NEW IMMUNOSTIMULATORS AND USE THEREOF IN IMMUNOTHERAPY

Abstract

The present invention relates to a new family of iNKT stimulators, referred to as 6″-O-PEGm-NHR-GalCer, which are potent analogues of KRN7000. These iNKT stimulators can advantageously be used in therapy, in particular for the prevention and/or treatment of many diseases requiring a stimulation of an immune response, such as cancer, viral, bacterial or parasitic diseases, autoimmune diseases or inflammatory diseases. The iNKT cell stimulator of the invention may be coupled to a biological carrier, such as a therapeutic and/or targeting agent, or be vectorized, for example in nanoparticles, to be specifically delivered to target cells.

Claims

1. An iNKT stimulator, wherein said iNKT stimulator consists of a compound of the following formula (II): ##STR00033## wherein X represents a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH.sub.2).sub.n— with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1, wherein R1 represents an alkyl, an aryl, an heterocyclic group, a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.p—R2 with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q—R2 with q being an integer from 1 to 24, wherein R2 represents a functional reacting group allowing coupling to a carrier D or represents L-D, wherein L is a linker and D is a carrier.

2. The iNKT stimulator according to claim 1, wherein said iNKT stimulator is selected from the group consisting of: a compound of the following formula (V): ##STR00034## a compound of the following formula (VI): ##STR00035## a compound of the following formula (VII): ##STR00036##  and a compound of the following formula (VIII): ##STR00037##

3. A conjugate comprising at least one iNKT stimulator coupled to at least one carrier, wherein said conjugate: (i) comprises a compound of the formula (II) ##STR00038## wherein X represents a PEG fragment —[CH2-CH2-O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1 and wherein R1 represents a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.p-L-D with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q-L-D with q being an integer from 1 to 24, wherein L is a linker and D is a carrier, or (ii) is obtained by coupling to one carrier D at least one iNKT stimulator of the above formula (II), wherein X represents a PEG fragment —[CH2-CH2-O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1, wherein R1 represents a PEG fragment —[CH2-CH2-O].sub.p—R2 with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q—R2 with q being an integer from 1 to 24 and wherein R2 represents a functional reacting group allowing coupling to said carrier D.

4. The iNKT stimulator according to claim 1, wherein said carrier D is a therapeutic agent and/or a targeting agent.

5. The iNKT stimulator according to claim 1, wherein said carriers D is an antibody, antibody fragment, sugar, lectin, affitin, growth factor, antigen, a protein, peptide, glycoprotein, aptamer, loaded cell, virus and/or carbohydrate.

6. A vector comprising: at least one iNKT stimulator consisting of a compound of the following formula (II): ##STR00039## wherein X represents a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1, wherein R1 represents an alkyl, an aryl, an heterocyclic group, a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.p—R2 with p being an integer from 1 to 24 or an alkyl chain —(CH2)-R2 with q being an integer from 1 to 24, wherein R2 represents a functional reacting group allowing coupling to a carrier D or represents L-D, wherein L is a linker and D is a carrier, at least one conjugate comprising at least one iNKT stimulator coupled to at least one carrier, wherein said conjugate: (i) comprises a compound of the formula (II) ##STR00040## wherein X represents a PEG fragment —[CH2-CH2-O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1 and wherein R1 represents a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.p-L-D with p being an integer from 1 to 24 or an alkyl chain —(CH2)-L-D with q being an integer from 1 to 24, wherein L is a linker and D is a carrier, or (ii) is obtained by coupling to one carrier D at least one iNKT stimulator of the above formula (II), wherein X represents a PEG fragment —[CH2-CH2-O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1, wherein R1 represents a PEG fragment —[CH2-CH2-O].sub.p—R2 with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q-R2 with q being an integer from 1 to 24 and wherein R2 represents a functional reacting group allowing coupling to said carrier D, and/or at least one iNKT stimulator consisting of a compound of the following formula (IV) ##STR00041##

7. The vector according to claim 6, wherein said vector is a nanoparticle.

8. A pharmaceutical composition comprising: (i) at least one iNKT stimulator according to claim 1; at least one conjugate comprising at least one iNKT stimulator coupled to at least one carrier, wherein said conjugate: (i) comprises a compound of the formula (II) ##STR00042## wherein X represents a PEG fragment —[CH2-CH2-O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1 and wherein R1 represents a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.p-L-D with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q-L-D with q being an integer from 1 to 24, wherein L is a linker and D is a carrier, or (ii) is obtained by coupling to one carrier D at least one iNKT stimulator of the above formula (II), wherein X represents a PEG fragment —[CH2-CH2-O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1, wherein R1 represents a PEG fragment —[CH2-CH2-O].sub.p—R2 with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q-R2 with q being an integer from 1 to 24 and wherein R2 represents a functional reacting group allowing coupling to said carrier D; at least one vector comprising: at least one iNKT stimulator consisting of a compound of the following formula (II): ##STR00043## wherein X represents a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1, wherein R1 represents an alkyl, an aryl, an heterocyclic group, a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.p—R2 with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q-R2 with q being an integer from 1 to 24, wherein R2 represents a functional reacting group allowing coupling to a carrier D or represents L-D, wherein L is a linker and D is a carrier, at least one conjugate comprising at least one iNKT stimulator coupled to at least one carrier, wherein said conjugate: (i) comprises a compound of the formula (II) ##STR00044## wherein X represents a PEG fragment —[CH2-CH2-O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1 and wherein R1 represents a PEG fragment —[CH.sub.2—CH.sub.2—O].sub.p-L-D with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q-L-D with q being an integer from 1 to 24, wherein L is a linker and D is a carrier, or (ii) is obtained by coupling to one carrier D at least one iNKT stimulator of the above formula (II), wherein X represents a PEG fragment —[CH2-CH2-O].sub.m with m being an integer from 1 to 24, an alkyl chain —(CH2).sub.n- with n being an integer from 1 to 24 or a branched hydrocarbon group, wherein R represents —CO—R1, —CONR1, —COOR1, —CSR1, —CSNR1 or —CSOR1, wherein R1 represents a PEG fragment —[CH2-CH2-O].sub.p—R2 with p being an integer from 1 to 24 or an alkyl chain —(CH2).sub.q-R2 with q being an integer from 1 to 24 and wherein R2 represents a functional reacting group allowing coupling to said carrier D; and/or at least one iNKT stimulator consisting of a compound of the following formula (IV) ##STR00045## and/or at least one iNKT stimulator consisting of a compound of the following formula (IV): ##STR00046## and (ii) at least one pharmaceutically acceptable vehicle.

9. A method of prevention and/or treatment of a disease requiring a stimulation of an immune response in a subject in need thereof, wherein said method comprises administering to said subject an effective dose of the iNKT stimulator according to claim 1.

10. The method according to claim 9, wherein said disease requiring a stimulation of an immune response is cancer, an autoimmune disease, an inflammatory disease, a viral infection, a bacterial infection and/or a parasitic disease.

11. (canceled)

12. The conjugate according to claim 3, wherein said carrier D is a therapeutic agent and/or a targeting agent.

13. The conjugate according to claim 3, wherein said carriers D is an antibody, antibody fragment, sugar, lectin, affitin, growth factor, antigen, a protein, peptide, glycoprotein, aptamer, loaded cell, virus and/or carbohydrate.

14. A method of prevention and/or treatment of a disease requiring a stimulation of an immune response in a subject in need thereof, wherein said method comprises administering to said subject an effective dose of the iNKT stimulator of claim 2.

15. A method of prevention and/or treatment of a disease requiring a stimulation of an immune response in a subject in need thereof, wherein said method comprises administering to said subject the conjugate according to claim 3.

16. A method of prevention and/or treatment of a disease requiring a stimulation of an immune response in a subject in need thereof, wherein said method comprises administering to said subject the conjugate according to claim 4.

17. A method of prevention and/or treatment of a disease requiring a stimulation of an immune response in a subject in need thereof, wherein said method comprises administering to said subject the vector according to claim 6.

18. A method of prevention and/or treatment of a disease requiring a stimulation of an immune response in a subject in need thereof, wherein said method comprises administering to said subject the pharmaceutical composition according to claim 8.

19. The conjugate according to claim 3, wherein said carrier D is a therapeutic agent and/or a targeting agent.

20. The conjugate according to claim 3, wherein said carriers D is an antibody, antibody fragment, sugar, lectin, affitin, growth factor, antigen, a protein, peptide, glycoprotein, aptamer, loaded cell, virus and/or carbohydrate.

Description

FIGURES

[0225] FIG. 1: Expression of CD1d on HeLa cells in comparison with their respective CD1d transfectants.

[0226] FIG. 2: IFN-γ, IL-2 and IL-13 secretions by IKNT cells after activation with HEK293 (A) or HeLa (B) cells and their respective CD1d transfection models, loaded with KRN7000.

[0227] FIG. 3: IFN-γ, IL-2 and IL-13 secretions by IKNT cells after activation with HEK293 (A) or HeLa (B) cells and their respective CD1d transfection models, loaded with 6-PEG.sub.3-NH.sub.2-GalCer (2a).

[0228] FIG. 4: IFN-γ secretion after activation of iNKT cells by either PBMCs or non-CD1d-HEK293 cells loaded with 6-PEG.sub.3-NH.sub.2-GalCer (2a).

[0229] FIG. 5: CD1d-dependency of the iNKT cells activation observed using both HEK293 CD1d-transfected (left panel A) and control HEK293 cells (right panel B) as antigen presenting cells. IL-2 secretion induced by 6-PEG.sub.3-NH.sub.2-GalCer (2a) in the presence or absence of anti-CD1d antibody.

[0230] FIG. 6: RT-PCR analysis of CD1d expression on cells lines used as antigen presenting cells (SW620, non-transfected HEK293 and HEK293 transfected to express CD1d).

[0231] FIG. 7: Cytotoxicity induced by iNKTs on HeLa-CD1d cells (expressed as percentage of dead cells) after activation with 6-PEG.sub.3-NH.sub.2-GalCer (2a) or KRN7000.

[0232] FIG. 8: iNKT phenotyping. Expansion of iNKT cells in PBMC in the presence of KRN7000 or 6-PEG.sub.3-NH.sub.2-GalCer (2a) expressed as the ratio CD8+/CD4+.

[0233] FIG. 9: Cytotoxicity induced by iNKTs on HeLa-CD1d spheroid models, after activation with 6-PEG.sub.3-NH.sub.2-GalCer (2a) or KRN7000. Experiment protocol (A) and cells death analysis by flow cytometry (B).

[0234] FIGS. 10 and 11: IFN-γ and IL-13 secretion by iNKTs after activation with 6-PEG.sub.3-NHAc-GalCer (2b), 6-PEG.sub.3-NH.sub.2-GalCer (2a), 6-PEG.sub.3-NHBz-GalCer (2c) and KRN7000.

[0235] FIG. 10: IFN-γ secretion with HeLa-CD1d cells (A) and non CD1d HeLa cells (C) and IL-13 secretion by iNKTs after activation with HeLa-CD1d cells (B) and non CD1d HeLa cells (D).

[0236] FIG. 11: IFN-γ secretion with HEK293-CD1d cells (E) and non CD1d HEK293 cells (G) and IL-13 secretion by iNKTs after activation with HEK293-CD1d cells (F) and non CD1d HEK293 cells (H).

[0237] FIG. 12: IFN-γ secretion by iNKTs after activation with HeLa cells loaded with 6-PEG.sub.3-NHAc-GalCer (2b), 6-PEG.sub.3-NH.sub.2-GalCer (2a), 6-PEG.sub.3-NHBz-GalCer (2c), 6-Mal-PEG-GalCer (3b), 6-Mal-PEG.sub.6-GalCer (3a) and KRN7000.

[0238] FIG. 13: EGFR recognition by cetuximab purified after the coupling (“Coupled Cetuximab”) or the “mock” coupling (“Cetuximab alone”) reactions.

[0239] FIG. 14: IFN-γ secretion by iNKTs after activation with HeLa-CD1d cells (A) or non transfected HeLa cells (B) loaded with Cetuximab+6-PEG.sub.3-NH.sub.2-GalCer (2a) (left panel) or with 6-PEG.sub.3-NH.sub.2-GalCer (2a) alone (right panel).

TABLE-US-00001 TABLE 1 Figure 14, A, left panel a b c Concentration of coupling product 10 μg/ml 1 μg/ml 0.1 μg/ml Starting concentration .sup. 10.sup.−7M .sup. 10.sup.−8M .sup. 10.sup.−9M Observed concentration 10.sup.−10M 10.sup.−11M 10.sup.−12M

TABLE-US-00002 TABLE 2 Figure 14, B, left panel a b c Concentration of coupling product 10 μg/ml 1 μg/ml 0.1 μg/ml Starting concentration   .sup. 10.sup.−7M   .sup. 10.sup.−8M   .sup. 10.sup.−9M Observed concentration <10.sup.−10M <10.sup.−10M <10.sup.−10M

[0240] FIG. 15: IFN-γ secretion by iNKTs after activation with HeLa-CD1d cells (C) or non transfected HeLa cells (D) loaded with the complex Cetuximab 6-Mal-PEG.sub.6-GalCer (3a) formed by TRAUT activation (left panel) or with 6-Mal-PEG.sub.6-GalCer (3a) alone (right panel).

TABLE-US-00003 TABLE 3 Figure 15, C, left panel a b c Concentration of coupling product 10 μg/ml 1 μg/ml 0.1 μg/ml Starting concentration   10.sup.−7M   10.sup.−8M   10.sup.−9M Observed concentration >10.sup.−8M >10.sup.−8M >10.sup.−8M

TABLE-US-00004 TABLE 4 Figure 15, D, left panel a b c Concentration of coupling product 10 μg/ml 1 μg/ml 0.1 μg/ml Starting concentration   10.sup.−7M   10.sup.−8M   10.sup.−9M Observed concentration >10.sup.−6M >10.sup.−6M >10.sup.−6M

[0241] FIG. 16: Activity of fractions A and B on iNKT cells compared to 6-Mal-PEG.sub.6-GalCer 3a after loading on non-CD1d HeLa cells.

EXAMPLES

Example 1: Synthesis of 6-PEG-NHR-GalCer 2 and 6-Mal-PEG-GalCer 3

[0242] The synthesis of compounds 6-PEG.sub.3-NHR-GalCer of formula IV (2a), V (2b), VI (2c) (R=H, Ac and PhCH.sub.2CO, respectively) and 6-Mal-PEG.sub.n-GalCer VII (3a) and VIII (3b) is based on conventional chemical pathway described in the literature.

[0243] The synthesis of key protected 6″OH-Galcer intermediate to produce 6-PEG.sub.3-NHR-GalCer 2 and 3 was achieved from galactose and phytosphingosine precursors, by slightly modified established chemical procedures (see Scheme 1 below).

##STR00027## ##STR00028##

From the protected 6″-OH-Galcer, all 6-PEG.sub.3-NHR-GalCer 2 and 6-Mal-PEG.sub.n-GalCer 3 are accessible (see Schemes 2 and 3 below).

##STR00029## ##STR00030##

##STR00031##

Example 2: Activation of iNKTs by 6-PEG.SUB.3.-NHR-GalCer 2 and 6-Mal-PEG-GalCer 3

[0244] To analyse ability of 6-PEG.sub.3-NHR-GalCer 2 and 6-Mal-PEG.sub.n-GalCer 3 to activate iNKTs, several types of presenting cells were used, such as HEK293 or HeLa cells transfected to express CD1d molecule on their membrane. Non-transfected cells were also used as negative control “at first glance” since presentation of glycolipids to iNKT cells is known to be dependent on CD1d.

[0245] CD1d expression on HEK293 and HeLa+/−CD1d was analyzed by flow cytometry (FIG. 1). Cells were labeled with an anti-CD1d-FITC or with the associated isotype control for 20 min and washed to be read on Accuri C6 Flow Cytometer. A shown in FIG. 1, in both cases non transfected cells appeared negative, notably when compared with their CD1d-transfected counterparts.

[0246] These cells were then used as antigen presenting cells (APC) to compare the iNKT cell activation potency of the canonical α-GalCer (KRN7000) ligand to that of the analogue 6″-modified with an aminoPEG linking arm 6-PEG.sub.3-NH.sub.2-GalCer 2a. APC were loaded with glycolipids after co-incubation at various concentrations overnight. Next day, cells were washed and co-cultured with iNKTs (2 APC for 1 iNKT). After 6 hours, supernatants were collected, and cytokines secretions (IFN-γ and IL-2 for Th1 panel and IL-13 for Th2 panel) was measured by ELISA (FIGS. 2 and 3)

[0247] Firstly, canonical ligand, KRN7000, was presented to iNKT cells by HEK293 (FIG. 2A) or HeLa (FIG. 2B) cells and the associated CD1d transfected model. In both cases, KRN7000 induced IFN-γ, IL2 and IL13 releases (Log IC50 are recapitulated in table 1), in the presence of a high expression of CD1d on CD1d-transfected cells, underscoring the CD1d-dependency of the glycolipid recognition by iNKT cells. When 6-PEG.sub.3-NH.sub.2-GalCer 2a was used (FIGS. 3A and 3B), we observed a much stronger IFN-γ, IL13 and IL-2 secretions at almost 100 fold higher than KRN7000 (See comparisons in table 1).

[0248] But more surprisingly, in absence of CD1d (tests carried out on non-CD1d Hela and HEK293 cells), still a significant dose response secretion of cytokines was detected while KRN7000 remains very poorly active or ineffective. These unexpected results were not observed when using SW620 cells as control (absence of CD1d, data not shown).

[0249] A similar cytokine release profile was confirmed with IFN-γ released from PBMC cells, which represent a closer human medium, compared from non-CD1d-HEK293 cells (see FIG. 4).

[0250] This result indicates that 6-PEG.sub.3-NH.sub.2-GalCer 2a appears among the most powerful activators of h-iNKT known to date. These biological outcomes of the 6-PEG.sub.3-NH.sub.2-GalCer 2a makes this novel candidate very interesting in the context of immunotherapy against cancer regarding the development of the clinical trial with KRN7000.

TABLE-US-00005 TABLE 5 KRN7000 6-PEG.sub.3-NH.sub.2-GalCer Delta LogEC50 LogEC50 LogEC50 HEK293-CD1d IFN-γ −8.447 −10.78  2.333 IL-2 −8.680 −10.23  1.55  IL-13 −7.956 −10.49  2.534 HeLa-CD1d IFN-γ −8.653 −10.53  1.877 IL-2 −9.315 −10.81  1.495 IL-13 −7.841  −9.930 2.089

Example 3: “Non-CD1d” Tumor Cells: A Wrong Paradigm

[0251] In light of the intriguing observations of an INKT activation on “non-CD1d” tumor cells, it was investigated how 6-PEG.sub.3-NH.sub.2-GalCer 2a could activate iNKT cells when seemingly CD1d negative cells. Non-transfected HeLa and HEK293 cells were thus used as auto-antigens presenting cells. The hypothesis made was about the existence of a pool of CD1d, even very low, at the surface of the non-CD1d tumor cells but sufficient to be recognized by the high potent 6-PEG.sub.3-NH.sub.2-GalCer a2 glycolipid allowing an activation of iNKT, while KRN7000 fails to achieve this goal.

[0252] It was then checked whether iNKT cells activation might be provided by a very low expression of CD1d on the proper non-CD1d tumor cells that cannot be detected by flow cytometry. To this aim, the same activation experiments were performed but in presence of an anti-CD1d antibody. HEK293 cells and HEK293-CD1d were loaded with 6-PEG.sub.3-NH.sub.2-GalCer a2 as described previously. Before co-culture with iNKTs cells, loaded presenting cells were co-incubated with the anti-CD1d antibody for 1 hours, iNKTs were added directly in suspension and co-cultured 6 hours before cytokine analysis in supernatant (ELISA). If activation signal was provided by presentation of the modified glycolipid through CD1d, it was expected to observe a blockage, or at least a decrease, of cytokine secretion after blocking the signal with the anti-CD1d antibody.

[0253] When CD1d-HEK293 positive cells were used, only an incomplete inhibition was observed in the presence of the antibody (see FIG. 5A). This is likely due to the very high potency of 6-PEG.sub.3-NH.sub.2-GalCer 2a combined with the high level of CD1d expression on the transfected cells. However, the cytokine signal was almost completely eliminated when using the “non CD1d” cells (see FIG. 5B). This experiment clearly suggests the existence of an, until now, unknown low level of CD1d molecules on non-transfected HEK293 cells.

[0254] To confirm this hypothesis, the most sensitive method to detect low signal of CD1d is to determine the ARN expression of CD1d by using PCR. Three cells lines were analysed, HEK293 with CD1d as positive control, SW620 that do not activate iNKTs cells with 6-PEG.sub.3-NH.sub.2-GalCer 2a as negative control and non-transfected HEK293 cells (see FIG. 6).

[0255] As shown in FIG. 6, HEK293-CD1d positive cells showed a strong CD1d signal whereas the SW620 cells proved to be CD1d-negative, consistent with their inability to function as antigen presenting cells of the 6-PEG.sub.3-NH.sub.2-GalCer compound 2a. Interestingly, for non-CD1d-HEK293 cells a significant signal was detected. This confirmed, contrary to the paradigm, that these cells express a low level of CD1d, not sufficient to induce iNKTs activation using KRN7000, but sufficient to be activate with 6-PEG.sub.3-NH.sub.2-GalCer 2a, 100 to 1000 fold more powerful.

[0256] These results clearly indicate that 6-PEG.sub.3-NH.sub.2-GalCer 2a unexpectedly turns out to be far more potent than KRN7000 although similarly dependent upon CD1d presentation.

[0257] Another interesting point is the ability of 6-PEG.sub.3-NH.sub.2-GalCer 2a to remain efficient even at a very low level of CD1d expression in tumor cells. This redefines the notion of non-CD1d cells in anticancer treatments, since these results showed that tumor cells previously considered as CD1d negative constitutively express a minor level of CD1d, sufficient to induce activation of iNKT cells when loaded with the very potent 6-PEG.sub.3-NHR-GalCer glycolipids.

[0258] This suggests that in order to initiate an immune response in the tumor environment, cancer cells might be used as presenting cells to activate iNKT cells in the presence of these potent glycolipids, bypassing the requirement for classical CD1d-antigen presenting cells (such as monocytes, macrophages or dendritic cells and lymphocyte B). To confirm this discovery, various other colon, lung, embryonic kidney, cervical cancer cells lines were screened by RT-PCR to define those that express small levels of CD1d and to confirm their ability to self-present the new potent iNKT agonist (see Table 6).

TABLE-US-00006 TABLE 6 IFN-γ secretion CD1d expression 10.sup.−7M Cell EGFR Cytometry PCR 6-PEG.sub.3- Line Origin expression detection detection KRN7000 NH.sub.2-GalCer SW480 Colon + Neg Neg NO NO SW620 Colon, + Neg Neg NO NO metastatic site: lymph node SW1116 Colon + Neg Low* Low High HCT116 Colon + Neg Low NO Low HT29 Colon + Neg Neg NO NO Meso13 Mesotheliome ? Neg? ? Low High Meso34 Mesotheliome ? Neg? ? Low High Meso225 Mesotheliome ? Neg? ? Low High HeLa Cervix + Neg Low Low High HEK293 Embryonic + Neg Low Low High kidney

[0259] Moreover, 6-PEG.sub.3-NH.sub.2-GalCer 2a is not only one of a strongest activator of iNKTs cells known for cytokine secretions, but that he is also able to induce a better iNKT cytotoxicity effect than KRN7000. iNKT were co-cultured with HeLa CD1 d transfected cells for 24 hours in presence of various concentration of glycolipids, then, mortality of target HeLa-CD1d cells was analysed by Flow Cytometry. As shown in FIG. 7, 6-PEG.sub.3-NH.sub.2-GalCer 2a appear at least, 10 fold more efficient than KRN7000 to induce cytotoxicity.

[0260] Comparative study was run to establish either a CD4 or CD8 orientation of the immune response can be induced using 6-PEG.sub.3-NH.sub.2-GalCer 2a vs KRN7000 (see FIG. 8). PBMC were cultured in presence of an high dose of KRN or 6-PEG.sub.3-NH.sub.2-GalCer 2a, after ten days, phenotype of iNKTs was analysed by Flow Cytometry. Very interestingly it seems that 6-PEG.sub.3-NH.sub.2-GalCer 2a induces a higher cytotoxic CD8+ response from PBMC cells than KRN7000 which seems to not distinguish CD4+ to CD8+ cells stimulation. This result already established the immunocytotoxicity efficiency of 6-PEG.sub.3-NH.sub.2-GalCer 2a via iNKT stimulation. This ability was confirmed by an other cytotoxic assay (for 72 hours) on HeLa-CD1d 3D spheroid model (FIG. 9 A) an other more complex and more resistant model in which, one more time, 6-PEG.sub.3-NH.sub.2-GalCer 2a appear more efficient than KRN7000 to induce cell death (preliminary data, see FIG. 9B).

Example 4: Evaluation of 6-PEG.SUB.3.-NHR-GalCer 2b and 2c Analogues of 6-PEG.SUB.3.-NH.SUB.2.-GalCer 2a

[0261] In order to better understand the surprising iNKT stimulation potency of 6-PEG.sub.3-NH.sub.2-GalCer 2a and the influence of end terminal amino function of the 6″-O-PEG substituted KRN7000 analogues, it has been envisioned to block the NH.sub.2 group by an acetate 2b and a benzocarbonyl 2c groups. It was expected to establish if a suspected stabilisation of the ternary CD1d-Tumor cells/6-PEG.sub.3-NH.sub.2-GalCer/TCR-iNKT complex would be explained by the presence of a free amine at the end of the spacer 14 atoms chain or if the PEG sequence allows some variations at the terminal group without a loss of performance.

[0262] Two new protected NHR analogues of 6-PEG.sub.3-NH.sub.2-GalCer 2a were thus prepared, wherein R is an acetate 6-PEG.sub.3-NHAc-GalCer 2b or a benzoyl group 6-PEG.sub.3-NHCOBn-GalCer 2c. In same end, two activated derivatives 6-Mal-PEG.sub.n-GalCer 3a and 3b (n=6 or 1, respectively) were also prepared with the aim to be engaged in a linkage with a biological carrier.

[0263] Both end terminal NHR protecting groups were though to avoid or alter the interaction that could occurred with CD1d-tumor cells and the TCR receptor in the presence of a free NH.sub.2 terminal group.

[0264] 6-PEG.sub.3-NHR-GalCer analogues of 6-PEG.sub.3-NH.sub.2-GalCer 2a were evaluated on transfected CD1d and non-CD1d Hela and HEK293 cells for secretion of INF-γ and IL13 stimulation (see FIGS. 10 and 11).

[0265] Depending on the model, 6-PEG.sub.3-NHAc-GalCer 2b appears almost 10 to 100 fold more potent to activate iNKTs on CD1d transfect tumor cells (Log IC.sub.50>−12 for IFN-γ on HeLa CD1d transfected cells), than PEG.sub.3-NH.sub.2-GalCer 2a (Log IC50=−10.89) its self and almost 10.sup.4 higher than KRN7000 (Log IC50=−8.8) whereas 6-PEG.sub.3-NHCOBn-GalCer 2c have a closer cytokine secretion potency than KRN7000.

[0266] These data are confirmed on non-CD1d-tumor cells that PEG.sub.3-NH.sub.2-GalCer 2a and 6-PEG.sub.3-NHAc-GalCer 2b remains almost more potent to activate iNKTs than KRN7000 with 2 at 3 log of difference (FIGS. 11 G and H), while 6-PEG.sub.3-NHCOBn-GalCer 2c have again the same profile effect than KRN7000.

[0267] These data indicates that the activity of PEG.sub.3-NHR-GalCer derivatives is sensitive to the nature of NHR end terminal group displaying iNKT stimulation potency at a nearly pM range on transfected tumor cells and more interestingly at a sub nM range on non-CD1d-tumor cells when R is an acetate.

[0268] A comparison study was also run to evaluate the variation induces by the introduction of PEG.sub.n-maleimide (n=1 and 6) fragment at the end terminal position of the 6-PEG.sub.3-NH.sub.2-GalCer 2a (see FIG. 12).

[0269] Data shows that 6-Mal-PEG.sub.6-GalCer 3a presenting a maleimide activated function slightly decrease the activation of iNKT cells almost ten fold higher than KRN7000).

[0270] Regardless, these data already make the novel 6-Mal-PEG.sub.6-GalCer 3a as candidate for an association with a therapeutic antibody or other biological carriers to induce cumulative biological anticancer cytokines release and cytotoxic effects of the carrier close to the tumor environment.

Example 5: Elaboration of Non-Enzymatic Cleavable GalCer/Cetuximab Complexes

[0271] The synthesis of the 6-Mal-PEG.sub.6-GalCer 3a from 6-PEG.sub.3-NH.sub.2-GalCer 2a was optimized. Intermediate 2a presents a long bait featuring a suitable reactive maleimide ending group (dashed line box) aimed to react in situ with Traut activated Cetuximab antibody to achieve the covalent linkage between the two partners (Schema 4).

[0272] It has also been successfully experimented the one pot process to access the GalCer/Cetuximab complex C1 from 6-PEG.sub.3-NH.sub.2-GalCer 2a without purification of the maleimide 6-Mal-PEG.sub.6-Galcer intermediate 3a. For this purpose 6-PEG.sub.3-NH.sub.2-GalCer 2a was dissolved in phosphate buffer (PB) with 10% DMSO at 20° C. and then directly reacted with Maleimide-PEG.sub.6-succinimide linker (length of 6 PEG units was chosen in accordance with our previous results) to give 6-Mal-PEG.sub.6-Galcer 3a which was directly incubated with activated Traut cetuximab partner leading to the GalCer/Cetuximab complex C1.

##STR00032##

[0273] (i) Coupling of Glycolipids to Cetuximab and Purification

[0274] a) Coupling Conditions

[0275] The following conditions have been retained for coupling to cetuximab.

[0276] First cetuximab was modified by adding TRAUT functions, with a ratio of 100 TRAUT molecules for 1 antibody. It allowed addition of at least 4 TRAUT functions to cetuximab, as determined by the Ellman reaction previously investigated (data not shown). The mixture was washed and then 1/1 equivalent of 6-Mal-PEG.sub.6-GalCer 3a and activated cetuximab were incubated to initiate the linkage.

TABLE-US-00007 TABLE 7 Coupling conditions CONDITION FRACTION B FRACTION A Cetuximab + Modified Cetuximab with 6-PEG.sub.3-NH.sub.2- TRAUT + 6- GalCer 2a Mal-PEG.sub.6-GalCer 3a TRAUT ratio No TRAUT 1/100 Glycolipids Ratio 1/1 1/1 Coupling process No Yes Washing process Yes Yes

[0277] The main difficulty encountered in these experiments is to ensure the elimination of the unreacted glycolipids in the medium after the coupling reaction with the antibody. Various methods were tested: Protein A, exclusion chromatography, electrophoresis, filtrations. Success was encountered when the resulting fractions were washed 3 time by filtration on VivaSpin15 column (MWCO: 50 Kda).

[0278] Two different experimental conditions were used to validate the purification process (see Table 7). [0279] Fraction B: Cetuximab is not modified by TRAUT activation and 6-PEG.sub.3-NH.sub.2-GalCer 2a, which does not have chemical ability to link to the cetuximab, was added. This condition was used as a control to follow the elimination of unbound glycolipid under washing conditions. Considering the high reactivity of 6-PEG.sub.3-NH.sub.2-GalCer 2a on iNKT stimulation, the presence of remaining derivatives, even in trace, would be detected by a significant cytokine release; [0280] Fraction A: use of 6-Mal-PEG.sub.6-GalCer 3a and TRAUT activated Cetuximab to provide covalent linkage of glycolipid with the antibody and targeted GalCer/Cetuximab complex C1.

[0281] b) Purification and Activation of iNKT by GalCer/Cetuximab Complex

[0282] Experiments were run on two series of transfected-CD1d Hela cells and non-CD1d-Hela Cells and 3 diluted samples of fractions A and B (10 μg/ml, 1 μg/ml and 0.1 μg/ml) were evaluated. In each series of Hela cells stimulation activity of the glycolipid alone was previously evaluated as reference following IFN-γ secretion.

[0283] Starting Concentration=

[0284] Maximum theoretical concentration of free glycolipid (unbounded) that can remain in the diluted fractions after coupling reaction if the washing process is inefficient.

[0285] Observed Concentration=

[0286] Estimated theoretical concentration of glycolipid that have to be remained in the diluted fractions after the washing process to induce the level of observed cytokine secretion (based on the reaction control with free glycolipid).

[0287] The experiments show that the washing process provides an elimination of the glycolipids (up to 99.9%).

[0288] Results with fraction B used as control experiment: [0289] As shown in FIG. 14 A, left panel, after purification, the fraction resulting from the mixture of Cetuximab+6-PEG.sub.3-NH.sub.2-GalCer 2a, which cannot link together, lead to a decrease of cytokine release following the 1/10.sup.th dilutions. In this experiment using high sensitive CD1d-Hela cells, the concentration of the remaining 6-PEG.sub.3-NH.sub.2-GalCer 2a in the diluted fraction samples (observed concentration) appears at least to be 3 log lower than the starting concentration added in the mixture (Starting concentration). At a dilution of 0.1 μg/ml its activity become negligible leading to a lack of iNKT stimulation. [0290] Same experiment using non-CD1d Hela cells (FIG. 14 B, left panel) leads to the lack of cytokine release even at 10 μg/ml, indicating that in physiological model, unbounded 6-PEG.sub.3-NH.sub.2-GalCer 2a can be considered totally removed from the fraction B by the washing process, or at least as trace (<10.sup.−10M limit for detection on non transfected HeLa cells).

[0291] Results with fraction A=linked Cetuximab-GalCer complex C1:

As shown, the complex GalCer/Cetuximab C1 formed after TRAUT activation of the antibody in the presence of 6-Mal-PEG.sub.6-GalCer 3a induces IFN-γ cytokine release by iNKT cells from all diluted fractions (10 μg/ml, 1 μg/ml and 0.1 μg/ml). These surprising results were observed from both high sensitive transfected-CD1d cells and also from non-CD1d-cells (FIG. 15 C, left panel and 15 D, left panel, respectively).
The main interesting think is that iNKT activation is maintained despite the dilutions of the fractions in both cases. These results seems to indicate the activation of a pool of CD1d that could be surexpressed in the presence of Cetuximab-GalCer complex C1.

[0292] It should be kept in mind that starting concentration is the maximum theoretical concentrations estimated in the experiments are calculated in the case of a washing failure with no elimination of the Glycoplipd residues. However, it was known from previous experiences that almost 99.9% of glycolipids excess (unbounded) are eliminated after the first washing step. Thus, The theoretical concentrations estimated as reference after 3 washing steps are consequently largely overestimated and the “real” effect of the coupled Cetuximab-GalCer complex is thereby much more efficient in reality.

[0293] This latter experiment carried out from non-CD1d tumor cells indicates that coupled Cetuximab-GalCer complex C1 is able to induce a cytokine secretion at an upper level than the maximum release corresponding to a secretion induced by the 6-Mal-PEG.sub.3-GalCer 3a used in its free form at an upper concentration.

[0294] These observations have been confirmed form non-CD1d HEK293 cells and PBMC cells (Peripheral Blood Mononuclear Cells) (data not shown).

[0295] c) Mass Spectroscopy of GalCer/Cetuximab C1

[0296] GalCer/Cetuximab C1 was analysed by mass spectrometry (ESI) to show that at least one or two molecules of 6-Mal-PEG.sub.6-GalCer 3a analogue were linked to Cetuximab (see Table 8 below).

TABLE-US-00008 TABLE 8 heavy chain of complex C1 with 1 and 2 GalCer moieties linked Average molar mass calculated by deconvolution of the spectrum (Da) Retention time LC (min) Full Heavy Light Full Heavy Light antibody chain chain antibody chain chain Ab 153191 53005 23426 3.89 3.85-3.89 3.75 sample 155884 54220 23510 004 56160 23594 Ab 152481 52757 23425 3.84 3.81-3.87 3.7 sample 001 Δ 710 56160 23594 / / / 3403

[0297] Full antibody spectrum showed several major pics at 153191 Da et 155884 Da and deconvolution spectrum of pic at à 3.85 et 3.89 minutes of LC corresponds to the heavy chain of cetuximab linked to GalCer fragments (data not shown). The technique used for analysis was destructive for the antibody (leading to clear data only for linkage on heavy chain and unclear on light chain which is degraded), the maximum exact number of linked GalCer residues on the whole antibody cannot be not yet fully established.

[0298] (ii) Coupling of Glycolipids to Cetuximab and Purification (Compared to Coupling in the Absence of TRAUT)

[0299] a) Coupling Conditions

[0300] The following conditions have been retained for coupling to cetuximab.

[0301] First cetuximab was modified by adding TRAUT functions, with a ratio of 100 TRAUT molecules for 1 antibody. It allowed addition of at least 4 TRAUT functions to cetuximab, as determined by the Ellman reaction previously investigated (data not shown). The mixture was washed and then ½ equivalent of 6-Mal-PEG.sub.6-GalCer 3a and activated cetuximab were incubated to initiate the linkage.

TABLE-US-00009 TABLE 9 Coupling conditions CONDITION FRACTION B FRACTION A Cetuximab + Modified Cetuximab with 6-Mal-PEG.sub.6- TRAUT + 6- GalCer 3a Mal-PEG.sub.6-GalCer 3a TRAUT ratio No TRAUT 1/100 Glycolipids Ratio 1/2 1/2 Coupling process Yes Yes Washing process Yes Yes

[0302] The main difficulty encountered in these experiments is to ensure the elimination of the unreacted glycolipids in the medium after the coupling reaction with the antibody. Various methods were tested: Protein A, exclusion chromatography, electrophoresis, filtrations. Success was encountered when the resulting fractions were washed 3 time by filtration on VivaSpin15 column (MWCO: 50 Kda) follow by purification on Superdex 200 10/300 GL column for size exclusion chromatography.

[0303] Two different experimental conditions were used to validate the purification process (see Table 9). [0304] Fraction B: Cetuximab is not modified by TRAUT activation and 6-Mal-PEG.sub.6-GalCer 3a. This condition was used as a control to follow the elimination of unbound glycolipid under washing conditions. Considering the high reactivity of 6-Mal-PEG.sub.6-GalCer 3a on iNKT stimulation, the presence of remaining derivatives, even in trace, would be detected by a significant cytokine release; [0305] Fraction A: use of 6-Mal-PEG.sub.6-GalCer 3a and TRAUT activated Cetuximab to provide covalent linkage of glycolipid with the antibody and targeted GalCer/Cetuximab complex C1.

[0306] b) Mass Spectroscopy of GalCer/Cetuximab C1

[0307] GalCer/Cetuximab C1 was analysed by mass spectrometry (ESI) to show two molecules of 6-Mal-PEG.sub.6-GalCer 3a analogue were linked to Cetuximab (see Table 10 below).

TABLE-US-00010 TABLE 10 heavy chain with 1 and 2 GalCer moieties linked Cetuximab Alone Fraction B Fraction A Molecular Full antibody 152 583 152 533 156 065 Weight (Da) Heavy chain 52 904 52 904 53 232 + 55 541 Light chain 23 426 23 427 23 604 Delta from Full antibody / −50 3 482 Cetuximab Heavy chain / 0 328 + 2 637 Alone (Da) Light chain / 1 178 N Coupling Full antibody / 0 2, 2 Heavy chain / 0 0, 2 + 1, 7 Light chain / 0 0, 1

[0308] Full antibody spectrum showed several major pics at 152 583 Da alone et 156 056 Da when coupling. This results indicate on average two molecules of 6-Mal-PEG.sub.6-GalCer 3a (MW: 1 562 Da) are linked to the antibody. More precise study of heavy and light chains indicate that coupling mainly takes place on the heavy chain.

[0309] c) Activation of iNKT by GalCer/Cetuximab Complex

[0310] Experiments were run on non-CD1d-Hela Cells and 3 diluted samples of fractions A and B (10 μg/ml, 1 μg/ml and 0.1 μg/ml) were evaluated. Mass spectrometry analysis indicate that two molecules of 6-Mal-PEG.sub.6-GalCer 3a were linked per antibody, so when 10 μg/ml of Fraction A was used, it corresponds approximately to a concentration of 10.sup.−7M of equivalent 6-Mal-PEG.sub.6-GalCer 3a. In each series of Hela cells stimulation activity of the glycolipid alone was previously evaluated as reference following IFN-γ secretion.

[0311] Results with Fraction B Used as Control Experiment:

As shown in FIG. 16, after purification, the fraction resulting from the mixture of Cetuximab+6-Mal-PEG.sub.6-GalCer 3a, where no linkage was observed by mass spectrometry, no cytokine secretion was observed, indicating that washing process provide an elimination of unbounded glycolipids.

[0312] Results with Fraction A=Linked Cetuximab-GalCer Complex C1:

As shown, the complex GalCer/Cetuximab C1 formed after TRAUT activation of the antibody in the presence of 6-Mal-PEG.sub.6-GalCer 3a induces IFN-γ cytokine release by iNKT cells from all diluted fractions (10 μg/ml, 1 μg/ml and 0.1 μg/ml). These surprising results were observed on non-CD1d-cells.
These results seems to indicate that Cetuximab-GalCer complex C1 is able to vectorize and release 6-Mal-PEG.sub.6-GalCer 3a into the tumour cells allowing its presentation on the weak CD1d expression observed in HeLa cells leading to activation of iNKT cells.

[0313] (iv) Cetuximab in GalCer/Cetuximab Complexes is Still Able to Recognize EGFR

[0314] It was then assessed if after coupling with 6-Mal-PEG.sub.6-Galcer 3a, cetuximab was not altered and was still able to recognize EGFR. As shown in FIG. 13, cetuximab binding to cells expressing EGFR was not altered after coupling since there was no difference between native cetuximab (direct from the bottle) and the 3 conditions tested for coupling reactions. Results were confirmed on 3 different cells lines that also express EGFR (not shown).

[0315] (v) Cetuximab in GalCer/Cetuximab Complexes and Internalization

[0316] After EGFR recognition, Cetuximab is internalized, in clinical condition it allows to decrease surface expression of EGFR and reduces capacity of tumor cell to proliferate anarchically. Internalization of GalCer/Cetuximab complex C1 was followed by live microscopy for 21H. Complex C1 was labeled with a marker that only bright in red when it is internalized in acid endosome/lysosome. Most of HeLa cells were shown to have internalized complex C1 from 11H of co-incubation (data not shown). It was hypothesized that after internalization and support in lysosome, C1 complex release 6-Mal-PEG.sub.6-GalCer 3a under acid conditions, allowing loading on CD1d molecule and presentation to the surface of tumour cells, leading to the iNKT cells activation observed above in part iii).

[0317] (vi) ADCC Behaviour of GalCer/Cetuximab Complex

[0318] Another important function of cetuximab is its ability to induce ADCC. It was important to check the preservation of that behaviour from GalCer/Cetuximab complex C1. ADCC assays were performed on Hela cells and HCT116 cell line (FIGS. 14A and B respectively) because the latter proved to be the most responsive cell line among several cell lines that we tested in preliminary experiments and because HCT116 presents a Kras mutation. Target cells (HCT116 and HeLa cells) were coincubated for 1 hour with cetuximab alone (blue left panel) or Cetuximab-GalCer Complex C1 (red right panel). Then ADCC was initiated by addition of NK92-CD16+ cells (a conventionally cell line used for ADCC assay) follow by an incubation for 24 hours. Mortality of target cells was analysed by Flow Cytometry.

[0319] ADCC potency of Cetuximab is preserved in all conditions on both models showing that linkage of glycolipid fragment does not alter the behaviour of the antibody with respect to EGFR recognition.

[0320] (vii) Conclusion

[0321] GalCer/Cetuximab complex C1 with a covalent linkage are able to activate hiNKT cells to release cytokines using CD1d-tumor cells as antigen presenting partner. The important information is that carcinoma cells, previously considered as CD1d negative, can act as self-presenting cells through a very low pool of previously undetected CD1d, this behaviour being related to the exceptional potency of Cetuximab-GalCer complex C1. Additionally, GalCer derivative remains active despite its linkage to the antibody through a relatively short PEG spacer.

[0322] One hypothesis that can be made taking into account our data, is that Cetuximab achieves the vectorization of Cetuximab-GalCer complex C1 and the concentration of the glycolipids into the EGFR-tumor cells, probably through an internalization process. A subsequent release of glycolipids from the complex could then occur, maybe under acidic intracellular condition or antibody degradation, to allow its loading on an unknown internal pool of CD1d that can be consequently expressed at the surface of the tumor cell membrane. This CD1d-turnover appears to be able to mobilize iNKT cells and to induce cytokine stimulation restoring the immune response close to the tumor environment.

[0323] In that sense, Cetuximab-GalCer complex C1 can be regarded as potent drug candidate for immunotherapy providing enhance cytotoxic effect combining ADCC property of Cetuximab, or at least its cytotoxic effect, and a strong immunostimulation activity from iNKT.

CONCLUSION

[0324] The present application discloses a new potent immunostimulator 6-PEG.sub.3-NH.sub.2-GalCer 2a expressing an hiNKT stimulation potency nearly 1000 fold higher than that of parent KRN7000.

[0325] More interestingly, corresponding GalCer/Cetuximab complex C1 (see FIG. 15) keeps the ability to induce cytokines release by activating iNKT cells.

[0326] These data will lead to redefine the notion of CD1d negative cells since some tumoral cells, previously considered as CD1d negative (HeLa and HEK293), constitutively express CD1d at a sufficient level, albeit nearly undetectable, to effectively activate iNKT cells. iNKTs, themselves at low level in the experiment physiological level, when loaded with 6-PEG.sub.3-NH.sub.2-GalCer glycolipid 2a, induce cytokine release but as well when loaded with GalCer/Cetuximab complex C1. This suggests that the antibody can be already regarded as a good vehicle to carry the GalCer immunostimulator within the tumoral environment in order to initiate iNKT stimulation. This process can bypass the requirement for classical CD1d presenting cells (such as monocytes, macrophages or dendritic cells), cancer cells being able to self-present even when the glycolipid agonist is linked to the antibody.