Abstract
The present invention relates to the treatment of T-cell acute lymphoblastic leukemia (T-ALLs) by means of immunotherapy with a monoclonal antibody which recognizes the pre-TCR receptor which, due to its presence in all phases of T-ALL pre-TCR+ development, can also be used as a leukemia-initiating cell (LIC) biomarker and as a therapeutic target useful for monitoring minimal residual disease and identifying compounds inhibiting the disease.
Claims
1. A monoclonal antibody obtained by the hybridoma deposited with the European Collection of Cell Cultures under accession number 01080805, capable of specifically recognizing the TCRβ- and CD3-associated pTα chain of the pre-T cell receptor (pre-TCR), for use as a medicament.
2. The antibody according to claim 1, for use in the treatment and/or prevention of leukemia, preferably T-cell acute lymphoblastic leukemia (T-ALL), more preferably T-ALL pre-TCR+.
3. The antibody according to any one of claims 1 to 2, for use in the treatment and/or prevention of relapses of leukemia, preferably relapses of T-cell acute lymphoblastic leukemia (T-ALL), more preferably relapses of T-ALL pre-TCR+.
4. The antibody according to any one of claims 1 to 3, wherein said antibody is coupled to a toxin.
5. The antibody according to any one of claims 1 to 4, wherein said antibody is humanized.
6. A pharmaceutical composition comprising the monoclonal antibody obtained by the hybridoma deposited with the European Collection of Cell Cultures under accession number 01080805, capable of specifically recognizing the TCRβ- and CD3-associated pTα chain of the pre-T cell receptor (pre-TCR), for use in the treatment and/or prevention of leukemia, preferably T-cell acute lymphoblastic leukemia (T-ALL), more preferably T-ALL pre-TCR+.
7. The pharmaceutical composition according to claim 6, for use in the treatment and/or prevention of relapses of leukemia, preferably relapses of T-cell acute lymphoblastic leukemia (T-ALL), more preferably relapses of T-ALL pre-TCR+.
8. The pharmaceutical composition according to any one of claim 6 or 7, wherein the antibody is coupled to a toxin.
9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the antibody is humanized.
10. The pharmaceutical composition according to any one of claims 6 to 9, further comprising an acceptable pharmaceutical adjuvant, an acceptable pharmaceutical carrier, and/or another active ingredient.
Description
DESCRIPTION OF THE FIGURES
[0061] FIG. 1. Generation of human leukemia T-ALL in NSG (NOD scid gamma) mice transplanted with umbilical cord blood hematopoietic progenitor cells (HPCs) transduced with active Notch1 oncogene (ICN1). A) Diagram of the NSG mouse transduction and transplantation assay. B) Reconstitution kinetics of bone marrow (left) or peripheral blood (right) of different mice transplanted with CD34+ HPC cells transduced with ICN1.
[0062] FIG. 2. Analysis of pre-TCR expression in human T-ALL cells generated de novo after the expression of the ICN1 oncogene. A) Phenotype of ICN1+ leukemic cells for the expression of CD4, CD8, CD3, and TCRαβ in the bone marrow of transplanted mice (FIG. 1A) at 20 weeks post-transplantation. More than 45% of the ICN1+ CD4+CD8+ double-positive (DP) cells in the bone marrow express low levels of CD3 in the absence of TCRαβ, pre-TCR+ cell phenotype. B) The expression of pre-TCR is specific for ICN1+ leukemic cells, and not observed in ICN1-control cells. C) Pre-TCR+ leukemic cells are progressively expanded in vivo in animals with T-ALL until reaching >85% at 35-40 weeks post-transplantation.
[0063] FIG. 3. In vivo monitoring of T-ALL pre-TCR+ leukemia tumor progression in a preclinical model of xenotransplantation. A) Specific expression of pre-TCR in the T-ALL SUPT1 line, and absence thereof in the T-ALL HPB-ALL line, defined by the simultaneous binding of anti-CD3E and anti-pTα antibodies (UCHT1 and the antibody of the invention K5G3, respectively). B) Diagram of the xenotransplantation in NSG mice of primary T-ALL cells or SUPT1 cells transduced with luciferase for in vivo monitoring. C) Bioluminescence analysis of tumor progression in primary T-ALL or SUPT1 cells, as a pre-TCR+ leukemia/lymphoma model.
[0064] FIG. 4. In vivo progression in the serial transplantations of T-ALL pre-TCR+ cells generated de novo. (A) Heterogeneous TCRαβ+ and pre-TCR+ phenotype of leukemic cells generated in vivo from human ICN1+ HPC progenitors in the T-ALL pathogenesis model (FIG. 1). (B) Enrichment (>95%) of pre-TCR+ leukemic cells generated in (A) serial transplantations in NSG mice.
[0065] FIG. 5. Study of the molecular cell survival and proliferation pathways activated by the pre-TCR expressed in cells with leukemia-initiating capacity (LICs) of T-ALL generated de novo. A) 100% pre-TCR+ phenotype of T-ALL cells obtained from mouse bone marrow after 4 serial transplantations of a T-ALL generated de novo as described in FIG. 1. B) Western blot analysis of the activation of the indicated survival and proliferation pathways, after stimulation of T-ALL pre-TCR+ cells in (A) by cross-linking with antibodies recognizing the CD3ε subunit or the pTα chain of the pre-TCR (UCHT1 and the antibody of the invention K5G3, respectively).
[0066] FIG. 6. Analysis of in vivo progression in serial transplantations of primary T-ALL cells from patients and study of the function of their pre-TCR. (A) Enrichment in pre-TCR+ leukemic cells from a patient in serial transplantations in NSG mice. (B) Enrichment quantification (>65%). C) Activation of molecular survival and proliferation pathways by cross-linking of the pre-TCR with antibodies recognizing the CD3ε subunit or the pTα chain of the pre-TCR (UCHT1 and the antibody of the invention K5G3, respectively) in primary T-ALL cells.
[0067] FIG. 7. Separation of pre-TCR+ and pre-TCR− subpopulations of primary T-ALL cells and diagram of the in vivo assay of their LIC activity. A) Primary T-ALL17 cells isolated from a patient's blood were separated by flow cytometry in two populations: CD3+TCRαβ− (pre-TCR+) and CD3+TCRαβ+ (pre-TCR−), both with intracellular expression of TCR (TCRβic). B) The isolated pre-TCR− and pre-TCR+ populations were transplanted in increasing numbers (10-10.sup.5) in NSG mice in which the graft in the blood was analyzed on the indicated days.
[0068] FIG. 8. LIC activity of pre-TCR+ and pre-TCR− cells of a primary T-ALL leukemia in a patient. A) Frequency of cells with LIC activity in the T-ALL pre-TCR+ and pre-TCR− subpopulations from the same patient. B) Survival of mice transplanted with the T-ALL pre-TCR+ and pre-TCR− populations.
[0069] FIG. 9. Analysis of the in vivo progression of a primary T-ALL transplanted after the functional inhibition of the pre-TCR. A) Strategy for inhibiting the function of the pre-TCR expressed in T-ALL cells of patients by means of silencing the CMS adapter with shRNA-CMS lentiviral vectors. (B, E) Transduction efficiency measured by GFP expression. (C, F) Tumor progression in the indicated organs of T-ALL cells transduced with shRNA-CMS or shRNA-control (shsc) transplanted in Rag2−/− x γc−/− immunodeficient mice. (D) Silencing of CMS prevents tumor progression and splenomegaly induced by T-ALL cells.
[0070] FIG. 10. Analysis of the generation of T-ALL leukemia induced by the Notch1 oncogene in hematopoietic progenitors with a mutation affecting the function of the pre-TCR. A) Expression of the ckit marker (top) and the Notchl oncogene (ICN1) (bottom) in bone marrow progenitor cells of normal mice (wt for wild-type) or mutant mice (C80G) transduced with a lentiviral vector carrying ICN1 and GFP. B) Percentages and absolute numbers of wt or C80G ICN1+ cells grafted in the bone marrow, spleen, blood, and liver of NSG mice at 5 weeks post-transplantation. C) Absolute numbers of ICN1+ and ICN1− cells grafted in the bone marrow of the mice in (B).
[0071] FIG. 11. Survival of NSG mice transplanted with bone marrow progenitors with a normal or mutated pre-TCR function, transduced with ICN1. Survival after a primary transplantation (left) or secondary transplantation (right) at the indicated post-transplantation days is shown.
[0072] FIG. 12. Titration of the antibody of the invention K5G3 (anti-pTα) in human T-ALL SUPT1 and Jurkat cell lines. This figure shows the specific expression of pre-TCR in the SUPT1 line, measured by means of flow cytometry using anti-CD3ε antibody, UCHT1 (BD Biosciences), and increasing concentrations of the anti-pTα. antibody of the invention K5G3. Unlike UCHT1, K5G3 does not bind to the T-ALL Jurkat cell line which lacks pre-TCR, but expresses CD3-associated TCRαβ.
[0073] FIG. 13. Binding of the antibody of the invention K5G3 (anti-pTα) to human thymic pre-T progenitors and to primary T-ALL cells of patients. Analysis of the expression of pre-TCR using the anti-CD3ε antibody, UCHT1, and the antibody of the invention K5G3, anti-pTα , in (A) human thymus cells and (B) primary T-ALL cells obtained from a patient (T-ALL4). Phenotypic analyses in (A) show that subpopulations of human thymocytes expressing TCRαβ or TCRγδ, receptors in association with CD3, are not recognized by the anti-pTα, mAb, which demonstrates the specificity of the antibody of the invention K5G3.
[0074] FIG. 14. Study of the functionality of the human anti-pTα antibody of the invention, K5G3, in calcium mobilization assays. Isolation of pre-TCR+ cells from human thymus by means of the immunomagnetic selection of CD8+ TCRαβ− thymocytes (left) and calcium mobilization in isolated pre-TCR+ thymocytes after the cross-linking of the pre-TCR with the anti-CD3ε antibody, UCHT1, or with the anti-pTα antibody of the invention, K5G3 (right).
[0075] FIG. 15. Immunotherapy by means of the administration of the anti-pTα antibody of the invention, K5G3, in a preclinical model of human T-ALL xenotransplantation.
[0076] A) Diagram of the dosage regimen of the anti-pTα antibody of the invention or of a control isotypic antibody (IgG2a), in SCID mice in which disease has been established following transplantation from a T-ALL patient. B) Survival of treated animals with respect to controls. C) Percentage of T-ALL pre-TCR+ cells in the peripheral blood (PB) of treated and control animals.
[0077] FIG. 16. Effect of in vitro treatment with the anti-pTα antibody of the invention, K5G3, on the number of T-ALL pre-TCR+ cells. A) Diagram of the in vitro culture of T-ALL in the presence of the anti-pTαα antibody of the invention, K5G3, or of a non-specific antibody, IgG2a, used as a control. B) Heterogeneous TCRαβ+ (pre-TCR−) and pre-TCR+ (TCRαβ−) phenotype of the T-ALL leukemia cultured in (A). C) CD3ε expression levels in cells (B) after being cultured for 6 days in the presence of the anti-pTα antibody of the invention, K5G3, or of a non-specific antibody,IgG2a, used as a control. The superposition of both histograms where the selective reduction of pre-TCR+ cells is observed is shown below. D) Absolute numbers of TCRαβ+ and pre-TCR+ cells of the T-ALL leukemia in (B) after being cultured in vitro for 6 days in the presence of the anti-pTα antibody of the invention, K5G3, or of a control IgG2a. E) Absolute numbers and percentages of apoptotic cells recovered 6 days after culturing the T-ALL pre-TCR+ leukemia shown in FIG. 5A in the presence of the anti-pTα antibody of the invention, K5G3, or of a control IgG2a. Data±SEM derived from 3 experiments are shown.
[0078] FIG. 17. Effect of in vitro treatment with the human anti-pTα antibody of the invention, K5G3, on the internalization of pre-TCR in the SUPT1 line and in primary leukemias of patients. A) Flow cytometry of the expression levels of CD3ε and pTα components of pre-TCR in SUPT1 cells analyzed after 24 hours of in vitro incubation with an irrelevant mAb IgG2a (control) or with the anti-pTα mAb of the invention (K5G3). B) CD3ε expression levels representative of pre-TCR levels in SUPT1 cells (top) or in primary T-ALL leukemias of patients (T-ALL3, T-ALL17) treated as described in (A). The data shows relative fluorescence intensity (RFI) values±SEM of 3 experiments. C) Kinetics of the endocytosis and recycling of the CD3ε chain of the pre-TCR complex in SUPT1 cells treated for 30 min at 4° C. (time=0) with an irrelevant mAb IgG2a (control) or with the anti-pTα mAb of the invention (K5G3), and then cultured at 37° C. at the indicated times.
EXAMPLES
[0079] Next, the invention will be illustrated by means of assays carried out by the inventors which clearly show the effectiveness of the antibody of the invention for the proposed medical uses and the fact that the receptor which recognizes the antibody of the invention, i.e., the pre-TCR receptor, is a suitable therapeutic target to combat relapses in T-ALL patients.
Example 1
Generation of Human T-ALL Leukemias in NSG Mice by Means of the Transplantation of Umbilical Cord Blood HPCs Overexpressing Active Notch1 (ICN1)
[0080] In collaboration with the Transfusion Center of the Community of Madrid, umbilical cord blood samples have been obtained, from which hematopoietic progenitors (HPCs) were isolated by means of immunomagnetic selection with anti-CD34 antibodies. Selected cells were transplanted into NSG immunodeficient mice, after transduction thereof with a retroviral vector carrying the oncogenic form of Notch1 (ICN1) and GFP, as recently described (Garcia-Peydro et al., J Clin Invest. 2 July 2018; 128(7): 2802-2818; FIG. 1). In this model, the presence of human ICN1+ leukemic cells in peripheral blood and bone marrow aspirates from transplanted animals was analyzed over 8 months, which allowed identifying the in vivo generation of leukemia with characteristics of human T-ALL from 14-18 weeks in the bone marrow and from 30-36 weeks post-transplantation in the blood of transplanted mice (FIG. 1). This data demonstrates for the first time the de novo generation of a human T-ALL leukemia in vivo from healthy cells.
Example 2
Identification and In Vivo Monitoring of the Expression of Pre-TCR During the Progression of Human T-ALL in Transplanted Animals
[0081] In order to monitor the cell populations involved in the pathogenesis of human T-ALL and the molecules involved, the phenotype of cells carrying the ICN1 oncogene (ICN1+) in bone marrow aspirates was analyzed by means of flow cytometry as indicated in FIG. 2. The results obtained show that the pre-TCR complex is expressed early in the generation of leukemia and its expression on the surface of leukemic cells is maintained during the 10 months of T-ALL progression in transplanted animals. Likewise, the proportion of pre-TCR+ cells increases over time (FIG. 2), which indicates that during the T-ALL generation process, the differentiation of T-lymphocytes in the maturation stage in which pre-TCR is expressed, expanding the pre-TCR+ population, is blocked, which suggests a relevant role of this receptor in the progression of the disease. To establish the optimal conditions for the in vivo monitoring of the progression of pre-TCR+ human leukemias, a xenogeneic transplantation model was developed in NSG immunodeficient mice using, as a control of T-ALL pre-TCR+ cells, the SUPT1 cell line which co-expresses pre-TCR CD3ε and pTα molecules which are recognized by the mAb, UCHT1 (BD Biosciences), and the antibody of the invention (K5G3), respectively; meanwhile TCR+ leukemias, such as HPB-ALL, only express CD3ε (FIG. 3). The model was extended to the analysis of primary T-ALL pre-TCR+ leukemias from patients. The cells were transduced with a bicistronic lentiviral vector (pHRsin) carrying the GFP and luciferase gene, which makes it possible to follow the progression of leukemia and the infiltration of different peripheral organs in the transplanted mice. By means of this experimental strategy, it was possible to demonstrate the efficient in vivoexpansion of SUPT1 pre-TCR+ leukemia and primary T-ALL leukemias (FIG. 3).
Example 3
In Vivo Validation and Quantification of Leukemia-Initiating Activity (LIC) of Human Pre-TCR+ Leukemic Cells Generated De Novo in Animals and of Primary T-ALL Leukemias from Patients
[0082] To check the relevance of the pre-TCR complex in the in vivo expansion of T-ALL, serial transplantations of the leukemic cells generated in mice transplanted with ICN1-transduced human HPC progenitors were performed (FIG. 1). Cells from the bone marrow of these animals, showing a majority CD4+CD8+ DP phenotype and containing a significant pre-TCR+ subpopulation (35%), were transplanted into a second 6-8 week old NSG recipient mouse, sublethally irradiated with 1.5 Gy, and so on and so forth up to 3 transplantations. This assay allowed the LIC activity of pre-TCR+ cells in successive transplantations to be analyzed. Analysis of the phenotype of the T-ALL cells obtained from the bone marrow after 3 transplantations showed pre-TCR expression in the membrane of >95% of the leukemic cells that were expanded in the transplanted animals (FIG. 4), which demonstrates an in vivo proliferative advantage of pre-TCR+ cells over pre-TCR− cells. Subsequent biochemical analyses of the expanded cells corroborated the function of the pre-TCR complex in the activation of molecular pathways involved in cell survival and proliferation, in response to stimulation induced with anti-pre-TCR monoclonal antibodies (anti-CD3ε or the anti-pre-Tα antibody of the invention K5G3). These pathways include: Pi3K/Akt, Erk, y mTORC1/S6 (FIG. 5). Therefore, the LICs of the T-ALLs generated express a functional pre-TCR involved in cell proliferation.
[0083] Similar experiments were performed with peripheral blood primary leukemia cells from patients diagnosed with T-ALL (T-ALL17), using the mouse xenotransplantation model. Human T-ALL cells were obtained by Ficoll-Hypaque centrifugation and injected (10.sup.5) intravenously into 6-8 week old NSG mice, sublethally irradiated with 1.5 Gy. The in vivo expansion potential of the primary leukemic cells was analyzed by means of flow cytometry performed on bone marrow aspirates from mice at different weeks post-tranplantation. The study revealed that serial transplantations of T-ALL samples expressing very low levels of CD3 induce sequential enrichment in these cells, comprising the pre-TCR+ population, which demonstrates its in vivo proliferation advantage and its LIC activity. Biochemical assays subsequently corroborated the functionality of pre-TCR inducing the activation of survival and proliferation pathways also in primary T-ALLs (FIG. 6). Overall, this data demonstrates that human T-ALL pre-TCR+ cells exhibit LIC activity, and therefore confirms that pre-TCR can be a therapeutic target to combat relapses in patients.
[0084] To confirm that pre-TCR is a biomarker for cells with LIC activity, involved in the progression of leukemia-initiating cells, the two pre-TCR+ and pre-TCR− (TCRαβ+) subpopulations included in T-ALL17 leukemia were separated and the LIC activity of both subpopulations was analyzed separately in xenotransplantations with decreasing cell dilutions, according to the diagram shown in FIG. 7.
[0085] Next, the frequency of cells with LIC activity in the pre-TCR+ and pre-TCR− populations of the same patient was analyzed by means of monitoring the survival of transplanted animals by Kaplan-Meier estimation, using the ELDA program (http://bioinf.wehi.edu.au/software/elda/). The results showed that LIC cells are enriched about 40-fold in the pre-TCR+ population compared to the pre-TCR− population. Accordingly, mice transplanted with pre-TCR+ cells showed significantly decreased survival compared to mice receiving pre-TCR− cells from the same patient (FIG. 8).
Example 4
Analysis of the Function of Pre-TCR in T-ALL LIC Activity: CMS Adapter Silencing in Human T-ALL Leukemias and Analysis of the LIC Activity Thereof in In Vivo Preclinical Models
[0086] To directly determine the relevance of pre-TCR in T-ALL LIC activity, and therefore in the progression and relapse of T-ALL in patients, the impact of inhibition of pre-TCR function on the tumor progression of human T-ALL cells transplanted in NSG immunodeficient mice was analyzed. To that end, previous results published by the inventors (Navarro et al. Blood. 15 Dec. 2007; 110(13):4331-40), indicating that the function of pre-TCR is dependent on the CMS/CD2AP adapter that binds to pTα, were considered, and strategies involving CMS gene silencing mediated by short-hairpin(sh)RNA-CMS expressed in bicistronic lentiviral vectors also carrying GFP, or vectors carrying a control shRNA (shsc) were used (FIG. 9). Optimal silencing conditions were established using the T-ALL pre-TCR+ SUPT1 line, that were transferred to two primary leukemias (T-ALL5 and T-ALL9) from patients, The transduction efficiency of which was analyzed by means of GFP expression (FIG. 9). Modified T-ALL was transplanted intravenously and leukemic progression was analyzed in the transplanted animals, with a drastic inhibition of tumor progression being observed after CMS silencing (FIG. 9), which translated into a complete blockade of the induction of splenomegaly observed in animals transplanted with T-ALLs transduced with control vectors (FIG. 9). Therefore, intact pre-TCR signaling is required for the in vivo progression of human T-ALL cells, demonstrating that the inhibition of the function of the pre-TCR expressed in T-ALL cells efficiently blocks their LIC activity and their in vivo expansion, which indicates that the pre-TCR is a therapeutic target of T-ALL.
Example 5
Analysis of the Relevance of Pre-TCR as a Molecular Target of Cells with LIC Activity Involved in the Generation of Human T-ALL
[0087] Phenotypic analysis of leukemic cells generated in the human T-ALL leukemia induction model (FIG. 1) showed the majority expression of pre-TCR in all the generated leukemias (FIG. 2). More importantly, serial transplantations suggested an in vivo proliferative advantage of pre-TCR+ cells over pre-TCR− cells and the involvement of pre-TCR in the function of cells with LIC activity involved in the generation of T-ALL (FIG. 4).
[0088] In order to confirm the functional involvement of pre-TCR in the generation of T-ALL, the tumor potential of the Notch1 oncogene was subsequently examined in hematopoietic progenitors from the bone marrow of normal mice (wt for wild-type), or mice with a mutation (C80G) which prevents pre-TCR− mediated signaling (White et al. Science Signal. 2 Dec. 2014; 7(354):ra115). Progenitors from the bone marrow of normal or C80G mice transduced with an oncogenic form of GFP-associated Notch1 (ICN1) were transplanted into NSG immunodeficient mice, and the generation of T-ALL leukemia was analyzed at different times in the transplanted animals by monitoring GFP+ tumor cells in different organs.
[0089] Flow cytometric analysis demonstrated a similar expression of the ICN1 oncogene in normal and C80G cells. However, only normal cells showed the capacity of grafting and expanding in the bone marrow, blood, spleen, and other organs, such as the liver, in the transplanted animals 5 weeks post-transplantation, whereas mutant cells were not capable of generating leukemia in any of the organs analyzed at different times (FIG. 10). Accordingly, the survival of animals transplanted with mutant cells carrying the oncogene was not affected, with the survival of animals transplanted with normal cells, as well as the survival of second recipients transplanted with these cells, being dramatically reduced (FIG. 11). Therefore, it can be concluded that the function of pre-TCR is essential in the generation of T-ALL leukemia induced by the ICN1 oncogene, which demonstrates that the pre-TCR complex is an optimal target for therapeutic intervention.
Example 6
Analysis of the Prevalence of Pre-TCR Expression in Human Primary T-ALLs
[0090] The data obtained in Example 4 and Example 5 constitute proof of concept of the suitability of a new therapeutic strategy aimed at eliminating the function of pre-TCR and/or of leukemic cells carrying same. Therefore, the next objective was to evaluate the prevalence of pre-TCR expression in T-ALLs of patients and to determine the frequency of patients carrying pre-TCR+ LIC, who are potential beneficiaries of the proposed treatment. To that end, collaboration was established with the SEHOP Acute Leukemia Group, specifically, with Dr. Manuel Ramirez Orellana from Hospital Infantil Nino Jesus in Madrid and with Dr. Mireia Camas from Hospital Sant Joan de Déu in Barcelona, as well as with Dr. Antonio Perez Rodriguez from Hospital Universitario La Paz in Madrid, to determine pre-TCR expression in patients at diagnosis by means of flow cytometry. The samples were analyzed with the antibody of the invention. 13 T-ALL samples were analyzed, with pre-TCR expression being observed in 7 of them, which constitutes >50% of the samples analyzed (Table 1). Therefore, a significant number of patients with T-ALL could benefit from the proposed anti-pre-TCR treatment.
TABLE-US-00001 TABLE 1 Prevalence of pre-TCR expression in patients diagnosed with T-ALL Samples (a) TCR PRE-TCR IL7R CXCR4 T-ALL1 + − − ++ T-ALL2 + − ++ ND T-ALL3 − + ++ ND T-ALL5 − + + ++ T-ALL8 + − + ++ T-ALL9 + − + ++ T-ALL10 + − + ++ T-ALL17 + + + ++ T-ALL18 + + ++ ++ T-ALL26 − + ++ ++ T-ALL29 + + ND ND T-ALL30 − + ++ ++ T-ALL32 ++ − ++ ND (a) The expression of pre-TCR, TCR, IL-7 receptor (IL-7R), and chemokine receptor CXCR4 in the indicated primary samples, obtained from the peripheral blood or bone marrow of patients diagnosed with T-ALL, was analyzed.
Example 7
Study of the Functional Characteristics of the Antibody of the Invention
Production and Purification.
[0091] The antibody of the invention is a mouse IgG2a monoclonal antibody directed against the extracellular domain of human pTα, the specific component of pre-TCR which associates with the TCR chain and the CD3 complex. The antibody was produced as follows: the hybridoma, K5G3, deposited in the European Collection of Cell Cultures under accession number 01080805, was expanded in large quantities using a hollow fiber bioreactor (Fiber Cell Systems). The secreted monoclonal antibody was purified from the culture supernatant by means of affinity chromatography on a protein G Sepharose column (GE Healthcare), and the purified monoclonal antibody was subsequently analyzed in functional in vitro assays. The studies indicate that the antibody of the invention is capable of binding specifically to the pre-TCR expressed in the SUPT1 cell line in a concentration-dependent manner, but not to the TCR complex formed by a TCRα chain attached to the TCRβ chain and the CD3 complex in the T-ALL Jurkat cell line (FIG. 12). Despite low levels of pre-TCR expression in human pre-T cells, the monoclonal antibody of the invention is also capable of binding specifically to the pre-TCR expressed in human pre-T thymocytes, without binding to TCRαβ or TCRγδ cells of the thymus (FIG. 13), and inducing the activation of these thymocytes mediated by calcium mobilization, at levels similar to those induced by the anti-CD3ε monoclonal antibody UCHT1 (FIG. 14). Furthermore, as shown in FIGS. 5 and 6, the antibody of the invention is as effective as UCHT1 (or even more so) in inducing the activation of PI3K/AKT/mTOR and MAPK signaling pathways in primary human T-ALL pre-TCR+ cells.
Example 8
In Vivo Validation of the Efficacy of an Immunotherapy Targeting T-ALL Pre-TCR+ LICs in Preclinical Models
[0092] The success of treatments such as RITUXIMAB, based on the principle of specific recognition of a membrane protein of leukemia (B-ALL in this case), and the subsequent elimination of cells carrying the protein by a monoclonal antibody, suggests that the strategy of this invention may be valid. The fact that the chosen molecule, the pTα chain of pre-TCR, is selectively expressed during intrathymic development but absent in peripheral T-lymphocytes, represents an additional value of the strategy for preventing adverse effects due to the elimination of normal mature T-cells which could induce an aggressive immunodeficiency.
[0093] To analyze the efficiency of the proposed immunotherapy in vivo, SCID mice irradiated with sublethal doses (1.5 Gy) and transplanted with primary T-ALL cells from peripheral blood waste samples of T-ALL patients at diagnosis were used (FIG. 15). The SCID strain, unlike NSG, has intact complement-dependent cytotoxicity (CDC) function, as well as antibody-dependent cytotoxicity (ADCC) function. Animals transplanted with T-ALL were examined on different days post-transplantation and the presence of human leukemic cells in peripheral blood (PB) was determined. The disease was considered to be established when the animals presented >1% of leukemic cells in blood (>3 weeks post-transplantation). At this point, Treatment, consisting of the intraperitoneal administration of the antibody of the invention or of a isotypic control monoclonal antibody (200 μg/mouse), 2 times a week for 10 weeks, was started (FIG. 15). Throughout treatment, the graft in bone marrow aspirates, as well as infiltration into different lymphoid organs (thymus, spleen, and blood) and non-lymphoid organs (liver and brain) in the transplanted animals, were analyzed, observing a selective decrease in leukemic cells in animals treated with the antibody of the invention in various organs. Although treatment discontinuation resulted in leukemia progression, it should be noted that the survival of the treated animals was significantly greater than the survival of the control animals (from 70 to 110 days on average) (FIG. 15), obtaining similar results with another leukemia from a patient diagnosed with T-ALL, which validates the efficiency of the proposed immunotherapy.
[0094] The phenotypic analysis of the organs of the treated animals showed a selective effect on leukemic cells carrying pre-TCR, which decreased compared to pre-TCR− cells, which ensures the specificity of the treatment (FIG. 15). Additional preliminary analyses suggest that the antibody of the invention induces cell death by in vivo apoptosis of tumor cells through a CDC and/or ADCC induction mechanism.
Example 9
Study of the Capacity of the Anti-pTα Antibody of the Invention, K5G3, to Induce Pre-TCR Endocytosis and Apoptosis in Pre-TCR+ Cells
[0095] In vitro studies using heterogeneous primary T-ALL cells for pre-TCR and TCRαβ, expression which are cultured for several days in the presence of the anti-pTα antibody of the invention or an irrelevant antibody of the same isotype (IgG2a) indicate that the antibody of the invention not only induces the activation of pre-TCR+ leukemic cells (FIG. 5, FIG. 6), but also induces their specific disappearance, which correlates with the induction of cell death by apoptosis (FIG. 16).
[0096] Furthermore, in vitro incubation studies of SUPT1 pre-TCR+ cells with the anti-pTα antibody of the invention for short periods of time had demonstrated that this antibody also induces the internalization of pre-TCR from the cell surface (Navarro et al. Blood. 15 Dec. 2007; 110(13):4331-40). The results shown in FIG. 17 corroborate this data in SUPT1 cells and extend said data to primary T-ALL pre-TCR+ leukemias obtained from patients, with a significant decrease in expression levels, and therefore of the percentage of pre-TCR+ cells, being detected in both cases after short incubation periods. Furthermore, endocytosis kinetics studies by means of flow cytometry corroborated the internalization capacity of the anti-pTα antibody of the invention within a few minutes of binding to its target, i.e., pre-TCR, in the cell membrane (FIG. 17), so it is a molecular mechanism which can be exploited from a therapeutic viewpoint, since therapies based on the use of antibodies conjugated to toxins/drugs (ADC for antibody-drug conjugates) are particularly effective in inducing cell lysis in cases where the antibody is internalized together with the corresponding molecular targets.
