ANTI-SIRPa ANTIBODIES AND THEIR THERAPEUTIC APPLICATIONS

Abstract

The present invention provides new anti-SIRPa antibodies able to specifically antagonize the interaction between SIRPa and CD47, without affecting the interaction between SIRPg and CD47, and their uses.

Claims

1. An anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic that specifically binds to at least one peptide of amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 within human SIRPa, in particular, that specifically binds to the peptide of amino acid sequence set forth in SEQ ID NO: 3 within human SIRPa and to at least another peptide of amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 within human SIRPa; and/or that specifically binds to a conformational epitope comprising at least one peptide selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72 within human SIRPa.

2. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to claim 1, that specifically binds to the peptides of amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 within human SIRPa; and/or that specifically binds to a conformational epitope consisting of the peptide of amino acid sequence set forth in SEQ ID NO: 73 and the peptide of SIR amino acid sequence within human SIRPa.

3. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to claim 1 or 2, which comprises: a) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and/or b) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16, HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17 SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21, LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 22 and LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 23.

4. An anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic, which comprises: a) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and/or b) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16, HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17 SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21, LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 22 and LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 23.

5. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 4, which is an anti-SIRPa antagonist antibody, which inhibits the binding of human CD47 to human SIRPa.

6. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 5, which does not specifically bind to human SIRPg.

7. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 6, which does not specifically bind to human T-cells, in particular CD3+ T-cells.

8. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 7, which does not inhibit the proliferation of human T-cells.

9. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 8, which does not inhibit the binding of human CD47 to human SIRPg.

10. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 9, which comprises: a heavy chain variable domain comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, and/or a light chain variable domain comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, in particular, which comprises: a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33, and a heavy chain variable domain comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, in particular SEQ ID NO: 29, SEQ ID NO: 30, and more particularly SEQ ID NO: 30.

11. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 10, which comprises: a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 24, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 31, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 26, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 26, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 27, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 27, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 28, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 28, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 30, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 30, and a light chain variable domain comprising or consisting of amino acid sequence set forth in SEQ ID NO: 33.

12. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 11, wherein said antibody is a humanized monoclonal antibody, in particular wherein the antibody light chain constant domain is derived from a human kappa light chain constant domain, more particularly wherein the light chain constant domain consists of the sequence of SEQ ID NO: 35, and wherein the antibody heavy chain constant domain is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain, in particular from a human IgG4 heavy chain constant domain, more particularly wherein the antibody heavy chain constant domain consists of the sequence with SEQ ID NO: 34.

13. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 12, or an anti-human SIRPa antagonist antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic, which inhibits the binding of human CD47 to human SIRPa, and which does not bind specifically to human SIRPg, and/or which does not bind specifically to human T-cells, and/or which does not inhibit the proliferation of human T-cells, and/or which does not inhibit the binding of human CD47 to human SIRPg, for use as a medicament.

14. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 12, or an anti-human SIRPa antagonist antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic, which inhibits the binding of human CD47 to human SIRPa, and which does not bind specifically to human SIRPg, and/or which does not bind specifically to human T-cells, and/or which does not inhibit the proliferation of human T-cells, and/or which does not inhibit the binding of human CD47 to human SIRPg, for use in the prevention or treatment of a disease selected from the group consisting of a cancer (in particular inflammatory cancer and cancer with infiltrated myeloid cells, particularly with infiltrated MDSCs and/or TAM cells), an infectious disease, a chronic inflammatory disease, an auto-immune disease, a neurologic disease, a brain injury, a nerve injury, a polycythemia, a hemochromatosis, a trauma, a sceptic shock, a chronic infectious disease (in particular Pseudomonas and CMV), fibrosis, atherosclerosis, obesity, type II diabetes and a transplant dysfunction, or for use in vaccination.

15. The anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined in any one of claims 1 to 14, for its use according to claim 14, wherein said anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic is administered to a patient presenting a SIRPa-positive tumor.

16. A pharmaceutical composition comprising at least one anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined in any one of claims 1 to 14, and a pharmaceutically acceptable carrier.

17. A combination product comprising: at least one anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined in any one of claims 1 to 14, and at least one second therapeutic agent selected from the group consisting of chemotherapeutic agents, radiotherapy agents, immunotherapeutic agents, cell therapy agents, antibiotics and probiotics; in particular immunotherapeutic agents selected from the group consisting of checkpoint blocker or activator of adaptive immune cells, particularly selected from the group consisting of anti-PDL1, anti-PD1, anti-CTLA4, anti-CD137, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1/3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonist, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists, for simultaneous, separate or sequential use as a medicament.

18. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic according to any one of claims 1 to 12.

19. A vector comprising a nucleic acid molecule according to claim 18.

20. An isolated host cell comprising a nucleic acid molecule according to claim 18 and/or a vector according to claim 19.

21. A polypeptide, particularly an antigen, comprising or consisting of the epitope sequence of human SIRPa consisting of SEQ ID NO: 3, in particular an antigen comprising or consisting of the epitope sequence of human SIRPa consisting of SEQ ID NO: 3 and at least one epitope sequence of human SIRPa selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, more particularly an antigen comprising or consisting of the epitope sequences of human SIRPa consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, said polypeptide consisting of a sequence of up to 300 amino acids.

22. A method of manufacturing an antibody as defined in any one of claims 1 to 12 comprising immunizing a non-human animal, in particular a non-human mammal, against at least one antigen as defined in claim 21 and/or against at least one antigen comprising or consisting of at least one peptide selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73 and the peptide of SIR amino acid sequence, said antigen consisting of a sequence of up to 300 amino acids, and in particular collecting the resulting serum from said immunised non-human animal to obtain antibodies directed against said antigen.

23. An in vitro or ex vivo method to determine a SIRPa positive cells in a subject from a biological sample previously obtained from said subject, comprising: i) determining in vitro the expression and/or the level of expression of SIRPa, in a biological sample previously obtained from said subject using the anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined in any one of claims 1 to 14.

24. An in vitro or ex vivo method of diagnosis, in particular a method of diagnostic suitable for use in personalized medicine, more particularly in a companion diagnosis, wherein an anti-SIRPa antibody or an antigen-binding fragment thereof or an antigen-binding mimetic according to any one of claims 1 to 14 is used for the detection of SIRPa+ cells in a sample previously obtained from a subject and optionally for the quantification of the expression of SIRPa.

25. Use of an anti-SIRPa antibody or an antigen-binding fragment thereof or an antigen-binding mimetic according to any one of claims 1 to 14, in the manufacture of a medicament suitable for use in a diagnostic test, in particular for use in personalized medicine, or in a companion diagnostic test.

26. In vitro or ex vivo use of at least one anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined in any one of claims 1 to 14 in a method wherein SIRPa is used as a biomarker that is predictive for the response to a treatment in a subject, in particular in a cancer subject.

27. An in vitro or ex vivo method of predicting the response of a cancer subject to a treatment, in particular with anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined in any one of claims 1 to 14, comprising: determining the expression level of SIRPa in a tumor sample previously obtained from a subject, in particular with anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined in any one of claims 1 to 14, and comparing the expression level of SIRPa to a value representative of an expression level of SIRPa in a non-responding subject population, wherein a higher expression level of SIRPa in the tumor sample of the subject is indicative for a patient who will respond to the treatment.

Description

FIGURE LEGENDS

[0474] FIG. 1A, B, C, D. Binding analyses of anti-SIRPa antibodies by ELISA assay (human SIRPa-His coating and anti-human kappa detection). [0475] Assessment by ELISA on immobilized SIRPa-His of chimeric (.diamond-solid.), HALA () HFLA (*), HFLB (+), HEFLA (.box-tangle-solidup.), HEFLB (.square-solid.), SIRP29 (), Kwar23 () on A; of HCLA (), HCLB (x), HELA (), HELB () on B; of HALB (), HBLA (_), HBLB () on C. Revelation was performed with a donkey anti-human antibody and revealed by colorimetry at 450nm using TMB substrate. ED50 is the concentration of the indicated antibody to reach 50% of the signal in this assay. Binding of m18D5 clone (.square-solid.) (n=4), SE5A5 commercial clone (.box-tangle-solidup.) (n=7), 6G10 clone () (n=3) and 12D7 clone () (n=4) on D.

[0476] FIG. 2. Affinity analysis by Biacore of anti-SIRP antibodies on human SIRPa recombinant protein. SIRPa-His recombinant protein was immobilized onto a CM5 chip at 5 g/ml (500RU) and the indicated antibodies were added at different concentration. Values were measured after an association period (ka) of 3 min followed by a dissociation period of 10 min (kd) to determine affinity constant (KD).

[0477] FIG. 3A, B, C, D, E, F, G. Binding analyses of anti-SIRPa antibodies on human monocytes (homozygote for SIRPa variant 1 (v1/v1)). [0478] (A, B) Assessment by cytofluorometry on human monocytes v1/v1 (previously stained with human Fc Receptor Binding Inhibitor antibody) of chimeric (.diamond-solid.), HALA () HFLA (*), HFLB (+), HEFLA (.box-tangle-solidup.), HEFLB (.square-solid.), SIRP29 (), Kwar23 (). Revelation was performed with a PE labeled mouse anti-human Fc mAb on Cantoll cytometer, values corresponding to percentage of stained monocytes. ED50 is the concentration of the indicated antibody to reach 50% of the signal in this assay. A corresponds to the percentage of monocytes v1/v1 stained. B corresponds to the mean of fluorescence intensity (MFI) of monocytes v1/v1. [0479] (C, D) Binding study of SIRPa antibodies on human monocytes by Flow cytometry (FACS): different anti-SIRPa antibodies were tested: m18D5 (.square-solid.) (n=1), SE7C2 (.box-tangle-solidup.) (n=2), 12D7 () n=2), 6G10 (.diamond-solid.) (n=4): C represents the Mean Fluorescence Intensity (MFI) of the different antibodies over a dose response. D represents the percentage of stained monocytes over antibody dose response. Statistical analysis were performed when it was possible. [0480] (E, F, G) SIRPa variants binding in the population by anti-h SIRPa antibodies: The capacity of different anti-hSIRPa antibodies to bind SIRPa variants in the 32 volunteers was measured by FACS with a PE-anti mouse IgG. All clones were tested at 10 g/ml: m18D5 (.square-solid.), 12D7 (.Math.), 6G10 (.diamond-solid.) and commercial antibodies SE5A5 () SE7C2 ().E represents the homozygote Variant 1 volunteers (n=16). F represents the homozygote variant 2 volunteers (n=8). G represents the heterozygote V1/V2 volunteers (n=8).

[0481] FIG. 4A, B, C. Competition of anti-SIRPa antibodies with CD47 on SIRPa. [0482] (A) Assessment by ELISA on immobilized SIRPa-His of chimeric (.diamond-solid.), HFLA (*), HFLB (+), HEFLA (.box-tangle-solidup.), HEFLB (.square-solid.), SIRP29 (), Kwar23 () at different concentrations incubated with constant concentration of biotinylated CD47-Fc (6 g/ml). Revelation was performed with streptavidin peroxidase to detect CD47 molecule and revealed by colorimetry at 450 nm using TMB substrate. (B) The results of a second experiment are given with the IC50 values. IC50 is the concentration of the indicated antibody to inhibit 50% of the signal in this assay. [0483] (C) Antagonist activity study of anti-SIRPa antibodies on SIRPa-CD47 interaction by ELISA: The different anti-SIRPa antibodies were tested over a dose response: m18D5 clone (.square-solid.) (n=1), commercial antibody SE5A5 (.box-tangle-solidup.) (n=2) and m12D7 () n=2). The figure represents the percentage of CD47 positive SIRPa-CD47 interactions measured by ELISA during a dose response of anti-hSIRPa antibodies.

[0484] FIG. 5A, B, C. Competition of anti-SIRPa antibodies with CD47 on human monocytes. [0485] (A, B) Assessment by cytometry on human monocytes (v1/v1) of chimeric (.diamond-solid.), HFLA (*), HFLB (+), HEFLA (.box-tangle-solidup.), HEFLB (.square-solid.) at different concentrations incubated with constant concentration of biotinylated CD47-Fc (10 g/ml). Revelation was performed with PhycoErythrin-streptavidin to detect CD47 molecule and revealed by Cantoll cytometer. IC50 is the concentration of the indicated antibody to inhibit 50% of the signal in this assay. A corresponds to the percentage of positive cells. B corresponds to the mean of fluorescence intensity. [0486] (C) Antagonist activity study of anti-SIRPa antibodies on Sirpa-CD47 interaction by FACS: The different anti-SIRPa antibodies were tested over a dose response: m18D5 clone (.square-solid.) (n=1), commercial antibody SE7C2 (.box-tangle-solidup.) (n=2) and m12D7 () n=2). C represents the percentage of CD47 positive cells measured by FACS after competition with anti-hSIRPa antibodies.

[0487] FIG. 6A, B. (A) Affinity analysis by Blitz of anti-SIRP antibody on human SIRPa recombinant protein pre-incubated or not with SP-D ligand. SIRPa-His recombinant protein was immobilized onto a NINTA biosensor at 10 g/ml and the SP-D ligand was added at 100 g/ml (saturating concentration). Then anti-SIRPa antibody was added at 20 g/ml and affinity values were deduced after an association period (ka) of 120 sec followed by a dissociation period of 120 sec (kd) to determine affinity constant (KD). (B) Affinity analysis by Blitz of anti-SIRP antibody on human SIRPa recombinant protein pre-incubated with mouse 18D5 antibody. SIRPa-His recombinant protein was immobilized onto a NINTA biosensor at 10 g/ml and the anti-SIRPa antibody was added at 20 g/ml (saturating concentration). Then SP-D ligand was added at 100 g/ml and affinity values were deduced after an association period (ka) of 120 sec followed by a dissociation period of 120 sec (kd) to determine affinity constant (KD).

[0488] FIG. 7A, B. (A) Affinity analysis by Blitz of anti-SIRP antibodies on human SIRPb recombinant protein. SIRPb-His recombinant protein was immobilized onto a NINTA biosensor and the indicated antibodies were added at 20 g/ml. Values were deduced after an association period (ka) of 120 sec followed by a dissociation period of 120 sec (kd) to determine affinity constant (KD). (B) Binding analysis of anti-SIRP antibodies (human SIRPb-His coating and anti-human kappa detection). Assessment by ELISA on immobilized SIRPb-His of HEFLB (.square-solid.), SIRP29 (), Kwar23 (), B4B6 (.diamond-solid.) and IgG4 Ab control (). Revelation was performed with a donkey anti-human antibody with the exception of B4B6 revealed with a mouse antibody and revealed by colorimetry at 450 nm using TMB substrate.

[0489] FIG. 8A, B. (A) Affinity analysis by Blitz of anti-SIRP antibodies on human SIRPg recombinant protein. SIRPg-His recombinant protein was immobilized onto a NINTA biosensor and the indicated antibodies were added at 10 g/ml. Values were deduced after an association period (ka) of 120 sec followed by a dissociation period of 120 sec (kd) to determine affinity constant (KD). (B) Binding analysis by ELISA assay of anti-SIRP antibodies on SIRPg (human SIRPg-His coating and anti-human kappa detection). Assessment by ELISA on immobilized SIRPg-His of HEFLB (.square-solid.), SIRP29 (), Kwar23 (), LSB2-20 () and IgG4 Ab control () Revelation was performed with a donkey anti-human antibody and revealed by colorimetry at 450 nm using TMB substrate.

[0490] FIG. 9. Affinity analysis by Blitz of CD47 on human SIRPg recombinant protein pre-incubated with anti-SIRP antibodies. SIRPg-His recombinant protein was immobilized onto a NINTA biosensor at 10 g/ml and the indicated antibodies were added at 20 g/ml (saturating concentration). Then, CD47Fc was added at 100 g/ml and affinity values were deduced after an association period (ka) of 120 sec followed by a dissociation period of 120 sec (kd) to determine affinity constant (KD).

[0491] FIG. 10A, B, C. Geometric mean fluorescence intensity measured by flow cytometry on (A) peripheral human CD3+ T cells, (B) red blood cells or (C) platelets after staining with different monoclonal antibodies and revealing with secondary anti-IgG fluorescent antibody.

[0492] FIG. 11A, B. (A) Percentage of positive peripheral human CD3+ T cells after staining with different monoclonal antibodies. (B) The table indicates the value of % of positive cells in duplicate experiments.

[0493] FIG. 12A, B, C, D, E. Human T cells isolated from peripheral blood mononuclear cells from healthy volunteers were stimulated with (A) (C) anti-CD3+ anti-CD28 beads at a 1:1 ratio for 3 days or (B) (D) with allogeneic dendritic cells (DC) at a 5 T cell: 1 DC ratio for 5 days or (E) with different concentrations of tuberculin unpurified protein derivative (PPD) for 5 days. Antibodies were added at day 0 of the culture. Proliferation was measured by incorporation of H3-thymidine during the last 12 h of culture.

[0494] FIG. 13. Mouse CD8+ T cells were isolated from splenocytes of naive mice. CD8 T cells were stimulated with anti-CD3+ anti-CD28 beads at a 1:1 ratio for 3 days. Antibodies were added at day 0 of the culture. Proliferation was measured by incorporation of H3-thymidine during the last 12 h of culture.

[0495] FIG. 14A, B. Human T cells isolated from peripheral blood mononuclear cells from healthy volunteers were stimulated with allogeneic dendritic cells (DC) at a 5 T cell: 1 DC ratio for 5 days. Antibodies were added at day 0 of the culture. Proliferation was measured by incorporation of H3-thymidine during the last 12 h of culture.

[0496] FIG. 15A, B, C, D, E. (A) Anti-tumor effect of anti-SIRPa (P84 clone) i.p. administration three times a week for 4 weeks (300 g/injection) in combination or not with two injection (day 4 & 8) of anti-4-1-BB mAb (3H3 clone, 100 g/injection) or with injections (twice a week) of anti-PDL-1 (10F.9G2 clone, 200 g/injection, treatment during 4 weeks) in an orthotropic model of murine hepatoma (2.5.10{circumflex over ()}6 of Hepa 1.6 cells injected through the portal vein on day 0). Mice were considered cured when they survived three times longer than the time necessary to all control mice died. (B) Tumor infiltrating cells were analyzed at day 13 after tumor inoculation. (C) Tumor infiltrating cells and spleen cells were analyzed at day 13 after tumor inoculation. (D) Mice previously cured in the hepatoma model by anti-SIRPa+anti-4-1BB injection or SIRPa mutant mice treated with anti-4-1BB were rechallenged by Hepa 1.6 cells injection in the spleen (2.5.10{circumflex over ()}6 cells/mouse). Naive mice were injected in parallel in the same route in order to compare tumor development with rechallenged mice. Mice were then left untreated. (E) Mice previously cured in the hepatoma model by anti-SIRPa+anti-4-1BB were rechallenged by Hepa 1.6 cells injection in the spleen (2.5.10{circumflex over ()}6 cells/mouse) and their spleen was harvested 30 days after rechallenge. Splenocytes and T-cell splenocytes were isolated. Naive mice were injected intravenously with vehicle, whole splenocytes (10.10{circumflex over ()}6/mouse) or T-cell purified from splenocytes (2.5.10{circumflex over ()}6/mouse) and all received Hepa1.6 cell injection in the spleen (2.5.10{circumflex over ()}6 cells/mouse). Mice were then left untreated and considered cured when they survived three times longer than the time necessary to all control mice died.

[0497] FIG. 16. Mice previously cured in the hepatoma model by anti-SIRPa+anti-PDL1 injection were rechallenged by Hepa 1.6 cells injection in the spleen (2.5.10{circumflex over ()}6 cells/mouse). Naive mice were injected in parallel in the same route in order to compare tumor development with rechallenged mice. Mice were then left untreated.

[0498] FIG. 17. Anti-tumor effect of anti-SIRPa (P84 clone) i.p. administration three times a week for 4 weeks (200 g/injection) in an orthotopic model of murine mammary carcinoma (0.25.10{circumflex over ()}6 of 4T1 cells injected in the mammary gland). The tumor development was evaluated by measuring the diameter of the tumor and calculated according to the formula: =(0,52*(d.sup.2)){circumflex over ()}1,5. Mice were euthanized when tumor development was nearly 1000 mm.sup.3 according to the ethical guidelines.

[0499] FIG. 18A, B, C. Immune cells phenotype analysis in spleen, tumor and lymph nodes of mice treated with anti-SIRPa (P84 clone) or ctrl mAb i.p. three times a week for two weeks (200 g/injection) in an orthotropic model of murine mammary carcinoma (0.25.10{circumflex over ()}6 of 4T1 cells injected in the mammary gland). Immune cell analysis were performed two weeks after tumor inoculation.

[0500] FIG. 19A, B. Anti-SIRPa (P84 clone), anti-CD47 (MIAP410 clone) and irrelevant isotype control were administered intraperitoneally at day 0 and day 2 at 12 mg/kg in C57Bl/6 mice. Blood samples were collected at day 0 and day 3 in EDTA containing tubes and blood count was performed with a XS-800i hematology analyzer (Sysmex). The level of hemoglobin (A) and the percentage of hematocrit U (B) were evaluated at day 3. The dotted-lines represent normal range values in the C57Bl/6 mice for each parameter.

[0501] FIG. 20A, B. (A) Flow cytometry analysis of anti-SIRPa (HEFLB, grey) and anti-CD47 (B6H12, black) mAb as compared to a control mAb (dotted line) on human platelets freshly isolated from the blood of healthy donors. (B) Human platelet aggregation measurement using optical aggregometer in the presence of a 50 g/ml of control mAb, anti-SIRPa (HEFLB), anti-CD47 (B6H12) or anti-integrin IIb as positive control of inhibitor of aggregation. Antibodies were evaluated on non-activated and ADP-activated platelets.

[0502] FIG. 21A, B, C. Allogeneic CD4+ T cells were cultured in a 1:1 ratio with CD14+ myeloid cells extracted from (A) fresh or (B) frozen ovarian cancer ascites with 10 g/ml of control antibody (white), anti-SIRPa HEFLB (black) or anti-CD47 mAb (grey). Proliferation was measured at day 5 by .sup.3H-thymidine incorporation. (C) Alternatively, allogeneic T cells were cultured in a 5:1 ratio with allogeneic dendritic cells and different ratio of CD14+ myeloid cells extracted from ovarian cancer ascites in the presence of 10 g/ml of antibody as in (A).

EXAMPLES

[0503] In the following Examples, the antibody 18D5 (or m18D5) corresponds to the mouse antibody 18D5, the chimeric antibody corresponds to the chimeric mouse/human 18D5 antibody, and the antibodies HALA, HALB, HBLA, HBLB, HCLA, HCLB, HELA, HELB, HFLA, HFLB, HEFLA and HEFLB correspond to specific humanized 18D5 variants. The antibodies 6G10 and 12D7 belong to the Applicant; these antibodies have been obtained by the same method than m18D5 and are used as control. These control antibodies are IgG2a mouse monoclonal anti-human SIRPa antibodies.

[0504] In addition, commercial antibodies were used for comparison. The first one is an anti-SIRPa antibody, named SE7C2 (Santa Cruz sc-23863); the second antibody is an antibody able to recognize both SIRP a/B and is named SE5A5 (BioLegend BLE323802); and the third one is an anti-human SIRPa antibody named Kwar23 (Creative Biolabs). An anti-human SIRPa antibody named SIRP29 from University of Toronto described in the PCT application WO2013056352 was also used for comparison.

Example 1. Binding Analyses of the Anti-SIRPa Antibodies on SIRPa by ELISA

[0505] Method: The binding activity of the anti-SIRPa antibodies was assessed by ELISA. For the ELISA assay with the chimeric antibody, the humanized antibodies, SIRP29 and Kwar23, a recombinant hSIRPa (Sino Biologicals, Beijing, China; reference 11612-H08H) was immobilized on plastic at 0.5 g/ml in carbonate buffer (pH 9.2) and the purified antibody was 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.

[0506] For the ELISA assay with the mouse antibodies, a recombinant hSIRPa (Sino Biologicals, Beijing, China; reference 10975-H08H) was immobilized on plastic at 0.5 g/ml in carbonate buffer (pH 9.2) and the purified antibody was added to measure binding. After incubation and washing, peroxidase-labeled goat anti-mouse Fc chain (Jackson Immunoresearch; reference 115-036-071) was added and revealed by conventional methods.

[0507] Results: As shown in FIG. 1A, 1B and 1C, the binding activity of the different anti-SIRPa antibodies on SIRPa as measured by ELISA showed effective concentrations (EC50) of 2.9 ng/ml for the chimeric antibody, 3.9 ng/ml for HALA, 5.1 ng/ml for HFLA, 4.0 ng/ml for HFLB, 7.1 ng/ml for HEFLA, 4.4 ng/ml HEFLB in a first experiment, 4.06 ng/ml for the chimeric antibody, 5.60 ng/ml for HCLA, 5.59 ng/ml for HCLB, 4.61 ng/ml for HELA, 4.13 ng/ml for HELB in a second experiment, and 2.74 ng/ml for the chimeric antibody, 2.53 ng/ml for HALB, 2.68 ng/ml for HBLA, 2.95 ng/ml for HBLB in a third experiment. Those results indicate that the antibodies of the invention tested are good SIRPa binders by ELISA as compared to other known anti-SIRPa antibodies SIRP29 (3.7 ng/ml) and Kwar23 (3.3 ng/ml). Those results indicate that the epitope recognized by all the antibodies of the invention is accessible when SIRPa is coated on a plastic well.

[0508] As shown in FIG. 1D, the binding activity of different anti-SIRPa antibodies on SIRPa as measured by ELISA showed an effective dose (ED50) of 0.16 nM (24 ng/ml) for SE5A5 and 0.06 nM (9 ng/ml) for the clone m18D5. The clones 6G10 and 12D7 did not seem to bind SIRPa by ELISA assay. Those results indicate that the clone m18D5 is a good SIRPa binder by ELISA compared to a commercial antibody and indicate that the epitope recognized by this clone is accessible when SIRPa is coated on a plastic well compared to clones 6G10 and 12D7.

Example 2. Biosensor Affinity Measurement of the Anti-SIRPa Antibodies for SIRPa

[0509] Method: Recombinant hSIRPa (Sino Biologicals, Beijing, China; reference 11612-H08H) was immobilized into a CM5 sensor chip (GeHealthcare; France) at 5 g/ml (500RU) and antibodies were applied at different concentrations with a flow rate of 40 l/min. Analysis was performed with a BIAcore 3000 (Biacore, GeHealthcare). Values were measured after an association period (ka) of 3 min followed by a dissociation period of 10 min (kd) to determine affinity constant (KD).

[0510] Results: As shown in FIG. 2, the antibodies of the invention have a strong affinity (KD) for SIRPa (from 1.93e-10 M to 3.67e-10 M), which is equivalent to the affinity of the known anti-SIRPa antibodies SIRP29 and Kwar23 and better than the affinity of the commercial anti-SIRPa antibodies SE7C2 and SE5A5.

Example 3. SIRPa Binding Assay on Human Monocytes by Cytofluorometry

[0511] Method: To measure the binding of the anti-SIRPa antibodies on human monocytes, human Fc Receptor Binding Inhibitor (BD pharmingen; USA; reference 564220) was first added for 30 min at room-temperature to block human Fc receptors on human monocytes to reduce background. Then, an antibody was incubated for 30 min at 4 C., and washed before stained 30 min at 4 C. with PE-labelled anti-human IgG Fc (Biolegend; USA; reference 409303). For the mouse antibodies, a PE-labelled anti-mouseIgG (Jackson Immunoresearch; reference 715-116-151) was used. Samples were analyzed on BD LSRII or Canto II cytofluorometer.

[0512] Results: As shown in FIG. 3, the results indicate a strong binding of the anti-SIRPa antibodies of the invention on human monocytes and a binder binding (as measured with the MFI (Median Fluorescent Intensity)) that the known anti-SIRPa antibodies Kwar23, SE7C2 and SE5A5.

Example 4. Competitive Analysis Between CD47 and the Anti-SIRPa Antibodies by Antagonist ELISA Assay

[0513] Method: For competitive ELISA assay, recombinant hSIRPa (Sino Biologicals, Beijing, China; reference 11612-H08H) was immobilized on plastic at 0.5 g/ml in carbonate buffer (pH 9.2). For the chimeric antibody, the humanized antibodies, SIRP29 and Kwar23, a purified antibody (at different concentrations) was mixed with 6 g/ml final (fix concentration) of biotinylated Human CD47Fc (AcroBiosystems interchim; France; reference: #CD7-H82F6) to measure competitive binding for 2 h at 37 C. After incubation and washing, peroxidase-labeled streptavidin (Vector laboratoring; USA; reference SA-5004) was added to detect Biotin-CD47Fc binding and revealed by conventional methods.

[0514] For the mouse antibodies, a purified antibody (at different concentrations) was mixed with 0.04 g/ml of CD47Fc (Sino Biologicals, Beijing, China; reference 12283-H02H) to measure competitive binding for 2 h at 37 C. After incubation and washing, peroxidase-labeled donkey anti-human Fc chain (Jackson Immunoresearch; reference 709-035-149) was added to detect CD47Fc binding and revealed by conventional methods.

[0515] Results: As shown in FIG. 4, the antibodies of the invention have an antagonist activity on the SIRPa-CD47 interaction. In particular, it is observed that the chimeric antibody, HFLA, HFLB, HEFLA and HEFLB have a better antagonist activity as compared to the antagonist activity of SIRP29 and the commercial anti-SIRPa antibody SE5A5.

Example 5. Competitive Analysis Between CD47 and the Humanized Anti-SIRPa Antibodies on Human Monocytes by Antagonist Cytofluorometry Assay

[0516] Method: To measure the competition between CD47 and the humanized anti-SIRPa antibodies on human monocytes, a purified antibody was added on monocytes for 15 min at 4 C., then mixed with 5 g/ml final of biotinylated Human CD47Fc (AcroBiosystems interchim; France; reference: #CD7-H82F6) and incubated for 30 min at 4 C. to measure competitive binding antibody. After incubation and washing, PE-labelled streptavidin (BDBiosciences; USA; reference 554061) was added for 15 min at 4 C. to detect Biotin-CD47Fc binding and analyzed on BD LSRII or Canto II cytofluorometer.

[0517] To measure the competition between CD47 and the mouse anti-hSIRPa antibodies on human monocytes, a purified antibody was added on monocytes for 15 min at 4 C., then mixed with 5 g/ml final of CD47Fc (Sino Biologicals, Beijing, China; reference 12283-H02H) and incubated for 15 min at 4 C. to measure competitive binding antibody. After incubation and washing, FITC-labelled anti-human Fc (Beckman Coulter; reference IM1627) was added for 15 min at 4 C. to detect CD47Fc binding and analyzed on BD LSRII or Canto II cytofluorometer.

[0518] Results: As shown in FIG. 5, the antibodies of the invention have an antagonist activity on SIRPa-CD47 interaction on human monocytes.

Example 6. Blitz Method Competition With SP-D

[0519] Method: This method was performed with a Blitz (Fort Bio; USA; reference C22-2 No 61010-1).

[0520] Condition A: SIRPa+Anti-SIRPa antibody+Surfactant Protein D (SP-D). In a first step, SIRPa (His) recombinant protein (Sino Biologicals, Beijing, China; reference 11612-H08H) was immobilized at 10 g/ml by histidine tail into a Ni-NTA biosensor (Fort Bio; USA; reference 18-0029) for 30 seconds. In a second step, anti-SIRPa antibodies were added at 20 g/mL (saturating concentration) for 120 seconds. Then, human SP-D (R et D Systems; USA; reference 1920-SP-050) was associated at 100 g/mL, in competition with anti-SIRPa antibodies, for 120 seconds. The dissociation of SP-D was made in kinetics buffer for 120 seconds. Analysis of data was made with the Blitz pro 1.2 software, which calculated association constant (ka) and dissociation constant (kd) and determined the affinity constant KD (ka/kd). Condition B: SIRPa+Surfactant Protein D (SP-D)+Anti-SIRPa antibody. In a first step, Sirp-a (His) recombinant protein (Sino Biologicals, Beijing, China; reference 11612-H08H) was immobilized at 10 g/ml by histidine tail into a Ni-NTA biosensor (Fort Bio; USA; reference 18-0029) for 30 seconds. In a second step, human SP-D (R et D Systems; USA; reference 1920-SP-050) was added at 100 g/mL for 120 seconds. Then, anti-SIRPa antibodies were associated at 20 g/ml (saturating concentration) for 120 seconds. The dissociation of anti-SIRPa antibody was made in kinetics buffer for 120 seconds. Analysis data was made with the Blitz pro 1.2 software, which calculated association constant (ka) and dissociation constant (kd) and determined the affinity constant KD (ka/kd).

[0521] Results: As shown in FIG. 6, the binding of the anti-SIRPa antibody 18D5 does not block the binding of SP-D to SIRPa and the binding of SP-D does not block the binding of 18D5 to SIRPa. Thus, the antibody of the invention does not inhibit the interaction between SIRPa and SP-D.

Example 7. Affinity of the Anti-SIRPa Antibodies for SIRPb by Blitz Method

[0522] Method: This method was performed with a Blitz (Fort Bio; USA; reference C22-2 No 61010-1). Recombinant hSIRPb-His (Antibodies-online; USA; reference ABIN3077231) was immobilized at 10 g/ml by histidine tail into a Ni-NTA biosensor (Fort Bio; USA; reference 18-0029) for 30 seconds. Then, an anti-SIRPa antibody was associated at 20 g/mL for 120 seconds. The dissociation of anti-SIRPa antibody was made in kinetics buffer for 120 seconds. Analysis of data was made with the Blitz pro 1.2 software, which calculated association constant (ka) and dissociation constant (kd) and determined the affinity constant KD (ka/kd).

[0523] Results: As shown in FIG. 7A, the antibodies of the invention have a lower affinity for SIRPb as compared to SIRPa. In particular, it is noted that the chimeric antibody, HFLA, HFLB, HEFLA, HEFLB have a reduced affinity for SIRPb as compared to SIRP29 and Kwar23.

Example 8. ELISA Binding of Anti-SIRP Antibodies on SIRPb

[0524] Method: For activity ELISA assay, recombinant hSIRPb-His (Antibodies-online; USA; reference ABIN1466557) was immobilized on plastic at 1 g/ml in carbonate buffer (pH 9.2) and a purified antibody was 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.

[0525] Results: As shown in FIG. 7B, the anti-SIRPa antibodies have a low affinity for SIRPb. It must be indicated that the revelation was performed with a donkey anti-human antibody for all antibodies except for B4B6 (revealed with a mouse antibody), which may explain that the signal obtained for the anti-SIRPb antibody B4B6 is lower than the signal obtained for the anti-SIRPa antibodies.

Example 9. Affinity Analysis of the Anti-SIRPa Antibodies for SIRPg by Blitz Method

[0526] Method: This method was performed with a Blitz (Fort Bio; USA; reference C22-2 No 61010-1). Recombinant hSIRPg-His (Sino Biologicals, Beijing, China; reference 11828-H08H) was immobilized at 10 g/ml by histidine tail into a Ni-NTA biosensor (Fort Bio; USA; reference 18-0029) for 30 seconds. Then, an anti-SIRPa antibody was associated at 20 g/mL for 120 seconds. The dissociation of anti-SIRPa antibody was made in kinetics buffer for 120 seconds. Analysis of data was made with the Blitz pro 1.2 software, which calculated association constant (ka) and dissociation constant (kd) and determined the affinity constant KD (ka/kd).

[0527] Results: As shown in FIG. 8A, the anti-SIRPa antibodies of the invention have a low affinity for SIRPg. This affinity is slightly weaker than the affinity of the known anti-SIRPa antibodies SIRP29 and Kwar23. However, the kinetics properties differ between anti-SIRPa antibodies, SIRP29 and Kwar23, with a high dissociation rate constant (Kd) for anti-SIRPa antibodies as compared to SIRP29 and Kwar23. In particular, HFLB has the lowest affinity for SIRPg with a KD value of 1.036e-7 M that equals to a 2-log difference as compared to the KD values of SIRP29 and Kwar23.

Example 10. ELISA Binding of the Anti-SIRP Antibodies on SIRPg

[0528] Method: For activity ELISA assay, hSIRPg-His (Sino Biologicals, Beijing, China; reference 11828-H08H) was immobilized on plastic at 1 g/ml in carbonate buffer (pH 9.2) 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.

[0529] Results: As shown in FIG. 8B, the anti-SIRPa antibody HEFLB does not bind SIRPg while the known anti-SIRPa antibodies SIRP29 and Kwar23 show a significant binding to SIRPg.

Example 11. Blitz Method Competition With CD47 for SIRPg: SIRPg+Anti-SIRPa Antibody+CD47

[0530] Method: This method was performed with a Blitz (Fort Bio; USA; reference C22-2 No 61010-1). In a first step, hSIRPg-His (Sino Biologicals, Beijing, China; reference 11828-H08H) was immobilized at 10 g/ml by histidine tail into a Ni-NTA biosensor (Fort Bio; USA; reference 18-0029) for 30 seconds. In a second step, an anti-SIRPa antibody was added at 20 g/ml (saturating concentration) for 120 seconds. Then, human CD47Fc ((Sino Biologicals, Beijing, China; reference 12283-H02H) was associated at 100 g/mL, in competition with anti-SIRPa antibodies, for 120 seconds. The dissociation of CD47Fc was made in kinetics buffer for 120 seconds. Analysis data was made with the Blitz pro 1.2 software, which calculated association constant (ka) and dissociation constant (kd) and determined the affinity constant KD (ka/kd).

[0531] Results: As shown in FIG. 9, the anti-SIRPa HEFLB of the invention does not compete with the binding of CD47 to SIRPg. At the opposite, the other known antibodies SIRP29 and, in particular, kwar23 compete with the binding of CD47 to SIRPg.

Example 12. Binding to Blood Cells by Flow Cytometry

[0532] Method: The experiment was realized to analyze the binding of the anti-SIRPa antibodies on human blood cells. CD3-positive T lymphocytes, red blood cells and platelets were extracted from purified blood from healthy volunteers. Cells were then stained for 30 min at 4 C. with 10 micrograms/ml of each tested antibody, washed and then stained with a secondary fluorescent anti-IgG antibody for another 30 min at 4 C. After washes, cells were analyzed on a CANTO II (BD Bioscience) flow cytometer.

[0533] Results: As shown in FIG. 10, the T cells, the red blood cells and the platelets are positive for CD47, which is expressed ubiquitously, and they were stained with the B6H12 antibody. The SIRP29 and the Kwar23 antibodies, like LSB2.20 (specific anti-SIRP antibody), bind to T cells that are known to express SIRP. However, the anti-SIRPa humanized 18D5 antibody does not bind to the T cells (same results obtained with four different 18D5 humanized variants tested). Red blood cells and platelets do not express SIRPa and, thus, they were not revealed with the humanized 18D5 antibody and the other anti-SIRPa antibodies. This result shows the specificity of the humanized 18D5 antibody for SIRPa on live cells as compared to the known anti-SIRPa antibodies.

[0534] As shown in FIG. 11, the T-cells are not stained by the humanized 18D5 antibody (same results obtained with five different 18D5 humanized variants tested) and with the chimeric 18D5 (data not shown) whereas more than 70% of T cells are stained by SIRP29 and Kwar23.

Example 13. Human CD3+ T Cell Proliferation

[0535] Method: hPBMC were isolated from buffy coat of healthy volunteers. CD4 or CD8 T cells were selected by positive selection using an AutoMACS (Miltenyi) and plated in 96-round well plate (50 000 cells/well). The proliferative signals were provided by either anti-CD3/anti-CD28 coated microbeads (LifeTechnologies) at a 1 bead for 1 T cell ratio during three days, or allogeneic mature dendritic cells generated in vitro at a 5 T cell for 1 mDC during 5 days or with different concentrations of tuberculin unpurified protein derivative (PPD) for 5 days. Antibodies targeting the SIRPa/CD47 pathway were added from the beginning of the proliferation test at a saturating concentration (10 g/ml). Proliferation was measured by incorporation of H3-thymidine during the last 12 h of culture.

[0536] Results: As shown in FIG. 12, the anti-SIRPa antibody HALA and HEFLB variants do not inhibit the T cell proliferation when T cells are stimulated with anti-CD3+ anti-CD28 beads (A) (C) or with allogenic dendritic cells (B) (D) or with PPD (E), whereas the anti-SIRPa Kwar23 inhibits T cell proliferation when T cells are stimulated with allogenic dendritic cells. As expected, the anti-CD47 antibodies and the anti-SIRPg antibody are inhibitors of the T cell proliferation.

Example 14. Mouse CD8+ T Cell Proliferation

[0537] Method: Splenocytes were isolated from naive mice. CD8 T cells were selected by positive selection using an AutoMACS (Miltenyi) and plated in 96-round well plate (50 000 cells/well). The proliferative signals were provided by anti-CD3/anti-CD28 coated microbeads (LifeTechnologies) at a 1 bead for 1 T cell ratio during three days. A mouse anti-SIRPa antibody (P84) and an anti-CD47 antibody (MIAP310) targeting the SIRPa/CD47 pathway were added from the beginning of the proliferation test at a saturating concentration (10 g/mL). Proliferation was measured by incorporation of H3-thymidine during the last 12 h of culture.

[0538] Results: As shown in FIG. 13, there is no inhibition of the anti-SIRPa or anti-CD47 antibody on the proliferation of mouse T cells. This result is explained by the fact that mice does not express the SIRPg gene. Thus, mice can be used as a model to predict the in vivo effects of a specific anti-SIRPa antibody that does not bind SIRPg. In contrast, anti-CD47 or non-selective anti-SIRPa antibodies in vivo preclinical efficacy, in particular on adaptive immunity and generation of memory T lymphocytes, is not predictive of human situation.

Example 15. Human T Cell Proliferation

[0539] Method: hPBMC were isolated from buffy coat of healthy volunteers. CD4 or CD8 T cells were selected by positive selection using an AutoMACS (Miltenyi) and plated in 96-round well plate (50 000 cells/well). The proliferative signals were provided by either anti-CD3/anti-CD28 coated microbeads (LifeTechnologies) at a 1 bead for 1 T cell ratio during three days, or allogeneic mature dendritic cells generated in vitro at a 5 T cell for 1 mDC during 5 days. Antibodies were added from the beginning of the proliferation test at a saturating concentration (5 g/mL for anti-CD47 and anti-SIRPa antibodies and 2.5 g/mL for the anti-PD-1/PD-L1 antibodies and the recombinant 4-1BBL). Proliferation was measured by incorporation of H3-thymidine during the last 12 h of culture.

[0540] Results: As shown in FIG. 14, the anti-PD-1/PD-L1 antibodies and the recombinant 4-1BBL have a boost effect on the proliferation of human T cells, while anti-CD47 has a negative effect on the proliferation of human T cells. In particular, the anti-CD47 antibody prevents the human T-cell immunostimulatory efficacy of anti-PD-1/PD-L1 or 4-1BB agonist agents. The anti-SIRPa HEFLB has no significant effect on the proliferation of T cells.

Example 16. Anti-Tumor Effects in Mice

[0541] Method: Mice were anesthetized with a cocktail of xylazine/ketamine. After a laparotomy, tumoral Hepa 1.6 cells were injected through the portal vein (2,5.106 cells/100 L) in PBS. The treatment was started 4 days after tumor injection. The agonistic anti-4-1BB monoclonal antibody (3H3) was injected two times at d4 and d8 after Hepa 1.6 cells (Hepatocarninoma cells, HCC) injection intraperitoneally in PBS (100 g/injection). The anti-PDL1 monoclonal antibody was injected twice a week during 4 weeks intraperitoneally in PBS (200 g/injection). The antagonistic anti-SIRPa antibody (P84) was injected three time a week during four weeks intraperitoneally in PBS (300 g/injection).

[0542] The anti-tumor response was evaluated in the orthotopic model of HCC thirteen days after the tumor inoculation. At this time, the tumor and the spleen were collected in order to phenotype the immune cells that infiltrated the tumor or in the systemic way. Splenocytes and non-parenchymal cells (NPC) of the liver which are the infiltrating immune cells were stained with four different mixes for flow cytometry acquisition.

[0543] Results: As shown in FIG. 15A, the anti-SIRPa antibody alone significantly prolongs survival in a fraction of mice (28%). In combination with anti-4-1BB or anti-PDL1 antibodies, anti-SIRPa antibody allows a very high response rate of mice surviving even after treatment withdrawal.

[0544] As shown in FIG. 15B, the combination of anti-SIRPa with a co-stimulatory agent (e.g. anti-4-1BB) or T-cell checkpoint inhibitor (e.g. anti-PDL1) modifies the tumor microenvironment by decreasing the regulatory and immunosuppressive immune cells (Tregs, Mo-MDSC) while increasing accumulation of effector memory CD8+ T cells in combination with anti-4-1BB. The Mo-MDSC are characterized by a high expression of Ly6C and no Ly6G among the CD11b positive-and MHC class II negative-population.

[0545] As shown in FIG. 15C, the combination of anti-SIRPa with a co-stimulatory agent (e.g. anti-4-1BB) or T-cell checkpoint inhibitor (e.g. anti-PDL1) modifies the cell composition of the tumor microenvironment and in periphery in the spleen, by decreasing the frequency of immature and nave B cells while increasing accumulation of memory and plasmablast cells. Similarly, accumulation of cytolytic (CD27-negative) NK cells is induced the tumor and periphery by the anti-SIRPa combination with anti-41BB or anti-PDL1.

[0546] Altogether, anti-SIRPa modifies the tumor and peripheral immunity in particular adaptive (T-cell, Tregs, B-cells) and innate (MDSC, Macrophages, NK cells) immune cells contributing to tumor elimination and long-term protection.

Example 17. Anti-Tumor Effects in Mice Previously Cured

[0547] Method: Mice previously cured in the hepatoma model by anti-SIRPa+anti-4-1BB injection or SIRPa mutant mice treated with anti-4-1BB were rechallenged by Hepa 1.6 cells injection in the spleen (2.5.10{circumflex over ()}6 cells/mouse). Mice were anesthetized with 3% of isoflurane in the air. After incision on the flank of the mice and isolation of the spleen, tumoral Hepa 1.6 cells were injected into the spleen (2,5.106 cells/50 L) in PBS. Naive mice were injected in parallel in the same route in order to compare tumor development with rechallenged mice.

[0548] Results: As shown in FIG. 15D, all the cured mice survived when rechallenged and, at the opposite, all nave mice died. This result demonstrates that memory T cells were induced under anti-SIRPa therapy or absence of SIRPa signals (SIRPa mutant mice) and still persist on the long-term in cured mice.

Example 18. Anti-Tumor Effects of T-Cell Splenocytes or Whole Splenocytes Collected From Mice Previously Cured

[0549] Method: Cured anti-SIRPa+anti-4-1BB rechallenged mice were euthanized and the spleen was collected. After red blood cell lysis, splenocytes were extracted and CD3 positive T cells were isolated from a part of splenocytes with an AutoMACS. After anesthesia, mice were injected with either T-cell splenocytes (2.5.10.sup.6 cells/100 L) or whole splenocytes (10.10.sup.6 cells/100 L) or excipient alone (PBS) intravenously. All mice received Hepa 1.6 cells through the portal vein as described previously (2.5.10.sup.6 cells/100 L).

[0550] Results: As shown in FIG. 15E, the splenocytes and isolated T lymphocytes collected from cured mices has a high positive effect on the survival of mice. This results indicate that memory T cells are present in the splenocytes of the cured mice after treatment of the hepatoma and are responsible on the long-term adaptive immune memory.

Example 19. Anti-Tumor Effects in Mice Previously Cured

[0551] Method: Mice previously cured in the hepatoma model by anti-SIRPa+anti-PDL-1 injection were rechallenged by Hepa 1.6 cells injection in the spleen (2.5.10{circumflex over ()}6 cells/mouse). Mice were anesthetized with 3% of isoflurane in the air. After incision on the flank of the mice and isolation of the spleen, tumoral Hepa 1.6 cells were injected into the spleen (2.5.10.sup.6 cells/50 L) in PBS. Naive mice were injected in parallel in the same route in order to compare tumor development with rechallenged mice.

[0552] Results: As shown in FIG. 16, all the cured mice survived when rechallenged and, at the opposite, all nave mice died. This result suggests that memory T cells are still present in cured mice. This result demonstrates that memory T cells were induced under anti-SIRPa therapy and still persist on the long-term in cured mice.

Example 20. Effects of the Growth of a Tumor in a Mammary Carcinoma Model

[0553] Method: Mice were anesthetized with 3% of isoflurane in the air. Mice were shaved on the abdomen and 4T1 cells were injected in the mammary gland with an insulinic syringe (30 Gauges) in 50 L of PBS. The antagonistic anti-SIRPa antibody (P84) or a control antibody was injected three time a week during four weeks intraperitoneally in PBS (200 g/injection).

[0554] Results: As shown in FIG. 17, the anti-SIRPa antibody significantly (p<0.01) reduces the growth of the tumor in the mammary carcinoma model as compared to a control antibody.

[0555] FIG. 18 shows the immune cell analysis two weeks after inoculation. Anti-SIRPa has a positive effect on myeloid and non-myeloid cells (T and NK cells) both in tumor and in periphery (spleen) with a dramatic decrease of Tregs and accumulation of memory T cells.

Example 21. Effects of SIRPa Antibodies on the Concentration of Hemoglobin and on the Hematocrit

[0556] Method: Anti-SIRPa (P84 clone), anti-CD47 (MIAP410 clone) and irrelevant isotype control were administered intraperitoneally at day 0 and day 2 at 12 mg/kg in C57Bl/6 mice. Blood samples were collected at day 0 and day 3 in EDTA containing tubes and blood count was performed with a XS-800i haematology analyzer (Sysmex). The level of hemoglobin (left) and the percentage of hematocrit (right) were evaluated at day 3.

[0557] Results: As shown in FIG. 19, the anti-SIRPa antibody has no toxic effect on the concentration of hemoglobin and on the hematocrit. At the opposite, the anti-CD47 antibody induces a decrease of the concentration of hemoglobin and of the hematocrit in accordance with anemia observed during phase 1 in man.

Example 22. Platelet Aggregation

[0558] Method: Blood was collected from healthy donor volunteers into Vacuette collection tubes (Greiner Bio-One) buffered with sodium citrate. Platelet rich plasma (PRP) and platelet poor plasma (PPP) were obtained by centrifugation for 10 minutes at 200 g and 15 minutes at 3 500 g, respectively. The working PRP was adjusted to 3.10.sup.8 platelets.L.sup.1. Inhibition Assays: mAb were pre-incubated with PRP for a final concentration of 40 or 50 g.mL.sup.1 test antibodies. After 3 minutes without stirring, platelet aggregation was initiated with ADP 5 M addition. Aggregation was determined by measuring the transmission of light through the sample at 37 C. with continuous stirring using a standard optical aggregometer (TA-8V Thrombo-Aggregometer, SD Innovation SAS, Frouard, France). The transmission of PPP was set as 100%. Aggregation was recorded under stirring for a total of 5 minutes. Induction Assays: Platelet aggregation was directly initiated by mAb addition (50 g.mL.sup.1). Aggregation was recorded under stirring for a total of max. 10 minutes.

[0559] Results: As shown in FIG. 20, in contrast to anti-CD47 antibodies, anti-SIRPa antibodies does not bind to human red blood cells or platelets. Consequently, anti-CD47 induces in vitro human platelets aggregation while anti-SIRPa antibodies does not. Similarly, anti-SIRPa antibodies does not disturb reversible ADP-induced human platelets aggregation while anti-integrin alpha 2b completely abrogates it.

Example 23. Proliferation of Allogeneic T cells by SIRPa-Blocking CD14+ Cells From a Cancer Ovarian Ascitis

[0560] Method: Allogeneic CD4 T cells were isolated by positive selection using an AutoMACS (Miltenyi) from hPBMC of a buffy coat of a healthy volunteer. CD4 were plated in 96-round well plate (50 000 cells/well). CD14+ cells were isolated by the same method from the ascitis of a cancer ovarian patient. The CD14+ cells were plated with the allogeneic CD4 T cells at a 1:1 ratio for 5 days. In some conditions, human LPS-matured allogeneic monocyte-derived dendritic cells (moDC) were added at a 1:5 ratio to stimulate T cells and analyzed the immunosuppressive action of different ratio of CD14+ MDSC purified from the ascite. Antibodies targeting the SIRPa/CD47 pathway were added from the beginning of the proliferation test at a saturating concentration (10 g/mL). Proliferation was measured by incorporation of H3-thymidine during the last 12 h of culture.

[0561] Results: As shown in FIGS. 21A and 21B, Fresh and frozen human myeloid cells (TAM) purified from ovarian cancer ascites are hypo-stimulating allogeneic human T lymphocytes. In contrast to anti-CD47 antibodies, anti-SIRPa antibodies modifies myeloid cells properties allowing human T-cell activation and proliferation.

[0562] As shown in FIG. 21C, human myeloid cells (MDSC) purified from ovarian cancer ascites can suppress human T-cell proliferation induced by allogeneic moDC at 1:1 and 2:1 myeloid to T-cell ratio. In contrast to anti-CD47 antibodies, anti-SIRPa antibodies does not potentiates the immunosuppression induced by human MDSC.