PRIMING OF AN IMMUNE RESPONSE

Abstract

The present invention relates to a technology and method of priming of an immune response using invariant chain linked antigen, when these are used to prime a subsequent booster immunization using any suitable vacci.

Claims

1. A nucleic acid construct comprising sequences encoding a. at least one variant or fragment of an invariant chain operatively linked to b. at least one antigenic protein or peptide or an antigenic fragment of said protein or peptide, herein said at least one variant or fragment of an invariant chain does not comprise the LRMK amino acid residues of the KEY region, and wherein the antigenic protein or peptide or antigenic fragment of said protein or peptide is derived from Hepatitis B virus.

2.-7. (canceled)

8. The nucleic acid construct according to claim 1, wherein one, two, three or four of the LRMK amino acid residues of the KEY region of the at least one variant or fragment of an invariant chain are deleted.

9.-10. (canceled)

11. The nucleic acid construct according to claim 1, wherein the first 17 amino acids of the at least one variant or fragment of an invariant chain are deleted (Δ17Ii).

12.-19. (canceled)

20. The nucleic acid construct according to claim 1, wherein the operative linker between the at least one variant or fragment of an invariant chain and the antigenic protein or peptide or an antigenic fragment of said protein or peptide is a direct link.

21.-24. (canceled)

25. The nucleic acid construct according to claim 1, wherein the at least one variant or fragment of an invariant chain and at least one antigenic protein or peptide or an antigenic fragment of said protein or peptide encoding sequence is preceded by a promoter enabling expression of the construct.

26. The nucleic acid construct according to claim 25, wherein the promoter is selected from the group of constitutive promoters, inducible promoters, organism specific promoters, tissue specific promoters and cell type specific promoters, CMV promoter, SV40 promoter, and RSV promoter.

27.-28. (canceled)

29. A delivery vehicle comprising the nucleic acid construct according claim 1.

30. The delivery vehicle according to claim 29, wherein the vehicle is selected from the group of: RNA based vehicles, DNA based vehicles/vectors, lipid based vehicles, polymer based vehicles and virally derived DNA or RNA vehicles.

31. The delivery vehicle according to claim 30, wherein said delivery vehicle is a pegylated vector or vehicle.

32. The delivery vehicle according to claim 30, wherein said lipid based vehicle is a liposome.

33.-50. (canceled)

51. A method for increasing the potency of a vaccine comprising the steps of a. providing the nucleic acid construct according to claim 1, b. priming the immune system of a subject by administering the nucleic acid construct of step a) thereby stimulating an immune response in said subject, and c. boosting the immune response of step b) by administering a suitable vaccine.

52.-68. (canceled)

69. The nucleic acid construct claim 1, wherein the operative linker between the at least one variant or fragment of an invariant chain and the antigenic protein or peptide or an antigenic fragment of said protein or peptide is a link mediated by a spacer region.

70. The delivery vehicle of claim 29 wherein the delivery vehicle is an adenoviral vector.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0425] FIG. 1: DNA-priming with an Ii chain based naked DNA vaccine significantly augments the generation of virus-specific CD8.sup.+ T cells upon subsequent boosting with a highly efficient viral vector. Mice were gene-gun immunized twice 3 weeks apart with DNA-IiGP, DNA-GP or left untreated. Three weeks after last immunization, all the mice were injected in the right hind footpad with 2×10.sup.7 IFU Ad5-IiGP, and 4 weeks later the animals were sacrificed, and splenocytes were analyzed as described in FIG. 1. Numbers of epitope-specific IFN-γ.sup.+CD8.sup.+ T cells are presented as mean±SE (n=5 mice/group). * denotes statistical significance relative to mice vaccinated with Ad5-IiGP only (Mann-Whitney rank-sum test). Results from one of two similar experiments are depicted.

[0426] FIG. 2: Location of the domains and the tested mutations in the Ii sequence. Domains in WT Ii are depicted above the bar. ESS; endosomal sorting signal, TM; transmembrane domain, KEY; peptide presentation enhancing region, CLIP; class-II-associated invariant chain peptide, TRIM; trimerization domain. Extent of deletion mutations and substitutions in Ii is marked below the bar. A; Ad-Δ17IiGP, b; Ad-IiLTMGP, c; Ad-IiUTMGP, d; Ad-Δ501iGP, e; Ad-Ii1-201GP, f; Ad-Ii1-118GP, g; Ad-Ii1-105GP, h; Ad-IiCLIPGP, i; Ad-IiKEYGP, j; Ad-Ii51-118GP.

[0427] FIG. 3: Ii dramatically increases cell surface presentation of the SIINFEKL/H-2kb OVA derived epitope. Bone Marrow derived Dendritic Cells were transfected with Ad-OVA, Ad-IiOVA or Ad-IiGP (negative control), and surface stained for MHC class II (stains mature dendritic cells) and with a SIINFEKL/H-2kb specific antibody (OVA epitope).

[0428] FIGS. 4A and 4B: Ii works only in cis. FIG. 4A) Expression of Ii from Ad-IiGP and Ad-Ii+ GP vectors; Ii expression was normalized to GAPDH in COST cells infected with 50 mol of Ad-IiGP and Ad-Ii+GP. FIG. 4B) TCR318 GP33 restricted T-cell proliferation in response to Ad-GP, Ad-IiGP or Ad-Ii+GP transduced BMDCs (bone marrow derived dendritic cell).

[0429] FIG. 5: N-terminal deletions and substitutions does not affect Ii stimulatory capacity. TCR 318 GP33 restricted T-cells proliferation in response to Ad-GP, Ad-IiGP, Ad-Δ17IiGP, Ad-IiITMGP, Ad-IiUTMGP, Ad-Δ501iGP transduced BMDCs (bone marrow derived dendritic cell).

[0430] FIG. 6: C-terminal deletions and substitutions does not affect Ii stimulatory capacity. TCR 318 GP33 restricted T-cells proliferation in response to Ad-GP, Ad-IiGP, Ad-Ii1-205GP, AdIi1-118GP and Ad-Ii1-105GP transduced BMDCs (bone marrow derived dendritic cell culture system).

[0431] FIG. 7: Only a N- and C-terminal deletion reduces Ii stimulatory capacity. TCR 318 GP33 restricted T-cells proliferation in response to Ad-GP, Ad-IiGP, Ad-IiCLIPGP, Ad-IiKEYGP and Ad-Ii51-118GP transduced BMDCs (bone marrow derived dendritic cell culture system).

[0432] FIG. 8: Dose-response of Ad-IiGP and Ad-GP vaccines. Groups of mice were vaccinated with the indicated vaccines in the indicated strains. 14 days after vaccination mice were sacrificed, and splenocytes stimulated with the indicated epitopes. Total number of specific CD8+ splenocytes was determined by intracellular staining and FACS analysis. The data shows that Ad-IiGP induces responses at very low dosages, and thus priming with a low dose Ad-IiGP (or any antigen) and subsequent boosting with a higher dose Ad-IiGP (or any antigen) may be applicable for homologous prime-boost regimens.

[0433] FIG. 9: Comparison of Ad-GP, Ad-IiGP and Ad-IiCLIPGP for MHC class II presentation (stimulation of CD4.sup.+ T-cells). SMARTA GP61-80 restricted T-cells proliferation in response to Ad-GP, Ad-IiGP and Ad-IiCLIPGP transduced BMDC's show an increased MHCII antigen presentation of Ad-IiCLIPGP.

[0434] FIGS. 10A to 10C: Comparison of Ad-GP, Ad-IiGP, Ad-GPLamp-1 and Ad-liΔ17GP in an in vivo time-course study.

[0435] FIGS. 11A and 11B: Comparison of Ad-GP, Ad-IiGP, Ad-IiΔ17GP, Ad-IiKEYGP, Ad-IiCLIPGP, Ad-Ii1-117GP and Ad-Ii1-199GP in vivo responses.

[0436] FIG. 12: Ad-GP is capable of priming a subsequent Ad-IiGP boost. 3 Groups of C57BL/6 mice were vaccinated with Ad-GP. 60 days later these mice were either left undisturbed, vaccinated with Ad-GP or vaccinated with Ad-IiGP. A 4.sup.th group of mice were included which were vaccinated with Ad-GP. 120 days after the first vaccinations, mice were sacrificed and antigen specific cells recognizing the indicated epitopes where quantitated by ex vivo restimulation with said peptides and intracellular staining for interferon-γ production.

[0437] FIG. 13: Ad-IiGP is not capable of priming a subsequent Ad-GP or Ad-IiGP boost. 3 Groups of C57BL/6 mice were vaccinated with Ad-IiGP. 60 days later these mice were either left undisturbed, vaccinated with Ad-GP or vaccinated with Ad-IiGP. A 4.sup.th group of mice were included which were vaccinated with Ad-IiGP. 120 days after the first vaccinations, mice were sacrificed and antigen specific cells recognizing the indicated epitopes where quantitated by ex vivo restimulation with said peptides and intracellular staining for interferon-γ production. This shows that Ad-IiGP priming can not be boosted with Ad-IiGP, whereas DNA-IiGP priming can be boosted with Ad-IiGP (see FIG. 1).

[0438] FIG. 14: Dose-response of Ad-GP and AdIi-Gp vaccines. Groups of mice were vaccinated with the indicated vaccine in the indicated strains. 14 days after vaccination mice were sacrificed, and splenocytes stimulated with the indicated epitopes. Total number of CD8+ splenocytes was determined by intracellular staining and FAGS analysis.

[0439] FIG. 15: The Mannose receptor coupled to a variant of invariant chain comprising residues 50 to 215 (Ii50-215), further coupled to an adenoviral fiber protein. The adenoviral fiber protein (Ad fiber) may stem from any serotype of adenovirus. The mannose receptor may be one or more domains from the Mannose receptor. The Ii may be a variant of or full length Ii. Ag=Antigen.

EXAMPLES OF THE INVENTION

[0440] The invention will now be further illustrated with reference to the following examples. It will be appreciated that what follows is by way of example only and that modifications in detail may be made while still falling within the scope of the invention.

Example 1: Priming with an Ii Chain Based Naked DNA Vaccine Significantly Augments the Generation of Virus-Specific CD8.SUP.+.T Cells Upon Subsequent Boosting with an Optimized Viral Vector

[0441] Priming with a naked DNA vaccine (i.e. a nucleic acid construct) is shown to augment the immune response raised by subsequent immunization with Ad5 (adenovirus serotype 5) vector. Priming with DNA-IiGP (DNA construct expressing LCMV (lymphocytic choriomeningitis virus) glycoprotein (GP) fused to invariant chain (Ii)) is herein demonstrated to significantly enhance the CD8.sup.+ T-cell response induced by the same gene construct delivered in an adenovirus serotype 5 vector (Ad5-IiGP), providing a strong argument for the inclusion of Ii chain based DNA-constructs in future heterologous immunization (“prime-boost”) protocols.

[0442] Our study shows that the immunoenhancing effect of Ii chain linkage is not limited to the Ad5 vector, but is relevant on a DNA platform as well. Furthermore, given the fact that Ii chain enhances presentation of more than one epitope, this places Ii chain based DNA vaccines as very promising candidates for various heterologous prime-boost regimes.

Results & Discussion

[0443] One way to improve the induced T-cell memory is through heterologous prime-boost regime e.g. naked DNA priming followed by a vector boost. Thus having in our laboratory the appropriate vector, replication deficient adenovirus expressing LCMV GP fused to p31 Ii chain (Ad5-IiGP) this possibility was tested experimentally. First, we performed standard DNA vaccination, gene-gun-vaccination twice 3 weeks apart with DNA-IiGP or DNA-GP. Three weeks after the second DNA-vaccination, both groups of mice and matched controls were immunized by inoculation of 2×10.sup.7 IFU Ad5-IiGP in the right hind footpad, and 4 weeks later the number of virus-specific CD8.sup.+ T cells in the spleen was enumerated by way of ICCS for IFN-γ and flow cytometry. Mice primed with the fused DNA construct contained significantly more GP.sub.33-41 and GP.sub.276-286-specific IFN-γ.sup.+ CD8.sup.+ T cells than did unprimed mice, and a similar trend was noted for GP.sub.92-101-specific cells, although in this case the difference was not statistically significant. In contrast, priming with naked DNA encoding GP in the absence of Ii had little effect on the level of GP-specific memory CD8.sup.+ T cells induced by subsequent immunization with Ad5-IiGP (FIG. 1). It should be noted that the observed effect of including Ii does not reflect non-specific augmentation of the immunoreactivity of vaccinated mice, as DNA priming with a vector including only Ii, but no GP, had no effect on the level of GP-specific CD8.sup.+ T cells in mice subsequently inoculated with the adenoviral vector (data not shown).

[0444] We have shown that use of the improved DNA-vector as a part of a heterologous prime-boost regime will significantly augment the response induced by an already optimized viral vector (Hoist et al., 2008). This strongly indicates that even very immunogenic vector based immunization may be further improved through initial priming of the host with an Ii chain based naked DNA vaccine. Altogether, since Ii chain fusion to the antigen will lead to priming for a broad CD8.sup.+ T cell response, Ii chain based DNA vaccines should represent a clear advantage with regard to prevention strategies against rapidly mutating viruses as part of heterologous prime-boost regimes.

Materials and Methods

Mice.

[0445] C57BL/6 (B6) wild type mice were obtained from Taconic M&B (Ry, Denmark). Perforin deficient B6 mice were bred locally from breeder pairs originally obtained from The Jackson Laboratory (Bar Harbor, Me.). Seven- to 10-week-old mice were used in all experiments, and animals from outside sources were always allowed to acclimatize to the local environment for at least 1 week before use. All animals were housed under specific pathogen free conditions as validated by screening of sentinels. All animal experiments were conducted according to national guidelines.

DNA Vaccine Construction and Immunization Procedure.

[0446] The DNA vaccines are produced using the eukaryotic expression vector pACCMV.pLpA containing either the murine invariant chain followed by GP of LCMV or LCMV GP alone. The constructs were generated as recently described (Hoist et al., 2008). The E. coli strain XL1-blue (Stratagene, USA) was transformed with the constructs by electroporation. DNA sequencing using cycle sequencing, Big Dye Terminator and ABI310 genetic analyzer (ABIprism, USA) identified positive clones. Primers were obtained from TAG, Copenhagen, Denmark. Large scale DNA preparations were produced using Qiagen Maxi Prep (Qiagen, USA).

Gene-Gun Immunization.

[0447] DNA was coated onto 1.6 nm gold particles in a concentration of 2 μg DNA/mg gold, and the DNA/gold complexes were coated onto plastic tubes such that 0.5 mg gold was delivered to the mouse pr. shot (1 μg DNA pr. shot). These procedures were performed according to the manufacturer's instructions (Biorad, CA, USA) (Bartholdy et al., 2003). Mice were immunized on the abdominal skin using a hand held gene-gun device employing compressed Helium (400 psi) as the particle motive force. Unless otherwise mentioned, mice were immunized twice with an interval of 3-4 weeks and then allowed to rest for 3 weeks before further challenge/investigation.

Virus.

[0448] LCMV of the Armstrong strain clone 13 was used in most experiments. Unless otherwise stated, mice to be infected received a dose of 10.sup.5 pfu of clone 13 in an i.v. injection of 0.3 ml, or 20 pfu in 0.03 ml in the right hind footpad (f.p.). For i.c. injection mice received 20 pfu of neurotropic Armstrong clone 53b in a volume of 0.03 ml. Replication deficient adenovirus encoding invariant chain linked GP (Ad5-IiGP) was produced and titrated as recently described (Hoist et al., 2008).

Virus Titration.

[0449] Organ virus titers were assayed by an immune focus assay as previously described (Battegay et al., 1991).

In Vivo Depletion of CD4.sup.+ and CD8.sup.+ T Cells.

[0450] The anti-CD4 (clone GK1.5) and anti-CDS mAbs (clone 53.6.72) were used. Mice to be depleted of cells received a dose of 200 μg. in a volume of 0.3 ml PBS intraperitoneally on days −1 and 0 relative to infection; for sham treatment purified rat IgG (Jackson ImmunoResearch) was used instead. The efficiency of cell depletion was verified by flow cytometry.

Survival Study.

[0451] Mortality was used to evaluate the clinical severity of acute LCMV induced meningitis. Mice were checked twice daily for a period of 14 days or until 100% mortality was reached.

Assay of LCMV-Specific Footpad Swelling Reaction.

[0452] Mice were infected locally in the right hind footpad as described above, and the local swelling reaction was followed until day 14 p.i. Footpad thickness was measured with a dial caliper (Mitutoyo 7309, Mitutoyo Co., Tokyo, Japan), and virus-specific swelling was determined as the difference in thickness of the infected right and the uninfected left foot (Christensen et al., 1994).

Cell Preparations.

[0453] Spleens from mice were aseptically removed and transferred to Hanks' balanced salt solution (HBSS). Single cell suspensions were obtained by pressing the organs through a fine sterile steel mesh. The cells were washed twice with HBSS, and cell concentration was adjusted in RPMI 1640 containing 10% fetal calf serum (FCS), supplemented with 2-mercaptoethanol, L-glutamin, and penicillin-streptomycin solution.

mAb for Flow Cytometry.

[0454] The following mAbs were all purchased from PharMingen (San Diego, Calif.) as rat anti-mouse antibodies: FITC-conjugated anti-CD44, Cy-Chrome conjugated anti-CD8a, Cy-Chrome conjugated anti-CD4 and Phycoerythrin (PE)-conjugated anti IFN-γ.

Flow Cytometric Analysis.

[0455] For visualization of LCMV-specific (interferon-γ producing) CD8.sup.+/CD4.sup.+ T cells, 1-2×10.sup.6 splenocytes were resuspended in 0.2 ml complete RPMI medium supplemented with 10 units murine recombinant IL-2 (R&D Systems Europe Ltd, Abingdon, UK), 3 μM monensin (Sigma Chemicals co., St Louis, Mo.) and 1 μg/ml relevant peptide and incubated for 5 hours at 37° C. The following peptides were used: for CD8.sup.+ T cells GP33-41, GP276-86, GP92-101, GP118-125, and NP396-404 for control; for CD4.sup.+ T cells GP61-80. After incubation, cells were surface stained, washed, permeabilized and stained with IFN-γ specific mAb as described previously (Andreasen et al., 2000; Christensen et al., 2003). Isotype matched antibody served as control for non-specific staining. Cells were analyzed using a FAGS Calibur (Becton Dickinson, San Jose, Calif.), and at least 10.sup.4 live cells were gated using a combination of low angle and side scatter to exclude dead cells and debris. Data analysis was conducted using Cell-Quest software.

Example 2: Enhanced CD8.SUP.+ T-Cell Activation of Ii Linked Antigen is Independent of Native Ii

[0456] The Ii sequence contains multiple regions with functions in antigen processing including: a cytoplasmic sorting domain and trimerization domain, a cytoplasmic and proximal membrane signalling domain, cytoplasic, intramembrane and periplasmic trimerization domains, the “key” motif involved in unlocking MHC molecules to facilitate binding of exogenous peptides, binding motifs for MHC class I and II in the CLIP region, a periplasmic glycosylation site as well as a structurally unidentified region of interaction with CD44 and Macrophage migration Inhibitory Factor (MIF) (FIG. 2).

[0457] Ii linkage increases the antigen presentation on both MHC class I and II. By using Ad-IiOVA (OVA is ovalbumin) or Ad-OVA transduction of Bone Marrow derived Dendritic Cells (BMDC), we found that Ii linkage did indeed induce a dramatic increase in MHC class I restricted antigen presentation, as measured by direct staining with an antibody directed against the SIINFEKL OVA epitope presented on H-2Kb (FIG. 3). The increased MHC class I restricted antigen expression from the Ii linked sequences works directly on the APC independently of MHC class II, CD4.sup.+ T cells, and any other cell type and can be directly measured in dendritic cell cultures.

Ii in cis

[0458] To establish whether Ii works only in cis or also in trans, an additional reading frame into the adenoviral vector was established by synthesizing a phosphoglycerate kinase (pGK) promoter with a 13-globin polyadenylation signal and cloning this into the E3 region of the adenoviral backbone. This vector could then be used for recombination with the shuttle vector used to create the Ad-GP vector (which expresses LCMV GP from the E1 reading frame under control of the human CMV promoter and SV40 polyA). The new vector expresses LCMV GP from the adenoviral E1 region and Ii from the E3 region (Ad-Ii+GP). The promoter was verified for the induction of green fluorescent cells by transfection into COS7 cells, and a measurement of Ii mRNA expression in Ad-IiGP and Ad-Ii+GP infected COS7 cells confirms that Ii is at least as efficiently expressed from the pGK promoter as from the CMV promoter (FIG. 4A). Comparing of TCR318 cells stimulated with Ad-GP, Ad-IiGP and Ad-Ii+GP infected BMDC's clearly show that Ii must be linked to the antigen to have any effect (FIG. 4B). It would have been surprising if Ii expression in trans had shown efficacy as the BMDC cultures used for the stimulation already express Ii.

N-Terminal Alterations:

[0459] Starting from the N-terminal, we made 1) a deletion of the first 17 amino acids (Ad-Δ17IiGP), which removes the Leucine based endosomal sorting signals, 2) a replacement of the first half of the transmembrane segment (Ad-IiLTMGP), with the corresponding segment from the chemokine receptor CCR6 TM6, 3) a replacement of the second half of the transmembrane segment with the corresponding CCR6 TM6 segment (Ad-IiUTMGP), and finally 4) a complete deletion of the first 50 amino acids (Ad-Δ50IiGP). The latter deletion removed the entire cytosolic, TM and membrane proximal region. None of these mutations had any effect on the ability of the remaining Ii sequence to enhance stimulation of CD8.sup.+ T cells (FIG. 5).

C-Terminal Alterations:

[0460] From the C-terminus we made deletions of the last 14 aa (Ad-Ii1-201GP, this removes the C-terminal glycosylation signal), the last 97 aa (Ad-Ii1-118GP), and the last 110 aa (Ad-Ii1-105GP). No effect on the ability of the remaining Ii sequence to enhance stimulation of CD8+ T cells was observed by the 1-201, whereas only inconsistent and minor trends of reductions could be seen from the 1-105 mutation and the 1-118 mutations (FIG. 6).

Mutations:

[0461] We also attempted to make point mutations in the reported MHC class I binding site of the CLIP region (Ad-IiCLIPGP: a double M to A point mutation—M91A M99A —designed to abolish Ii interaction with MHC class I molecules) and the KEY motif (Ad-IiKEYGP: a LRMK (SEQ ID NO: 5) to AAAA (SEQ ID NO: 6) quadriple point mutation which would destroy the Ii-Key segment). None of these mutations were key to the Ii mediated enhanced stimulation of CD8+ T cells. The only interesting data came when we combined N- and C-terminal truncations. Thus when we tested a 51-118 variant (Ad-Ii51-118GP), a pronounced reduction in CD8+ T cells stimulatory capacity was observed, but the mutant was still superior to the Ad-GP (FIG. 7).

Example 3

[0462] In one embodiment of the invention, a non-human glycosyltransferase combined with glycosyl-binding proteins coupled to Ii is provided. Ii may be full length or a variant, wherein the variant may be a truncated version of Ii comprising residues number 50 to 215. This variant has full activity despite the lack of a transmembrane domain. Optionally, an adjuvant or one or more translocation domain may be further provided. In FIG. 15 is provided a schematic drawing of an embodiment wherein the Mannose receptor (a calcium-dependent lectin often targeted in vaccines) is coupled to a variant of invariant chain comprising residues 50 to 215 (Ii50-215), further coupled to an adenoviral fiber protein. The adenoviral fiber protein (Ad fiber) may stem from any serotype of adenovirus. The mannose receptor may be one or more domains from the Mannose receptor.

[0463] In one specific example, an Adenovirus expressing Egghead (a protein from Drosophila) in one reading frame, and expressing the Mannose receptor (or domains from the Mannose receptor) coupled to a variant of Ii having full activity without a transmembrane region such as the Ii50-215 variant further couplet to and adenoviral fiber protein in another reading frame is provided.

[0464] The glycosyltransferase such as Egghead and the glycosyl-binding proteins such as Mannose receptor may be expressed from different reading frames in the same Adenoviral vector, or the glycosyltransferase such as Egghead and the glycosyl-binding proteins such as Mannose receptor may be expressed from different Adenoviral vectors administered simultaneously.

[0465] Egghead couples Mannose on all glycosylated ER (endoplasmatic reticulum) proteins. The mannosylation of secreted proteins may thus cause the binding of mannosylated protein to the Mannose receptor-Ii-Ad fiber complex (as shown in FIG. 15). The Adenoviral fiber of the complex causes the secreted proteins linked to said complex to be taken up by other cells, activating these to become immune-stimulating and providing access of the complex to the cytosol where Ii may exert its effects.

[0466] This technology may be used to construct a vaccine that may be administered directly into for example cancers.

REFERENCE LIST

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