Method for Treating CD127-Positive Cancers by Administering an Anti-CD127 Agent

Abstract

The invention pertains to the field of immunotherapy. The present invention provides a new use of anti-CD127 agent, in particular anti-CD127 antibodies or related compounds for the treatment and/or the prevention of cancer.

The invention relates to a method for treating a patient having a CD127-positive cancer, in particular a CD127-positive leukemia, by administering to the patient a therapeutic dose of an anti-CD127 agent, the anti-CD127 agent having the capability to enhance the Antibody Dependent Cellular Phagocytosis (ADCP) activity of macrophages targeting CD127-positive cancer cells, and that does not have Antibody Dependent Cytotoxic Activity (ADCC), in particular on immune cells, more particularly on T cells.

Claims

1. A method for treating a patient having a CD127-positive cancer by enhancing the phagocytosis of CD127-positive tumor cells, in particular by macrophages, wherein the method comprises the administration to the patient of an effective amount of an anti-CD127 agent, in particular an anti-CD127 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic, that has Antibody Dependent Cellular Phagocytosis (ADCP) activity on CD127-positive tumor cells, in particular by macrophages cells, and that does not have Antibody Dependent Cytotoxic Activity (ADCC), in particular on immune cells, more particularly on T cells.

2. The method according to claim 1, wherein the CD127-positive cancer is Leukemia, in particular is Acute Lymphoblastic Leukemia (ALL), more particularly is T-cell ALL or B-cell ALL, more preferably is B-cell ALL.

3. The method according to claim 1, wherein the CD127-positive cancer is selected from the group consisting of CD127 overexpressing Acute Lymphoblastic Leukemia (ALL), CD127 and/or JAK-STAT pathway mutated ALL, BCR-ABL1-like ALL, and B cell precursor ALL bearing one the following cytogenetics: t(1;19), t(12,21), MLL-rearrangements, hyperdiploid karyotypes, trisomy 4 and trisomy 10.

4. The method according to claim 1, wherein the CD127-positive cancer is treated by the phagocytosis of CD127-positive tumor cells, in particular by macrophages.

5. The method according to claim 1, wherein the anti-CD127 agent is an anti-CD127 antibody or antigen-binding fragment thereof, comprising a constant chain belonging to the subclass of IgG1, IgG2, IgG3 or IgG4, in particular the subclass of mammalian IgG1, IgG2, IgG3 or IgG4, more particularly the subclass of mammalian IgG4.

6. The method according to claim 1, wherein the anti-CD127 agent is an anti-CD127 antibody or antigen-binding fragment thereof, which comprises: a VH chain comprising at least the following amino acid sequences: VHCDR1 SEQ ID No. 3; VHCDR2 SEQ ID No. 4; VHCDR3 SEQ ID No. 5 or SEQ ID No. 6; and a VL chain comprising at least the following amino acid sequences: VLCDR1 SEQ ID No. 7 or SEQ ID No. 8; VLCDR2 SEQ ID No. 9 or SEQ ID No. 10; VLCDR3 SEQ ID No. 11.

7. The method according to claim 1, wherein the anti-CD127 antibody or antigen-binding fragment thereof is an antagonist of the IL7-R signaling pathway induced by the binding of IL7 to CD127.

8. The method according to claim 1, wherein the method further comprises the administration of at least one second therapeutic agent selected from the group consisting of an anti-CD3 agent, in particular anti-CD3 antibody, anti-CD19 agent, in particular an anti-CD19 antibody, and anti-CD47 agent, in particular an anti-CD47 antibody, more particularly an anti-CD47 antagonist agent, even more particularly an anti-CD47 antagonist antibody, an inhibitor of the tyrosine/kinase pathway, Dexamethasone, rituximab, trastuzumab, cetuximab, Arranon (Nelarabine); Asparaginase Erwinia chrysanthemi (or Erwinaze), Asparlas (or Calaspargase Pegol-mknl); Besponsa (Inotuzumab Ozogamicin); Blinatumomab (or Blincyto); and Cerubidine (or Daunorubicin Hydrochloride or Rubidomycin); Clofarabine (or Clolar); Cyclophosphamide; Cytarabine; Dasatinib (or Sprycel); Doxorubicin Hydrochloride; Gleevec (Imatinib Mesylate); Iclusig (Ponatinib Hydrochloride); Inotuzumab Ozogamicin; Imatinib Mesylate; Kymriah (or Tisagenlecleucel); Marqibo (Vincristine Sulfate Liposome); Mercaptopurine (or Purinethol or Purixan); Methotrexate Sodium (or Trexall); Nelarabine; Oncaspar (or Pegaspargase); Ponatinib Hydrochloride; Prednisone; Purinethol (Mercaptopurine); Vincristine Sulfate, Vincristine Sulfate Liposome, and more particularly Dexamethasone.

9. The method according to claim 8, wherein the second therapeutic agent is Dexamethasone.

10. The method according to claim 8, wherein the administration of the anti-CD127 agent and the second therapeutic agent is simultaneous, separate or sequential.

11. A method for treating Acute Lymphoblastic Leukemia (ALL) in a patient by enhancing the Antibody Dependent Cellular Phagocytosis of ALL cells, in particular by macrophages, in particular T-cell ALL or B-cell ALL, more particularly CD127 overexpressing ALL, CD127 and/or JAK-STAT pathway mutated ALL, BCR-ABL1-like ALL, and B cell precursor ALL bearing one the following cytogenetics: t(1;19), t(12,21), MLL-rearrangements, hyperdiploid karyotypes, trisomy 4 and trisomy 10, wherein the method comprises the administration to the patient of an effective amount of an anti-CD127 agent, in particular an anti-CD127 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic, that has Antibody Dependent Cellular Phagocytosis (ADCP) activity on CD127-positive tumor cells, in particular by macrophages cells, and that does not have Antibody Dependent Cytotoxic Activity (ADCC), in particular on immune cells, more particularly on T cells.

12. The method according to claim 11, wherein the anti-CD127 agent is an anti-CD127 antibody or antigen-binding fragment thereof, which comprises: a VH chain comprising at least the following amino acid sequences: VHCDR1 SEQ ID No. 3; VHCDR2 SEQ ID No. 4; VHCDR3 SEQ ID No. 5 or SEQ ID No. 6; and a VL chain comprising at least the following amino acid sequences: VLCDR1 SEQ ID No. 7 or SEQ ID No. 8; VLCDR2 SEQ ID No. 9 or SEQ ID No. 10; VLCDR3 SEQ ID No. 11.

13. The method according to claim 11, wherein the method further comprises the administration at least one second therapeutic agent selected from the group consisting of an anti-CD3 agent, in particular anti-CD3 antibody, anti-CD19 agent, in particular an anti-CD19 antibody, and anti-CD47 agent, in particular an anti-CD47 antibody, more particularly an anti-CD47 antagonist agent, even more particularly an anti-CD47 antagonist antibody, an inhibitor of the tyrosine/kinase pathway, Dexamethasone, rituximab, trastuzumab, cetuximab. Arranon (Nelarabine); Asparaginase Erwinia chrysanthemi (or Erwinaze), Asparlas (or Calaspargase Pegol-mknl); Besponsa (Inotuzumab Ozogamicin); Blinatumomab (or Blincyto); and Cerubidine (or Daunorubicin Hydrochloride or Rubidomycin); Clofarabine (or Clolar); Cyclophosphamide; Cytarabine; Dasatinib (or Sprycel); Doxorubicin Hydrochloride; Gleevec (Imatinib Mesylate); Iclusig (Ponatinib Hydrochloride); Inotuzumab Ozogamicin; Imatinib Mesylate; Kymriah (or Tisagenlecleucel); Marqibo (Vincristine Sulfate Liposome); Mercaptopurine (or Purinethol or Purixan); Methotrexate Sodium (or Trexall); Nelarabine; Oncaspar (or Pegaspargase); Ponatinib Hydrochloride; Prednisone; Purinethol (Mercaptopurine); Vincristine Sulfate, Vincristine Sulfate Liposome, and more particularly Dexamethasone

14. The method according to claim 13, wherein the administration of the anti-CD127 agent and the second therapeutic agent is simultaneous, separate or sequential.

15. The method according to claim 13, wherein the second therapeutic agent is dexamethasone.

16. A method for treating a patient having a CD127-positive cancer, wherein the method comprises the steps of: a) Determining if the patient has CD127-positive tumor cells, b) When the patient has a CD127-tumor cells, administrating to the patient an effective amount of an anti-CD127 agent, in particular an anti-CD127 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic, that has Antibody Dependent Cellular Phagocytosis (ADCP) activity on CD127-positive tumor cells, in particular by macrophages cells, and that does not have Antibody Dependent Cytotoxic Activity (ADCC), in particular on immune cells, more particularly on T cells.

17. The method according to claim 16, wherein the CD127-positive cancer is selected from the group consisting of Acute Lymphoblastic Leukemia (ALL), in particular T-cell ALL or B-cell ALL, more particularly CD127 overexpressing ALL, CD127 and/or JAK-STAT pathway mutated ALL, BCR-ABL1-like ALL, and B cell precursor ALL bearing one the following cytogenetics: t(1;19), t(12,21), MLL-rearrangements, hyperdiploid karyotypes, trisomy 4 and trisomy 10.

18. The method according to claim 16, wherein an effective amount of a second therapeutic agent selected from the group consisting of an anti-CD3 agent, in particular anti-CD3 antibody, anti-CD19 agent, in particular an anti-CD19 antibody, and anti-CD47 agent, in particular an anti-CD47 antibody, more particularly an anti-CD47 antagonist agent, even more particularly an anti-CD47 antagonist antibody, an inhibitor of the tyrosine/kinase pathway, Dexamethasone, rituximab, trastuzumab, cetuximab. Arranon (Nelarabine); Asparaginase Erwinia chrysanthemi (or Erwinaze), Asparlas (or Calaspargase Pegol-mknl); Besponsa (Inotuzumab Ozogamicin); Blinatumomab (or Blincyto); and Cerubidine (or Daunorubicin Hydrochloride or Rubidomycin); Clofarabine (or Clolar); Cyclophosphamide; Cytarabine; Dasatinib (or Sprycel); Doxorubicin Hydrochloride; Gleevec (Imatinib Mesylate); Iclusig (Ponatinib Hydrochloride); Inotuzumab Ozogamicin; Imatinib Mesylate; Kymriah (or Tisagenlecleucel); Marqibo (Vincristine Sulfate Liposome); Mercaptopurine (or Purinethol or Purixan); Methotrexate Sodium (or Trexall); Nelarabine; Oncaspar (or Pegaspargase); Ponatinib Hydrochloride; Prednisone; Purinethol (Mercaptopurine); Vincristine Sulfate, Vincristine Sulfate Liposome, is administrated to the patient.

19. The method according to claim 16, wherein an effective amount of Dexamethasone is administrated to the patient.

20. The method according to claim 19, wherein the administration of the second therapeutic agent is performed simultaneously, separately or sequentially with the administration of the anti-CD127 agent.

Description

LEGENDS OF THE FIGURES

[0160] FIG. 1. Minimal residual disease (MRD) eradication in patients derived xenografts (PDX) experiments. (A) and (B) correspond to the probability of survival of mice over days post-transplant of two PDX issued from two different pediatric patients having t(1;19) ALL. Mice are treated with two different anti-CD127 agents with ADCP capabilities (an antagonist anti-CD127 agent in red (N13B2-hVL6) and a neutral (i.e. not antagonist nor agonist) anti-CD127 agent in green, Effi-3-VH3VL3), and a negative control (blue).

[0161] FIG. 2. Overt leukemia development in patients derived xenografts (PDX) experiments. (A) and (B) correspond to the probability of survival of mice over days post-transplant of two overt leukemia PDX issued from two different pediatric patients having t(1;19) ALL. Results illustrated in (A1) and (B1) correspond to mice treated with an antagonist anti-CD127 agent with ADCP capabilities (red, N13B2-hVL6) and a negative control (blue). Results illustrated in (A2) and (B2) correspond to mice treated with a neutral (i.e. not antagonist nor agonist) anti-CD127 agent with ADCP capabilities (green, Effi-3-VH3VL3) and a negative control (blue).

[0162] FIG. 3. Quantification of specific antibody binding of N13B2-hVL6 in a panel of tumor cell lines issued from different kinds of Acute Lymphoblastic Leukemias. Jurkat, HPB-ALL and DND41 correspond to three different T-cell ALL cell lines. 697, NALM6 and REH correspond to three different B-cell ALL cell lines. The specific binding N13B2-hVL6 was evaluated as the fold change of fluorescence intensity compared to that of an isotype control on each cell line.

[0163] FIG. 4. Normalized phagocytic index of tumor cells issued from different kinds of Acute Lymphoblastic Leukemia cell lines treated with an antagonist anti-CD127 agent (N13B2-hVL6).

[0164] FIG. 5. Phagocytosis of leukemia cells in a sample treated with N13B2-hVL6 with ADCP+ ADCC− capabilities as compared to a control. Leukemia cells (NALM6 cell line) are colored in red, while human M1 macrophages are colored in green. White arrows point to macrophages phagocytosing leukemia cells.

[0165] FIG. 6. Normalized phagocytosis index of leukemia cells in T-ALL models. Two T-ALL cell lineages (HPB-ALL on the left and DND41 IL7-R mutated on the right) have been treated with increasing doses of anti-CD127 antibodies (N13B2-hVL6, 1A11, and EFFI-3-VH3VL3).

[0166] FIG. 7. Normalized phagocytosis index of leukemia cells in B-ALL models. Three B-ALL cell lineages (697 t(1,19) BCP-ALL on the left, NALM6 DUX4 BCP-ALL in the middle and REH t(12;21) BCP-ALL on the right) have been treated with increasing doses of anti-CD127 antibodies (N13B2-hVL6, 1A11, HAL and EFFI-3-VH3VL3).

[0167] FIG. 8. Toxicity of anti-CD127 antibodies and anti-CD47 antibodies on macrophages. The viability of macrophages treated with an anti-CD127 antibody (N13B2-hVL6) or an anti-CD47 antibody (5F9) has been assessed with increasing doses of antibodies.

[0168] FIG. 9. Therapeutic windows for treating ALL with an anti-CD127 antibody or an anti-CD47 antibody. The phagocytic index of normal T cells versus diseased cells (REH model of B-ALL) has been compared in samples treated with increasing doses of an anti-CD127 antibody (N13B2-hVL6) or an anti-CD47 antibody (5F9).

[0169] FIG. 10. A. Phagocytosis of macrophages by macrophages (termed here “autophagocytosis” when treated with an anti-CD47 antibody (5F9), an anti-CD127 antibody (N13B2-hVL6) as compared to a negative control antibody (hlgG4). B. ADCC of human T cells by Natural Killer cells (NK) in presence of an anti-CD127 agent to be used according to the invention (N13B2-hVL6) and a positive control (an anti-CD127 antibody that is known to have an ADCC activity).

[0170] FIG. 11. Number of lymphocytes in the blood of healthy volunteers treated with N13B2-hVL6. (A) and (B) correspond to the lymphocyte counts measured in blood samples collected from healthy volunteers participating to the Single Ascending Dose cohort (SAD, 1 intra-venous injection) and to the Multiple Ascending Dose cohort (MAD, 2 intra-venous injections, 15 days apart), respectively.

[0171] FIG. 12. A. Example of resistance of T-ALL cells to Dexamathasone (HPB-ALL cell line, 48 h treatment). B. Induction of CD127 expression in a Dexamethasone dose-dependent manner in HPB-ALL cells (48 h treatment).

[0172] FIG. 13. Phagocytosis index of HPB-ALL T-ALL cells in response to N13B2-hVL6 with (red triangles) or without (black dots) Dexamethasone treatment (10 μM for 48 h).

[0173] FIG. 14. Phagocytosis index of leukemia cells treated with several anti-CD127 antibodies as defined in the present description. (A) on BCP-ALL cell line and (B) on REH t(12;21) BCP-ALL cell line. NB13B2-hVL6, VL3, VL4, VL5 and N13B2-h3 share the same subset of CDR domains (corresponding to HCDR1 of SEQ ID No.: 3; HCDR2 of SEQ ID No.: 4; HCDR3 of SEQ ID No.: 6; LCDR1 of SEQ ID No.: 8; LCDR2 of SEQ ID No.: 10 and LCDR3 of SEQ ID No.: 11) but have different framework sequences. N13B2-hVL6 has the heavy chain variable domain of SEQ ID No. 22, and the light chain variable domain of SEQ ID No. 26; VL2 has the heavy chain variable domain of SEQ ID No. 22, and the light chain variable domain of SEQ ID No. 21; VL3 has the heavy chain variable domain of SEQ ID No. 22, and the light chain variable domain of SEQ ID No. 23; VL4 has the heavy chain variable domain of SEQ ID No. 22, and the light chain variable domain of SEQ ID No. 24; VL5 has the heavy chain variable domain of SEQ ID No. 22, and the light chain variable domain of SEQ ID No. 25. N13B2alpha and beta are chimeric anti-CD127 antibodies which share closely related CDRs domains (with only one or two mutations within the 6 CDRs domains) with N13B2-hVL6 (CDRs of sequences SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11).

[0174] FIG. 15. Binding of N13B2-hVL6 and control ADCP+/ADCC+ anti-CD127 antibody to main FCγRs, namely A. CD16a, B. CD32a and C. CD64 assessed by ELISA.

EXAMPLES

Material and Methods

[0175] ALL patient samples, human leukemic cell lines. Leukemia patients were treated according to ALL-Berlin-Frankfurt-Münster (BFM) 2000 or 2009 protocols after informed consent in accordance with the Declaration of Helsinki. The study was approved by the ethical committee of the Christian-Albrechts-University Kiel (D437/17). Jurkat, HPB-ALL and DND41 T-ALL cell lines were purchased from ATCC. 697, NALM6 and REH B-ALL cell lines were purchased from DSMZ (Leibniz Institute, Germany). All cells were tested and found free from mycoplasma.

[0176] Mice. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice expressing Hc (NSG-Hc) mice were generated by backcrossing the intact Hc gene from the NOD-CBALs-Hc1/Lt congenic strain into the NSG strain, in collaboration with Lenny Shultz (Jackson Laboratories, Bar Harbor, USA). NSG-Hc mice were bred under pathogen-free conditions at Schleswig-Holstein Kiel University and xenografts were generated in accordance with governmental regulations (Schleswig-Holstein Ministerium für Energiewende, Landwirtschaft, Umwelt, Natur and Digitalisierung): leukemic cells were injected intravenously into female NSG-Hc mice (6-10 weeks of age) and leukemic engraftment was followed by detection of human CD45+/murine CD45−/human CD19+ cells in the peripheral blood via flow cytometry analysis. Animals were sacrificed when showing signs of overt leukemia (detection of >75% leukemic blasts in the peripheral blood or clinical signs of leukemia including loss of weight or activity, organomegaly, hindlimb paralysis). Mouse survival was assessed using Kaplan-Meyer log-rank statistics.

[0177] In minimal residual disease (MRD) experiments, mice were injected with 10,000 BCP-ALL patient derived xenograft cells (n=10) of E2A-PBX1 positive patients (n=2 patients) and antibody N13B2-HVL6 (5 mg/kg), EFFI-3-VH3VL3 (1 mg/kg) or vehicle were injected intravenously every 3 days starting from day 1 until day 21, when injections were applied every 14 days. Minimal residual disease was measured by PCR for patient-specific immunoglobulin/B-cell receptor rearrangements in bone marrow samples isolated from PDX mice.

[0178] In overt leukemia experiments, mice were injected with 1 million BCP-ALL patient derived xenograft cells (n=10) of E2A-PBX1 positive patients (n=2 patients). Once the leukemic engraftment (determined by detection of hCD45+/hCD19+/mCD45− cells in the peripheral blood) was superior to 1%, antibody N13B2-hVL6 (5 mg/kg), EFFI-3-VH3VL3 (1 mg/kg) or vehicle were injected intravenously every 3 days seven times, and subsequently every 14 days.

[0179] Antibodies used in treatments. All antibodies were generated at OSE and found free from endotoxin.

[0180] Phagocytosis assays. In vitro phagocytosis assays were performed by 1-hour coculture of 2.5×10.sup.4 human M1 macrophages labeled with CellTrackerGreen (ThermoFisher, Waltham, Mass., USA, 1/2000, 20 min at 37° C.) and 5×10.sup.4 leukemic cells labeled with CPD (ThermoFisher, 1/2000 10 min at 37° C.) in serum-free RPMI. Phagocytosis was analyzed by a CytoFLEX flow cytometer (Beckman, Brea, Calif., USA) and analysis using FlowJo software (TreeStar, BD Life Sciences Franklin Lakes, N.J., USAs). The phagocytic index was calculated as follows: fold change of percentage of CPD+ cells in CTG+ macrophages compared to the one detected by treatment with istotype control multiplied by the fold change in geometric mean in APC fluorescence (CPD) in CTG+ macrophages compared to the one detected with isotype control. The normalized phagocytic index defines the maximal response by each independent donor against each cell line as 100%, as described in Ring et al. PNAS 2017.

[0181] Visualization of engulfed leukemic cells (CPD+) by M1 human macrophages (CTG+) was investigated in parallel to flow cytometry analysis using a Nikon ECLIPSE Ti2 microscope using the NIS-Elements software (Nikon, Minato City, Tokyo, Japan).

[0182] Time-lapse microscopy experiments were performed in Ibidi 18-well plates coated with Poly-L-Lysine 0,001%. M1 human macrophages were labeled with pHrodo-SE (ThermoFisher) diluted at 1/333000 for 30 min at 37° C. and seeded at 0.1×106 cells per well. Images were taken every 5 minutes for 4 hours and every 15 minutes for 10 hours by a Nikon ECLIPSE Ti2 microscope using the NIS-Elements software (Nikon).

[0183] Phase I study. A First in Human, Phase 1, randomized, double blind, placebo-controlled, single center study (EUDRACT number 2018-001832-22) was conducted in 63 healthy adult male and female volunteers in order to evaluate the safety, tolerability, PK, pharmacodynamics and immunogenicity of single and repeat ascending doses of N13B2-hVL6. N13B2-hVL6 was either administered at single dose (0.002, 0.02, 0.2, 1, 4, or 10 mg/kg IV) or two doses were given 2 weeks apart (6 or 10 mg/kg) and blood samples were drawn in order to evaluate lymphocyte counts after treatment at each time point of the study.

[0184] Quantification of specific antibody binding to CD127. N13B2-h L6 and a corresponding isotype control (MOTA IgG4 S228P) were used to label cells (10 ug/mL each, 30 min at 4° C.). A secondary anti-human IgG Fc [HP6017] Mouse IgG2a, κ PE antibody (BioLegend, San Diego, Calif., USA, cat #409304) was used to detect the level of binding of N13B2-hVL6 to the different cell lines. The fold change of receptor occupancy of N13B2-hVL6 (FC RO) in FIG. 3 was calculated as the fold change of Geometric Mean PE fluorescence of N13B2-hVL6 labelled cells compared to that of isotype control labelled cells.

[0185] ADCC assay. 1 million human freshly isolated T cells were labelled with 15 uL .sup.51Cr (5 mCi/ml, PerkinElmer, Waltham, Mass., USA, cat #NEZ030001MC) for 1 h at 37° C., 5% CO2. T cells were then washed until radioactivity (measured by radioactive gamma counter) was absent in the supernatant. 25 μl/well of T cells-.sup.51Cr target cells at 0.4 million cells/mL (10,000 cells/w) were seeded on P96-microtiter plate (flat bottom). 25 μl/well of anti-hCD127 antibodies were added in triplicate at 200 ng/mL (100 ng/mL final concentration) and left to incubate for 15 min at RT. Eventually, 50 μl of NK cells at 2 million cells/mL (100,000 cells/w) (ratio 10 NK cells:1 T cell) were added and incubated for 4 hours at 37° C., 5% CO2. As positive control of cytotoxicity, 75 μL of SDS 10% was added in 3 wells for 10 min before reading. Eventually, 25 μl/w supernatant were placed into radioactive reader plate and 100 μL/w Microscint Scintillant (PerkinElmer cat #60136211) were added. Release of .sup.51Cr in supernatant was measured by radioactive gamma counter in counts per minutes (cpm). Specific ADCC in FIG. 10 B corresponds to sample well mean cpm (triplicates).

[0186] ELISA binding to FcγR. For binding ELISA assay, recombinant hCD64/FcγRI (R&Dsystems, Minneapolis, Minn., USA; reference 1257-FC-050) or hCD32a/FcγRIIa (R&Dsystems, Minneapolis, Minn., USA; reference 1330-CD-050) or hCD16a/FcγRIIIa (R&Dsystems, Minneapolis, Minn., USA; reference 4325-FC-050) was immobilized on plastic at 2 μg/ml in borate buffer (pH9) and purified antibody were added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035-149) was added and revealed by conventional methods.

Results

Anti-CD127 Agent Effect in Minimal Residual Disease Analysis in a PDX Experiment

[0187] Minimal residual disease (MRD) is the name given to small numbers of leukemic cells that remain in the patient during treatment or after treatment when the patient is in remission (no symptoms or signs of disease). It is the major cause of relapse in leukemia. As illustrated on FIG. 1A, mice with a leukemia patient-derived xenograft (PDX) treated with an anti-CD127 agent having ADCP capabilities over CD127-positive tumor cells all survive during the entire time of the experiment (160 days), while all mice that have been treated with a control were dead after 80 days post-transplantation. Further, it should be noted that 100% of mice were MRD negative, illustrating the anti-leukemic effect of the anti-CD127 agent administrated to the mice, and the potential to fully and definitively treat leukemia. The same result is illustrated on FIG. 1B, wherein mice received a xenograft from another patient. Again, it is shown that mice treated with an anti-CD127 agent having ADCP capabilities towards CD127-positive tumor cells survive, on the contrary to mice treated with a control compound, and further that most of the mice (between 80% and 90% treated with the anti-CD127 agent are MRD negative.

Effect of Administering an Anti-CD127 Agent in an Overt Leukemia Model

[0188] Overt leukemia is the setting where animals are treated when the disease is already well established in the host animal (presence of leukemic blasts over 1 to 5% in the peripheral blood). As illustrated in FIG. 2, PDX mice that have developed an overt leukemia and that were treated with an anti-CD127 agent as defined in the present application survived longer than mice treated with a control compound. In the first xenograft experiment (FIGS. 1A1 and 1A2), the mice treated with the anti-CD127 agent survived between 25% and 50% longer than mice treated with the control antibody. It should be noted that these results were obtained irrespectively of the antagonistic property of the anti-CD127 agent; indeed, even the mice treated with a neutral (i.e. not antagonistic nor agonistic) anti-CD127 agent survived longer than untreated mice. The same results have been obtained in a second experiment (FIGS. 2B1 and 2B2). In this second experiment, it can be seen that some mice treated with anti-CD127 agents that have ADCP capabilities towards CD127-positive tumor cells survived more than 200 days after transplant, twice longer than mice treated with a control. This survival rate is observed irrespectively of the antagonistic capability of the anti-CD127 agent administered to the mice; the same result is observed for mice treated with an antagonistic anti-CD127 agent (FIGS. 2A1 and 2B1) and for mice treated with a neutral (i.e. not antagonist nor agonist) anti-CD127 agent (FIG. 2A2 and 2B2).

In Vitro Effect on the Phagocytosis of Tumor Cell When an Anti-CD127 Agent is Administered

[0189] As illustrated on FIG. 3, CD127 expression (assessed by specific N13B2-hVL6 binding to CD127) is variable in different examples of T-cell ALL (HPB-ALL, and CD127 mutated DND41) and B-cell ALL (697, NAML6, and REH) cell lines, or is absent (Jurkat T-ALL cell line), as defined by using an isotype control as a negative control for the evaluation of CD127 expression level. As illustrated on FIG. 4, wherein no cell pre-treatment has been performed, the phagocytosis of CD127 positive tumor cells is enhanced by an anti-CD127 agent, namely N13B2-hVL6 in all ALL lines expressing CD127, the level of phagocytosis achieved associating with the level of CD127 expression (probed by measurement of specific N13B2-hVL6 binding to CD127). As shown on FIG. 5, the administration of N13B2-hVL6 leads to the phagocytosis of leukemia cells by macrophages, illustrating the positive effect of the anti-CD127 agent to induce, sustain, or enhance the phagocytosis of tumor cells. On FIGS. 6 and 7, the capability of several different anti-CD127antibodies to enhance phagocytosis of tumor cells issued from five ALL cell lines expressing CD127 has been tested. Four anti-CD127 antibodies, namely N13B2-hVL6 (with ADCP capability but no ADCC capability: ADCP+/ADCC−), EFFI-3-VH3VL3 (both in-house antibodies), HAL (initially designed by Pfizer and produced in-house) and 1A11 (initially designed by GlaxoSmithKline and produced in-house) all three having ADCP and ADCC capabilities (ADCP+/ADCC+) have been administered at increasing doses on two T-cell ALL cell lines (HPB-ALL and DND41 IL7R mut.) and three B-ALL cell lines (697 t(1;19), NAML6 (DUX4) and REH t(12;21)) in presence of human macrophages. The phagocytosis of tumor cells by macrophages has been assessed according to the method described here above. On FIG. 6, it is shown that all three anti-CD127 antibodies enhance the phagocytosis of T-ALL tumor cells by macrophages. While it may be considered that the anti-CD127 antibody EFFI-3-VH3VL3 is less efficient to enhance the phagocytosis of tumor cells by macrophages, it should be noted that this antibody is less affine for its target CD127 than the other tested antibodies. It can be seen that the anti-CD127 antibody N13B2-hVL6 is very efficient in enhancing the phagocytosis of tumor cells by macrophages. Similar results are illustrated on BALL-cell lines on FIG. 7. The anti-CD127 agents are all able to enhance the phagocytosis of B-ALL tumor cells by macrophages. The anti-CD127 antibody N13B2-hVL6 is the most efficient to enhance the phagocytosis of B-ALL tumor cells.

[0190] To sum up, these results illustrate that while all anti-CD127 agents tested are efficient to enhance the phagocytosis of CD127-positive tumor cells by macrophages through the ADCP mechanism of action, irrespectively of the type of ALL, including CD127 mutated ALL, N13B2-HVL6 (ADCP+ADCC−) demonstrated the strongest ADCP capability against CD127-positive tumor cells by macrophages, at levels that can surpass that of the reference anti-CD47 antibody 5F9 antibody (see FIG. 9).

In Vitro Toxicity Effect on Macrophages and Healthy T Cells When an Anti-CD127 Agent of the Invention is Administered, and Lack of ADCC Activity on Human T Cells

[0191] The toxicity (i.e. deleterious effect like cell apoptosis or other mechanisms leading to the loss of viable cells) of an anti-CD127 antibody (N13B2-hVL6) or an anti-CD47 antibody (5F9) on macrophages has been assessed and the results are illustrated on FIG. 8. As illustrated, the overall number of live macrophages is not impacted by the dose of anti-CD127 antibody added; it means that the anti-CD127 antibody does not lead to a reduction of the overall number of macrophages, irrespectively of its dosage. On the contrary, when an anti-CD47 agent is administered, the overall number of macrophages is drastically reduced with the administered doses, suggesting that the anti-CD47 agent has a toxic effect on macrophages that leads to their depletion.

[0192] According to these results, the ADCP+/ADCC− N13B2-hVL6 antibody does not have a negative impact on the overall population of macrophages, and does not have any adverse effect on their capability to phagocytose tumor cells, unlike other agents currently used in the treatment of ALL.

[0193] The phagocytosis of tumor cells (from the REH cell line) and normal T cells by macrophages has been assessed in presence of an ADCP+/ADCC− anti-CD127 antibody (N13B2-hVL6) and an anti-CD47 antibody (5F9), which is a positive control for potent induction of phagocytosis. The results are illustrated on FIG. 9. The anti-CD127 antibody does not have any significant impact on the phagocytosis of normal T cells by macrophages. A similar result is obtained when an anti-CD47 agent is administered. However, the administration of an ADCP+/ADCC− anti-CD127 antibody (N13B2-hVL6) leads to a higher increase in phagocytosis of the tumor cells than that of an anti-CD47 agent. These results mean that the anti-CD127 agents are more likely to enhance the phagocytosis of tumor cells while leaving the normal T cells unharmed at different doses. By combining the results illustrated on FIGS. 8 and 9, the inventors show for the first time that the ADCP+/ADCC− anti-CD127 agents of the invention do not lead to macrophage depletion, do not lead to normal T cell phagocytosis, while they greatly enhance CD127-positive tumor cells phagocytosis by macrophages. These results are further confirmed by the data illustrated on FIG. 10A, which correspond to the phagocytosis of macrophages by macrophages (termed here “autophagocytosis”) in presence of an ADCP+/ADCC− anti-CD127 antibody (N13B2-hVL6) or an anti-CD47 antibody (5F9). In presence of the anti-CD47 antibody, the macrophages have an autophagocytosis activity, due to the expression of CD47 by macrophages. When the anti-CD127 antibody is administered, there is no autophagocytosis of macrophages. These results clearly illustrate once again the lack of toxicity of the anti-CD127 antibody. Further, the ADCC of human T cells by Natural Killer cells induced by the anti-CD127 agent used in the invention has been assessed. The results are illustrated on FIG. 10B. As illustrated, the anti-CD127 agent which has ADCP capability and which does not induce ADCC does not lead to lymphodepletion, contrary to the positive control, which is an antibody that binds to the same target, but which is known for enhancing ADCC activity.

Lack of In Vivo Toxicity on Healthy Lymphocytes in Humans When an Anti-CD127 Agent is Administered

[0194] The toxicity (i.e. deleterious effect like cell apoptosis or other mechanisms leading to the loss of viable cells) of an ADCP+/ADCC− anti-CD127 antibody (N13B2-hVL6) on human lymphocytes in vivo has been assessed during a phase 1 clinical trial (EUDRACT number 2018-001832-22) and the results are illustrated on FIG. 11. The administration of single dose of N13B2-hVL6 (0.002, 0.02, 0.2, 1, 4, or 10 mg/kg IV or 1 mg/kg SC) or two doses given 2 weeks apart (6 or 10 mg/kg) was safe and well-tolerated. In all subjects exposed to N13B2-hVL6 up to 10 mg/kg (single and double doses) no clinically significant lymphopenia was reported after N13B2-hVL6 administration.

Effect of Administering an Anti-CD127 Agent Alone or in Combination in a CD127 Positive ALL Cell Line

[0195] As discussed in the description of the invention, several forms of leukemias are resistant to current treatment. As an example, dexamethasone is used to treat different forms of leukemias, but several T-cell ALL and B-cell ALL are known to be resistant to dexamethasone, such as the HPB-ALL cell line (see FIG. 12A) Interestingly, in response to increasing concentrations of dexamethasone treatment, CD127 expression was increased in this cell line in a dose dependent fashion (FIG. 12B).

[0196] The ADCP+/ADCC− anti-CD127 antibody N13B2-hVL6 has been administered to the T-ALL HPB-ALL cell line in presence or absence of dexamethasone. As illustrated in FIG. 13, a synergetic effect of the combination of the anti-CD127 N13B2-hVL6 antibody and dexamethasone can be observed in HPB-ALL cells. These results mean that the use of anti-CD127 agents with ADCP+/ADCC− capabilities is efficient to enhance the phagocytosis of tumor cells by macrophages, and may be efficient to treat CD127-positive cancer, but it also means that these ADCP+/ADCC− anti-CD127 agents may be useful to treat patient that have a CD127-positive cancer that is resistant to current therapies, like dexamethasone therapy.

Effect on the Phagocytosis of Tumor Cells by Macrophage in Presence of Different Anti-CD127 Agents

[0197] Several anti-CD127 antibodies corresponding to their definition in the description of the invention have been tested to assess their capabilities to enhance the phagocytosis of tumor cells by macrophages. As illustrated on FIG. 14A, anti-CD127 antibodies which share the same CDR domains but different frameworks and on FIG. 14B, anti-CD127 antibodies which share close related CDR domains (with only one or two mutations within the 6 CDRs domains) have all the same capability to enhance the phagocytosis of tumor cells by macrophages.

Effect on the Binding to FCγR of Different Anti-CD127 Agents

[0198] Investigation of N13B2-hVL6 to main activating FCγR by ELISA technology (FIG. 15) indicates that, contrary to a positive control ADCP+/ADCC+ anti-CD127 antibody, N13B2-hVL6 does not bind efficiently to CD16a (FIG. 15A), CD32a (FIG. 15B) or CD64 (FIG. 15C), further highlighting the unexpected capacity of N13B2-hVL6 to induce robust ADCP in CD127 positive tumor cells. These three CD markers bind antibodies through the Fc domain of the antibodies, thereby inducing ADCC and ADCP. As illustrated, the antibody that has an IgG1 domain binds to these three CD markers, which was intended, and may accordingly induce cell clearance mechanisms through ADCC and ADCP. But the anti-CD127 antibodies that are IgG4 do not bind to these markers, which can explain the lack of ADCC capability of these antibodies. Nonetheless, the ADCP capabilities of these antibodies are thus unexpected, because ADCP mechanism is especially mediated by these three CD markers.