Medicament for use in a method of inducing or extending a cellular cytotoxic immune response

Abstract

The present invention relates to a medicament for use in a method of extending a cellular cytotoxic immune response against an antigen-comprising protein, the method comprising the step of: i) administering to a patient having T cells activated against an antigen a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and an adjuvant which supports a Th-1-mediated response, wherein the peptide is derived from the antigen-comprising protein, thereby re-activating the activated T cell, wherein the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell of step i) is administered in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.

Claims

1. A method of inducing and amplifying antigen-specific CD8.sup.+ T cells in a patient, the method comprising the steps of (A) and (B), wherein step (A) is selected from (A)(i), (A)(ii), or (A)(iii), and step (A) is followed by step (B): (A) (i) (1) administering to said patient a delivery system comprising: (a) a molecule that binds to a receptor on the surface of an antigen-presenting cell, and (b) an antigen-comprising protein bound to the molecule of (a) and wherein upon binding of the molecule of (a) to the receptor, the protein of (b) is internalized and processed in the antigen-presenting cell and the antigen comprised in the protein is presented on the surface of the antigen-presenting cell, thereby activating CD8.sup.+ T cells in the patient, and (2) administering a further adjuvant which supports a Th-1-mediated response, or (ii) (1) activating one or more CD8.sup.+ T cells obtained from said patient in vitro with an antigen-comprising protein or a fragment thereof comprising the antigen, (2) re-transferring one or more CD8.sup.+ T cells obtained in step (1) to said patient, and (3) optionally administering a further adjuvant which supports a Th-1-mediated response, or (iii) (1) administering to said patient an antigen-comprising protein or a fragment thereof comprising the antigen, or a nucleic acid comprising a nucleic acid sequence encoding an antigen-comprising protein or a fragment thereof comprising the antigen, which is capable of expressing the antigen-comprising protein or a fragment thereof comprising the antigen in said patient, thereby activating CD8.sup.+ T cells, and (2) administering a further adjuvant which supports a Th-1-mediated response, and (B) administering to the patient a reactivator comprising: (1) primary peptide-loaded major histocompatibility complex class I (MHC I) presenting cells, wherein the MHC I presenting cells do not comprise monocyte-derived dendritic cells, and wherein the peptide is derived from the antigen-comprising protein of step (A), and (2) an adjuvant which supports a Th-1-mediated response, thereby re-activating the activated CD8.sup.+ T cells of step (A), and wherein the reactivator is administered in a time frame of from 5 to 12 days after the CD8+ T cells were activated against the antigen in step (A), and is administered only once to said patient during the time frame, and is administered intravenously to said patient, and wherein the MHC I presenting cells are obtained from said patient and peptide-loaded in vitro between 2 h and 3 days prior to administration to the patient.

2. The method of claim 1, wherein said reactivator is administered in a time frame of from 5 to 9 days after the T cells were activated against the antigen.

3. The method of claim 1, wherein the delivery system (A)(i) is administered.

4. The method of claim 1, wherein said molecule that binds to said receptor on the surface of said antigen-presenting cell of (A)(i) is a ligand to the receptor or an antibody or antibody fragment against the receptor.

5. The method of claim 1, wherein said method further comprises administering complexed interleukin 2 (IL-2cx), complexed interleukin 15 (IL-15cx), complexed interleukin 4 (IL-4cx), complexed interleukin 7 (IL-7cx), or a IL-2 mutein.

6. The method of claim 3, wherein said MHC I presenting cells are lymphocytes.

7. The method of claim 6, wherein said lymphocytes are B cells or T cells.

8. The method of claim 1, wherein the receptor on the surface of said antigen-presenting cell of (A)(i) is a receptor on the surface of cross-presenting dendritic cells.

9. The method of claim 8, wherein said receptor on the surface of said cross-presenting dendritic cells is chemokine (C motif) receptor 1 (XCR1), nectin-like molecule 2, CD205, or a c-type lectin (CLEC).

10. The method of claim 5, wherein administering of said complexed interleukin 2 (IL-2cx), complexed interleukin 15 (IL-15cx), complexed interleukin 4 (IL-4cx), complexed interleukin 7 (IL-7cx), or a IL-2 mutein is performed after administering to said patient said MHC I presenting cells coll.

11. The method of claim 1, wherein the antigen of said antigen-comprising protein is an immunogen, a pathogen-derived antigen, or a tumor antigen.

12. The method of claim 9, wherein said receptor on the surface of a dendritic cell is a c-type lectin (CLEC).

13. The method of claim 12, wherein said c-type lectin (CLEC) is CLEC9A.

14. The method of claim 9, wherein said receptor is XCR1.

15. The method of claim 4, wherein said receptor is XCR1 and wherein said molecule is an anti-XCR1 antibody or antigen-binding fragment thereof or chemokine (C motif) ligand 1 (XCL1) or a functionally active variant thereof.

16. The method of claim 1, wherein the MHC I presenting cells comprise at least 110.sup.6 cells.

17. The method of claim 16, wherein the MHC I presenting cells comprise at least 810.sup.7 cells.

Description

FIGURES

(1) FIG. 1 shows induction of cytotoxic activity after targeting of antigen into XCR1.sup.+ DC

(2) FIG. 2 shows protection from infection or from seeding of cancer cells by the induced cytotoxic activity

(3) FIG. 3 shows amplification of CD8.sup.+ T cell cytotoxicity obtained by injection of syngeneic lymphocytes loaded with antigenic peptide SIINFEKL (SEQ ID NO: 11)

(4) FIG. 4 shows highly synergistic amplification of cytotoxic CD8.sup.+ T cells by co-application of peptide-loaded cells and complexed IL-2

(5) FIG. 5 shows synergistic effects of antigen targeting and co-application of peptide-loaded cells and complexed IL-2 in the treatment of established tumors

(6) FIG. 6 shows that the low frequency of cytotoxic CD8.sup.+ T cells after priming using various modes of vaccination can be strongly amplified with ADAS. (A) C57BL/6 animals were injected on day 0 with 200 g of soluble, non-targeted ovalbumin (OVA), or with 5 g of mAb MARX10-OVA, DEC-205-OVA, 33D1-OVA, MOPC21-OVA; in all cases, 10 g poly I:C were co-injected as adjuvant. On day 5, blood samples were taken and the frequencies of SIINFEKL-specific CD8.sup.+ T cells determined by flow cytometry using a specific tetramer. (B) On day 5, the immune response to the OVA-derived peptide SIINFEKL was amplified with the ADAS procedure (injection of 1010.sup.6 syngeneic splenocytes loaded with SIINFEKL together with 50 g poly I:C as adjuvant). On day 10, the animals were sacrificed and the frequencies of SIINFEKL-specific CD8.sup.+ T cells were determined in the spleen using the tetramer. (C) C57BL/6 Batf3-KO animals were immunized as described in (A) and ADAS-treated, as described in (B), and the frequencies of SIINFEKL-specific CD8.sup.+ T cells determined in the spleen on day 10.

(7) FIG. 7 (A) R9-SIINFEKL polypeptide does not externally bind to the MHC-I groove: C57BL/6 splenocytes were incubated at a density of 510.sup.6 cells/ml with SIINFEKL peptide at 1 M for 2 h at 37 C., 5% CO.sub.2 in complete RPMI1640 culture medium, or at a density of 210.sup.6 cells/ml with R9-SIINFEKL polypeptide at 1 M for 4 h. Cells were washed twice, and stained with anti-SIINFEKL-H2K.sup.b mAb (clone 25-D1.16) to determine the efficiency of peptide loading to the MHC-I groove. In addition, cells were co-stained with various lineage markers to identify different splenic cell populations and analyzed by flow cytometry. (B) R9-SIINFEKL can be used to transport antigen into the cytoplasmic compartment of primary cells and thus allow loading of MHC-I with derived peptides: C57BL/6 splenocytes were incubated at a density of 210.sup.6 cells with R9-SIINFEKL polypeptide at various concentrations (1-M) for 7 h as above. Thereafter, the cells were washed twice, stained with mAb 25-D1.16 to determine the efficiency of peptide loading to the MHC-I groove, co-stained with lineage markers, and analyzed by flow cytometry. With 30 M polypeptide a significant cell death was observed (not shown). (C) R9-SIINFEKL loaded primary cells can be used for ADAS: C57BL/6 mice were injected i.v. on day 0 with 1010.sup.6 R9-SIINFEKL loaded (5 M for 7 h) splenocytes, or for comparison with 1010.sup.6 SIINFEKL-loaded (2 M for 2 h) splenocytes, or with 2 g MARX10-OVA (all with 10 g poly I:C) for priming, and the frequency and cytotoxic potential (granzyme B, KLRG1, not shown) of CD8.sup.+ T cells were determined on day 5. Alternatively, C57BL/6 mice were primed with 2 g MARX10-OVA and 10 g poly I:C, and subjected to ADAS on day 5 by injection i.v. with SIINFEKL-loaded (2 M for 2 h) splenocytes and 50 g poly I:C (positive control). In parallel, C57BL/6 mice were primed i.v. with 2 g MARX10-OVA, or 2 g 33D1-OVA, or 2 g 1D3-OVA, or 200 g untargeted OVA (all together with 10 g poly I:C) on day 0, and subjected to ADAS by injection i.v. of R9-SIINFEKL-loaded (5 M for 7 h) syngeneic splenocytes and 50 g poly I:C. The ADAS-induced expansion of SIINFEKL-specific CD8.sup.+ T cells was determined on day 10 by flow cytometry using a specific tetramer. All CD8.sup.+ T cells exhibited markers indicative of cytotoxicity (granzyme B, KLRG1, not shown).

(8) FIG. 8 Administration of antigen can be dissociated from application of adjuvant in the priming step: (A) C57BL/6 mice were injected i.v. on day 0 with 2 g MARX10-OVA together with 10 g poly I:C as adjuvant, mixed in one solution. Alternatively, mice were injected on day 1 with 10 g of poly I:C and on day 0 with 2 g MARX10-OVA. Alternatively, mice were injected on day 0 with 2 g MARX10-OVA and on day 1 with 10 g poly I:C. In each experimental group, blood samples were taken on day 5 and the frequencies of SIINFEKL-specific CD8.sup.+ T cells determined by flow cytometry using a specific tetramer. (B) The mice described in (A) were subjected to the ADAS amplification procedure (i.v. injection of splenocytes externally loaded with SIINFEKL together with 50 g of poly I:C) and the frequencies of SIINFEKL-specific CD8.sup.+ T cells in the spleen were determined by flow cytometry. Administration of antigen can be dissociated from application of adjuvant in the ADAS procedure.

(9) FIG. 9 Highly synergistic amplification of cytotoxic CD8.sup.+ T cells by combining ADAS and administration of complexed IL-15: C57BL/6 animals were primed on day 0 with 2 g MARX10-OVA and 10 g poly I:C and analyzed for the total number of SIINFEKL-specific CD8.sup.+ T cells in the spleen on day 5 (prime) using a specific tetramer and flow cytometry. Some of the primed animals were on day 5 subjected to ADAS only (injection of 10106 SIINFEKL-loaded splenocytes together with 50 g poly I:C), some received ADAS and were injected i.p. with IL-2cx (as described in Examples 4 and 5) on days 6, 7, and 8, other mice received ADAS on day 5 and were injected i.p. with complexed IL-15 (IL-15cx) on day 6, 7, and 8. In all ADAS-treated groups, the total number of SIINFEKL-specific cytotoxic CD8.sup.+ T cells was determined on day 9. The dose of IL-15cx for 1 mouse was generated by incubating 2 g IL-15 (Peprotech #210-15) with 9.3 g sIL-15R-Fc (R&D, #551-MR-100) at 37 C. for 20 min, PBS was added to 500 l and the solution injected i.p.

(10) FIG. 10 ADAS can amplify resting memory CD8.sup.+ T cells in an antigen-specific manner: C57BL/6 mice were on day 1 adoptively transferred with 2,000 or 10,000 of OT-I T cells and primed on day 0 with MARX10-OVA and 10 g poly I:C. ADAS (injection of 1010.sup.6 SIINFEKL-loaded splenocytes together with 50 g poly I:C) was performed in all animals on day 5, and animals were analyzed for the frequency of (A) OT-I T cells and (B) endogenous, Thy 1.1-negative SIIFEKL-specific CD8.sup.+ T cells on days and 40. Another group of mice was subjected to ADAS again on day 69, and analyzed for the frequency of (A) OT-I T cells and (B) endogenous SIINFEKL-specific CD8.sup.+ T cells on day 74.

(11) FIG. 11 In vivo amplification of in vitro-activated antigen-specific CD8.sup.+ T cells by ADAS: Splenocytes were isolated from OT-I mice and cultured in complete medium with SIINFEKL peptide at 1.4 nM for 3 days. Thereafter, cells were washed with PBS and 510.sup.5 OT-I T cells (as determined by flow cytometry) were adoptively transferred into nave C57BL/6 mice and the animals were treated by ADAS at various time points after transfer. Shown is the effect on the frequency of transferred CD8.sup.+ T cells when ADAS was performed on day 5 after adoptive transfer. The proportion of adoptively transferred CD8.sup.+ T cells of all CD8.sup.+ T cells was determined in the blood on day 9 using flow cytometry. The frequencies in the blood correspond in these experiments to the frequencies in the spleen of the animals.

EXAMPLES

Example 1

Induction of Cytotoxic Activity after Targeting of Antigen into XCR1.SUP.+ .DC (FIG. 1)

(12) The model antigen OVA was recombinantly fused to the XCR1-specific mAb MARX10 (Bachem et al. 2012) or recombinantly fused to the chemokine ligand XCL1, which specifically binds to the XCR1-receptor (Hartung et al. 2015). When MARX10-OVA or XCL1-OVA were injected i.v. into nave C57BL/6 mice at low levels, the antigen was targeted into XCR1+DC. If this antigen priming occurred in the presence of an adjuvant (3 g LPS, CpG, or 10 g poly I:C), substantial cytotoxic activity was induced (shown are data with poly I:C as adjuvant). This cytotoxic activity was tested by injecting the primed animals on day 6 i.v. with target cells (splenic lymphocytes), which were previously loaded with SIINFEKL (SEQ ID NO: 11) in vitro, an OVA-derived peptide. The target cells were labeled after loading with the fluorophore CFSE to a high degree, while non-loaded control splenic lymphocytes were labeled with CFSE to a low degree. As shown in FIG. 1A, priming animals with OVA+adjuvant resulted in cytotoxic activity which eliminated almost all SIINFEKL-loaded target cells. FIG. 1B shows a dose-response curve obtained with various amounts of antigen targeted to XCR1.sup.+ DC. Identical results were obtained after using MARX10-SIINFEKL or XCL1-SIINFEKL for targeting of the immunogenic peptide into XCR1+DC.

Example 2

Protection from Infection or from Seeding of Cancer Cells by the Induced Cytotoxic Activity (FIG. 2)

(13) C57BL/6 mice were primed with MARX10-OVA (containing 2 g of OVA) and adjuvant (10 g of poly I:C) or were left untreated. Five days later, all mice were infected with 110.sup.6 CFU (=5LD50) of a L. monocytogenes strain, into which the peptide sequence SIINFEKL (SEQ ID NO: 11) has been engineered recombinantly (Foulds et al., 2002, J. Immunol. 168, 1528-1532). While all untreated mice died within 3 to 4 days, the induced level of cytotoxicity by antigenic priming fully protected all animals from disease (FIG. 2A).

(14) C57BL/6 mice were primed with MARX10-OVA or XCL1-OVA (each containing 0.16 g of OVA) and adjuvant (3 g LPS) i.v., control animals were injected with PBS. Seven days later, all animals were injected with 510.sup.5 EG.7 cells, an aggressive syngeneic tumor line engineered to express OVA (Moore et al. 1988).

(15) While PBS-treated animals all exhibited strong tumor growth after 14 days, none of the immunized animals had any tumor tissue at the site of injection or elsewhere, indicating that the induced level of cytotoxicity protected the animals from tumor seeding (FIG. 2B).

Example 3

Amplification of CD8.SUP.+ T Cell Cytotoxicity Obtained by Injection of Syngeneic Lymphocytes Loaded with Antigenic Peptide SIINFEKL (SEQ ID NO: 11) (FIG. 3)

(16) C57BL/6 mice were primed with MARX10-OVA (containing 2 g OVA) and an adjuvant (poly I, CpG, or LPS, shown are the data with 10 g poly I:C) on days 20, 15, 10, 7, 5, or 3. On day 0, the primed animals were injected i.v. with 1010.sup.6 splenocytes (FIG. 3A) which were loaded before injection with SIINFEKL (SEQ ID NO: 11) in vitro (Antigen-Dependent Amplification System, ADAS). Together with the peptide-loaded cells an adjuvant was injected (various amounts of LPS or poly I:C), shown are data with 50 g of poly I:C. On day 10 animals were sacrificed and the total number of SIINFEKL-specific CD8.sup.+ T cells was determined in the spleen using SIINFEKL-specific tetramers and flow cytometry (FIG. 3A). The results demonstrated that the system used for amplification of antigen-specific CD8.sup.+ T cells was effective in a narrow timeframe between days 5 and 9 after the initial antigenic stimulation (FIG. 3A).

(17) C57BL/6 mice were primed with MARX10-OVA (containing 2 g OVA) and an adjuvant (10 g poly I:C). Five days later, 1010.sup.6 splenic lymphocytes, or purified T cells, dendritic cells, or B cells which were loaded before injection with SIINFEKL (SEQ ID NO: 11) in vitro were injected i.v. into the primed animals. Together with the peptide-loaded cells an adjuvant was injected (50 g poly I:C). On day 10 after priming with MARX10-OVA, the animals were sacrificed and the total number of SIINFEKL-specific CD8.sup.+ T cells was determined in the spleen using SIINFEKL-specific tetramers and flow cytometry. The results showed that the amplification of the cytotoxic CD8.sup.+ T cell response can be achieved with various cell populations expressing MHC-I on the cell surface. The amplified cells expressed high levels of effector molecules (TNF-, IFN-, granzyme B).

Example 4

Highly Synergistic Amplification of Cytotoxic CD8.SUP.+ T Cells by Co-Application of Peptide-Loaded Cells and Complexed IL-2 (FIG. 4)

(18) C57BL/6 mice were primed with MARX10-OVA (containing 2 g OVA) and an adjuvant (10 g poly I:C). One group of primed animals was injected on days 1, 2, and 3 after priming with complexed IL-2 (IL-2cx) obtained by incubating IL-2 (2 g) and the anti-IL-2 mAb JES6-5H4 (10 g, Sander et al. 1993) overnight at 4 C., and sacrificed on day 6 (Primed+IL-2cx). Another group of mice was injected on day with SIINFEKL-loaded splenocytes (1010.sup.6) and adjuvant (50 g poly I:C) and sacrificed on day 10 (Primed+ADAS). Another group of mice was injected on day with SIINFEKL-loaded splenocytes (1010.sup.6) and adjuvant (50 g poly I:C), and with IL-2cx on days 6, 7, 8, and 9, and sacrificed on day 10 (Primed+ADAS+IL-2cx). At the end of each experiment, the total number of SIINFEKL-specific CD8.sup.+ T cells was determined in the spleen using SIINFEKL-specific tetramers and flow cytometry. The experiment determined a highly synergistic effect of ADAS and IL-2cx in the amplification of antigen-specific cytotoxic CD8.sup.+ T cells.

Example 5

Synergistic Effects of Antigen Targeting and Co-Application of Peptide-Loaded Cells and Complexed IL-2 in the Treatment of Established Tumors (FIG. 5)

(19) C57BL/6 mice were injected s.c. with 510.sup.5 EG.7 cells, an aggressive syngeneic tumor line engineered to express OVA (Moore et al. 1988). On day 6 after tumor injection mice were primed with MARX10-OVA (containing 2 g OVA) and adjuvant (10 g poly I:C), one group was left untreated. Six days after priming (day 11), one group of mice was injected with SIINFEKL-loaded splenocytes (1010.sup.6) and adjuvant (50 g poly I:C) (Primed+ADAS). Another primed group of mice was injected on days 12, 13, 14, 15, 16, 17, 18, 19, 21, and 23 with IL-2cx only (Primed+IL-2cx). Another primed group of mice was injected on day 11 with SIINFEKL-loaded splenocytes (1010.sup.6) and adjuvant (50 g poly I:C), and with IL-2cx on days 12, 13, 14, 15, 16, 17, 18, 19, 21, and 23 (Primed+ADAS+IL-2cx). On day 25 all mice were sacrificed (some control animals had to be sacrificed earlier, because the tumor became >1 cm in diameter). All data represent the mean average size of tumors (in mm.sup.2) in each treatment group (n=6). The results demonstrate that priming of the tumor-injected mice alone with targeted OVA was not effective. In contrast, either IL-2cx or ADAS alone were effective by strongly reducing the tumor mass for approximately 7-10 days in primed mice, but could not prevent the outgrowth of the tumor thereafter. When a combination of ADAS and IL-2cx was applied to primed animals, the tumor mass was strongly reduced and the tumor was fully controlled until the end of the experiment (some mice had fully rejected the tumor tissue).

Example 6

Various Modes of Immunization, Result in a Low-Frequency of Primed Antigen-Specific CD8+ T Cells. These can be Generally Strongly Expanded and Differentiated to Killer CD8+ T Cells with the ADAS Procedure

(20) MAb MARX10 (Bachem et al., Front Immunol 2012, EP2641915A1) recognizes XCR1, the lineage marker for XCR1.sup.+ DC, Mab DEC-205 (NLDC-145, Kraal et al., 1986, obtained from Biolegend) recognizes the CD205 molecule expressed on murine XCR1.sup.+ DC, mAb 33D1 (Nussenzweig et al., 1982, obtained from ATCC) recognizes the DCIR2 molecule on SIRP.sup.a DC, mAb 1D3 recognizes CD19 on B cells (Krop et al., 1996, obtained from ATCC), mAb MOPC-21 (Potter et al., J Natl Cancer Inst 1961, obtained from Biolegend) does not recognize any molecule in the mouse and is therefore used as an IgG1 isotype control. XCL1 is the chemokine ligand for XCR1 and can be used for targeting of antigen to XCR1+DC in the mouse and in the human (Hartung et al., 2015).

(21) The antigen-binding regions of the heavy and light chains of mAb DEC-205, 33D1, 1D3, MOPC-21 were identified by mass spectrometry and grafted onto the backbone of mAb DEC-205 by standard recombinant techniques, as described previously for mAb MARX10 (Hartung et al., 2015). This backbone has been modified previously to minimize binding to Fc-receptors. All constructs were then modified in such a way as to accommodate OVA as a C-terminal fusion protein to each of the antibodies, as described previously for mAb MARX10 (Hartung et al., 2015). XCL1-OVA was generated as described previously (Hartung et al., 2015). C57BL/6 animals were injected on day 0 with a high amount (200 g) of soluble, non-targeted OVA, or with 5 g of mAb MARX10-OVA, DEC-205-OVA, 33D1-OVA, MOPC21-OVA; in all cases, 10 g poly I:C were co-injected as an adjuvant. On day 5, blood samples were taken and the frequencies of OVA-specific CD8 T cells determined by flow cytometry using a H-2K.sup.b tetramer loaded with SIINFEKL and capable to bind to SIINFEKL-specific CD8.sup.+ T cells. As shown in FIG. 6A, all modes of antigen application, either non-targeted, or targeted to XCR1 DC, to SIRP.sup.+ DC, or to B cells induced an initial expansion of SIINFEKL-specific CD8.sup.+ T cells (priming), resulting in a frequency of approximately 2-3% of all CD8.sup.+ T cells in the blood. On day 5, the immune response to the OVA-derived peptide SIINFEKL was amplified with the ADAS procedure (injection of 1010.sup.6 SIINFEKL-loaded syngeneic splenocytes together with 50 g poly I:C). On day 10, the animals were sacrificed and the frequency of SIINFEKL-specific CD8.sup.+ T cells determined in the spleen. In addition, several markers indicative of cytotoxicity were determined (KLRG1, perforin, granzyme B, data not shown). In all cases, the ADAS procedure amplified the initial frequency of SIINFEKL-specific CD8.sup.+ T cells approximately tenfold (FIG. 6B) and induced a phenotype indicative of cytotoxic T cells (not shown).

(22) In parallel, Batf3-KO animals on the C57BL/6 background (animals which lack XCR1.sup.+ DC, Hildner et al., 2008) were primed with the non-targeted or targeted OVA reagents, as above. On day 5 the Batf3-KO animals were also treated by the ADAS procedure, as above. On day 10, all animal were sacrificed and the frequencies of SIINFEKL-specific CD8.sup.+ T cells were determined in the spleen. While priming, followed by ADAS gave high frequencies of SIINFEKL-specific CD8.sup.+ T cells in all C57BL/6 animals (FIG. 6B), no substantial SIINFEKL-specific response could be observed after ADAS in any of the Batf3-KO animals (FIG. 6C).

(23) Several conclusions can be drawn from these experiments. Immunization using high levels of non-targeted protein, when applied together with a Th1 adjuvant, will result in an initial frequency of antigen-specific cytotoxic CD8.sup.+ T cells, as demonstrated by us and others previously (Hartung et al., 2015). Targeting of antigen into DC makes this primary immunization much more effective, since only low amounts of antigen are required to achieve the same effect (Hartung et al, 2015, Caminschi et al., 2012). Surprisingly, even targeting of antigen to B cells (in this case via CD19, a surface molecule specifically expressed on B cells) was similarly effective to targeting of antigen to DC. This effect can either be explained by transfer of antigen from B cells to DC (Allan et al., 2006), or by unspecific binding of the targeting anybody to Fc-receptors on DC. The latter effect is most likely responsible for the efficiency of priming when using MOPC-21, an isotype control antibody which does not recognize any antigen in the mouse immune system. These results are fully compatible with earlier results, in which targeting of antigen to marginal metallophilic macrophages via the surface receptor Siglec-1 also led to the generation of low-frequency cytotoxic CD8.sup.+ T cells, but not in Batf3-KO animals (Backer et al., 2010). Our experiments with Baf3-KO animals are in line with the general assumption that priming of nave T cells has to occur by DC. In particular, these experiments indicate that XCR1.sup.+ DC are required for this initial CD8.sup.+ T cell priming. Thus, targeting of antigen into XCR1.sup.+ DC promises to be the most effective way of targeting protein antigens, nucleic acids coding for antigens, or viral systems coding for antigens in order to achieve a good primary CD8.sup.+ T cell response (priming).

(24) Together, these results indicate that targeting of antigen using antibodies or targeting using receptor ligands (e.g. XCL1-OVA) to conventional DC, skin DC or other DC, such as monocyte-derived DC, pDC, to macrophages or other cells is far more efficient for induction of an initial cytotoxic response compared to the application of non-targeted antigen. It can be anticipated that all kinds of priming with protein antigens (for example, but no limited to, by employing liposomes, nanoparticles, and other systems as antigen carriers (Saroja et al., 2011) will give similar results, as long as the protein is applied in the context of a Th1 adjuvant. It is also well documented in the literature that a similarly low initial frequency of cytotoxic CD8.sup.+ T cells can be induced by non-protein immunization, such as, but not limited to, application or injection of DNA or DNA-based vaccines, or RNA or RNA-based vaccines, into the body using a variety of systems, either targeted or non-targeted (Saroja et al., 2011, Koup et al., 2011, Ulmer et al., 2012, Kramps et al., 2013) Similar priming of CD8+ T cells can be achieved with viral systems or attenuated viruses (Draper et al., 2010). Injection or application of RNA or DNA-based preparations or non-replicating viral systems or attenuated viruses does not necessarily require an additional Th1 adjuvant, since these agents are in many cases self-adjuvanted; i.e. these agents also represent Th1 adjuvants themselves.

(25) It is clear that in essentially all ways of vaccine delivery into the body, the initial CD8.sup.+ cytotoxic response will be relatively weak. Such a weak response will in many cases be insufficient to prevent infection, to treat an infection, or to eradicate cancerous tissue. Therefore, there is a need for a system which can strongly amplify the initial priming.

(26) In our experiments, we demonstrate that in all cases in which the initial CD8 cytotoxic response (priming) is insufficient to clear the infection or the tumor, it can be amplified using the ADAS procedure.

(27) Also immunization of patients with tumor tissue or dead cells will lead to a priming of the CD8.sup.+ T cell compartment (de Gruijl et al., 2008) and thus will make these cells susceptible to the ADAS procedure.

(28) Alternatively, primary CD8.sup.+ T cell activation can be achieved by isolating pre-existing tumor or pathogen-specific CD8.sup.+ T cells from a patient, activating and expanding them in vitro, and injecting them back into the patient. These adoptively transferred (re-injected) CD8.sup.+ T cells will be re-activated and further expanded and differentiated to cytotoxic T cells by the ADAS procedure.

(29) The ADAS amplification can be in all cases be further augmented synergistically by subsequent injections of complexed IL-2 (Examples 4, 5) or complexed IL-15 (see Example 9).

(30) In our experiments we used poly I:C as adjuvant. Similar results will be achieved with all type of Th1 adjuvants, such as, but not limited to, poly I:C, RIG-I agonists, and TLR8 agonists.

Example 7

Delivery of Antigen into the Cytoplasmic Compartment of Primary Cells for ADAS Leads to an Effective Loading of MHC-I with Antigen-Derived Peptides

(31) We have demonstrated that external loading of the MHC-I of a variety of primary cells (e.g. B cells, T cells, DC, splenocytes) with antigen-derived peptide (e.g. SIINFEKL) can be used for ADAS. We have anticipated that the same procedure will also work with a variety of methods introducing whole protein into primary cells or expressing a protein inside the cell, as described above.

(32) To further illustrate this concept we have used a stretch of 9 arginine residues as a cell-penetrating peptide (CPP, Milletti 2012, Bechara et al., 2013) to transport an antigen-comprising protein (polypeptide) into primary cells. The entire sequence of this 37 aa-polypeptide (termed R9-SIINFEKL) is RRRRRRRRRGYPYDVPDYALEQLESIINFEKLTEWTS (SEQ ID No. 13).

(33) R9-SIINFNEKL needs intracellular processing by the proteasome before a derived antigenic peptide (SIINFEKL) is presented on the cell surface in the context of MHC-I. Thus, the system can serve as a model for introducing a whole protein into a primary cell which is then processed by the proteasome into fragments, some of which are then presented on the cell surface in the context of MHC-I.

(34) This polypeptide cannot bind directly, externally, into the groove of the MHC-I. To prove this point, we incubated splenocytes of C57BL/6 mice with the polypeptide R9-SIINFEKL at 1 M for 4 h 37 C. in complete medium. In parallel, splenocytes were incubated with the peptide SIINFEKL, which can directly bind to MHC-I externally, at 1 M for only 2 h. After incubation, the cells were washed and analyzed by flow cytometry using mAb 25-D1.16, which recognizes SIINFEKL in the context of MHC-I H2K.sup.b (Porgador et al., 1997). While incubation of splenocytes with SIINFEKL gave a strong signal with macrophages, B cells, T cells, pDC, and DC, as expected (since SIINFEKL externally binds to MHC-I), no signal was obtained with the R9-SIINFEKL polypeptide (FIG. 7A). This experiment directly demonstrated that the R9-polypeptide cannot directly fit into the MHC-I groove and serve as antigen.

(35) In the next step, splenocytes were incubated for 7 h at increasing concentrations (1-30 M) of the R9-SIINFEKL polypeptide. After incubation and washing, the amount of SIINFEKL-loaded MHC-I was determined by staining with mAb 25-D1.16. As shown in FIG. 7B, all examined primary splenic cells exhibited a dose-dependent signal. Although not directly measured, it can be assumed that also the MHC-II was loaded in a similar manner. This experiment demonstrated that a polypeptide, once transported into a cytoplasmic compartment of a primary cell by a CPP, will be processed and presented in the context of the MHC-I (and MHC-II).

(36) From this experiment, it can be deduced that the same procedure will also work with a whole protein, which has also to be processed before being presented in the context of the MHC-I (and MHC-II). In fact, a similar loading of the MHC-I with SIINFEKL has been demonstrated with whole OVA, to which a stretch of 9 arginine residues has been fused N-terminally fused using standard recombinant techniques. In that experiment, OVA was correctly processed and presented in the context of MHC-I, as determined by staining with mAb 25-D1.16 (Mitsui et al. 2006). Thus, our experiment, which is in line with the literature, demonstrates that the introduction of non-processed polypeptides or proteins into the cell using a CPP will effectively load the MHC-I (and MHC-II) of this cell.

(37) From this experiment, one can deduce that any type of internal loading of primary cells with proteins and unprocessed peptides will lead to it an effective MHC-I presentation of peptides derived from this material.

(38) Such an internal loading of primary cells could similarly be achieved with other methods introducing peptides or proteins into cells, for example by, but not limited to, electroporation, or by introducing nucleic acids coding for a peptides and proteins by a variety of methods such as, but not limited to, transfection, lipofection, transduction, injection, ballistic injection, infection.

(39) We then tested the biological potency of primary cells internally loaded using R9-SIINFEKL. To this end, splenocytes from C57BL/6 mice were incubated with R9-SIINFEKL at 5 M for 7 hours, and washed. They were then injected on day 0 i.v. into nave C57BL/6 animals and were compared to i.v. injection of SIINFEKL-loaded splenocytes, or to the i.v. injection of MARX10-OVA. All preparation were applied together with poly I:C as adjuvant. As can be seen in FIG. 7C, all methods of antigenic delivery induced a low frequency of SIINFEKL-specific cytotoxic CD8 T cells, as assessed with a specific tetramer and by phenotypic analysis (expression of granzyme B, KLRG1), MARX10-OVA being the most efficient method of antigen delivery.

(40) In the next step, the R9-SIINFEKL loaded splenocytes were assessed for their capacity in the ADAS procedure. In this experiment, priming by MARX10-OVA and ADAS with SIINFEKL-loaded splenocytes served as the positive control and gave around 20% of SIINFEKL-specific cytotoxic CD8.sup.+ T cells (FIG. 7C). R9-SIINFEKL-loaded splenocytes were similarly effective in the ADAS procedure after priming with MARX10-OVA, 33D1-OVA, 1D3-OVA or high amounts of non-targeted OVA as SIIFEKL-loaded splenocytes (compare also FIG. 7A). This experiment demonstrated that any type of effective internal loading of primary cells with antigen will be efficient for ADAS. This loading could be with unprocessed polypeptides, whole proteins, or with nucleic acids coding for peptides or proteins, or using infectious agents, or viral or bacterial vector systems recombinantly modified to encode a desired polypeptide or protein.

Example 8

Application of Th1 Adjuvant can be Dissociated in Time from Application of Antigen Both in the Priming Step and in the ADAS Procedure

(41) It is currently generally assumed that antigen has to be applied together with an antigen in order to achieve immunization of the host (Cohn et al. 2014). Therefore, antigen is usually mixed with the adjuvant and applied together. It is currently generally assumed that the adjuvant should ideally be even physically linked to the antigen to achieve optimal results. Therefore, it was very surprising for us to realize that a dissociation of antigen administration from adjuvant delivery in the priming and ADAS procedures leads to good and even better immune responses.

(42) C57BL/6 mice were injected i.v. on day 0 with 2 g MARX10-OVA together with 10 g poly I:C as adjuvant, mixed in one solution. Alternatively, mice were injected on day 1 with 10 g of poly I:C and on day 0 with 2 g MARX10-OVA. Alternatively, mice were injected on day 0 with 2 g MARX10-OVA and on day 1 with 10 g poly I:C. In each experimental group, blood samples were taken on day 5 and the frequency of SIINFEKL-specific CD8.sup.+ T cells determined by flow cytometry. As can be seen in FIG. 8A, joint administration of antigen and adjuvant on day 0 gave a priming frequency of around 2% of all CD8.sup.+ T cells. In contrast, application of adjuvant one day before antigen appeared ineffective. Very surprisingly, administration of antigen on day 0 and application of adjuvant on day 1 was most effective, giving a frequency of around 4% of all CD8.sup.+ T cells on day 5.

(43) When all experimental groups were treated by the ADAS procedure (i.v. injection of splenocytes externally loaded with SIINFEKL and 50 g of poly I:C), the group with the joint application of antigen and adjuvant had around 15% SIINFEKL-specific cytotoxic CD8.sup.+ T cells. Clearly the best result was achieved with the group in which adjuvant was applied 1 day after antigen (around 30% SIINFEKL-specific CD8.sup.+ T cells). Interestingly, even in the group in which poly I:C was applied 1 day before antigen, there was a low level of SIINFEKL-specific T cells (around 5%), indicating that a certain priming has been achieved even in this group (which then became measurable through the amplification achieved in the ADAS procedure).

(44) These experiments clearly demonstrate that application of antigen and adjuvant can be dissociated in time in the priming procedure. In fact, the results indicate that application of adjuvant some time after administration of antigen is advantageous over a joint application of the components. Our results indicate that application of adjuvant even several days after application of antigen will be effective. The exact time frame cannot be determined in the human and has to be estimated also as 1-2, possibly up to 3 days.

Example 9

Highly Synergistic Amplification of Cytotoxic CD8.SUP.+ T Cells by Combining ADAS and Administration of Complexed IL-15

(45) We have demonstrated that injection of complexed IL-2 (IL-2cx=murine IL-2 complexed with an antibody blocking the binding of IL-2 to its high affinity receptor CD25) on days 1, 2, and 3 after priming with MARX10-OVA (2 g) and poly I:C (10 g) on day 0, did not substantially raise the number of primed CD8.sup.+ T cells on day 6. However, when the animals were primed with MARX10-OVA (2 g) and poly I:C (10 g) on day 0 and subjected to ADAS (injection of 1010.sup.6 splenocytes loaded with SIINFEKL together with 50 g poly I:C) on day 5, injection of IL-2cx on days 6, 7, 8, and 9 dramatically raised the number of SIINFEKL-specific CD8.sup.+ T cells in the spleen on day 10 (Example 4 and FIG. 4).

(46) Without ADAS, injection of IL-2cx on days 6, 7, 8, and 9 did not substantially raise the number of SIINFEKL-specific CD8.sup.+ T cells in the spleen (not shown), but was effective in reducing a tumor burden. Obviously, the ADAS procedure reactivates the primed CD8.sup.+ T cells in such a manner that they become highly sensitive to the action of IL-2cx and this results in a strong expansion of cytotoxic CD8.sup.+ T cells. However, the exact molecular mechanism leading to this highly heightened sensitivity to IL-2cx remains undetermined.

(47) C57BL/6 animals were primed on day 0 with 2 g MARX10-OVA and 10 g poly I:C and subjected to ADAS (injection of 1010.sup.6 SIINFEKL-loaded splenocytes together with 50 g poly I:C) on day 5. As described earlier, the ADAS procedure raised the level of SIINFEKL-specific CD8.sup.+ T cells from around 200,000 on day 5 to around 210.sup.6 cells after ADAS on day 10 (FIG. 9). ADAS-treated animals were then injected on days 6, 7, and 8 with complexed IL-15 (IL-15cx), or for comparison with IL-2cx (as described above). On day 9, the total number of SIINFEKL-specific CD8.sup.+ T cells was determined in the spleen using flow cytometry. As can be seen in FIG. 9, repeated injection of IL-15cx was similarly effective to the repeated injection of IL-2cx in strongly amplifying the number of SIINFEKL-specific CD8.sup.+ T cells after the ADAS procedure. IL-15cx could be injected, like IL-2cx, i.v., s.c., i.p., or into a tumor to achieve this effect. Injection of IL-15cx also increased the cellular levels of granzyme B and expression of KLRG1, indicating augmented cytotoxicity (not shown). The dose of IL-15cx for 1 mouse was generated by incubating 2 g IL 15 (Peprotech #210-15) with 9.3 g sIL-15R-Fc (R&D, #551-MR-100) at 37 C. for 20 min, PBS was added to 500 l and the solution injected i.p.

(48) Surprisingly, this experiment thus revealed that IL-15cx could strongly amplify the levels of ADAS-reactivated antigen-specific CD8.sup.+ T cells, similar to the action of IL-2cx. From this experiment it can be deduced that in all cases in which injection of IL-2cx was beneficial for the augmentation of the cytotoxic immune response (as described above) also IL-15cx will be effective.

Example 10

The ADAS Procedure can be Used to Re-Activate and Expand Memory CD8.SUP.+ T Cells in an Antigen-Specific Manner

(49) With nave animals, which were primed with 2 g MARX10-OVA and 10 g poly I:C on day 0, we could demonstrate that ADAS is rather ineffective for an amplification of the response when performed on day 3, but becomes effective thereafter, and continues to be effective to a certain extent at least until day 20 (Example 3 and FIG. 3), and is most effective between days 4 and 9 in mice.

(50) In these experiments, ADAS was used to amplify freshly activated nave antigen-specific CD8.sup.+ T cells. The fact that ADAS only optimally works in a certain time window following primary activation of CD8.sup.+ T cells indicated that these T cells must be in a particular activation stage in order to be sensitive for the ADAS amplification.

(51) We therefore wondered whether ADAS could also be performed on resting memory CD8.sup.+ T cells, which have a clearly different activation status compared to freshly activated CD8.sup.+ T cells. To this end, C57BL/6 mice were on day 1 adoptively transferred with 2,000 or 10,000 of OT-I CD8.sup.+ T cells, which bear a T cell receptor specific for SIINFEKL presented in the context of H-2K.sup.b. These transferred OT-I T cells bore the genetic marker Thy1.1 (CD90.1), which made it possible to discriminate them from endogenous T cells (compare Dorner et al., 2009). On day 0, the animals were primed with MARX10-OVA together with 10 g poly I:C. ADAS (injection of 1010.sup.6 SIINFEKL-loaded splenocytes together with 50 g poly I:C) was performed on day 5, resulting in a frequency of 30% of OT-I T cells of all CD8.sup.+ T cells in the spleen on day 10 for both groups of adoptively transferred animals (FIG. 10A). When animals from the same experimental groups were analyzed on day 40, the frequency of their OT-I CD8.sup.+ T cells had declined to 1-2%, as expected (FIG. 10A). On day 69, another ADAS amplification was performed (injection of 1010.sup.6 splenocytes loaded with SIINFEKL together with 50 g poly I:C) and the frequency of OT-I T cells was determined in the spleen 5 days later (day 74). Through the ADAS amplification, the frequency of OT-I T cells again rose to 30-60% of all CD8.sup.+ T cells in the spleen.

(52) In all animals adoptively transferred with OT-I T cells, we also analyzed the response of endogenous CD8.sup.+ T cells, since these could be identified as CD8 T cells negative for the Thy 1.1 marker, but staining with the SIINFEKL-tetramer. As can be seen in FIG. 10B, the endogenous, SIINFEKL-reactive CD8.sup.+ T cells showed a behavior similar to the OT-I T cells. In particular, the ADAS procedure could highly amplify endogenous SIINFEKL-specific memory T cells and these T cells bore all phenotypic markers typical of cytotoxic CD8.sup.+ T cells (granzyme B positivity, KLRG1-expression, data not shown).

(53) These experiments determined that the ADAS procedure can reactivate and massively expand resting memory CD8.sup.+ T cells in an antigen-specific manner, so that they again become CD8.sup.+ cytotoxic effectors. Since the ADAS procedure generates a state of T cells in which they are responsive to IL-2cx and IL-15cx, both cytokine preparations will further strongly augment the response of memory CD8.sup.+ T cells to ADAS.

(54) In summary, ADAS can not only can amplify freshly activated nave CD8+ T cells in an antigen-specific manner in a certain time window after activation, but also resting memory CD8.sup.+ T cells, i.e. CD8.sup.+ T cells in memory state.

Example 11

ADAS is Also Effective with In Vitro Activated, Adoptively Transferred CD8.SUP.+ T Cells

(55) Splenocytes of OT-I mice were in vitro activated with the peptide SIINFEKL for 3 days, without adjuvant. This activation of CD8.sup.+ T cells could, however, also be done in the presence of a Th1 adjuvant. After culture, the cells (at this time composed of 97% OT-I CD8.sup.+ T cells) were transferred into syngeneic C57BL/6 mice on day 0. ADAS (injection of 1010.sup.6 SIIFEKL-loaded splenocytes and 50 g poly I:C i.v.) was applied on either day 0, 1, 2, 3, 4, 5, or 6. ADAS on days 1, 2 or 3 did not have any significant effect on the frequency of the adoptively transferred CD8.sup.+ T cells in the host animals. Clear amplification of the transferred CD8 T cells could, however, be observed with ADAS applied on day 4, and optimal amplification was seen with ADAS on day 5 (FIG. 11). At the same time, the expression of the surface marker KLRG1, indicative of maturation of the transferred CD8.sup.+ T cells to effector cells, rose from 30-40% with ADAS on day 1 to 80-90% on days 5 and 6. These data allow to conclude that the ADAS procedure is capable to strongly amplify a population of in vitro-activated and adoptively transferred antigen-specific CD8.sup.+ T cells. At the same time, ADAS induces a further differentiation of these CD8.sup.+ T cells to cytotoxic effector cells. The optimal time point for ADAS may differ in the human, since experiments in the mouse do not allow a precise prediction in the human. Thus, the period for optimal ADAS effects on the adoptively transferred CD8+ T cells may be more extended. It can also be anticipated that ADAS is not only effective within the short time frame after adoptive transfer of from 0 h to 14 days, but also when the adoptively transferred CD8.sup.+ T cells have returned to the memory state after a prolonged period of time.

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