USE OF ANTI-HUMAN SIRPA V1 ANTIBODIES AND METHOD FOR PRODUCING ANTI-SIRPA V1 ANTIBODIES

Abstract

The invention is in the field of immunotherapy. The present invention provides antibodies useful in therapeutic and diagnostic applications targeting human SIRPa, said antibodies enhancing the cross-presentation of antigens to T cells. The invention also provides antibodies against specific isoforms of SIRP a and able to disrupt the interaction between those isoforms of SIRP a and human CD47 for use in treating or preventing a disease, or diagnostic applications, and methods for producing and/or selecting anti-human SIRPa antibodies that bind with different affinities to various isoforms of human SIRP members.

Claims

1. An anti-human SIRPa antibody, antigen-binding fragment thereof or modified antibody thereof, which comprises: (a) heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, wherein: HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID No: 9, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID No: 10 or SEQ ID No: 11, HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID No: 12, or SEQ ID No: 13, or SEQ ID No: 14 or SEQ ID No: 15; and (b) a light chain variable domain comprising the amino acid sequence set forth in SEQ ID No: 16 or in SEQ ID No: 17, for use in the treatment or the prevention of a disease, in particular a cancer, or for use in therapeutic vaccination against a disease, in particular against a cancer, wherein the antibody, antigen-binding fragment thereof, or modified antibody thereof, enhances cross-presentation of an antigen expressed in said disease, in particular in said cancer, and is involved in eliciting a T cell response suitable for the treatment of said disease.

2. The anti-human SIRPa antibody or antigen-binding fragment thereof or modified antibody thereof for use according to claim 1, which: binds specifically to human SIRPa v1 and inhibits the binding of human CD47 to human SIRPa v1, does not prevent or inhibit the binding of human CD47 to human SIRPg; and in particular does not bind specifically to human SIRPg.

3. An anti-human SIRPa antibody or antigen-binding fragment thereof or modified antibody thereof, which comprises: a) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, wherein: HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID No: 9, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID No: 10 or SEQ ID No: 11, HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID No: 12, or SEQ ID No: 13, or SEQ ID No: 14 or SEQ ID No: 15; and b) a light chain variable domain comprising of the amino acid sequence set forth in SEQ ID No: 16 or in SEQ ID No: 17; wherein the anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic inhibits the binding of human CD47 to human SIRPa v1 and does not prevent or inhibit the binding of human CD47 to human SIRPa v2; for use in the prevention and/or treatment of a disease in a subject that is SIRPa v1 positive.

4. The anti-human SIRPa antibody or antigen-binding fragment thereof or modified antibody thereof for use according to anyone of claims 1 to 3, wherein the heavy chain variable domain comprises or consists of the amino acid sequence set forth in SEQ ID No: 18, or in SEQ ID No: 19, or in SEQ ID No: 20; or in SEQ ID No: 21; or in SEQ ID No: 22; or in SEQ ID No: 23; in particular the heavy chain variable domain comprises or consists of the amino acid sequence set forth in SEQ ID No: 20; wherein the light chain variable domain comprises or consists of the amino acid sequence set forth in SEQ ID No: 17.

5. The anti-human SIRPa antibody or antigen-binding fragment thereof modified antibody thereof for use according to any one of claims 1 to 4, wherein the disease is selected from the group consisting of a cancer, in particular inflammatory cancer and cancer with infiltrated myeloid cells, particularly with infiltrated Dendritic Cells and/or MDSCs and/or TAM cells, cancer metastasis, in particular breast cancer metastasis, melanoma, or wherein the use according to claim 1 or 4 is for therapeutic vaccination against one of these diseases, in particular a therapeutic vaccination against melanoma.

6. The human anti-SIRPa antibody or antigen-binding fragment thereof modified antibody thereof for use according to any one of claims 1 to 5, which has the following properties: it does not bind specifically to human SIRPa v2; and it does not inhibit the binding of human CD47 to human SIRPg; in particular it does not bind specifically to human SIRPg; and it binds with human SIRPa v1 with an affinity of at least 10E-9 M; and it enhances the cross-presentation of at least one antigen by Antigen Presenting Cells to human T cells, in particular by dendritic cells, in particular to human CD8+ T cells; and optionally at least one of the following properties: it does not inhibit the activation and/or the proliferation of human T cells in vivo; and/or it enhances the activation of macrophages.

7. The human anti-SIRPa antibody or antigen-binding fragment thereof or modified antibody thereof for use according to any one of claims 1 to 6, wherein the disease is a cancer, and wherein at least one antigen selected from the group consisting of antigens from Human Papilloma Virus, Epstein-Barr Virus, Merkel cell polyomavirus, Human Immunodeficiency Virus, Human T-cell Leukemia Virus, Human Herpes Virus 8, Hepatitis B virus, Hepatitis C virus, HCV, HBC, Cytomegalovirus, or from the group of single-point mutated antigens derived from the group consisting of the antigens of ctnnb1 gene, casp8 gene, her2 gene, p53 gene, kras gene, nras gene, or tumor antigens, in particular tumor antigens issued or derived from the group consisting of ras oncogene, BCR-ABL tumor antigens, ETV6-AML1 tumor antigens, melanoma-antigen encoding genes (MAGE), BAGE antigens, GAGE antigens, ssx antigens, ny-eso-1 antigens, cyclin-A1 tumor antigens, MART-1 antigen, gp100 antigen, CD19 antigen, prostate specific antigen, prostatic acidic phosphatase antigen, carcinoembryonic antigen, alphafetoprotein antigen, carcinoma antigen 125, mucin 16 antigen, mucin 1 antigen, human telomerase reverse transcriptase antigen, EGFR antigen, MOK antigen, RAGE-1 antigen, PRAME antigen, wild-type p53 antigen, oncogene ERBB2 antigen, sialyl-Tn tumor antigen, Wilms tumor 1 antigen, mesothelin antigen, carbohydrate antigens, B-catenin antigen, MUM-1 antigen, CDK4 antigen and ERBB2IP antigen, in particular Melan-A melanoma tumor-associated antigen (TAA), is expressed or has been detected in the subject.

8. A pharmaceutical composition comprising the antibody, antigen-binding fragment thereof, or modified antibody thereof for use according to any one of claims 1 to 7, and further comprising at least one pharmaceutical vehicle.

9. Use of a polypeptide, in particular the use of an antigen, comprising or consisting of the epitope of human SIRPa v1 consisting of SEQ ID No: 1; SEQ ID No: 4 or SEQ ID No: 25 in the production or in the selection of an anti-human SIRPa v1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic or modified antibody thereof which binds specifically to human SIRPa v1, and which inhibits the binding of human CD47 to human SIRPa v1 and which does not prevent or inhibit the binding of human CD47 to human SIRPa v2 and to human SIRPg, in particular which enhances the cross-presentation of an antigen by Antigen Presenting Cells, in particular dendritic cells, to human T cells, in particular CD8+ T cells.

10. A method of preparing an anti-human SIRPa v1 antibody, said method comprising immunizing a non-human animal, in particular a non-human mammal, with at least one antigen as defined in claim 9 or with at least one antigen comprising or consisting of the epitope of human SIRPa v1 consisting of SEQ ID No: 1, SEQ ID No: 4 or SEQ ID No: 25; and in particular collecting the resulting serum or B cells from said immunized non-human animal to obtain antibodies directed against said antigen.

11. The use according to claim 9 or the method according to claim 10, further comprising a step of selection and recovery of the anti-human SIRPa v1 antibody, said step of selection comprising at least one of the following steps: a. Testing the binding capability of the antibody to human SIRPa v1, in particular the binding capability of the antibody to the antigen recited in claim 7; and/or b. Testing the capability of the antibody to decrease or inhibit the binding of human CD47 to human SIRPa v1; and/or c. Testing the capability of the antibody to prevent or inhibit the binding of human CD47 to human SIRPa v2, and/or d. Testing the capability of the antibody to prevent or inhibit the binding of human CD47 to human SIRPg; and optionally: e. Testing the capability of the antibody to bind to amino acid residues D and V located respectively on positions 130 and 132 of SIRPa of SEQ ID No: 3; or located respectively on positions 100 and 102 of SIRPa of SEQ ID No: 24; f. Testing the binding capability of the antibody to human SIRPa v2; and/or g. Testing the binding capability of the antibody to human SIRPg; and wherein the recovered antibody has the following properties: 1. It binds specifically to human SIRPa v1; 2. It decreases or inhibits the binding of human CD47 to human SIRPa v1; and 3. It does not decrease or inhibit the binding of human CD47 to human SIRPg; and and optionally at least one of the following properties: 4. It binds to human SIRPa v1 with an affinity of at least 10E-9 M; and/or 5. It binds to amino acid residues D and V located on positions 130 and 132 respectively of SIRPa of SEQ ID No: 3; or located on positions 100 and 102 respectively of SIRPa of SEQ ID No: 24; and/or 6. It does not specifically bind to human SIRPg; and/or 7. It does not specifically bind to human SIRPa v2.

12. The use according to claim 9 or the method according to claim 10 or 11, wherein the obtained antibodies bind specifically to human SIRPa v1 with an affinity of at least 1E-9 M and which does not specifically bind to human SIRPa v2, in particular wherein the recovered anti-human SIRPa v1 antibody is an antagonist of the binding of human CD47 to human SIRPa v1.

13. An in vitro or ex vivo method of assessing the likelihood of effectiveness of a treatment with an anti-human SIRPa antibody or antigen-binding fragment thereof or an antigen-binding antibody mimetic or a modified antibody thereof in a human subject, more particularly wherein an anti-human SIRPa v1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody is to be administered to a human subject, said method comprising the determination of the presence of SIRPa v1 in a biological sample previously obtained from the subject, said SIRPa v1 being in particular detected by an anti-human SIRPa v1 antibody as defined in any one of claims 1 to 7, the composition according to claim 8, or produced as defined in claim 9, or a compound selected according to claim 10 or 11, and wherein the presence of SIRPa v1 in the biological sample is indicative that the treatment is likely to be effective.

14. An in vitro or ex vivo method of assessing the likelihood of effectiveness of a treatment with an anti-human SIRPa antibody or antigen-binding fragment thereof or an antigen-binding antibody mimetic or a modified antibody in a human subject, more particularly wherein an anti-human SIRPa v1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody is to be administered to a human subject, said method comprising determining the SIRPa alleles of the subject, in particular determining if at least one SIRPa allele of the subject comprises a nucleic acid sequence encoding the amino acid residues of SEQ ID No: 1 or SEQ ID No: 4 or SEQ ID No: 25, and wherein the presence of a nucleic acid sequence encoding the amino acid residues of SEQ ID No: 1 or SEQ ID No: 4 or SEQ ID No: 25 within a SIRPa allele in a biological sample previously obtained from the subject is indicative that the treatment is likely to be effective, in particular the determination of a SIRPa alleles is performed on the biological sample by polymerase chain reaction using primers suitable for amplifying a portion of SIRPa gene or SIRPa transcript comprising the SNP 15 and SNP 16 of human SIRPa, or comprising SNP 17 of human SIRPa, and/or a portion comprising the nucleic acid sequence encoding the amino acid residues DDV within SEQ ID No: 1, SEQ ID No: 4 or SEQ ID No: 25.

15. A combination of compounds comprising: (i) an antibody, an antigen-binding fragment thereof, or a modified antibody thereof as defined in any one of claims 1 to 7; and/or the composition according to claim 8; and (ii) at least one antigen selected from the group consisting of antigens from Human Papilloma Virus, Epstein-Barr Virus, Merkel cell polyomavirus, Human Immunodeficiency Virus, Human T-cell Leukemia Virus, Human Herpes Virus 8, Hepatitis B virus, Hepatitis C virus, HCV, HBC, Cytomegalovirus, or from the group of single-point mutated antigens derived from the group consisting of the antigens of ctnnb1 gene, casp8 gene, her2 gene, p53 gene, kras gene, nras gene, or tumor antigens, in particular tumor antigens issued or derived from the group consisting of ras oncogene, BCR-ABL tumor antigens, ETV6-AML1 tumor antigens, melanoma-antigen encoding genes (MAGE), BAGE antigens, GAGE antigens, ssx antigens, ny-eso-1 antigens, cyclin-A1 tumor antigens, MART-1 antigen, gp100 antigen, CD19 antigen, prostate specific antigen, prostatic acidic phosphatase antigen, carcinoembryonic antigen, alphafetoprotein antigen, carcinoma antigen 125, mucin 16 antigen, mucin 1 antigen, human telomerase reverse transcriptase antigen, EGFR antigen, MOK antigen, RAGE-1 antigen, PRAME antigen, wild-type p53 antigen, oncogene ERBB2 antigen, sialyl-Tn tumor antigen, Wilms tumor 1 antigen, mesothelin antigen, carbohydrate antigens, B-catenin antigen, MUM-1 antigen, CDK4 antigen and ERBB2IP antigen, in particular Melan-A melanoma tumor-associated antigen (TAA), or any particular mutated antigen (neo-antigen or neo-epitope); for use in the treatment or the prevention of a disease, in particular a cancer, or for use in therapeutic vaccination against a disease, in particular against a cancer, wherein the antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or modified antibody, enhances cross-presentation of an antigen expressed in said disease, in particular a cancer, and is involved in eliciting a T cell response suitable for the treatment of said disease, in particular for treating a disease within a SIRPa v1-positive subject, in particular for treating or preventing a disease wherein CD47 is over-expressed.

16. A combination of compounds comprising: (i) an antibody, an antigen-binding fragment thereof, an antigen-binding antibody mimetic or a modified antibody as defined in any one of claims 1 to 7; and/or the composition according to claim 8; and (ii) 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, anti-VISTA, anti-OX40, anti-CD40 agonist, CD40-L, TLR agonists, anti-ICOS, ICOS-L, STING agonist, IDO inhibitor, oncolytic virus and B-cell receptor agonists; for use in the treatment or the prevention of a disease, in particular a cancer, or for use in therapeutic vaccination against a disease, in particular against a cancer, in particular in a SIRPa v1-positive subject, in particular for treating or preventing a disease wherein CD47 is over-expressed.

17. A combination of compounds for use according to claim 15 or 16, wherein the combination is suitable to elicit an immune response within a subject, in particular the combination elicits a T cell response suitable to treat the disease, said activation comprising (i) activating T cells, in particular CD8.sup.+ T cells; and/or (ii) enhancing the cross-presentation of at least one antigen by dendritic cells to CD8.sup.+ T cells; and/or (iii) enhancing macrophages polarization, in particular M1 macrophages polarization; or (i), (ii) and (iii).

18. The combination of compounds for use according to any one of claims 15 to 17, wherein the disease is selected from the group consisting of cancer, in particular inflammatory cancer and cancer with infiltrated myeloid cells, particularly with infiltrated dendritic cells or 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 infectious disease, fibrosis, atherosclerosis, obesity, type II diabetes, melanoma and a transplant dysfunction, in particular melanoma.

19. A method of increasing the cross-presentation of an antigen by antigen presenting cells, in particular dendritic cells, to T cells, in particular to CD8+ T cells, said method comprising the administration to a subject of a compound selected from the group consisting of anti-human SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody, said compound having at least the following properties: 1. It does not specifically bind to human SIRPa v2; and 2. It binds to human SIRPa v1 with an affinity of at least 1E-9 M; and 3. It decreases or inhibits the binding of human CD47 to human SIRPa v1; and 4. It does not prevent or inhibit the binding of human CD47 to human SIRPg, said compound having optionally at least one of the following properties: i) It does not inhibit the T cell proliferation; and/or ii) It does not inhibit the T cell activation; and/or iii) It enhances the activation of macrophages.

20. The method of increasing the cross-presentation of an antigen according to claim 19, wherein the administered compound is an anti-human SIRPa antibody or antigen-binding fragment thereof or modified antibody thereof, which comprises: (a) heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, wherein: HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID No: 9, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID No: 10 or SEQ ID No: 11, HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID No: 12, or SEQ ID No: 13, or SEQ ID No: 14 or SEQ ID No: 15; and (b) a light chain variable domain comprising the amino acid sequence set forth in SEQ ID No: 16 or in SEQ ID No: 17.

21. A method for assessing the likelihood of effectiveness of a treatment with an anti-human SIRPa v1 antibody or antigen-binding fragment thereof, or modified antibody thereof, within a human subject, said method comprising: the determination of the presence of human SIRPa v1 in a biological sample previously obtained from the human subject; and when the human subject is SIRPa v1-positive; the administration of a therapeutic amount of: an anti-SIRPa antibody or antigen-binding fragment thereof, or modified antibody thereof, as defined in any one of claims 1 to 8; or of the combination according to any one of claims 15 to 18; or a compound selected according to claim 11 or 12.

22. The method according to claim 21, wherein the presence of SIRPa v1 in the biological sample is indicative that the treatment is likely to be effective in the human subject.

23. The method according to claim 21 or 22, wherein the human subject to be treated has a cancer or is likely to develop a cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0262] FIG. 1. SIRP family sequences alignment. Alignment of membrane distal domain protein sequence of SIRPa variants v1 and v2, SIRPb and SIRPg and localization of SNPs into the protein sequence (numbered arrows from 1 to 16). Grey rectangle corresponds to the peptide signal sequences, bold underlines represent linear epitopes recognized by an anti-human SIRPa antibody, the grey dashed rectangles represent the conformational epitopes recognized by an anti-human SIRPa v1 antibody. SEQ ID NO. 32 to 35 correspond respectively to the sequences referenced in FIG. 1 as P78328_SIRPA_HUMAN_Variant1; SIRPA_HUMAN_Variant; O00241_SIRPB_HUMAN; and Q9P1W8_SIRPG_HUMAN.

[0263] FIG. 2. Binding of SIRPa antibodies against human SIRPa variants V1 and V2 expressed by blood monocytes. Healthy donors already sequenced for the SIRPa exon 3 were selected. By Fluorescence-activated cell sorting (FACS), the binding of in-house anti-SIRPa antibody (Anti-SIRPa v1-FITC on the left hand side of the drawing) and the commercial SE7C2 (on the right hand side of the drawing), were tested on blood monocytes from donors selected from SIRPa V1/V1 and SIRPa V2/V2 homozygous as well as SIRPa V1/V2 heterozygous.

[0264] FIG. 3. Antagonist activity of an anti-human SIRPa v1 antibody on blood monocytes from donors that are SIRPa v1/v1 homozygous donors, SIRPa v1/v2 heterozygous donors or SIRPa v2/v2 homozygous donors. Competition assay by FACS of one anti-human SIRPa v1 antibody on the SIRPa-CD47 interaction using homozygote or heterozygote donor cells. A dose response of anti-human SIRPa v1 antibody was used to compete with the binding of CD47 to SIRPa variants from different donors. The percentage of the CD47 binding are normalized to the control, and results are represented for each cell donors: V2/V2 homozygote (triangle), V1/V2 heterozygote (square) and homozygote V1/V1 (circle).

[0265] FIG. 4. SIRPa variants binding study using anti-human SIRPa v1 antibody and Kwar antibody by Elisa. Assessment of the binding by ELISA on immobilized SIRPa mutated variants (SIRPa from v1 to v8, see Table 2 in example 1) linked to the mouse Fc domain. Anti-human SIRPa v1 antibody and Kwar antibody (known to bind both SIRPa v1 and SIRPa v2) were compared for their ability to bind the different SIRPa mutated variants. Revelation was performed with a donkey anti-human antibody and revealed by colorimetry at 450 nm using TMB substrate. ED50 (ng/ml) is the concentration of the indicated antibody to reach 50% of the signal in this assay.

[0266] FIG. 5. Mouse antigen cross-presentation by dendritic cells (DCs). Mice DCs were preloaded with Ovalbumin antigen and then cultured with transgenic T cells from OTI or OTII mice (Transgenic for their TCR expression dedicated to OVA MHC I or II). T cell proliferation was measured by thymidine incorporation (OTI CD8+ T or OTII CD4+ T cells) in different conditions: control with no Ag (empty circle) or Isotype Antibody control (crosses), mice anti-SIRPa antibodies P84 (square) and MY1 (round) or anti-CD47 antibody (triangle). A: results obtained on OTI CD8 positive T cells. B: results obtained on OT-II CD4 positive T cells.

[0267] FIG. 6. Human antigen cross presentation by dendritic cells. Monocytes from HLA-A2+ HV were phenotyped for SIRPa and both homozygous (v1/v1) and heterozygous (v1/v2) monocytes were used in two type of experiments. A. IL-2 expression in CD8+ T cells. Results obtained with SIRPa v1 homozygote immature DCs are represented with a round and those obtained with SIRPa v1/v2 heterozygote immature DCs with a square. Melan-A loaded immature DCs were used to activate the Melan-A/HLA-A2+ specific thymoma clone that was measured by IL-2 secretion after 48 h of culture. B. IFNg expression in CD8+ T cells stimulated with Melan-A loaded iDC. iDCs were previously incubated during the loading phase with different antibodies: anti-SIRPa v1 (round), anti-SIRPa/g (square), anti-SIRPa v1+anti-SIRPa/g (diamond), B6H12 (inverted triangle) and CC2C6 (triangle). The expression of IFNg was evaluated by flow cytometry.

[0268] FIG. 7. Allogenic response of T cells (CD4 and CD8 positive cells) to anti-human SIRPa v1 and anti-human CD47 antibodies in presence of dendritic cells. 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. A: Proliferation measured by incorporation of H.sup.3-thymidine during the last 12 h of culture. B: Percentage of IFNg secretion measured by ELISA. Results were normalized to the control conditions. Anti-SIRPa v1 (square) corresponds to cells treated with an in-house antibody specific to SIRPa v1, which does not bind SIRPa v2 and SIRPg. CD47 mAb (triangle) corresponds to cells treated with an antibody that binds to CD47.

[0269] FIG. 8. Macrophage polarization bioassay comparing V1/V1 and V1/V2 donor monocytes treated with anti-SIRPa v1 antibody. MIP-1 a/CCL-3 and MIP-1b/CCL-4 secretion were measured by ELISA in supernatant of cells not treated (cercle) or treated with an anti-human SIRPa v1 antibody (square). A: results obtained with cells from homozygote donors V1/V1. B: results obtained with cells from heterozygote donors V1/V2.

[0270] FIG. 9. Binding analysis of anti-SIRPa antibodies on human monocytes homozygote for SIRPa v2 by FACS and determination of the ED50 of the antibody against SIRPa v2 by ELISA. Assessment by cytofluorometry on human monocytes v2/v2 (previously stained with human Fc Receptor binding inhibitor) of chimeric 18D5 antibody or SIRP29 antibody or different humanized anti-SIRPa antibodies. Revelation was performed with a PE labeled mouse anti-human Fc mAb on Cantoll cytometer. ED50 is the concentration of the indicated antibody to reach 50% of the signal. A: Mean of Fluorescence Intensity of positive cells v2/v2 stained. B: determination of the ED50 by ELISA for each antibody. The ED50 was not detectable for most of the humanized 18D5 antibodies

[0271] FIG. 10. Competition 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 s followed by a dissociation period of 120 s (kd) to determine affinity constant (KD). KD affinity of CD47 to SIRPg was determined in different conditions: in presence of the LSB2.20 antibody (an antibody that binds specifically to SIRPg); the kwar antibody, and the SIRP29 antibodies.

[0272] FIG. 11. Effect of anti-SIRPa antibodies on mice metastasis model of Mammary cancer. The 4T1 mammary cancer model was used to study the efficiency of mice anti-SIRPa (p84 antibody and MY-1 antibody) on lung metastasis. The figure represents the numbers of lung metastasis nodules in mice treated or not with anti-SIRPa antibodies. Mice treated with the isotopic control are represented with a diamond, mice treated with the p84 antibody are represented with a circle and those treated with the MY-1 antibody are represented with a square. Results are significant between control and each anti-SIRPa antibody.

EXAMPLES

Example 1: Identification of the Epitope which Allows the Production of Anti-Human SIRPa v1 Antibodies

[0273] Human SIRPa was previously described to present some level of polymorphism in IgV domain 1 which interacts with CD47. This polymorphism is mainly located in the exon 3 of SIRPa gene (SEQ ID No: 27) (Takenaka et al., 2007). On 37 different donor's genomic sequences from different origin, Takenaka et al. identified 10 different sequences/alleles, with 2 principal alleles: variant 1 (v1) and variant 2 (v2). Other alleles differ from V1 or V2 sequences by only 1 or 2 SNPs. Therefore, the SIRPa family is sub-divided into two sub-families: SIRPa v1 isoforms and SIRPa v2 isoforms. These different alleles lead to slightly different proteins, but all variants bind similarly CD47 ligand. Allelic frequency of v1 in Takenaka et al. is 78% (89% for V1 and V1-like) while genotype frequency for homozygous V1/V1-like donors is 65%. 24% of their 37 donors presented a heterozygous genotype.

[0274] Coding DNA and Protein Sequences of SIRPA V1 and V2:

[0275] V1 of human SIRPa Sequence was obtained on ncbi (gene ID: 140885).

[0276] Genomic DNA reference sequence of SIRPa v1 exon 3 (transcript SIRPA-201 ID ENST00000400068.7) (SEQ ID No: 27):

TABLE-US-00001 GAGTGGCGGGTGAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTG TTGGTTGCAGCTGGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCT GATCCCTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGGACCAGGCCGGG AATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACTGTT TCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCGGTAA CATCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAG GGAGCCCCGATGACGTGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCT GTGCGCG

[0277] Amino acid sequence of one SIRPa v1 (Genbank reference NP_001035111.1 (UniProtKB: P78324) (SEQ ID No: 3):

TABLE-US-00002 MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGET ATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRN NMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSA PVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDP VGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETI RVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETAS TVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVS AHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKK AQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNH TEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQ VPRK

[0278] Amino acid sequence of one SIRPa v2 (SEQ ID No: 28):

[0279] Variant 2 protein sequence was referenced in Takenaka et al., 2007. The coding DNA sequence was obtained on ncbi (GenBank: BC075849.1). No gene sequence has been found for this variant on ncbi database.

TABLE-US-00003 MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVSVAAGES AILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRE NMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSAP VVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPV GESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIR VPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETAST VTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSA HPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKA QGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHT EYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQV PRK

[0280] Genomic DNA reference sequence of SIRPa v2 exon 3 (SEQ ID No: 31):

TABLE-US-00004 GAGTGGCGGGTGAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTA TCAGTTGCAGCTGGAGAGTCGGCCATTCTGCACTGCACTGTGACCTCCCT GATCCCTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGGACCAGCCCGGG AATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACTGTT TCAGAGTCCACAAAGAGAGAAAACATGGACTTTTCCATCAGCATCAGTAA CATCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAG GGAGCCCTGACACGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTG CGTGC

[0281] The amino acid sequences of the D1 domain of SIRPa v1, SIRPa v2, SIRPb and SIRPg were aligned as illustrated on FIG. 1.

[0282] Genotype Frequency Analysis by Bioinformatic

[0283] To explore more data on SIRPa polymorphism in human, Data from 1000 Genomes project (1 KG, >2500 different human genomes) were used to identify relevant SNPs in human SIRPa exon 3. Inventors identified that SIRPa SNPs segregated in 2 haplotype blocks using Haploview (data not shown).

[0284] To explore genotype frequency and analyze the differences between homozygous and heterozygous donors, inventors first identified 18 SNPs (Table 1) within human SIRPa exon 3 (the known polymorphic exon from literature and the exon responsible of the binding with the ligand) and retained SNPs associated with a codon change (change in amino acid sequence). SNPs were numbered from 1 to 18. 1 SNP was not analyzed since not known in the literature. SNP7 and SNP18 were not analyzed either in terms of genotype frequency since both correspond to synonymous codon (no change at protein level). The inventors then identified SNPs allele and genotype frequencies with the 1000 genomes phase 3 project which includes more than 5000 donors from 5 super populations: n=1030 East Asian (EAS), n=1010 European (EUR), n=1338 African (AFR), n=704 Ad Mixed American (AMR) and n=988 South Asian (SAS). All individuals from 1000 genomes phase 3 project are being considered.

TABLE-US-00005 TABLE 1 SNPs identification on SIRPa variants and their amino acid positions Amino acid Nucleotide position and mutations mutations (SIRPa v1 - (SIRPA v1 - SIRPA v2) SIRPA v2) Rs number SNP1 g-a 44 L-S rs386811660 SNP2 a-t 50 T-S rs17855609 SNP3 a-g 51 A rs17853846 SNP4 c-t 52 T-I rs17855610 SNP5 g-a 54 R-H rs17855611 SNP6 c-t 57 A-V rs17855612 SNP7 t-c 60 L rS17853847 SNP8 g-c 75 G-A rs1057114 (=rs72620874) SNP9 c-g 95 D-E rs138283486 SNP10 c-t 96 L-S rs149634649 SNP11 t-c 97 T rs146163282 SNP12 a-g 100 N-E rs17855613 SNP13 c-a 101 N rs17855614 SNP14 c-a 107 R-S rs17855615 SNP? g-a 109 G-S SNP15 + gt - ac 132 V - T rs115287948 SNP16 rs114499682 SNP17 = gt - ac 132 V - T s386811663 (SNP15 + SNP16) SNP18 c - t 145 R rs6136375

[0285] Then, inventors clustered physically neighbouring SNPs with roughly similar frequency (<1% difference) and plotted mean frequencies of these clustered SNPs on Pie Chart according to population genetics. They finally performed a correlation with in-house anti-human SIRPa v1 antibody epitope (since it displays low binding on V2/V2 donors and poorly block CD47 binding on V2/V2, data not shown) determined by two methods (linear or conformational methods). They also took into consideration SNPs associated with a mutation in SIRP gamma (since in-house antibody does not bind to SIRP gamma). Finally, inventors also reinforced this analysis by comparing sequence differences between human SIRPa V1, human SIRPa V2 and mutations found in cynomolgus (Macaca fascicularis) or rhesus (Macaca mulatta) monkeys since the in-house antibody has no cross-reactivity with monkey.

[0286] Anti-SIRPa Binding Relationship with SNPs:

[0287] Method: The binding activity of the anti-SIRPa antibodies was assessed by ELISA. For the ELISA assay, the in-house anti-human SIRPa v1 antibody and Kwar antibody were tested on 8 different mutated hSIRPa (SIRPA v1 to v8; see table 2). The different variants of mutated hSIRPa (SIRPa v1 to v8) were immobilized on plastic at 0.5 g/ml in carbonate buffer (pH9.2) 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.

[0288] Results: In order to evaluate the impact of human SIRPa SNPs on the in-house anti-human SIRPa v1 antibody binding capabilities, 8 different recombinant human SIRPa proteins were generated (Table 2). The extracellular domains of these SIRPa proteins were fused with mouse Fc from IgG2a, and the binding capability of the in-house anti-human SIRPA v1 antibody to those SIRPa variants was assessed (FIG. 4). The in-house antibody loses its binding property in ELISA when SNPs 15+16 are mutated in the V1 sequence (FIG. 4A). In the experiments disclosed in the present application, the anti-human SIRPa v1 antibody used corresponds to an antibody with a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID No: 20; and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID No: 17. Other SNPs mutation did not alter the in-house antibody binding property. In comparison, KWAR23 (which is described to bind both V1 and V2) binding properties were not modified with any one of the SNPs mutations (FIG. 4B), confirming that SIRPa V8 (SNPs15+16) protein is functional and that an antigen comprising an epitope localized in the surrounding of SNPs 15+16 allows the production of anti-SIRPa v1 antibodies which do not recognize or bind specifically to SIRPa v2.

TABLE-US-00006 TABLE 2 SIRPa recombinant proteins SIRPa V1 SIRPa V2 SIRPa V3 (SNP1 + 2 + 3 + 4 + 5 + 6) SIRPa V4 (SNP8) SIRPa V5 (SNP9 + 10 + 11) SIRPa V6 (SNP12 + 13) SIRPa V7 (SNP14 + ?) SIRPa V8 (SNP15 + 16)

[0289] Impact of SIRPA Polymorphism on Antibodies PropertiesAntagonist Assay SIRPA-Cd47 by FACS on Monocytes

[0290] METHOD: Human monocytes (purified by elutriation from human PBMC at DTC platform, Nantes, and frozen in DMSO at 10M/ml at 80 C. or liquid nitrogen) were thawed in 40 mL complete RPMI medium. Then immediately centrifuged at 1000 rpm during 10 minCells are resuspended in 10 mL of RPMI medium and counted on Malassez cell. Frozen human monocytes are thawed, diluted samples are added and biotinylated CD47Fc is added afterwards. Biotinylated CD47Fc is then revealed with a streptavidin-PE and fluorescence is measured by flow cytometry. The following protocol was applied: Put 100,000 human monocytes per well on V-bottom P96 plate; Centrifuge 1 min at 2500 rpm and empty wells by flicking the plate; Wash cells 2 times with 200 L PSE (Centrifuge 1 min at 2500 rpm and empty wells); Prepare 8 dilutions of the sample beginning with 10 g/mL (concentrated 2, final concentration 5 g/mL) and diluting by steps of 3; Add 12.5 L of sample/well on cells and mix; Incubate 15 min on ice; Prepare CD47Fc biotinylated solution concentrated 2, determined in function of paragraph and add 12.5 L of this solution/well on cells and mix.Incubate 30 min on ice; Add 175 L PSE/w-Centrifuge 1 min at 2500 rpm and empty wells; Wash cells 2 times with 200 L PSE (Centrifuge 1 min at 2500 rpm and empty wells). An intermediate concentration is chosen for CD47Fc biotinylated. Here, 5 g/mL CD47Fc biotinylated are took for the antagonist test. This step has to be made each time because percentage of CD47 positive cells depends of individual donor; Dilute streptavidin-PE at 1/1000, Add 25 L/w-Incubate 15 min on ice; Add 175 L PSE/w; Centrifuge 1 min at 2500 rpm and empty wells; Wash cells 2 times with 200 L PSE (Centrifuge 1 min at 2500 rpm and empty wells); Transfer stained cells in V-bottom P96 canto plate and read on BD canto II.

[0291] RESULTS: Inventors analyzed the binding properties of the in-house anti-SIRPa v1 antibody by flow cytometry on blood monocytes from healthy donors already sequenced for the SIRPa exon 3 (FIG. 2). They selected V1/V1 homozygous donors, V2/V2 homozygous donors as well as V1/V2 heterozygous donors. FIG. 3 shows that the anti-human SIRPa v1 antibody binds significantly less on V2/V2 donors. In comparison, the inventors found that a commercial anti-SIRPa mAb (clone SE7C2) binds only the V2 protein. SE7C2 data show that V2/V2 donors express similar level of SIRPa protein at their surface as compared to V1/V2 donors, confirming that reduced binding of the anti-human SIRPa v1 antibody on V2/V2 is not due to lower expression but low binding of this antibody to this SIRPa v2 protein (FIG. 2).

[0292] Then the inventors analyzed by flow cytometry the in-house anti-human SIRPA v1 antibody antagonist property to prevent the binding of recombinant human CD47 protein binding on blood monocytes from healthy donors (FIG. 3). To study the impact of SIRPa polymorphism on the antagonistic effect on the binding of CD47-Fc, an antagonistic assay was performed on frozen human monocytes genotyped for SIRPa. The data illustrated on FIG. 3 show that the anti-human SIRPa v1 antibody greatly antagonizes CD47 binding on V1/V1 donors (n=3) while it only reduces by half CD47 binding on heterozygous V1/V2 donors (n=5). Weak (25%) antagonist action of the anti-human SIRPa v1 antibody is observed on homozygous V2/V2 donors (n=3). As shown on FIG. 3, the in-house antibody recognized SIRPa V1 and very weakly recognized SIRPa V2. It indicates that the in-house antibody has a strong antagonistic effect on binding of CD47-Fc on human monocytes which were homozygous V1/V1 for SIRPa, a very weak antagonistic effect on homozygous V2/V2 human monocytes, and an intermediary antagonistic effect on heterozygous V1/V2 human monocytes.

[0293] Conclusion

[0294] Polymorphism of SIRPa was not described to affect CD47 binding but can affect recognition by anti-human SIRPa monoclonal antibodies. Multiple variants have been determined by Takenaka et al. with a high sequence homology with V1 or V2 sequences. Other variants represent different combinations between V1 and V2 sequences and can be considered as V1-like or V2-like variants depending on their majority genotype. These results were confirmed with data from 1000 genomes which show there are two major variants for SIRPa: V1 and V2. Variant 1 is the most frequent in worldwide population except in East Asian super population. The SNPs analysis in the present invention showed that SIRPa V1 allele frequencies (V1/V1 and V1/V2) relevant for the epitope allowing the production and/or the selection of anti-human SIRPa v1 antibodies is between 76-86% in the US and EU and V1/V1 homozygous patients represent between 40 and 50% of US and EU populations (based on SNPs 15 and 16 frequencies). The inventor analysis on n=184 donors showed that the in-house anti-human SIRPa v1 antibody strongly binds 83-86% of v1 donors (V1/V1 and V1/V2). V1/V1 homozygous donors represent between 40 and 50% of analyzed cohort. While CD47 antagonist assay showed in an interestingly manner that the in-house antibody prevented only half of the binding, functional assays on macrophage polarization presented on FIG. 8 and tumor-antigen cross-presentation by human dendritic cells presented FIG. 6 showed that the in-house anti-human SIRPa v1 antibody has the same biological efficacy on both V1/V1 and V1/V2 donors.

Example 2: Comparison of SIRPa-CD47 Interaction Blockade on Immune Cross Presentation Between Mouse and Human

[0295] Mouse Cross-Presentation (FIG. 5)

[0296] DRUGS: the allosteric antagonist monoclonal antibody targeting the domain 2 of the mouse SIRPa (P84 clonerat IgG1) was purified from hybridoma. The orthosteric antagonist monoclonal antibody targeting the domain 1 of the mouse SIRPa (MY-1 clonemouse IgG2a) (Garcia et al., 2011) was reengineered into an IgG1 Fc domain from the parental hybridoma. Both anti-SIRPa antibodies block the signaling through SIRPa in myeloid cells. The isotype control mouse IgG1 (3G8 clone) was purified. The surrogate antagonistic anti-mouse CD47 monoclonal antibody (MIAP410 clone) was purchased from BioXCell (#BE0283).

[0297] MOUSE SIRPa EXPRESSION BY SPLENIC DCs: natural DC were isolated from the spleen of nave mice by CD11c positive magnetic selection and separated for their CD8 expression by cell sorting with a BD FACS ARIA II. As described in the literature, CD8+/+DC, which are the best antigen-presenting cells (APC) for cross-presentation, express low to negative levels of SIRPa while CD8/DC express SIRPa (and are known to less efficiently cross-present antigens).

[0298] ANTIGEN PRESENTATION FUNCTION BY SPLENIC DCs: According to the literature, the SIRPa low/neg CD8+/+DC are the best antigen-presenting cells (APC) for the antigen (Ag) cross-presentation compared to CD8/DC (expressing high level of SIRPa) (Haan et al., 2000; Hochrein et al., 2001). However, the two subtypes of DC loaded and presented exogenous Ag on MHC class II molecules equivalently. Inventors showed that the protocol used to evaluate the role of SIRPa on Ag cross-presentation with OVA and OT-IT cells reproduces these DC's properties (data not shown). Indeed, the CD8+/+DC induced a better proliferation of OT-I T cells (from CD8+ Ovalbumin-specific TCR-transgenic mice) than CD8/DC indicating a better Ag processing, loading and presentation on MHC class I molecules whereas the exogenous antigen presentation is high for both subtypes of splenic DCs as they observed with the OT-II T cell (from CD4+ Ovalbumin-specific TCR-transgenic mice) proliferation. Thus, expression of SIRPa is inversely correlated with the capacity of dendritic cells to cross-present antigen to CD8+ T cells, suggesting that SIRPa represses cross-presentation in mice.

[0299] MOUSE ANTIGEN CROSS-PRESENTATION: CD8+/+ and CD8/DC were loaded with ovalbumin (OVA) overnight in the presence of GM-CSF. Then, CD8+ T cells isolated from the spleen of the OT-I transgenic mice and CD4+ T cells isolated from the spleen of the OT-II transgenic mice were cultured with OVA-loaded DC subtypes for 3 days. The transgenic mice express TCR specific of OVA MHC I (OT-I) and II (OT-II). Proliferation was evaluated by H3-thymidin incorporation during the last 16 hours of culture. Anti-SIRPa mAb was added at 10 g/ml during the incubation of DC with OVA protein and during T cell proliferation with OVA-loaded DC. This protocol allows to evaluate the impact of SIRPa blockade during protein processing by DCs and then presentation of OVA peptide by the MHC class I molecules by the DC to CD8 OT-I T cells and by the MHC class II molecules to CD4 OT-II T cells highlighting the antigen cross-presentation and the exogenous antigen presentation respectively.

[0300] Inventors shows that the blockade of SIRPa or CD47 in mouse potentiated the antigen presentation by SIRPa+ splenic dendritic cells. The CD8+/+DC (SIRPa low/neg) were not affected by the blockade of the SIRPa/CD47 pathway regarding their ability to cross-present OVA to OT-I T cells (not shown). However, the blockade of either SIRPa by P84 or MY-1 blocking antibodies or CD47 by MIAP410 enhances Ag cross-presentation by CD8/SIRPa+ DC reflected by CD8+ OT-I proliferation increase (FIG. 5A).

[0301] EXOGENOUS ANTIGEN PRESENTATION: the inventors analyzed the effect of the SIRPa/CD47 blockade on exogenous Ag presentation, they found that Ag presentation by SIRPa low/neg-CD8a+/+DC was not modified by the blockade of the SIRPa/CD47 pathway (not shown). Similar to cross presentation process, SIRPa/CD47 blockade on SIRPa positive CD8a/DC increased exogenous antigen processing and presentation on MHC class II molecules as measured by CD4+OT-II cell proliferation (FIG. 5B).

[0302] Conclusion

[0303] Inventors demonstrated that blocking the SIRPa/CD47 pathway (with anti-SIRPa or anti-CD47 mAbs) in mouse increased both MHC-I antigen cross-presentation (T CD8 response) and MHC-II antigen presentation (T CD4 response). Those results confirmed what was suggested by others such as (Liu et al., 2016, 2015; Xu et al., 2017).

[0304] Human Cross-Presentation (FIG. 6)

[0305] DRUGS: the antagonistic monoclonal antibody targeting the human SIRPa (in-house anti-human SIRPA v1 antibodyhuman IgG4) was generated and purified by inventors. The isotype control human IgG4 was purchased from Biolegend (QA16A15 clone). The antagonistic monoclonal antibody targeting the human CD47 (B6H12 clone) was purchased from BioXCell (#BE0019-1) and CC2C6 clone from BioLegend (#TBD2) were used. In some experiments, the anti-SIRPa:g antibody (clone SIRP29 from WO201356352) was used alone or in combination with the in-house anti-SIRPa V1 antibody.

[0306] MELAN-A SPECIFIC RESPONSE: the cross-presentation by human cells was evaluated by the presentation of a long peptide (25-mer, which implies that the peptide cannot dock onto Class I MHC molecules without being processed) of the Melan-A melanoma tumor-associated antigen (TAA) by HLA-A2+ DCs to TCR-specific T cells that recognize specifically HLA-A2/Melan-A complexes. Two different TCR-specific T cells were used to evaluate the antigen cross-presentation. The first was a T lymphocyte clone from a melanoma patient which is specific for these HLA-A2/Melan-A complexes (kind gift from Dr. N. Labarrire, Univ. Nantes, France, Vignard at al., J. Immunol 2005). The second clone was a transgenic murine thymoma cell line transduced with the TCR of the same melanoma patient's T-cell clone and transfected with the human CD8 co-receptor. DCs were generated in vitro from blood monocytes of HLA-A2+ healthy volunteers (HV; volunteers from the Etablissement Franais de Sang, Nantes). Meanwhile, monocytes were phenotyped for the polymorphism of SIRPa. After 7 days of culture with GM-CSF and IL-4, immature DCs (iDCs) were induced. Then iDCs were loaded overnight with the long 25-mer peptide of Melan-A in the presence of antagonistic antibodies targeting the SIRPa/CD47 pathway and finally cultured independently with the two different Melan-A/HLA-A2 specific T cell clones. The human T cell clone from the melanoma patient was cultured with Melan-A-loaded iDC for 5 hours and T cell activation evaluated by flow cytometry by intracellular staining of IFNg.

[0307] The TCR transgenic thymoma clone was cultured with Melan-A-loaded iDC for 48 hours and T cell activation was evaluated by ELISA for IL-2 secretion.

[0308] To validate the protocol, Melan-A loaded iDCs from HLA-A2 negative donors were used as a negative control, as well as unloaded-iDCs from HLA-A2+HV. Results (not shown) showed that only HLA-A2+ Melan-A loaded-iDC were able to induce IFNg secretion by the human T cell clones. The secretion of IL-2 by murine thymoma cells was only measured in HLA-A2 positive donors and compared to HLA-A2-Melan-A loaded DCs (data not shown) demonstrating the specificity of the two different HLA-A2/Melan-A specific T cell clones.

[0309] Monocytes from HLA-A2+HV were phenotyped for SIRPa and both V1 homozygous and V1/V2 heterozygous monocytes were included in 2 different experiments.

[0310] THYMOMA CELL CLONE ENGINEERED IN VITRO: after 48 hours of stimulation of the TCR-transgenic thymoma cell clone with the Melan-A loaded human iDCs, IL-2 secretion was measured. In this first read-out of the antigen cross-presentation, inventors observed an increase of IL-2 secretion by the thymoma clone in most of donors when SIRPa was blocked by a specific anti-SIRPa antibody (in-house anti-human SIRPa v1 antibody) during the loading of Melan-A and the stimulation assay (FIG. 6A). No difference was observed between V1 homozygous and V1/V2 heterozygous donors of DCs.

[0311] HUMAN T CELL CLONE FROM MELANOMA PATIENT: after 5 hours of stimulation of the CD8 human T cell clones with Melan-A loaded human iDC, the expression of IFNg was evaluated by flow cytometry. FIG. 6B represents the IFNg expression in CD8+ T cells. SIRPa or CD47 were blocked during the loading of Melan-A and during T cell stimulation. Various antibodies were tested: an anti-human SIRPa v1; an anti-SIRPa/g (SIRP29), anti-CD47 (B6H16 and CC2C6). FIG. 6B shows that SIRPa blockade by in-house anti-human SIRPa v1 antibody led to an increase of IFNg expression by tumor-antigen-specific human T-cells induced by iDC from homozygote or heterozygote donors. Interestingly, inventors showed that positive effect of SIRPa blockade on Ag presentation was not restricted to SIRPa v1 homozygous iDCs since similar increase was observed in most V1/V2 heterozygous donors. Unexpectedly, in contrast to mouse cross-presentation, inventors found in human that the two anti-CD47 mAbs had no positive impact on most donors IFNg secretion and worst strongly suppress basal level of T-cell activation in the majority of the donors. More surprisingly, an anti-SIRPa which is not SIRPa-specific but binds as well SIRPg and inhibits the binding of CD47 to SIRPg, does not induce the IFNg expression by T cells indicating that the cross presentation by iDC to T cells is specific of the in-house anti-human SIRPa v1 antibody. Unusually, the addition of the non-selective SIRPa/g antibody to the selective in-house SIRPa v1 antibody prevents increased IFNg secretion by selective blockade of SIRP alpha and not gamma. These results obtained in human with different anti-CD47 antibodies and anti-SIRPa/g antibody were not predictable regarding results in mice models and could explain some interesting results from inventors showing that polyclonal human T-cell proliferation and human mixed lymphocyte reaction were strongly inhibited in human by different anti-CD47 mAbs and anti-SIRPg (Piccio et al., 2005) but not by the specific anti-SIRPa v1 antibody of the invention.

[0312] Conclusion

[0313] The two different clones used to evaluate antigen cross-presentation showed similar results with SIRPa blockade on iDCs during Ag loading and Ag presentation to T cells. Inventors showed for the first time that the human SIRPa has an inhibitory role on antigen cross-presentation, measured by IFNg or IL2 secretion, which can be alleviated by selective anti-SIRPa mAbs. This was not predictable because the blockade of CD47 with different anti-CD47 antibodies or non-selective anti-SIRPa/g antibody did not present this effect on human T cells which find explanation by considering previous observations on the immunosuppressive properties of anti-CD47 mAbs in human but not in mouse. Previous art shows that SIRPa inhibits mouse DC maturation. For the first time, inventors disclosed that a specific anti-SIRPa antibody could be a therapeutic compound to potentiate Ag cross-presentation in human. While anti-CD47 mAbs in mouse have similar potentiating effect than mice anti-SIRPa, the effect of human anti-SIRPa on human DCs cross-presentation was not predictable. Our understanding of the immune mechanism mediated by SIRPa-CD47 interaction led us to presume that the immunosuppressive properties of anti-CD47 in human is due to its interaction with SIRPg which is not express in mice. Surprisingly, the genetic statute of DCs donors on SIRPa V1 homozygote or heterozygote did not show any differences on human T cell activation.

Example 3: Impact of SIRP/CD47 Blockade on Polyclonal Stimulation

[0314] 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 (Life Technologies) 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 targeting the SIRPa/CD47 and/or the SIRPg/CD47 pathways were added from the beginning of the proliferation test at a saturating concentration (10 g/mL). Proliferation was measured by incorporation of H.sup.3-thymidine during the last 12 h of culture. Anti-CD47 antibody (commercial references: B6H12), anti-SIRPa antibodies (HEFLB as referred in the patent (WO2017178653)).

[0315] RESULTS: to investigate the immunosuppressive effect of the blockade of CD47 on human T lymphocytes, PBMC were isolated from 3 different healthy volunteers and a part of the cells was irradiated (35 Gy). Each donor (responder) has been included in Mixte Lymphocyte Reaction with each of the two remaining donors (irradiated stimulators). Blocking antibodies were added at the beginning of the MLR and T cell proliferation was measured by thymidine incorporation in the last 16 h of a five-days-culture. Inventors found a strong inhibition of human T cell proliferation with anti-CD47 mAb whereas anti-SIRPa (in-house anti-human SIRPa v1 antibody) did not significantly differ from control conditions (FIG. 7). IFNg secretion which also reflects T cell activation was dosed in the supernatant of the 4-days culture. Inventors observed also a dramatic inhibition of the secretion of the cytokine with anti-CD47mAb (B6H12) indicating and confirming previous results from the inventors and other groups that CD47 is important for T cell activation in human.

Example 4: Effect of Anti-SIRPa Antibody on Macrophage Polarization Bioassay Using Monocytes from Healthy Donors: Bioassay Measuring Chemokine Production: MIP-1a/CCL-3 and MIP-1b/CCL-4

[0316] To investigate the impact of in-house anti-human SIRPa v1 antibody on macrophage polarization and activation, the secretion of some chemokines, more specifically MIP-1a/CCL-3 and MIP-1b/CCL-4, were measured in the supernatant of fresh or frozen human monocytes genotyped homozygous V1 for SIRPa or heterozygous V1/V2 cultured with GM-CSF to induce non-polarized immature macrophages (FIG. 8). Two sources of recombinant CD47-Fc were tested at 10 g/ml: SinoBiological #12283-H02H and R&D systems #4670-CD-050. Quantification of MIP-1a/CCL-3 and MIP-1b/CCL4 was realized by ELISA in culture supernatants (R&D systems: Human CCL3/MIP-1 alpha DuoSet ELISA #DY270 and Human CCL4/MIP-1 betaDuoSet ELISA #DY271). Altogether, results showed that in the presence of coated CD47-Fc, in-house anti-human SIRPa v1 antibody, significantly increased the secretion of MIP-1a and MIP-1b, both on homozygous for V1 SIRPa (FIG. 8A) or heterozygous (V1/V2 genotype) donors (FIG. 8B).

[0317] Conclusion

[0318] Inventors identified that M1-associated chemokines MIP-1a/CCL-3 and MIP-1b/CCL-4 secretion is significantly increased with in-house anti-human SIRPa v1 antibody in the presence of coated recombinant CD47-Fc. CD47-Fc induces functional suppression of human myeloid cells, in particular by decreasing MIP-1a/CCL-3 and MIP-1b/CCL-4 basal secretion. Blocking SIRPa-CD47 interaction by an anti-SIRPa antibody restores myeloid function as shown by secretion of MIP-1a and MIP-1b in the supernatant. The genotype of the donors regarding of the SIRPa V1 allele expression has no impact on the induction of those chemokines, suggesting a threshold effect on the SIRPa blockade to observe a functional effect.

Example 5: SIRPa Binding Assay on Human Monocytes v2/v2 by Cytofluorometry

[0319] 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 mm 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-mouse igG (Jackson immunoresearch; reference 715-116-151) was used. Samples were analyzed on BD LSRII or Canto II cytofluorometer.

[0320] RESULTS: As shown in FIG. 9, the results indicate a binding of the SIRP29 antibody and the chimeric 18D5 antibody parent antibody of the invention on human monocytes SIRPa v2/v2 and no binding for all the humanized anti-SIRPa antibody (as measured with the MFI (Median Fluorescent Intensity) indicating that the humanization of the antibodies induces the loss of the SIRPa v2 specificity.

Example 6: Competition Analysis of the Anti-SIRPa Antibodies on the CD47-SIRPg Interaction by Blitz

[0321] METHOD: This assay was performed with a Blitz (Forte 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 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 different antibodies, for 120 seconds (LSB2.20, Kwar or SIRP29 antibodies). 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).

[0322] RESULTS: As shown in FIG. 10, in normal condition the affinity of the CD47 to SIRPg is around 6.10.sup.8 M, Kwar23 and SIRP29 significantly reduces the binding of CD47 to SIRPg, while the LSB2.20 a commercial anti-SIRPg antibody, does not disrupt the interaction CD47-SIRPg. These results underline the specificity of the in-house anti-SIRPa v1 antibody of the invention against the SIRPa compare to the antibodies of the prior art.

Example 7: Mammary 4T1 Preclinical Model: A Metastasis Model

[0323] This orthotopic and syngeneic 4T1 preclinical model was used to evaluate the effect of two different specific anti-mice SIRPa monotherapies in a model where myeloid cell infiltration is important and well described. Indeed, it was previously reported that 4T1 model is predominantly infiltrated by CD11 b+ myeloid cells (DuPr et al., 2007), in particular MDSC (Markowitz et al., 2013).

[0324] This model is known to induce metastasis. The inventors have therefore collected the liver and the lung and numbered the metastases of mice treated or not with mouse anti-SIRPa antibodies. The isotype control and anti-SIRPa antibodies were used at 8 mg/kg from D4 to D28 (3 times/week). The blockade of SIRPa by two different antibodies targeting specifically SIRPa (and disrupting the binding of CD47 to SIRPa) demonstrated a potent clinical effect in monotherapy on the tumor development of an aggressive model of TNBC. Inventors also analyzed lung and liver metastasis after sacrifice by comparing P84 (Ref: MABS164 an Anti-SHPS-1 Antibody, clone P84 from Merck Millipore) and MY1-mG1 (described in Yanagita et al.) surrogates. Control mice developed lung metastases while no mice treated with MY1-mG1 or P84 did so. The FIG. 11 shows results on lung metastasis for P84 and MY-1 anti-SIRPa mAbs, both showing the same efficacy on lung metastasis.

[0325] These results in a mice model that does not express SIRPg on their cells underline the importance to target specifically SIRPa for cancer applications and more particularly for metastasis treatment or prevention. In human where cells express SIRPg, it will be useful to target specifically SIRPa without disrupting the CD47-SIRPg interaction such as the antibodies of the invention.

BIBLIOGRAPHY

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