity was calculated according to the following formula: (experimental release−spontaneous release)×100/(maximum release−spontaneous release).
In Vivo Antibody-Induced Cytotoxicity
Cytotoxic activity of anti-LAG-3 antibodies against LAG-3+ cells was evaluated in vivo. ConA-activated (48 h) LEW.1 W splenocytes were labelled with the CFSE and transferred (108 cells) into irradiated (4.5 Gy, day −3) LEW.IA recipients, together with anti-LAG-3 antibodies. On day 1, recipients were sacrificed and the presence of CFSE-positive cells evaluated by flow cytometry in lymphoid organs and in the blood.
Immunostaining
Graft samples were embedded in Tissue Tek (OCT Compound, Torrance, Calif., USA), snap-frozen in liquid nitrogen, cut into 5 μm sections and fixed in acetone. Endogenous biotin activity was blocked using the Dako biotin blocking system (Dako, Trappes, France). Sections were then labelled by a three-step indirect immunoperoxidase revelation. The area of each immunoperoxidase-labeled tissue section infiltrated by cells was determined by quantitative morphometric analysis. Positively stained cells on each slide were counted by morphometric analysis using point counting analysis (14) with a 121-intersection squared grid in the eyepiece of the microscope. Briefly, the percentage of the area of each graft section occupied by cells of a particular antigenic specificity (area infiltrate) was calculated as follows: [number of positive cells under grid intersection/(total number of grid intersections=121)]×100. The graft sections were examined at a magnification of ×400. The accuracy of the technique is proportional to the number of points counted. Thus, to maintain a SE of <100, 15 fields were counted for each labeled section. Results are expressed as the percentage of the area of the tissue section infiltrated by leukocytes (determined with OX1, OX30 labeling) and the phenotypic composition of the infiltrate and subpopulations which are related to the percentage of total leukocytes and are expressed as the percentage of leukocytes.
Graft Infiltrating Cell Extraction Staining
Dilacerated hearts were digested with collagenase D (2 mg/ml; Boehringer Mannheim) for 10 min at 37° C. Cells were then collected by extraction through a stainless steel mesh. The resulting suspension was then clarified by Ficoll isolation.
Quantitative RT-PCR
Real-time quantitative PCR was performed in an Applied Biosystems GENAMP™ 7700 Sequence Detection System using SYBR™ Green PCR Core Reagents (Applied Biosystems, Foster City, Calif.). The following oligonucleotides were used in this study: rat LAG-3: upper primer is 5′-ATATGAATTCACAGAGGAGATGAGGCAG-3′ (SEQ ID NO. 2) and lower primer is 5′-ATATGAATTCTCCTGGTCAGAGCTGCCT-3′ (SEQ ID NO. 3) Rat INFg: upper primer is 5′-TGGATGCTATGGAAGGAAAGA-3′ (SEQ ID NO. 4) and lower primer is 5′-GATTCTGGTGACAGCTGGTG-3′ (SEQ ID NO. 5) Rat HPRT: upper primer is 5′-CCTTGGTCAAGCAGTACAGCC-3′ (SEQ ID NO. 6) and lower primer is 5′-TTCGCTGATGACACAAACATGA-3′ (SEQ ID NO. 7) A constant amount of cDNA corresponding to the reverse transcription of 100 μg of total RNA was amplified in 25 MI of PCR mix containing 300 nM of each primer; 200 μM dATP, dGTP, dCTP; 400 μM dUTP; 3 mM MgCl2; 0.25 U of uracyl-N-glycosylase; 0.625 U of AmpliTaq Gold DNA polymerase. The mix was subjected to 40 cycles of amplification. The real-time PCR data were plotted as the ΔRn, fluorescence signal vs. the cycle number. The ΔRn values were calculated by the Applied Biosystems 7700 sequence detection software using the formula: ΔRn=(Rn+)−(Rn−), where Rn+ is the fluorescence signal of the product at any given time, Rn− is the mean fluorescence signal during cycles 3-13 and referred to as the baseline. The Ct value is defined as the cycle number at which the ΔRn crosses a threshold. The threshold is set above the background fluorescence to intersect the exponential portion of the amplification curve of a positive reaction. The Ct is inversely proportional to the log amount of template within the PCR.
Statistical Analyses
Statistical significance was evaluated using a Mann-Whitney test for the comparison of two groups. Graft survival was evaluated by Kaplan-Meier analysis using the log rank test.
Results
LAG-3 mRNA expression in rejected allograft and lymphoid organs LAG-3 is expressed by activated T cells in inflamed lymphoid organs and tissues (7). In order to see if LAG-3 is also expressed in rejected allografts, hearts grafts from LEW.1W to LEW.IA rat recipients were analyzed on day 5 (rejection occurring on day 6). Messenger RNA for LAG-3 was analyzed and compared with allografts receiving a tolerance-inducing regiment (anti-CD28 antibodies+CSA, as described (16)) and with isografts. Rejected allografts presented a 7-fold and a 25-fold accumulation of LAG-3 mRNA as compared with tolerated and syngeneic grafts, respectively (
Mechanism of Action of Anti-LAG-3 Antibodies
Anti-LAG-3 antibodies were produced in rabbits by immunization with a synthetic peptide from the extra-loop of LAG-3 Ig-like N-terminal domain, involved in the interaction of LAG-3 with Class II (ref PNAS Huard 1997). Post-immune serum, as well as the IgG fraction, stained <1% of rat spleen cells and 400 of rat spleen cells activated for 48 h with ConA, PMA+ionomycin or PHA. Pre-immune serum was negative (data not shown). In order to characterize the effect of anti-LAG-3 antibodies on LAG-3+ cells, complement and ADCC-dependent cytotoxicity was assayed in vitro. Fifteen % of ConA-activated spleen cells were lysed in the complement-dependent cytotoxicity assay (
In vivo, the depleting activity of anti-LAG-3 antibodies was estimated by measuring the fate of CFSE-labeled activated T cells adoptively transferred to irradiated rat recipients. One day after the injection of therapeutic doses of anti-LAG-3 immune serum, only half the amount of CFSE+/CD4+ and CFSE+/CD8+ cells could be recovered from the spleen, as compared with similar injections of pre-immune serum (
Anti-LAG-3 Antibodies Delay Heart Allograft Rejection
From preliminary pharmacokinetic observations, we established that two i.v. injections of 600 μl of anti-LAG-3 rabbit serum on days 0 and 3 resulted in the maintenance of anti-LAG-3 binding activity in recipient's serum for at least 2 weeks. This treatment delayed cardiac allograft rejection from 6 days in untreated and control-treated recipients to a median of 27 days. All recipients, however, eventually rejected their graft within 10 weeks (
Anti-LAG-3 Antibodies Inhibit Ongoing Acute Heart Allograft Delay Rejection
In order to investigate whether anti-LAG-3 antibodies might serve as a treatment of an ongoing acute allograft rejection, we grafted LEW.1W hearts into LEW.1A allogenic recipients that we maintained untreated over 3 or 4 days. At that time, recipients received an injection of 600 microliter of control or anti-LAG-3 rabbit serum. Control-treated recipients rejected the allografts on day 5 whereas anti-LAG-3 antibodies-treated recipients rejected only on day 11 (Table 1).
Table 1: Heart graft recipients were treated on day 3 or 4 with control or anti-LAG-3 antibodies. Rejection was monitored by daily heart palpation.
Material and Methods
Mice were immunized 3 times with hLAG-3-transfected CHO cells (107 cells, intra-peritoneal injection), followed by a boost i.v. injection with 10 μg IMP321, the clinical-grade hLAG-31 g recombinant protein. Three days after the boost, splenocytes were fused with the X63.AG8653 fusion partner to yield hybridoma cells. Supernatants from hybridomas were screened for their specific binding (FACS analysis) on hLAG-3-tranfected CHO versus wild type (wt) CHO cells.
One murine IgG2a antibody (580.1 E MM H12, called A9H12) was selected, subcloned to yield a stable cell line and further characterized for its potency to deplete LAG-3+ cells through CDC (complement-dependent cytotoxicity) and ADCC (antibody-dependent cell-mediated cytotoxicity), given that the murine IgG2a Pc region is known to be the most efficient Fc isotype in mice at delivering these activities, even on heterologous cells (i.e. CHO cells or human PBMCs). Similarly, a second IgM antibody (31 G11 E8, called 31 G11) was also selected.
Results
Dose-dependent binding of A9H12 was first compared to the reference LAG-3-specific 17B4 mAb on hLAG-3-tranfected CHO cells and on LAG-3+ in vitro activated human T cells (
For CDC testing, the target cells used in this assay were LAG-3+ CHO cells compared to wt CHO cells (
The CDC bioassay was also performed on PBMCs stimulated with the superantigen SEB. The cytotoxicity of A9H12 and 31G11 were analysed on both activated (namely CD25+/LAG-3+ cells) and non-activated (namely CD25-/LAG-3-cells) CD4+ helper T and CD8+ cytotoxic T cells. Only activated CD4+ and CD8+ T cells were specifically killed by A9H12 and 31G11 (
For ADCC testing, PMBCs were stimulated for one day with IL-2 to serve as effector cells and LAG-3+ CHO cells were labelled with the vital dye CFSE to serve as target cells. In the presence of A9H12, PBMCs were able to kill a significant percentage of LAG-3+ CHO cells and this effect was increased with the number of effector cells (
Material and Methods
Animals and Materials
Male DBA/1 (H-2q) mice, 8-10 weeks old, were obtained from Janvier Laboratories. All animal experiments were performed according to local guidelines. BovineCII (joint cartilage) was purchased from BioCol. Incomplete Freund's adjuvant was provided by Sigma. Heat-killed M. tuberculosis H37Ra was purchased from Difco Laboratories.
Induction of Collagen-Induced Arthritis (CIA)
The induction and assessment of CIA were performed as previously described in two publications (13, 15). Complete Freund's adjuvant was prepared by mixing 100 mg heat-killed Mycobacterium tuberculosis in 13.3 ml IFA (final concentration 7.5 mg/ml). Bovine CII was dissolved at 3 mg/ml in 10 mm acetic acid overnight at 4° C. An emulsion was formed by mixing 2 volumes of CII with 1 volume of CFA. The CII solution and the emulsion with CFA were always freshly prepared. Male DBA/1 mice were intra-dermally injected at the base of the tail with a total of 100 μl of emulsion containing 200 μg CII and 250 μg M. tuberculosis on day 1 (D1). The injection was repeated at day 21 (D21). As control, three mice were injected with the emulsion of CFA without CII.
Clinical Assessment of Arthritis
Mice were examined for signs of arthritis three times a week from day 22. The disease severity was determined with the following scoring system for each limb: score 0=normal; score 1=swelling of footpad or joint; score 2=swelling of footpad and 1 or 2 joints; score 3=swelling of footpad and 3 or 4 joints; score 4=swelling of footpad and all joints. Each paw was graded, and the 4 scores were summed so that the maximum possible score was 16 per mouse. Incidence was expressed as the percentage of mice with an arthritis score ≥1.
Results
CIA was induced by i.d. injections of bovine type II collagen (CII) emulsified in CFA containing 250 μg M. tuberculosis. After one injection, 4 out of 22 mice had developed arthritis at D21. Two weeks after the second injection, at D35, 80-90% of the mice had developed clinical signs of arthritis (
Table 2: Mean clinical scores (±SEM) over 55 days. Male DBA/1 mice (n=10) were injected i.d. with bovine type II collagen (200 mg) emulsified in CFA containing 250 mg M. tuberculosis at D1 and D21.
Our results show that with the CIA protocol used, it is possible to obtain a high percentage (80-90%) of mice developing signs of arthritis. This experimental protocol provides a model to evaluate the therapeutic effect of depleting LAG-3 antibodies (specific for mouse LAG-3) in auto-immune diseases.
200 μg of the depleting LAG-3 mAb (A9H12 or 31 G11) are injected i.p. or i.v. on day 15 and 25. Both a significant decrease in arthritis incidence and a significant lowering of mean clinical scores are involved.
Materials and Methods
A new murine mAb with depleting properties, A9H12, has been shown to recognize also baboon and macaque monkey LAG-3+ cells with high avidity and had been chosen as our lead depleting therapeutic mAb (ImmuTune IMP731).
A9H12 has been chimerized with a human IgG1 Fc region using standard genetic engineering and PCR protocols, to give CDC (complement-dependent cytotoxicity) and ADCC (antibody-dependent cell cytotoxicity) properties.
The A9H12 VH and VL cDNA sequences derived from A9H12 hybridoma cell mRNA were fused upstream of the human CH1-hinge-CH2-CH3 IgGI domains and Ckappa chains, respectively (
The two light and heavy IMP731 chimeric chains were independently cloned into separate expression plasmids (
The two IMP731 light and heavy chains were then cloned together with the PGK (or SRalpha, not shown) promoter in a head-to-tail situation for coordinated expression of the two IMP731 chains from the same integration site (
The DNA and protein sequences for the kappa region of IMP731 are set forth in
Results
Dose-dependent binding of IMP731 was first assessed on hLAG-3-tranfected CHO cells (
For complement dependent cytotoxicity (CDC) testing, the target cells used in this assay were LAG-3+ CHO cells (
For antibody-dependent cell-mediated cytotoxicity (ADCC) testing, PBMCs were stimulated for one day with IL-2 to serve as effector cells and LAG-3+ CHO cells were labelled with the vital dye CFSE to serve as target cells. In the presence of IMP731, PBMCs were able to kill a high percentage of LAG-3+ CHO cells (
It appeared that binding and functional activities of IMP731 were similar to the parental A9H12 murine mAb produced by hybridoma cells. For instance, IMP731 has the same high affinity as the parent antibody, as assessed by BIACORE™ analysis.
Introduction
More than 80% of the population is cytomegalovirus (CMV) seropositive and more than 50% has a high frequency of circulating CMV-reactive T cells that can be analyzed in short-term in vitro assays in response to a CMV protein recall antigen. This is therefore a convenient setting in which to analyze IMP731-dependent antibody-dependent cellular cytotoxicity (ADCC) on subsets of antigen-specific T cells physiologically activated through peptide-dependent T-cell receptor triggering, using defined synthetic peptides (i.e. no antigenic contaminants) from the sequence of a CMV protein.
In addition, this experimental setting mimics closely the situation encountered in auto-immune diseases where circulating antigen-specific T cells are periodically stimulated by auto-immune recall antigens. For example in the case of psoriasis, the recall antigens are skin antigens that are presented to the immune system in the form of MHC-class I- and class II-restricted peptides.
Purpose
To assess the potency of IMP731 in killing activated antigen-specific human T cells stimulated with a CMV peptide pool and thus to estimate the lowest dose likely to have a pharmacological effect in man.
Experimental Design
PBMCs from CMV-positive donors were thawed, washed in complete RPMI 1640 supplemented with 10% FCS and seeded at 106/ml in 24-well plate (1.75 ml/well) in the presence of a CMV peptide pool (BD Biosciences). The CMV peptide pool consists of a mix of 13815-mers with 11 amino acid overlaps spanning the entire sequence of the pp65 protein (CMV AD169 strain). This peptide pool will induce MHC class II-restricted CD4 peptide-specific T cell activation and proliferation but will also activate MHC class I restricted CD8 peptide-specific T cells through the internalization and processing of 15-mers into 9-mers. Together the entire T cell repertoire specific for the pp65 protein will be activated. The final concentration of each peptide was 0.875 μg/ml. The cells were incubated at 37° C. for 5 days. As CD8 T cells proliferate more rapidly than CD4 T cells, there will always be more CD8 than CD4 activated T cells at day 5.
For ADCC testing, the cells were seeded in a 96-well U-bottom plate (180 μl/well, containing 150-175×103 cells/well) in the CMV peptide-containing medium and 20 μl of 10× dilutions of IMP731 or its isotype-matched control (human IgG1, Chemicon) were added. After 4 hr at 37° C., the cells were stained with CD3-PE, CD4-PE-Cy7, CD8-APC-Cy7, CD25-APC (all from BD Biosciences) and FITC-conjugated anti-LAG-3 mAb (17B4 antibody, 1 μg/point) for 30 min at 4° C. After centrifuging, the cells were suspended for 15 min at room temperature in culture medium containing 2.5 μl 7-AAD (viability dye), before FACS analysis. After exclusion of dead cells based on size/granularity and 7-AAD staining, the percentages of LAG-3+ and CD25+ (another T cell activation marker) in the CD3+CD4+ and the CD3+CD8+ cells populations were analyzed.
Results
The stimulation of PBMCs with the CMV peptide pool induced the expression of the activation marker CD25 and LAG-3 (see
The observed effect is not due to a competition between IMP731 and the anti-LAG-317B4-FITC reagent since the binding of 17B4-FITC is not inhibited by a 3-fold excess of IMP731 (not shown). A putative internalization of the membrane LAG-3 induced by IMP731 was also excluded because the disappearance of activated T cells was also observed with an anti-CD25 antibody (see
Conclusions
IMP731 efficiently induced cell-mediated cytotoxicity of activated antigen-specific CD4 and CD8 human T cells at very low doses (1-10 ng/ml). From previous work we know that a concentration of 30 ng/ml IMP731 was measured at t=116 hr in the baboon injected with 0.1 mg/kg IMP731 (experiment showing a long-term suppression of DTH reaction). Therefore the concentration in the first few hours must have been much higher.
Based on this information, it is believed that a starting dose in human in the planned Phase Ia trial of 0.01 mg/kg could be pharmacologically active. It is possible that even such a very low dose could lead to >10 ng/ml serum concentration levels for a few hours and therefore be sufficient to deplete LAG-3+ T cells in patients.
IMP731 has been shown to be immunosuppressive after a single i.v. low dose (0.1 mg/kg) injection on a skin antigen-specific reaction (DTH) in baboon. This test is used as a surrogate in vivo assay for psoriasis inflammation. By continuing the DTH testing for five months after the injection of IMP731 it was possible to show that the immunosuppression was long-lasting corresponding to the “Campath Effect”.
The choice of psoriasis as an initial indication is supported by the work showing that psoriatic skin is massively infiltrated with LAG-3+ T cells compared to normal skin. The following describes the work which led to these results and conclusions.
IMP731 Suppresses DTH Reaction in Baboons
The half-life in 3 baboons injected (i.v.) with 0.1 mg/kg IMP731 showed similar pharmacokinetic profiles with the following elimination half-life: 16, 21 and 31 hours (
Delayed Type Hypersensitivity (DTH) reaction is an established model for skin inflammation (16-18). To assess the potency of IMP731 on skin tissue and their draining lymph nodes in baboons as a surrogate assay for the treatment of psoriasis lesions, 3 baboons were immunized with tuberculin and then their reactivity was assessed in a tuberculin-specific DTH reaction (see protocol in
Even a single dose as low as 0.1 mg/kg IMP731 is immunosuppressive as shown for Baboon #3 (
Decrease in LAG-3+ T Cells in Lymph Nodes
To quantify the systemic deletion of circulating LAG-3+ T cells following injection of IMP731, the number of LAG-3+ T cells in the DTH site draining lymph nodes 3 days after the tuberculin injection was analyzed. The mean percentage of live LAG-3+ cells in these immune-reactive lymph nodes was 3.5% for the first DTH and only 0.7% for the second DTH. This clearly indicates that IMP731 had killed 80% of LAG-3+ cells in the antigen-reactive lymph node at day 3. This mechanism of action explains why the immunosuppressive effect on the DTH is long-lasting (see below).
IMP731 Still Suppresses DTH Reaction in Baboons Five Months after Injection
This immunosuppressive effect is sustained over time as a third, fourth and fifth DTH reaction performed up to five months after the IMP731 injection is still inhibited (
IMP731 Suppresses DTH Reaction for Five Months Even after Two Booster Immunizations
This long duration of immunosuppressive effect was tested to determine whether it might be due to the deletion of tuberculin-specific memory T cells at the time of IMP731 injection. This is a possibility because CD45+ memory T cells (in both CD4 and CD8 subsets) are LAG-3+ in human. If so, the DTH would be suppressed following booster immunization. Accordingly, baboon #3 was injected with two BCG vaccines before performing the 5th DTH (
It is established that memory T cells may take up to a year to recover from deletion in human (the “Campath Effect”) (19). Therefore, it is likely that psoriasis patients can be treated with a low dose (e.g. 0.1 mg/kg) IMP731, which could be effective at deleting the activated memory T cells.
All references, including publications, patent applications and patents cited herein are hereby incorporated by reference, as if each were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 07290545.8 | Apr 2007 | EP | regional |
| 08750865.1 | Apr 2008 | EP | regional |
This application is a Continuation of U.S. application Ser. No. 16/212,732 filed Dec. 7, 2018, which is a Divisional of U.S. application Ser. No. 14/970,926, filed Dec. 16, 2015 (pending), which is a Continuation of U.S. application Ser. No. 12/823,795 (now U.S. Pat. No. 9,244,059), filed Jun. 25, 2010, which is a Continuation-in-part of U.S. application Ser. No. 12/598,128 (pending), filed Mar. 19, 2010 as a National Stage entry of International Application No. PCT/IB2008/001072, filed Apr. 30, 2008, which claims priority to European Patent Application Nos. 08750865.1, filed Apr. 30, 2008 and 07290545.8, filed Apr. 30, 2007, the disclosures of which are incorporated by reference in their entirety.
| Number | Date | Country | |
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| Parent | 14970926 | Dec 2015 | US |
| Child | 16212732 | US |
| Number | Date | Country | |
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| Parent | 16212732 | Dec 2018 | US |
| Child | 17333238 | US | |
| Parent | 12823795 | Jun 2010 | US |
| Child | 14970926 | US |
| Number | Date | Country | |
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| Parent | 12598128 | Mar 2010 | US |
| Child | 12823795 | US |
