IMMUNOMODULATORY COMPLEX AND USES THEREOF FOR THERAPY

Abstract

The invention relates to a molecular complex consisting of at least one ligand of a sulphated sugar of the glycosaminoglycan family linked to at least one ligand of a surface molecule of antigen-presenting cells or NK or NKT cells, for use as an immunomodulatory drug, in particular in the immunotherapy of cancer and infectious diseases.

Claims

1-16. (canceled)

17. An immunomodulatory composition, comprising at least one molecular complex, wherein said complex consists of at least one ligand of a sulfated sugar of the glycosaminoglycan family (first ligand) and at least one ligand of a surface molecule of antigen-presenting cells or of NK or NKT cells (second ligand), and wherein said ligands are bonded to one another and said complex is free of a specific antigen of the disease to be treated, and at least one pharmaceutically acceptable carrier, a carrier substance and/or an adjuvant.

18. The composition according to claim 17, wherein the first ligand is a heparan sulfate-binding peptide selected from the group consisting of: a peptide derived from the HIV Tat protein, comprising at least the basic region Tat49-57 (SEQ ID NO: 3) such as the peptides Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8) and Tat22-57C.sub.(22-37)S (SEQ ID NO:9); an R7 to R11 polyarginine peptide; and a peptide comprising the R domain of the diphtheria toxin (SEQ ID NO: 5) or at least the fragment DT453-467 (SEQ ID NO 7) of said domain, comprising the heparan sulfate binding region.

19. The composition according to claim 17, wherein the second ligand targets a surface molecule of antigen-presenting cells selected from the group consisting of: C-type lectin receptors, membrane-bound immunoglobulins, receptors for the constant region of immunoglobulins, and immune checkpoint molecules and ligands thereof.

20. The composition according to claim 17, wherein the second ligand is selected from the group consisting of: (i) antibodies directed against said surface molecules of antigen-presenting cells or of NK or NKT cells and fragments thereof containing at least the paratope; (ii) immunoglobulins, and fragments thereof comprising at least the Fc region; and (iii) proteins and protein fragments which bind to the Fc and/or Fab region of the antibodies.

21. The composition according to claim 20, wherein the immunoglobulins are IgG.

22. The composition according to claim 20, wherein the proteins and protein fragments which bind to the Fc and/or Fab region of the antibodies are selected from the group consisting of: protein A of S. aureus, the BB fragment thereof (SEQ ID NO: 1) and the ZZ derivative thereof (SEQ ID NO: 2).

23. The composition according to claim 17, wherein the at least one molecular complex is in the form of oligomers or a mixture of monomers and oligomers.

24. The composition according to claim 17, wherein the at least one molecular complex consists of a fusion protein between the first and the second ligand.

25. The composition according to claim 24, wherein the fusion protein comprises a first ligand selected from: Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8), Tat22-57C(22-37)S (SEQ ID NO 9), the R domain of the diphtheria toxin (SEQ ID NO: 5) or the fragment DT453-467 (SEQ ID NO 7) and a second ligand selected from: the BB fragment of the protein A (SEQ ID NO: 1) or the ZZ derivative thereof (SEQ ID NO: 2).

26. The composition according to claim 17, wherein the second ligand is an antibody or an antibody fragment and the first ligand forms a fusion protein with an immunoglobulin-binding element.

27. The composition according to claim 26, wherein said fusion protein comprises a first ligand selected from Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8), Tat22-57C(22-37)S (SEQ ID NO 9), the R domain of the diphtheria toxin (SEQ ID NO: 5) or the fragment DT453-467 (SEQ ID NO 7) and an immunoglobulin-binding element selected from the BB fragment of the protein A (SEQ ID NO: 1) or the ZZ derivative thereof (SEQ ID NO: 2).

28. The composition according to claim 26, wherein said fusion protein comprises a first ligand selected from Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8), Tat22-57C(22-37)S (SEQ ID NO 9), the R domain of the diphtheria toxin (SEQ ID NO: 5) or the fragment DT453-467 (SEQ ID NO 7) and an immunoglobulin-binding element which is the BB fragment of the protein A (SEQ ID NO: 1) and said fusion protein is complexed to the second ligand which consists of a whole immunoglobulin.

29. The composition according to claim 26, wherein said fusion protein comprises a first ligand selected from Tat49-57 (SEQ ID NO: 3), Tat37-57 (SEQ ID NO: 8), Tat22-57C(22-37)S (SEQ ID NO 9), the R domain of the diphtheria toxin (SEQ ID NO: 5) or the fragment DT453-467 (SEQ ID NO 7), and an immunoglobulin-binding element selected from the BB fragment of the protein A (SEQ ID NO: 1) or the ZZ derivative thereof (SEQ ID NO: 2) and wherein said fusion protein is complexed to the second ligand which is selected from an anti-RFcgamma I, II and/or III, anti-DEC-205, anti-DC-SIGN, anti-CD74, anti-CD275, anti-CD56, anti-CD335, anti-CD336, anti-CTLA-4, anti-PD-L1, anti-OX40 antibody, or a fragment of the preceding antibodies comprising at least the paratope.

30. The composition according to claim 17, which is an immunostimulatory composition.

31. The composition according to claim 17, which is for activating antigen-presenting cells, for activating NK or NKT cells, and/or for activating the secretion of the cytokines IL-6 and/or IL-12

32. The composition according to claim 31, wherein the antigen-presenting cells are dendritic cells or monocytes.

33. The composition according to claim 17, wherein the adjuvant is a CpG oligodeoxynucleotide, polyinosinic-polycytidylic acid or a mixture of CpG oligodeoxynucleotide(s) and polyinosinic:polycytidylic acid, and/or the carrier substance is a nanoparticle.

34. The composition according to claim 17, further comprising at least one immune checkpoint inhibitor.

35. The composition according to claim 34, wherein the immune checkpoint inhibitor is chosen from an anti-PD-1, an anti-PDL-1 and an anti-CTLA4.

36. A method of immunotherapy of cancer or infectious diseases, comprising the administration to a subject in need thereof of a therapeutically effective amount of the composition according to claim 17.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Other features, details and advantages of the invention will become apparent on reading the following detailed description which refers to exemplary implementations of the present invention, and on analyzing the appended drawings, in which:

[0097] FIG. 1

[0098] FIG. 1 shows the molecular weight and degree of homogeneity of molecular complexes produced in E. coli or in HEK cells. The proteins ZZ-DTRBD.sub.HEK, ZZ-DTRBD.sub.coli, BB-DTRBD.sub.HEK, BB-DTRBD.sub.coli and ZZ-Tat.sub.22-57C(22-37)S were analyzed by electrophoresis under denaturing conditions. The proteins were deposited in the presence of molecular weight markers. After electrophoretic migration, the proteins were stained with Coomassie blue.

[0099] FIG. 2

[0100] FIG. 2 shows the binding of various molecular complexes to heparin. Series dilutions of the proteins ZZfree, ZZ-DTRBD.sub.HEK, ZZ-DTRBD.sub.coli, BB-DTRBD.sub.HEK, BB-DTRBD.sub.coli and ZZ-Tat.sub.22-57C(22-37)S were respectively incubated at pH 7.2 in microtitration plate wells, previously adsorbed with rabbit IgG. After 4 hours, the plates were washed and biotinylated heparin was added. After 30 minutes of incubation, the binding of the heparin to the plates was detecting using streptavidin coupled to peroxidase and a substrate of this enzyme (ABTS). The heparin binding is considered to be significant when the optical density signal is at least equal to 50% of the signal measured when ZZ-Tat.sub.22-57C(22-37)S is incubated at 100 nM, pH 7.2, on the microtitration plates.

[0101] FIG. 3

[0102] FIG. 3 shows the cells types bonded by the fusion protein ZZ-DTRBD.sub.HEK in a population of mouse splenocytes. Splenocytes were incubated in the presence or absence of a fixed amount of ZZ-DTRBD.sub.HEK (100 nM). A series of fluorescent Ab specific to the CD11 b+pDC, cDC and CD8+ cDC (A); CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, monocytes (B) was then added. After 30 minutes at 4? C., the cells were washed and analyzed by flow cytometry. The percentage of cells bonded by ZZ-DTRBD.sub.HEK in the sub-population in question is shown.

[0103] FIG. 4

[0104] FIG. 4 shows that ZZ-DTRBD.sub.HEK induces the secretion of IL-6 and of IL-12 by splenocytes. A fixed amount of ZZ, DTRBD or ZZ-DTRBD.sub.HEK (1 ?M) was incubated with mouse splenocytes. After 24 h, the supernatants were taken off and the presence of IL-6 and IL-12 was evaluated by enzyme immunoassay.

[0105] FIG. 5

[0106] FIG. 5 shows that ZZ-Tat.sub.22-57C(22-37)S induces the secretion of IL-6 and of IL-12 by splenocytes. A fixed amount of ZZ, Tat.sub.CY49-57, ZZ+Tat.sub.CY49-57 or ZZ-Tat.sub.22-57C(22-37)S (1 ?M) was incubated with mouse splenocytes. After 24 h, the supernatants were taken off and the presence of IL-6 and IL-12 was evaluated by enzyme immunoassay.

[0107] FIG. 6

[0108] FIG. 6 shows that the proteins ZZ-DTRBD.sub.HEK, BB-DTRBD.sub.HEK induce the secretion of larger amounts of IL-6 and IL-12 by splenocytes than ZZ-DTRBD.sub.coli et BB-DTRBD.sub.coli. A fixed amount of ZZ-DTRBD.sub.HEK, ZZ-DTRBD.sub.coli, BB-DTRBD.sub.HEK and BB-DTRBD.sub.coli (1 ?M) was incubated with mouse splenocytes. After 24 h, the superatants were taken off and the presence of IL-6 and IL-12 was evaluated by enzyme immunoassay.

[0109] FIG. 7

[0110] FIG. 7 shows that ZZ-DTRBD.sub.HEK induces the secretion of IL-6 and of IL-12 by isolated murine dendritic cells. Dendritic cells were isolated by magnetic sorting. The dendritic cells were then incubated in the presence or absence of a fixed amount of ZZ-DTRBD.sub.HEK (0.6 UM). After 24 h, the supernatants were taken off and the presence of IL-6 and IL-12 was evaluated by enzyme immunoassay.

[0111] FIG. 8

[0112] [FIG. 8A-B] shows that the proteins ZZ-DTRBD.sub.HEK and ZZ-Tat.sub.22-57C(22-37)S are capable of inducing the expansion of dendritic cells in mice. Three groups of mice were injected three days apart with a PBS buffer in the absence or presence of a fixed amount of ZZ-DTRBD.sub.HEK (5 nmol per mouse) or ZZ-Tat.sub.22-57C(22-37)S (10 nmol per mouse). 24 h after the final injection, the animals were euthanized and the spleens were removed and splenocytes labeled with a cocktail of fluorescent Ab making it possible to distinguish the cDC-CD8+ (CD11c.sup.highB220-CD8.sup.+CD11b.sup.?) (A), cDC-CD11b+(CD11chighB220.sup.?CD8.sup.?CD11b.sup.+) (B) and the pDC (CD11c.sup.intCD317.sup.+CD11b.sup.+).

[0113] FIG. 8C shows the cells analyzed by flow cytometry. The number of positive cells has been shown as a percentage relative to the total number of living splenocytes. Statistical analysis was performed using a Kruskall-Wallis test (*p<0.05).

[0114] FIG. 9

[0115] FIG. 9 shows that ZZ-DTRBD.sub.HEK induces in vitro the secretion of IL-6 and of IL-12 by isolated human DCs. Isolated DCs originating from healthy human donors were incubated in the presence of a fixed amount of ZZ-DTRBD.sub.HEK (1 UM). After 24 h, the supernatants are collected to evaluate the presence of the cytokines IL-6 and IL-12 by enzyme immunoassay.

[0116] FIG. 10

[0117] FIG. 10 shows that mice injected with colorectal cancer cells exhibit slowed tumor growth when they are subsequently injected with ZZ-DTRBD.sub.HEK alone or in association with an adjuvant mixture. A: Sixteen C57BL/6 mice were injected subcutaneously with 0.5 M of MC38 cells. Three days later, eight mice were injected with the adjuvant mixture CpG-B 1018/Poly I:C. (30 ?g for each adjuvant) and eight were not injected (controls). A second injection was carried out three days later.

[0118] B: Eighteen C57BL/6 mice were injected subcutaneously with 0.5 M of MC38 cells. Three days later, six mice were injected with ZZ-DTRBD.sub.HEK in PBS buffer, six others were injected with ZZ-DTRBD.sub.HEK (2 nmol per mouse) in a PBS buffer containing the adjuvant mixture CpG-B 1018/Poly I:C (30 ?g for each adjuvant) and six were not injected (controls). Two additional injections were carried out three days apart. Tumor growth was monitored by measuring the tumors using calipers. Each injection is depicted by an arrow.

[0119] FIG. 11

[0120] FIG. 11 shows that mice injected with colorectal cancer cells exhibit slowed tumor growth when they are subsequently injected with ZZ-DTRBD.sub.HEK, ZZ-Tat.sub.22-57C(22-37)S in association with an adjuvant mixture. Thirty two C57BL/6 mice were injected subcutaneously with 0.5 M of MC38 cells. Three days later, a group of eight mice was injected with ZZ-DTRBD.sub.HEK (0.96 nmol per mouse) in 50 ?l of PBS containing the adjuvant mixture CpG-B 1018/PolyI:C (30 ?g for each adjuvant), another group with an anti-PD-1 Ab (50 ?l, 1.3 nmol per mouse), a third group with ZZ-Tat.sub.22-57C(22-37)S (0.96 nmol per mouse) in 50 ?l of PBS containing the adjuvant mixture CpG-B 1018/PolyI:C (30 ?g for each adjuvant), and a final group of eight mice was not injected (controls). Two additional injections were carried out three days apart. Tumor growth was monitored by measuring the tumors using calipers. Each injection is depicted by an arrow.

[0121] [FIG. 11 A] Kinetics of tumor growth.

[0122] [FIG. 11 B] Kinetics of tumor growth.

[0123] [FIG. 11 C] Survival of treated animals.

[0124] FIG. 12

[0125] FIG. 12 shows that the formation of the molecular complexes ZZ-Tat.sub.22-57C22-37)S, anti-DEC205/ZZ-Tat.sub.22-57C22-37)S and IgG/ZZ-Tat.sub.22-57C22-37)S induce, in vitro, the increase in the proportion of activated monocytes and CD4+ T lymphocytes. A fixed concentration (0.1 ?M) of anti-DEC205/ZZ-Tat.sub.22-57C22-37)S, IgG/ZZ-Tat.sub.22-57C22-37)S, anti-DEC205/ZZ, IgG/ZZ, anti-DEC205 Ab was incubated with human PBMCs. A fixed concentration (1 ?M) of ZZ-Tat.sub.22-57C22-37)S or ZZ was incubated with human PBMCs. After 24 h, the cells were collected and labeled using fluorescent Ab. The proportion of activated monocytes (CD3 CD14-) in the population of living PBMCs was evaluated using the marker CD69.

[0126] FIG. 12A The proportion of activated CD4+ T lymphocytes in the living CD4+ T lymphocyte population (CD3.sup.?CD4.sup.+) was evaluated by measuring the expression of the CD69 molecule.

[0127] FIG. 12B The cells were analyzed by flow cytometry.

[0128] FIG. 13

[0129] FIG. 13 shows that molecular complexes directed against receptors expressed by dendritic cells (anti-CD74/ZZ-Tat.sub.22-57C22-37)S, anti-CD209/ZZ-Tat.sub.22-57C22-37)S, et anti-CD275/ZZ-Tat.sub.22-57C22-37)S) induce the secretion of IL-6 by PBMCs. Human PBMCs were incubated in the presence or in the absence of free forms of the Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also molecular complexes targeting, respectively, the molecules CD74 (A), CD209 (B) and CD275 (C). After 24 h, the supernatants were taken off and the presence of IL-6 was evaluated by enzyme immunoassay.

[0130] FIG. 14

[0131] FIG. 14 shows that a molecular complex (anti-CD335/ZZ-Tat.sub.22-57C22-37)S) directed against the CD335 receptor expressed by NK and NKT cells induces an increase in the proportion of activated NK and NKT cells. PBMCs were incubated in the presence or in the absence of free forms of the Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also the molecular complexes anti-CD335/ZZ-Tat.sub.22-57C22-37)S, anti-CD335/ZZ. After 18 h, the cells were analyzed by cytometry to evaluate the expression of the co-stimulatory molecule CD69 at the surface of the NK cells (A) and NKT cells (B).

[0132] FIG. 15

[0133] FIG. 15 shows that molecular complexes directed respectively against two receptors expressed by NK and NKT cells induce an increase in the proportion of activated NKT cells. PBMCs were incubated in the presence or in the absence of free forms of the Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also the molecular complexes anti-CD56/ZZ-Tat.sub.22-57C22-37)S, anti-CD56/ZZ, anti-CD336/ZZ-Tat.sub.22-57C22-37)S, or anti-CD336/ZZ. After 18 h, the cells were analyzed by cytometry to evaluate the expression of the co-stimulatory molecule CD69 at the surface of the NKT cells.

[0134] FIG. 16

[0135] FIG. 16 shows that molecular complexes directed against receptors expressed by NK cells (anti-CD56/ZZ-Tat.sub.22-57C22-37)S, anti-CD335/ZZ-Tat.sub.22-57C22-37)S, et anti-CD336/ZZ-Tat.sub.22-57C22-37)S) induce the secretion of IL-6 by human PBMCs. PBMCs were incubated in the presence or in the absence of free forms of the Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also molecular complexes targeting, respectively, the molecules CD56 (A), CD335 (B) and CD336 (C). After 24 h, the supernatants were taken off and the presence of IL-6 was evaluated by enzyme immunoassay.

[0136] FIG. 17

[0137] FIG. 17 shows that molecular complexes containing anti-ICP Ab (anti-CTLA-4/ZZ-Tat.sub.22-57C22-37)S, anti-PD-L1/ZZ-Tat.sub.22-57C22-37)S, and anti-OX40/ZZ-Tat.sub.22-57C22-37)S) induce the secretion of IL-6 by human PBMCs. PBMCs were incubated in the presence or in the absence of free forms of the Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also molecular complexes targeting, respectively, the molecules CTLA-4 (A), PD-L1 (B) and OX40 (C). After 24 h, the supernatants were taken off and the presence of IL-6 was evaluated by enzyme immunoassay.

EXAMPLES

Example 1: Expression of Fusion Proteins in E. coli or in HEK Cells, Purification, Biochemical Characterization, and Study of the Capacity Thereof to Bind Heparan Sulfates

Materials and Methods

Production of the Different Molecular Complexes

[0138] The expression of the fusion protein ZZ-DTRBD in E. coli, referred to as ZZ-DTRBD.sub.coli, was previously described in the publication by Lobeck et al. (Infection and Immunity, 1998, 66, 418-423). The fusion protein ZZ-DTRBD.sub.coli (SEQ ID NO: 12) is encoded by a polynucleotide having the sequence SEQ ID NO: 11. The expression of the fusion protein ZZ-Tat.sub.22-57C(22-37)S in E. coli was produced according to a protocol similar to that described for the fusion ZZOVATat.sub.22-57S in the publication by Knittel et al. (Vaccine, 2016, 34(27):3093-3101). The fusion protein ZZ-Tat.sub.22-57C(22-37)Scoli (SEQ ID NO: 20) is encoded by a polynucleotide having the sequence SEQ ID NO: 19. For the expression in eukaryotic cells, HEK cells (2.5?10.sup.6 cells/ml in 250 ml 293F freestyle medium) were transfected with a pCDNA3.4 plasmid encoding ZZ-DTRBD.sub.HEK (400 ?g of DNA preparation resulting from maxiprep by transfection) in the presence of PEI (0.5 mg/ml). This plasmid comprises the polynucleotide having the sequence SEQ ID NO: 13 which encodes the fusion protein ZZ-DTRBD.sub.HEK (SEQ ID NO: 14). The cells were subsequently incubated for 24 h at 37? C. with stirring. 250 ml of Ex-Cell medium was then added. After 4 days of incubation at 37? C. with stirring, the culture superatants were recovered, filtered under sterile conditions, and a protease inhibitor cocktail was added.

[0139] After expression of the three proteins, the supernatants were respectively diluted by ? in 0.1% PBS-Tween then passed over an IgG sepharose column (IgG sepharose 6Fast flow #17-0969-02, Amersham) in order to purify the molecular complexes by immunoaffinity. The acidity of the fusion proteins eluted from the column was neutralized in 1 M Tris-HCl buffer, pH 8. The fusion proteins originating from the expression in E. coli, ZZ-DTRBD.sub.coli and ZZ-Tat.sub.22-57C(22-37)S, were subjected to a second purification cycle using a mono S 5/50 cation exchange column (GE Healthcare). The column was equilibrated with 0.05 M phosphate-citrate buffer, pH=5.5, for the purification of ZZ-DTRBD.sub.coli. The column was equilibrated with 0.05 M phosphate-citrate buffer, pH=4 for the purification of ZZ-Tat.sub.22-57C(22-37)S. The fusion proteins ZZ-DTRBD.sub.coli and ZZ-Tat.sub.22-57C(22-37)S were subsequently eluted with a linear gradient from 0 to 1 M NaCl. The proteins were finally concentrated in PBS and stored at ?20? C. until use. The molecular complexes BB-DTRBD.sub.HEK and BB-DTRBD.sub.coli were produced according to the same protocol as used for ZZ-DTRBD.sub.HEK and ZZ-DTRBD.sub.coli. The fusion protein BB-DTRBD.sub.HEK (SEQ ID NO: 18) is encoded by the polynucleotide sequence SEQ ID NO: 17. The fusion protein BB-DTRBD.sub.coli (SEQ ID NO: 16) is encoded by the polynucleotide sequence SEQ ID NO: 15.

Analysis of the Molecular Weight and of the Degree of Homogeneity of the Molecular Complexes ZZ-DTRBD.sub.HEK, ZZ-DTRBD.sub.coli, BB-DTRBD.sub.HEK and BB-DTRBD.sub.coli by Gel Electrophoresis

[0140] The proteins ZZ-DTRBD.sub.HEK, ZZ-DTRBD.sub.coli, BB-DTRBD.sub.HEK and BB-DTRBD.sub.coli and also the molecular weight markers were deposited on SDS-PAGE 4-12% gel under denaturing conditions and then subjected to electrophoretic migration. Following the migration, the presence of protein bands was revealed using Coomassie blue staining.

Binding of the Molecular Complexes to Heparin

[0141] In order to evaluate the binding of the molecular complexes to heparan sulfates, use was made of heparin, which is a sulfated sugar representing the heparan sulfate family.

[0142] The interaction was evaluated using an enzyme immunoassay technique. To this end, a series of microtitration plates was adsorbed beforehand with rabbit IgG (1 ?g/100 ?l/well in 0.1 M phosphate buffer, pH 7.2) then saturated with a buffer solution containing 0.3% bovine serum albumin (200 ?l/well in 0.1 M phosphate buffer, pH 7.2). Another series of microtitration plates was saturated with a buffer solution containing 0.3% bovine serum albumin (300 ?l/well in 0.1 M phosphate buffer, pH 7.2). The two series of plates were subsequently washed, and series dilutions (in 0.1 M phosphate buffer, pH 7.4, containing 0.1% bovine serum albumin) of the proteins ZZ-DTRBD.sub.HEK. ZZ-DTRBD.sub.coli, BB-DTRBD.sub.HEK and BB-DTRBD.sub.coli and ZZ-Tat.sub.22-57C(22-37)S and free ZZ were deposited in the wells. After 4 hours of incubation at ambient temperature, the plates were washed and 100 ?l of heparin-biotin (1 ?M) were added per well. After 1 hour at ambient temperature, the plates were washed and 100 ?l of streptavidin coupled to peroxidase (1/2000 dilution) was added. After 30 minutes of incubation, the plates were washed and a substrate (ABTS) was added. The staining was measured at 414 nm after 30 minutes of incubation. In order to eliminate the non-specific binding to albumin, the optical signal measured on the adsorbed plates solely with the bovine serum albumin was subtracted from the signal measured on the plates adsorbed with IgG. The heparin binding is considered to be significant when the optical density signal is greater than or equal to 50% of the signal measured when ZZ-Tat.sub.22-57C(22-37)S is incubated at 100 nM, pH 7.2, on the microtitration plates.

Results

[0143] The inventors previously constructed a fusion protein, referred to as ZZ-DTRBD, incorporating both a double ZZ domain derived from the protein A of Staphylococcus aureus and also the DTRBD domain derived from the diphtheria toxin (Lobeck et al., Infection and Immunity, 1998, 66, 418-423). ZZ can bind to the Fc region of immunoglobulins in a similar manner to the protein A. As for DTRBD, it binds to the diphtheria toxin receptor and also has a heparan sulfate binding site located in the region 453-467 (Knittel et al. J. Immunol., 2015, 194(8):3601-11; Knittel et al. Vaccine, 2016, 34(27):3093-3101). These characteristics enable ZZ-DTRBD to target different cell types bearing surface immunoglobulins and proteoglycan heparan sulfates via the interaction with the heparan sulfates.

[0144] Similarly, the inventors constructed a fusion protein, referred to as BB-DTRBD, by replacing the sequence encoding ZZ with a coding sequence referred to as BB. The BB protein corresponds to a double domain which, just like ZZ, is derived from the protein A of Staphylococcus aureus but has the particular feature of binding the Fc region and also the Fab region of immunoglobulins (Jansson 1998 FEMS Immunol. and Med. Microbiol., L?onetti et al. 1999. J. Exp. Med., 189, 1217-28).

[0145] The inventors also constructed a fusion protein, referred to as ZZ-Tat.sub.22-57C(22-37)S. It contains a double ZZ domain derived from the protein A of Staphylococcus aureus and a Tat.sub.22-57C(22-37)S derived from the transcriptional transactivator of HIV (WO 2011/092675, and Knittel et al. Vaccine, 2016, 34(27):3093-3101) which has a heparan sulfate binding site. These characteristics enable ZZ-Tat.sub.22-57C(22-37)S to target different cell types bearing surface immunoglobulins and proteoglycan heparan sulfates via the interaction with the HS.

[0146] The inventors expressed ZZ-DTRBD, BB-DTRBD and ZZ-Tat.sub.22-57C(22-37)S recombinantly using different expression systems. The complexes expressed in E. coli are referred to as ZZ-DTRBD.sub.coli, BB-DTRBD.sub.coli and ZZ-Tat.sub.22-57C(22-37)S and those expressed in HEK cells are named ZZ-DTRBD.sub.HEK and BB-DTRBD.sub.HEK. After expression, the complexes were purified using a column containing an IgG-bearing gel. Next, the proteins ZZ-DTRBD.sub.coli, BB-DTRBD.sub.coli and ZZ-Tat.sub.22-57C(22-37)S were subjected to ion-exchange chromatography in order to eliminate the contaminating LPS. Finally, certain characteristics of these complexes were evaluated by gel electrophoresis. As can be seen in FIG. 1, ZZ-DTRBD.sub.coli and BB-DTRBD.sub.coli migrate in a predominant band with a molecular weight of approximately 35 kDa which is close to the theoretical weight of the molecule (32230), indicating that this band corresponds to a monomer. Minor bands corresponding to degradation products are also present in these two molecular complexes. ZZ-DTRBD.sub.HEK and BB-DTRBD.sub.HEK migrate in several bands distributed substantially between 35 and 150 kDa, indicating that the expression in HEK cells leads to a heterogeneous mixture consisting of monomeric and oligomeric forms of ZZ-DTRBD. The molecular complex ZZ-Tat.sub.22-57C(22-37)S migrates in a predominant band, reflecting the homogeneity of the purified protein.

[0147] The molecular weight of approximately 25 kDa is slightly greater than that calculated for the theoretical weight of the molecule (MW=19 145). However, Tat and derivatives thereof tend to migrate abnormally (Kittiworakam et al. etc), strongly suggesting that the fusion protein ZZ-Tat.sub.22-57C(22-37)S expressed in E. coli and purified substantially consists of a monomer.

[0148] Next, the inventors studied the aptitude of free ZZ and of the five molecular complexes to bind heparin, which is a sulfated polysaccharide representing the heparan sulfate family. Enzyme immunoassay does not make it possible to observe a significant optical signal for free ZZ (FIG. 2). In contrast, an increase in the optical density on the basis of the incubated dose is measured for ZZ-DTRBD.sub.coli, BB-DTRBD.sub.HEK, BB-DTRBD.sub.coli, ZZ-DTRBD.sub.HEK and ZZ-Tat.sub.22-57C(22-37)S. This data therefore indicates that the presence of the DTRBD domain or of the Tat.sub.22-57C(22-37)S domain enables the molecular complexes to bind to heparin. Moreover, since the fusions ZZ-DTRBD.sub.HEK and BB-DTRBD.sub.HEK contain oligomers (see FIG. 1), this data indicates that the presence of oligomers does not disrupt the interaction.

Example 2: The Fusion Protein ZZ-DTRBD.SUB.HEK .is Capable of Binding Different Types of Cells of the Murine Immune System

Materials and Methods

Binding of ZZ-DTRBD to Cells of the Immune System

[0149] C57BI/6 mouse splenocytes are resuspended at 10?10.sup.6 cells/ml in PBS buffer, 0.5% BSA 2 mM EDTA. 100 ?l of the cell suspension are deposited in a 96-round-bottomed-well plate. 100 ?l of buffer, 0.5% BSA 2 mM EDTA are added per well in the absence or presence of ZZ-DTRBD.sub.HEK (100 nM). The mixtures are incubated for 30 minutes at 4? C. then washed twice in PBS 0.5% BSA 2 mM EDTA. 2 ?g per well of rabbit IgG are added to the cells, which are incubated for 20 minutes at 4? C. then washed twice with q.s. 200 ?l of PBS 0.5% BSA 2 mM EDTA. The cells are resuspended in 50 ?l of labeling buffer (PBS 0.5% BSA 2 mM EDTA) containing different mixtures of Ab from BioLegend. The mixtures are incubated for 20 minutes at 4? C. in darkness then the splenocytes are washed twice in q.s. 200 ?l of PBS 0.5% BSA 2 mM EDTA. The cells are then fixed for 30 minutes at ambient temperature. To this end, 100 ?l of 4% PFA buffer, then 100 ?l of PBS 0.5% BSA 2 mM EDTA were added before detection on BD FACSAria? cytometer.

[0150] Mixture 1 for the dendritic cells (duplicates): B220-FITC (#103206, 1/200), CD11 c-PE-Cy7 (#117318, 1/200), CD317-APC (#127016, 1/100), CD8a-PerCP-Cy5.5 (#100734, 1/200), CD11b-APC-Cy7 (#101226, 1/800), Donkey anti-rabbit-BV421 (#406410, 1/100), Live Dead Aqua (ThermoFischer #L34966, 1/1000).

[0151] Mixture 2 for the monocytes and T. B lymphocytes (duplicates): B220-FITC (#103206, 1/200), CD19-BV650 (#115541, 1/100), CD3-APC-Cy7 (#100222, 1/100), CD4-BV605 (#100451, 1/200), NK1.1-PE-Cy7 (#108714, 1/100), CD8a-PerCP-Cy5.5 (#100734, 1/200), CD11b-APC (#101212, 1/800), Ly6C-PE #128007, 1/800), Donkey anti-rabbit-BV421 (#406410, 1/100), Live Dead Aqua (ThermoFischer #L34966, 1/1000).

Results

[0152] To evaluate the capacity of a molecular complex to bind cells of the immune system, ZZ-DTRBD.sub.HEK was incubated in the presence of mouse splenocytes. It was thus possible to observe that this fusion protein preferentially binds to cDC-CD8+ cells which are specialized APCs (FIG. 3A). It also preferentially interacts with monocytes which may also serve as APCs (FIG. 3B). Finally, it binds to a lesser extent to lymphocytes. This data therefore indicates that the fusion protein preferentially targets APCs.

Example 3: The Fusion Proteins ZZ-DTRBD.SUB.HEK .and ZZ-Tat.SUB.22-57C(22-37)S .Induce In Vitro the Secretion of IL-6 and of IL-12 by Cells of the Immune System

Materials and Methods

[0153] C57BL/6 mouse splenocytes are resuspended at 2?10.sup.6 cells/ml in RPMI medium 10% FCS 1% penicillin/streptomycin. 100 ?l of the cell suspension are distributed in a 96-well plate in the presence or absence of the molecular complexes (ZZ, DTRBD, ZZ-DTRBD.sub.HEK, or ZZ-Tat.sub.22-57C(22-37)S), incubated at a final 1 ?M. After 24 h of incubation, the supernatants are collected for ELISA assay of the cytokines IL-6 and IL-12 carried out according to the manufacturer's instructions (R&D #DY406-05 and #DY419)

Results

[0154] The inventors wondered if the molecular complexes could induce the activation of cells of the immune system. Since cell activation can lead to cytokine secretion, the inventors decides to evaluate in vitro the presence of two cytokines in supernatants resulting from the incubation of mouse splenocytes with molecular complexes and different control proteins. The first cytokine is IL-6, because it represents inflammatory cytokines which are important for initiating the immune response. The second is IL-12 because it is crucial for inducing cellular immune responses. The inventors found these two cytokines in increased amounts in the supernatants resulting from the incubation with ZZ-DTRBD.sub.HEK, indicating that this molecular complex induces activation of the splenocytes (FIG. 4). In contrast, they did not find an increased amount of cytokines in the supernatants resulting from the incubation with free ZZ or free DTRBD, indicating that the double domain for binding to Ig and the heparan sulfate-binding domain cannot alone activate immune cells. These results therefore demonstrate that ZZ-DTRBD.sub.HEK is capable of inducing activation of cells of the immune system and indicate that the association of the double Ig-binding domain and of the DTRBD HS-binding domain is absolutely required for the effect.

[0155] The inventors proceeded according to a principle similar to that described in the previous paragraph, in order to evaluate whether ZZ-Tat.sub.22-57C(22-37)S is capable of activating cells of the immune system and if the association of ZZ and of the Tat domain involved in binding to heparan sulfates is absolutely required for the stimulatory effect. To this end, they used in particular a peptide, named Tat.sub.CY49-57, which contains the basic Tat region and thus represents the interaction of Tat and derivatives thereof with heparan sulfates. They incubated mouse splenocytes with ZZ, Tat.sub.CY49-57, ZZ+Tat.sub.CY49-57 and ZZ-Tat.sub.22-57C(22-37)S, respectively. They then evaluated the presence of IL-6 and IL-12 in the supernatants. They found these two cytokines in the supernatants resulting from incubation with ZZ-Tat.sub.22-57C(22-37)S but not in the supernatants resulting from incubation with ZZ, Tat.sub.CY49-57, ZZ+Tat.sub.CY49-57 (FIG. 5). These results therefore demonstrate that ZZ-Tat.sub.22-57C(22-37)S is capable of inducing the activation of cells of the immune system and that this characteristic is not shared by the free forms, ZZ and Tat.sub.CY49-57, indicating that the association of the double Ig-binding domain and of the basic Tat region responsible for the HS binding is absolutely required for the effect.

Example 4: A Molecular Complex i) Remains Capable of Triggering Secretion of IL-6 and IL-12 by Cells of the Immune System when the ZZ Domain Thereof is Substituted by BB, ii) has Increased Stimulatory Capacities when it Consists of Oligomeric Forms

Materials and Method

[0156] C57BL/6 mouse splenocytes are resuspended at 2?10.sup.6 cells/ml in RPMI medium 10% FCS 1% penicillin/streptomycin. 100 ?l of the cell suspension are distributed in a 96-well plate in the presence or absence of different molecular complexes (ZZ-DTRBD.sub.HEK, BB-DTRBD.sub.HEK, ZZ-DTRBD.sub.coli and BB-DTRBD.sub.coli) incubated at a final 1 ?M. After 24 h of incubation, the supernatants are collected for ELISA assay of the cytokines IL-6 and IL-12 carried out following the manufacturer's instructions (R&D #DY406-05 and #DY419).

Results

[0157] The work described in example 3 was carried out with two molecular complexes containing the ZZ double domain. This double domain, which binds the Fc of Ab, can thus target the Ab located at the surface of the APCs. The inventors then wondered if molecular complexes which can jointly target the Fc region and the Fab region of the Ig might also be capable of activating cells of the immune system. To this end, they focused on the BB double domain derived from the protein A of Staphylococcus aureus, which has the particular feature of being able to bind the Fc region of the Ab in the same way as Z, but also the Fab region of the Ab, unlike ZZ (Jansson B. et al., Fems Immunol. Med. Microbiol. 1998, 20:69-78). They prepared the complexes BB-DTRBD.sub.HEK and BB-DTRBD.sub.coli which are described in example 1, and compared them to ZZ-DTRBD.sub.HEK and ZZ-DTRBD.sub.coli for the capacity to induce secretion of the cytokines IL-6 and IL-12 in vitro when they are incubated with murine splenocytes.

[0158] Since the inventors noted that ZZ-DTRBD and BB-DTRBD are in oligomeric form following their expression in HEK cells (see FIG. 1) whereas they are monomeric after expression in E. coli, this work also made it possible to evaluate if the state of oligomerization could affect the capacity to induce the immune system.

[0159] The inventors observed that the supernatants resulting from incubation with ZZ-DTRBD.sub.HEK contained greater amounts of cytokines than those resulting from incubation with BB-DTRBD.sub.HEK (FIG. 6). Similarly, the supernatants resulting from incubation with ZZ-DTRBD.sub.coli contained greater amounts of cytokines than those resulting from incubation with BB-DTRBD.sub.coli. These results demonstrate that a molecular complex containing BB remains capable of inducing the secretion of cytokines in vitro, but to a lesser extent than a molecular complex containing ZZ. This data therefore indicates that the activation effect can be mediated by molecular complexes containing domains capable of binding different sites on Ab.

[0160] The comparison of the contents of cytokines in the supernatants based on the cell type used (i.e. HEK vs. E. coli) for producing the molecular complexes made it possible to demonstrate the difference in stimulatory efficacy depending on the type of production. Indeed, in the supernatants resulting from incubation with complexes originating from HEK cells (ZZ-DTRBD.sub.HEK, BB-DTRBD.sub.HEK) IL-6 and IL-12 are present in a greater amount than those resulting from incubation with complexes resulting from expression in E. coli (ZZ-DTRBD.sub.coli and BB-DTRBD.sub.coli) (FIG. 6). Since the proteins resulting from expression in HEK cells are mainly oligomeric (see FIG. 1), the inventors deduced from this that the immune system is more effectively induced when the molecular complexes have a high degree of oligomerization.

Example 5: Isolated Dendritic Cells Secrete IL-6 and IL-12 when they are Incubated in the Presence of ZZ-DTRBD.SUB.HEK

Materials and Methods

[0161] A C57BL/6J mouse is euthanized, then its spleen is recovered in RPMI medium 10% FCS 1% penicillin/streptomycin. The spleen is perfused with 3 ml collagenase D at 2 mg/ml in HBSS 0.5% BSA then incubated for 30 minutes at 37? C. The splenocytes are recovered and magnetic sorting for the dendritic cells (DC) is carried out according to the manufacturer's instructions (Miltenyi Biotec #130-100-875). The DC are centrifuged for 5 minutes at 4? C. at 390?g then resuspended at 2?10.sup.6 cells/ml in RPMI medium 10% FCS 1% penicillin/streptomycin. 100 ?l of the cell suspension (200 000 cells) are placed in a 96-flat-bottomed-well plate and 100 ?l of RPMI medium 10% FCS 1% penicillin/streptomycin is added in the absence or presence of ZZ-DTRBD.sub.HEK (final 0.6 ?M). The cells are incubated for 24 h at 37? C., then the supernatants are collected for ELISA assay of the cytokines IL-6 and IL-12 according to the manufacturer's instructions (R&D #DY406-05 and #DY419).

Results

[0162] The establishing of immune defense mechanisms depends on the collaboration between different cell partners. Among these, dendritic cells (DC) represent APCs playing a central role. This is because they contribute to activating other cell types, via direct interactions or via cytokines which they secrete. In order to evaluate if ZZ-DTRBD induces cytokine secretion by DCs. DCs were purified from mouse splenocytes C57BI/6. Next, these DCs were incubated for 24 h in the presence or absence of ZZ-DTRBD, then the supernatants were taken off in order to assay the presence of IL-6 and IL-12 (FIG. 7). The amount of IL-6 present in the supernatants resulting from incubation with ZZ-DTRBD.sub.HEK is approximately 5 times greater than that found in the superatants resulting from incubation without a molecular complex. A large amount of IL-12 (approximately 8700 pg/ml) is also detected in the supernatants resulting from incubation with ZZ-DTRBD.sub.HEK but virtually none in those resulting from incubation without a molecular complex. This data therefore indicates that ZZ-DTRBD causes the secretion of IL-6 and of IL-12 by murine dendritic cells and could thus contribute to establishing immune defense mechanisms.

Example 6: The Proteins ZZ-DTRBD and ZZ-Tat.SUB.22-57C(22-37)S .are Capable of Inducing Dendritic Cells in Mice

Materials and Methods

[0163] Three groups of eight C57BL/6 mice were injected three times, at three day intervals, with 100 ?l PBS in the absence or presence of ZZ-DTRBD.sub.HEK (5 nmol per mouse) or ZZ-Tat.sub.22-57C(22-37)S (10 nmol per mouse), respectively. 24 hours after the final injection, the animals were euthanized, the spleens were collected to recover the splenocytes. The cells were resuspended in 50 ?l of labeling buffer (PBS 0.5% BSA 2 mM EDTA) containing different mixtures of Ab. The cells were then incubated for 20 minutes at 4? C. in darkness then washed twice in PBS 0.5% BSA 2 mM EDTA. The cells were then fixed for 20 minutes at 4? C. in 100 ?l of 4% PFA buffer then washed in PBS 0.5% BSA 2 mM EDTA. They were finally resuspended in 200 ?l of PBS 0.5% BSA 2 mM EDTA then analyzing using a BD FACSAria? flow cytometer. The cells were analyzed by flow cytometry. The cDC-CD8+ are identified as CD11c.sup.highB220.sup.?CD8.sup.?CD11b.sup.?, the cDC-CD11b+ are identified as CD11c.sup.highB220.sup.?CD8.sup.?CD11b.sup.? and the pDC are identified as CD11c.sup.intCD317.sup.+CD11b.sup.+.

[0164] The mixtures of Ab used for the phenotype analysis of the dendritic cells are as follows: B220-FITC (#103206, 1/200), CD11c-PE-Cy7 (#117318, 1/200), CD317-APC (#127016, 1/100), CD11b-APC-Cy7 (#101226, 1/800), CD8a-PerCP-Cy5.5 (#100734, 1/200), Live Dead Violet (ThermoFischer #L34964, 1/1000).

Results

[0165] Since the molecular complexes bind preferentially to APCs in vitro and induce cytokine secretion, the inventors wondered if such complexes might induce the expansion of APCs in mice. In order to evaluate this aspect, the inventors injected three groups of 8 C57BI/6 mice with a PBS solution in the presence or absence of ZZ-DTRBD.sub.HEK or ZZ-Tat.sub.22-57C(22-37)S. They then euthanized the animals and sampled their spleen in order to evaluate the frequency of the different sub-populations of dendritic cells in the splenocytes. As can be seen in FIG. 8, the three murine DC populations (i.e. DC-CD8+, DC-CD11b+ and pDC) are present in the spleens of the group of mice injected solely with PBS. However, the frequency of these three cell types is significantly increased in the groups of animals injected with ZZ-DTRBD.sub.HEK or ZZ-Tat.sub.22-57C(22-37)S. This data therefore indicates that the molecular complexes are capable of inducing the expansion of the dendritic cells in vivo. Since these cells are central to initiating the immune response, these results strongly suggest that the complexes can thus promote the induction of immune response mechanisms.

Example 7: ZZ-DTRBD.SUB.HEK .Induces the Secretion of IL-6 and of IL-12 by Human Dendritic Cells In Vitro

Materials and Methods

Isolation of Human Dendritic Cells and Incubation for Studying the Secretion of IL-6 and IL-12

[0166] A leukocyte-platelet layer bag is diluted to ? in AIM V medium, then incubated overnight at ambient temperature with stirring. 15 ml of histopaque medium is then added to 4 leucosep tubes, to which are added 4?25 ml of diluted blood. The tubes are centrifuged unrestrictedly for 15 minutes at ambient temperature at 1000?g. The rings of peripheral blood mononuclear cells (PBMCs) are recovered and washed in PBS 2 mM EDTA without calcium or magnesium. The PBMCs are centrifuged at 150?g for 10 minutes at ambient temperature then a red blood cell-lyzing buffer (8.3 mg/ml NH4CI, 0.84 mg/ml NaHCO.sub.3, 0.1 mM EDTA) is added and incubated for 10 minutes at 4? C. 40 ml of PBS are added, then the cells are centrifuged for 10 minutes at ambient temperature and at 150?g. The DCs are then sorted according to the manufacturer's instructions (Miltenyi Biotec #130-091-379). The DC are centrifuged for 5 minutes at 4? C. at 390?g then resuspended at 2?10.sup.6 cells/ml in RPMI medium 10% FCS 1% penicillin/streptomycin. 100 ?l of the sorted DCs (200 000 cells) are placed in a 96-well plate and 100 ?l of RPMI medium 10% FCS 1% penicillin/streptomycin is added in the absence or presence of ZZ-DTRBD.sub.HEK (final 1 ?M). The cells are incubated for 24 h at 37? C., then the supernatants are collected for ELISA assay of the cytokines IL-6 and IL-12 according to the manufacturer's instructions (R&D #DY206-05 and #DY1270-05).

Results

[0167] To evaluate if ZZ-DTRBD.sub.HEK can induce in vitro the secretion of IL-6 and IL-12 by DCs originating from healthy human donors, after 24 h of incubation the presence of these two cytokines was evaluated in the culture supernatants of these cells. As can be seen in FIG. 9, IL-6 is found in an approximately 10-times greater amount in the supernatants resulting from the incubation in the presence of ZZ-DTRBD.sub.HEK than in the supernatants resulting from the incubation in the absence of the fusion protein. IL-12 is present in a large amount (2400 pg/ml) in the supernatants resulting from the incubation of the DCs in the presence of ZZ-DTRBD.sub.HEK and in a very low amount (8 pg/ml) in those resulting from the incubation in the absence of the molecular complex. All this data thus indicates that ZZ-DTRBD induces an increase in the secretion of IL-6 and IL-12 by human DCs. ZZ-DTRBD could thus contribute to establishing immune defense mechanisms in humans which are linked to the secretion of these cytokines.

Example 8: ZZ-DTRBD and ZZ-Tat.SUB.22-57C(22-37)S .Slow the Growth Progression of a Colorectal Tumor

Materials and Methods

Study of the Effect of the Adjuvant Mixture CpG/PolyI:C on the Tumor Growth of a Murine Colorectal Cancer Cell Line

[0168] Two groups of eight C57BL/6 mice were injected subcutaneously in the paw with 0.5 M of MC38 cells. The mice were then not injected (controls) or injected with the adjuvant mixture CpG-B 1018/Poly I:C (30 ?g for each adjuvant) three and six days later. Tumor growth was monitored by measuring the tumors using calipers. Upon reaching the cessation criterion, they were euthanized.

Study of the Effect of the Molecular Complex ZZ-DTRBD.SUB.HEK., Injected in the Absence or Presence of Adjuvant, on the Tumor Growth of a Murine Colorectal Cancer Cell Line

[0169] Three groups of six C57BL/6 mice were injected subcutaneously in the paw with 0.5 M of MC38 cells. Three, six and nine days later, the mice are either not injected (controls) or are injected with ZZ-DTRBD.sub.HEK (2 nmol per mouse) in the absence or presence of CpG-B 1018+Poly I:C. (30 ?g for each adjuvant per mouse). The mice are monitored individually for 15 days (tumor measurements using calipers) before euthanasia.

Comparison of the Effect of an Anti-PD-1 Ab and of the Molecular Complexes ZZ-DTRBD.sub.HEK Et ZZ-Tat.sub.22-57C(22-37)S on the Tumor Growth of a Murine Colorectal Cancer Cell Line

[0170] Four groups of eight C57BL/6 mice were injected subcutaneously in the paw with 0.5 M of MC38 cells. Three, six and nine days later, the mice are either not injected (controls) or are injected with the anti-PD-1 antibody (Euromedex #BE0146-100MG, clone RMP1-14), ZZ-DTRBD.sub.HEK or ZZ-Tat.sub.22-57C(22-37)S (0.96 nmol per mouse) in the presence of CpG-B 1018+Poly I:C (30 ?g for each adjuvant per mouse). The mice are monitored individually over time (tumor measurements using calipers). Upon reaching the cessation criterion, they are euthanized.

Results

[0171] In order to evaluate if the molecular complexes can have an impact on the growth of a tumor, studies were carried out in a syngeneic model of murine cancer. This model is based on a colon tumor cell line, referred to as MC38, and C57BI/6 mice. The induction of the cancer is caused by injecting 500 000 MC38 cells per mouse.

[0172] Firstly, the impact on tumor growth of the adjuvant mixture CpG1018/polyI:C was evaluated. As can be seen in FIG. 10A, compared to the untreated (control) group, tumor growth is virtually unaffected in the group of mice injected with the CpG1018/polyI:C mixture. This data therefore indicates that the adjuvant mixture alone cannot slow the growth of this colorectal tumor.

[0173] Secondly, the impact on tumor growth of ZZ-DTRBD.sub.HEK injected alone or in the presence of the adjuvant mixture CpG1018/polyI:C was evaluated. As can be seen in FIG. 10B, compared to the untreated (control) group, tumor growth is slowed on day 8 and on day 10 in the group injected with ZZ-DTRBD.sub.HEK without adjuvant. The effect is even greater in the group treated with ZZ-DTRBD.sub.HEK and the CpG1018/polyI:C adjuvant mixture. Indeed, in this group, tumor growth is slowed over the whole period of observation of the animals (day 6 to day 15). All of this data therefore indicates that ZZ-DTRBD.sub.HEK has an impact on the growth of the MC38 cell line and that this effect is amplified when it is mixed with the adjuvant CpG1018/polyI:C.

[0174] In another series of experiments, the effect on the growth of the MC38 tumor in C57BI/6 mice was compared for the following treatments: ZZ DTRBD.sub.HEK/CpG1018/polyI:C, ZZ-Tat.sub.22-57C(22-37)S/CpG1018/polyI:C, anti-PD-1 Ab. As can be seen in FIGS. 11A and 11B, the treatment with the anti-PD-1 Ab does not have a significant impact on tumor growth compared to the control group. In contrast, the mice injected respectively with ZZ-DTRBD.sub.HEK/CPG1018/polyI:C and ZZ-Tat.sub.22-57C(22-37)S/CpG1018/polyI:C exhibit decreased tumor growth. This slowing of tumor growth leads to an increased survival of the animals in these two groups (FIG. 11C).

Example 9: CD4+ Activated Monocytes and Lymphocytes can be Induced by Molecular Complexes Targeting, Respectively, the Surface Molecule DEC205 and Fc.SUB.gamma .Receptors

Materials and Methods

[0175] In order to prepare a molecular complex targeting the DEC205 molecule, an anti-DEC205 Ab was used (BioLegend; clone NLDC-145, ref BLE138202). It was incubated in the absence or presence of ZZ-Tat.sub.22-57C22-37)S at a fixed concentration (0.2 UM for each molecule) for 24 hours in RPMI medium without FCS. The capacity of the ZZ region to bind the Fc region of the anti-DEC205 Ab made it possible to form a non-covalent molecular complex, referred to as anti-DEC205/ZZ-Tat.sub.22-57C22-37)S. The same protocol was used to form a complex between the ZZ molecule and the anti-DEC205 Ab, referred to as anti-DEC205/ZZ.

[0176] In order to prepare a molecular complex targeting the FC.sub.gamma receptors, use was made of a non-specific human polyclonal Ab. This Ab was incubated with ZZ-Tat.sub.22-57C22-37)S or ZZ, following an identical protocol to that used for the anti-DEC205 Ab. It was thus possible to form a non-covalent molecular complex, referred to as IgG/ZZ-Tat.sub.22-57C22-37)S, and an IgG/ZZ complex.

[0177] Human PBMCs prepared as described in example 7 were then incubated in the absence or presence of the following compounds: anti-DEC205/ZZ-Tat.sub.22-57C22-37)S, IgG/ZZ-Tat.sub.22-57C22-37)S, anti-DEC205/ZZ, IgG/ZZ ZZ-DTRBD.sub.HEK, and the anti-DEC205 Ab and free IgG (final concentration of 0.1 UM for each compound). In these experiments, PBMCs were also incubated with ZZ-Tat.sub.22-57C22-37)S and free ZZ proteins at a concentration of 1 M. After 24 h, the cells were collected, labeled using fluorescent Ab making it possible to identify the monocytes (CD14, anti-CD14-BV605, Biolegend), the CD4+ T lymphocytes (CD4, anti-CD4-PerCP-Cy5.5, Biolegend) and the CD69 molecule (anti-CD69-BV785, Biolegend). After 30 minutes of incubation, the PBMCs were fixed using a solution containing 4% paraformaldehyde then analyzed by flow cytometry.

Results

[0178] The previous examples show that molecular complexes targeting Ab can effectively induce certain immune response mechanisms and effectively slow tumor growth. The Ab located at the surface of the APCs are the molecules targeted by these complexes. However, the APCs express a large number of other molecules which may also represent targets for molecular complexes according to the invention. The inventors thus wondered if molecular complexes targeting HS and receptors other than the immunoglobulins located at the APC surface might also activate these cells. In order to evaluate this aspect, they chose to evaluate two types of receptors. The first is the protein DEC205, which is a lectin selectively expressed in humans by monocytes and certain populations of dendritic cells (Kato M. et al. 2006, Int. Immunol., 18:857-869). The second is the FC.sub.gamma receptor, most forms of which are expressed by monocytes and certain populations of dendritic cells.

[0179] The inventors then prepared two molecular complexes which can respectively target these receptors. To target the DEC205 protein, they used a monoclonal Ab specific to this receptor, which they complexed to the fusion protein ZZ-Tat.sub.22-57C22-37)S. They referred to this molecular complex as anti-DEC205/ZZ-Tat.sub.22-57C22-37)S. In order to target the Fc receptors, they used a non-specific human polyclonal IgG Ab which can interact with these receptors via its Fc domain. They formed a molecular complex between this IgG and ZZ-Tat.sub.22-57C22-37)S, referred to as IgG/ZZ-Tat.sub.22-57C22-37)S. These two abovementioned molecular complexes thus have the capacity to bind APC receptors and heparan sulfates via the Tat.sub.22-57C22-37)S domain of ZZ-Tat.sub.22-57C22-37)S. In these molecular complexes, the two proteins are non-covalently associated via their respective Fc and ZZ domains. Therefore, ZZ can no longer target the immunoglobulins located at the surface of the APCs, because it is already interacting with the Ab. The inventors also prepared two complexes, respectively referred to as anti-DEC205/ZZ and IgG/ZZ, free of the Tat.sub.22-57C22-37)S region for binding to heparan sulfates, in order to use them as a control in subsequent activation experiments.

[0180] Since the DEC205 molecule and the Fc receptors are expressed by monocytes, the inventors then wondered if the molecular complexes might enable the activation of this sub-population of APCs. To this end, they incubated PBMCs in the absence or presence of a fixed concentration (0.1 ?M) of anti-DEC205/ZZ-Tat.sub.22-57C22-37)S, IgG/ZZ-Tat.sub.22-57C22-37)S, anti-DEC205/ZZ, IgG/ZZ, anti-DEC205, IgG, respectively. They also incubated the ZZ-Tat.sub.22-57C22-37)S and free ZZ proteins at a concentration of 1 ?M in order to evaluate the effect of ZZ-Tat.sub.22-57C22-37)S at an identical concentration to that which was found to be activating in examples 3, 4 and 5. After 24 h, they evaluated the proportion of activated monocytes and CD4+ T lymphocytes. As can be seen in FIG. 12A, ZZ does not modify the proportion of monocytes activated in the PBMCs, while it is significantly increased by ZZ-Tat.sub.22-57C22-37)S, indicating that this molecular complex makes it possible to activate human APCs. The proportion of cells activated is reduced with the anti-DEC205 Ab, free or included in the anti-DEC205/ZZ complex. In contrast, the proportion of activated monocytes is increased in the presence of the anti-DEC205/ZZ-Tat.sub.22-57C22-37)S complex. Similar behavior is observed when the CD4+ T lymphocytes are considered (FIG. 12B). This data therefore indicates that an Ab targeting APCs and not having the capacity to activate them may become capable of inducing them when it is included in a molecular complex which also makes it possible to target HS.

[0181] The analysis of the state of activation of the monocytes after incubation with free human IgG shows that this Ab makes it possible to increase the proportion of activated monocytes. The IgG/ZZ does not enable a significant increase in the proportion of activated cells. In contrast, the number of activated monocytes is more than two times greater when the PBMCs are incubated with the molecular complex IgG/ZZ-Tat.sub.22-57C22-37)S. Similar behavior is observed when the CD4+ T lymphocytes are considered (FIG. 12B). This data therefore indicates that an Ab targeting FC.sub.gamma receptors at the surface of APCs is capable of activating them but that the activation effect is increased when the Ab is included in a molecular complex making it possible to also target HS.

[0182] Interestingly, these results show a joint increase in the proportion of activated monocytes and T lymphocytes, indicating that the molecular complexes induce several cell actors which have a central role in immune defense mechanisms.

Example 10: Molecular Complexes Targeting Dendritic Cells Induce In Vitro the Secretion of IL-6 by Cells of the Immune System

Materials and Methods

[0183] In order to prepare molecular complexes targeting DCs, three antibodies specific to molecules expressed by DCs were used. The first Ab (BD reference 555538), referred to as anti-CD74 Ab, targets the CD74 molecule. The second Ab (BD reference 551186), referred to as anti-CD209 Ab, targets the CD209 molecule. The third Ab (BD reference 552501), referred to as anti-CD275 Ab, targets the CD275 molecule.

[0184] Each of these three Ab (30 nM for each molecule) was incubated in the absence or presence of ZZ-Tat.sub.22-57C22-37)S or ZZ (5 nM for each molecule), for 24 hours at 4? C. in RPMI medium 5% human AB serum. The capacity of the ZZ region to bind the Fc region of these Ab made it possible to form different non-covalent molecular complexes, referred to as anti-CD74/ZZ-Tat.sub.22-57C22-37)S, anti-CD209/ZZ-Tat.sub.22-57C22-37)S, anti-CD275/ZZ-Tat.sub.22-57C22-37)S, anti-CD74/ZZ, anti-CD209/ZZ, and anti-CD275/ZZ, respectively.

[0185] Human PBMCs are resuspended at 5?10.sup.6 cells/ml in RPMI medium 5% human AB serum. 100 ?l of the cell suspension was distributed in a 96-well plate in the presence or absence of free Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also the molecular complexes. After 24 h of incubation, the supernatants are collected for ELISA assay of the cytokine IL-6 carried out according to the manufacturer's instructions (R&D #DY406-05)

Results

[0186] The inventors wondered if molecular complexes targeting three molecules expressed by DCs could induce the activation of cells of the immune system. Since cell activation can lead to cytokine secretion, the inventors decided to evaluate in vitro the presence of IL-6. They found this cytokine in the supernatants resulting from incubation with ZZ-Tat.sub.22-57C22-37)S but not in the supernatants resulting from incubation with ZZ. They did not find any in the supernatants resulting from incubation of PBMCs with the free anti-CD74 Ab (FIG. 13A), indicating that this Ab cannot induce activation when it is in the free form. The inventors observed that the secretion of IL-6 is increased when the PBMCs are incubated with the anti-CD74/ZZ complex, indicating that the ZZ double domain enables activation mediated by this Ab (FIG. 13A). However, the secretion of this cytokine is even stronger with anti-CD74/ZZ-Tat.sub.22-57C22-37)S, indicating that the addition of the Tat region making it possible to bind HSPGs makes it possible to increase cell activation mediated by this complex.

[0187] The inventors observed that free anti-CD209 and anti-CD275 Ab are capable of inducing the secretion of IL-6 when they are incubated with PBMCs (FIGS. 13B and 13C). When these two Ab are respectively complexed to ZZ (anti-CD209/ZZ and anti-CD275/ZZ), the secretion of this cytokine is also observed. However, this secretion is increased when these two Ab are complexed to ZZ-Tat.sub.22-57C22-37)S, indicating that the activating power can be increased when the molecular targeting complexes are capable of binding HSPGs.

Example 11: A Molecular Complex Directed Against the CD335 Receptor Expressed by NK and NKT Cells Induces In Vitro an Increase in the Proportion of Activated NK and NKT Cells

Materials and Methods

[0188] In order to prepare molecular complexes targeting the CD335 molecule at the surface of NK and NKT cells, an Ab (Origene AM31284AF-N), referred to as anti-CD335 Ab, was used. This Ab was incubated at 30 nM in the absence or presence of ZZ-Tat.sub.22-57C22-37)S or ZZ (5 nM for each molecule), for 5 hours at 37? C. in RPMI medium, 5% human AB serum. The capacity of the ZZ region to bind the Fc region of these Ab made it possible to form two molecular complexes, referred to as anti-CD335/ZZ-Tat.sub.22-57C22-37)S and anti-CD335/ZZ, respectively.

[0189] Human PBMCs are resuspended at 5?10.sup.6 cells/ml in RPMI medium 5% human AB serum. 100 ?l of the cell suspension was distributed in a 96-well plate in the presence or absence of the anti-CD335 Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also the molecular complexes. After 18 h, the cells were collected, labeled using fluorescent Ab making it possible to identify the NK cells (CD56+, biolegend reference 362510 dilution to 1/100), NKT cells (CD56+ biolegend reference 362510; CD3+ cells; Miltenyi ref 130-113-136 dilution to 1/200), and the CD69 molecule (biolegend reference 310932, dilution to 1/100). After 30 minutes of incubation, the PBMCs were fixed using a solution containing 4% paraformaldehyde then analyzed by flow cytometry.

Results

[0190] The inventors wondered if molecular complexes targeting the CD335 molecule expressed at the surface of NK and NKT cells could induce the activation of these two cell types. To evaluate this, they used the anti-CD335 Ab in isolated form or included in a molecular complex. They incubated PBMCs in the absence or presence of the different mixtures. Since NK and NKT cells have a crucial role in immune defense mechanisms, after 18 h of incubation they evaluated the proportion of activated NK and NKT cells by monitoring the expression of the co-stimulatory molecule CD69.

[0191] As can be seen in FIG. 14, ZZ, the isolated anti-CD335 Ab and the anti-CD335/ZZ complex do not increase the proportion of NK (A) or NKT (B) cells expressing CD69 in PBMCs. Different behavior is observed with free ZZ-Tat.sub.22-57C22-37)S. Indeed, this molecular complex does not increase the proportion of NKT cells expressing CD69, but it does increase the percentage of NK cells expressing this marker, indicating that the association of ZZ and of the HS ligand in the ZZ-Tat.sub.22-57C(22-37)S complex makes it possible to increase the activation of NK cells. The molecular complex anti-CD335/ZZ-Tat.sub.22-57C(22-37)S has an impact on the activation of the two cell sub-populations. Indeed, it increases the proportion of NK and NKT cells expressing CD69. All this data therefore indicates that the anti-CD335 Ab does not have the capacity to activate the two cell types, but may become capable of inducing them when it is included in a molecular complex which also makes it possible to target HS.

Example 12: Molecular Complexes Respectively Directed Against the CD56 (A) and CD336 (B) Molecules Expressed by NK and NKT Cells Induce In Vitro an Increase in the Proportion of Activated NKT Cells

Materials and Methods

[0192] In order to prepare molecular complexes targeting the CD56 and CD336 molecules at the surface of the NK and NKT cells, the Ab anti-CD56 (Biolegend reference 304622) which targets the CD56 molecule, and the Ab anti-CD336 (Origene AM50346PU-N) which targets the CD336 molecule were used.

[0193] These Ab were incubated at 30 nM in the absence or presence of ZZ-Tat.sub.22-57C22-37)S or ZZ (5 nM for each molecule), for 5 hours at 37? C. in RPMI medium, 5% human AB serum. The capacity of the ZZ region to bind the Fc region of these Ab made it possible to form four molecular complexes, referred to as anti-CD56/ZZ-Tat.sub.22-57C22-37)S, anti-CD56/ZZ, anti-CD336/ZZ-Tat.sub.22-57C22-37)S, and anti-CD336/ZZ, respectively.

[0194] Human PBMCs are resuspended at 5?10.sup.6 cells/ml in RPMI medium 5% human AB serum. 100 ?l of the cell suspension was distributed in a 96-well plate in the presence or absence of the isolated forms of the anti-CD56 Ab, anti-CD336 Ab, anti-CD336 Ab. ZZ or ZZ-Tat.sub.22-57C(22-37)S and also the non-covalent molecular complexes. After 18 h, the cells were collected, labeled using fluorescent Ab described in example 11 which make it possible to identify the NKT cells (CD56+CD3+), and the CD69 molecule. After 30 minutes of incubation, the PBMCs were fixed using a solution containing 4% paraformaldehyde then analyzed by flow cytometry.

Results

[0195] The inventors wondered if molecular complexes targeting the expressed molecules CD56 and CD336 could induce the activation of NKT cells. To evaluate this, they used the anti-CD56 and anti-CD336 Ab in isolated form or included in a molecular complex. They incubated PBMCs in the absence or presence of the different mixtures. After 18 h of incubation, they evaluated the proportion of activated NKTs by monitoring the expression of the co-stimulatory molecule CD69.

[0196] As can be seen in FIG. 15, ZZ, ZZ-Tat.sub.22-57C22-37)S, the anti-CD56 Ab, the isolated anti-CD336 Ab and the anti-CD336/ZZ complex do not increase the proportion of NKT cells expressing CD69 in PBMCs. Different behavior is observed with the molecular complexes anti-CD56/ZZ-Tat.sub.22-57C22-37)S (FIG. 15A), anti-CD336/ZZ-Tat.sub.22-57C22-37)S (FIG. 15B). Indeed, an increase in the proportion of NKT cells expressing CD69 is observed when these two molecular complexes are incubated with PBMCs. All this data therefore indicates that the isolated anti-CD56 Ab and anti-CD336 Ab do not have the capacity to activate NKT cells, but may become capable of inducing them when they are included in a molecular complex which also targets HS.

Example 13: Molecular Complexes Targeting NK and NKT Cells Induce In Vitro the Secretion of IL-6 by Cells of the Immune System

Materials and Methods

[0197] In order to prepare molecular complexes targeting NK and NKT cells, the three antibodies described in examples 10 and 11 were used. Each of these three Ab (30 nM for each molecule) was incubated in the absence or presence of ZZ-Tat.sub.22-57C22-37)S of ZZ (5 nM for each molecule), for 24 hours in RPMI medium 5% human AB serum. The capacity of the ZZ region to bind the Fc region of these Ab made it possible to form different non-covalent molecular complexes, referred to as anti-CD56/ZZ-Tat.sub.22-57C22-37)S, anti-CD335/ZZ-Tat.sub.22-57C22-37)S, anti-CD336/ZZ-Tat.sub.22-57C22-37)S, anti-CD56/ZZ, anti-CD335/ZZ, and anti-CD336/ZZ, respectively.

[0198] Human PBMCs are resuspended at 5?10.sup.6 cells/ml in RPMI medium 5% human AB serum. 100 ?l of the cell suspension are distributed in a 96-well plate in the presence or absence of free Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also the molecular complexes. After 24 h of incubation, the superatants are collected for ELISA assay of the cytokine IL-6 carried out according to the manufacturer's instructions (R&D #DY406-05).

Results

[0199] The inventors wondered if molecular complexes targeting NK and NKT cells could induce the activation of cells of the immune system. Since cell activation can lead to cytokine secretion, the inventors decided to evaluate in vitro the presence of IL-6. They found this cytokine in the supernatants resulting from incubation with ZZ-Tat.sub.22-57C22-37)S but not in the supernatants resulting from incubation with ZZ. They did not find any IL-6 in the supernatants resulting from incubation of PBMCs with the free anti-CD56 and anti-CD335 Ab (FIGS. 16A and 16B), indicating that these two proteins cannot induce activation when they are in the free form. In contrast, they detected the cytokine in the superatants resulting from the incubation with the anti-CD336 antibody (FIG. 16C), indicating that, in its free form, this Ab has a stimulatory activity. The inventors observed, for the three Ab, increased II-6 secretion when the PBMCs are respectively incubated with the anti-CD56/ZZ-Tat.sub.22-57C22-37)S, (FIG. 16A), anti-CD335/ZZ-Tat.sub.22-57C22-37)S, (FIG. 16B) and anti-CD336/ZZ-Tat.sub.22-57C22-37)S complexes (FIG. 16C). This data therefore indicates that molecular complexes targeting NK cells and HSPGs can induce effective activation of immune cells.

Example 14: Molecular Complexes Targeting Immune Checkpoints (ICP) Induce in Vitro the Secretion of IL-6 by Cells of the Immune System

Materials and Methods

[0200] In order to prepare molecular complexes targeting ICPs, three antibodies specific to molecules considered to be ICPs were used. The first Ab (BioXCell reference BE0190), referred to as anti-CTLA-4 Ab, targets the CTLA-4 molecule. The second Ab (BioXCell reference BE0285), referred to as anti-PD-L1 Ab, targets the PD-L1 molecule. The third Ab (R&D reference MAB10542), referred to as anti-OX40 Ab, targets the OX40 molecule.

[0201] Each of these three Ab (30 nM for each molecule) was incubated in the absence or presence of ZZ-Tat.sub.22-57C22-37)S or ZZ (5 nM for each molecule), for 24 hours at 4? C. in RPMI medium 5% human AB serum. The capacity of the ZZ region to bind the Fc region of these Ab made it possible to form different non-covalent molecular complexes, referred to as anti-CTLA-4/ZZ-Tat.sub.22-57C22-37)S, anti-PD-L1/ZZ-Tat.sub.22-57C22-37)S, anti-OX40/ZZ-Tat.sub.22-57C22-37)S, anti-CTLA-4/ZZ, anti-PD-L1/ZZ, and anti-OX40/ZZ, respectively.

[0202] Human PBMCs were resuspended at 5?10.sup.6 cells/ml in RPMI medium 5% human AB serum. 100 ?l of the cell suspension was distributed in a 96-well plate in the presence or absence of free Ab, ZZ or ZZ-Tat.sub.22-57C(22-37)S and also the molecular complexes. After 24 h of incubation, the supernatants were collected for ELISA assay of the cytokine IL-6 carried out according to the manufacturer's instructions (R&D #DY406-05).

Results

[0203] The inventors wondered if molecular complexes targeting ICPs could induce the activation of cells of the immune system. Since cell activation can lead to cytokine secretion, the inventors decided to evaluate in vitro the presence of IL-6. They did not find any in the supernatants resulting from incubation of PBMCs with the three different free Ab or with free ZZ, indicating that these compounds cannot induce activation when they are in the free form (FIGS. 17A, 17B, and 17C). In contrast, they found this cytokine in supernatants resulting from incubation with ZZ-Tat.sub.22-57C22-37)S, demonstrating that the association of the double Ig-binding domain and the Tat.sub.22-57C22-37)S HS-binding domain enables the cells of the system to be activated. However, the inventors observed that 11-6 secretion is further increased when the PBMCs are respectively incubated with the anti-CTLA-4/ZZ-Tat.sub.22-57C22-37)S, (FIG. 17A), anti-PD-L1/ZZ-Tat.sub.22-57C22-37)S, (FIG. 17B) and anti-OX40/ZZ-Tat.sub.22-57C22-37)S complexes (FIG. 17C). In contrast, IL-6 is absent from the superatants resulting from incubation with anti-CTLA-4/ZZ, anti-PD-L1/ZZ, or anti-OX40/ZZ, indicating that the double ZZ domain does not contribute to activation mediated by these Ab. All this data therefore indicates that molecular complexes targeting ICPs and HSPGs can induce activation of immune cells.