DETOXIFIED LIPOPOLYSACCHARIDES (LPS), NATURALLY NON-TOXIC LPS, AND USES THEREOF
20230173064 · 2023-06-08
Assignee
Inventors
Cpc classification
A61K39/39
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2039/55572
HUMAN NECESSITIES
International classification
A61K39/39
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
Abstract
An enriched population of modified lipopolysaccharide (LPS) molecular species being: devoid of phosphate group at position C1 of the reducing end of their lipid A domain; and substituted at position C6′ of the non-reducing end of their lipid A domain by a hydrophilic moiety, with the proviso that the hydrophilic moiety is not a hydroxyl group. Also, compositions that include the enriched population of modified LPS and uses of naturally-occurring LPS molecular species and/or enriched population of modified LPS molecular species for treating and/or preventing cancer, inflammatory diseases or infectious diseases, and for stimulating an immune response or vaccinating a subject.
Claims
1-20. (canceled)
21. An enriched population of modified lipopolysaccharide (LPS) molecular species, comprising at least 80% of modified LPS molecular species, wherein said modified LPS molecular species: are devoid of phosphate group at position C1 of the reducing end of their lipid A domain; and are substituted at position C6′ of the non-reducing end of their lipid A domain by a hydrophilic moiety, with the proviso that said hydrophilic moiety is not a hydroxyl group.
22. The enriched population of modified LPS molecular species according to claim 21, wherein said modified LPS molecular species are substituted at position C6′ of the non-reducing end of their lipid A domain by a mono-, oligo- or poly-saccharide moiety.
23. The enriched population of modified LPS molecular species according to claim 21, wherein the modified LPS molecular species are of general Formula A ##STR00012## wherein: Y is —O— or —NH—; R.sub.2 is a phosphate group (—OPO.sub.3H.sub.2) or a hydroxyl group (—OH), optionally substituted; R.sub.3 is a monosaccharide, an oligosaccharide or a polysaccharide; optionally substituted; R.sub.6 is a fatty acid residue, optionally substituted; R.sub.7 is a hydrogen or a fatty acid residue, optionally substituted; Z is a hydroxyl group (—OH), optionally substituted, with the proviso that, if substituted, the substitution does not consist of a phosphate group (—OPO.sub.3H.sub.2), or Z is ##STR00013## wherein X is —O— or —NH—; R.sub.1 is a hydroxyl group (—OH), optionally substituted, with the proviso that, if substituted, the substitution does not consist of a phosphate group (—OPO.sub.3H.sub.2); R.sub.4 is a fatty acid residue, optionally substituted; and R.sub.5 is a hydrogen or a fatty acid residue, optionally substituted.
24. The enriched population of modified LPS molecular species according to claim 21, wherein the modified LPS molecular species are of general Formula B ##STR00014## wherein: X and Y are, independently from each other, —O— or —NH—; R.sub.1 is a hydroxyl group (—OH), optionally substituted, with the proviso that, if substituted, the substitution does not consist of a phosphate group (—OPO.sub.3H.sub.2); and R.sub.2 is a phosphate group (—OPO.sub.3H.sub.2) or a hydroxyl group (—OH), optionally substituted; R.sub.3 is a mono-, oligo- or poly-saccharide moiety, optionally substituted; R.sub.4 and R.sub.6 are, independently from each other, a fatty acid residue, optionally substituted; and R.sub.5 and R.sub.7 are, independently from each other, a hydrogen or a fatty acid residue, optionally substituted.
25. The enriched population of modified LPS molecular species according to claim 21, wherein the modified LPS molecular species are selected from the group consisting of LPS molecular species of Formulas (I)(1)(a), (I)(1)(b), (I)(1)(c), (I)(1)(d), (I)(1)(e), (I)(1)(f), (I)(2)(a), (I)(2)(b), (I)(2)(c), (I)(3)(a), (I)(3)(b), (I)(3)(c), (I)(5)(a), (I)(5)(b), (I)(6)(a), (I)(6)(b), (I)(7)(a), (I)(7)(b), (I)(7)(c), (I)(7)(d), (I)(8)(a), (I)(8)(b), (I)(8)(c), (I)(9)(a), (I)(9)(b), (I)(9)(c), (I)(9)(d), (I)(9)(e), (I)(9)(f), (II)(2)(a), (II)(2)(b), (II)(2)(c), (II)(2)(d), (III)(1)(a), (III)(1)(b), (III)(1)(c), (III)(1)(d), (IV)(1)(a), (IV)(1)(b), (IV)(1)(c), (V)(1)(a), (V)(1)(b), (V)(1)(c), (V)(1)(d), (V)(1)(e), (V)(1)(f), (V)(1)(g), (V)(1)(h), (V)(1)(i), (VI)(1)(a), (VI)(1)(b), (VI)(1)(c), (VI)(1)(d), (VI)(1)(e), (VI)(1)(f), (VI)(1)(g), (VI)(1)(h), (VI)(1)(i), (VI)(1)(j), (VI)(2)(a), (VI)(2)(b), (VI)(2)(c), (VII)(1)(a), (VII)(1)(b), (VII)(1)(c), (VIII)(1)(a), (VIII)(1)(b), (VII)(1)(c), (VII)(1)(d), (IX)(1)(a), (IX)(1)(b), (IX)(1)(c), (IX)(1)(d), (X)(1)(a), (X)(1)(b), (X)(1)(c), (X)(1)(d), (XI)(1)(a), (XI)(1)(b), (XI)(1)(c), (XI)(1)(d), (XIII)(2)(a), (XIII)(2)(b), (XIII)(3)(a), (XIII)(3)(b), (XIII)(4)(a), (XIII)(4)(b), (XIII)(5)(a), (XIII)(5)(b), (XIII)(5)(c), (XIII)(5)(d), (XIII)(6)(a), (XIII)(6)(b), (XIII)(6)(c), (XIII)(6)(d), (XIII)(6)(e), (XIII)(6)(f), (XIII)(7)(a), (XIII)(7)(b), (XIII)(7)(c), (XIII)(7)(d), (XIII)(7)(e), (XIII)(7)(f), (XIII)(8)(a), (XIII)(8)(b), (XIII)(8)(c), (XIII)(8)(d), (XIII)(8)(e), (XIII)(8)(f), (XIII)(9)(a), (XIII)(9)(b), (XIII)(9)(c), (XIII)(9)(d), (XIII)(9)(e), (XIII)(9)(f), (XIII)(9)(g), (XIII)(9)(h), (XIII)(9)(i), (XIII)(9)(j), and a combination thereof as shown in
26. The enriched population of modified LPS molecular species according to claim 21, wherein the modified LPS molecular species form particles having an average size smaller than about 200 nm in diameter in aqueous solutions, as can be assessed by dynamic light scattering (DLS) at 25° C.
27. A composition comprising: i. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (I)(1)(a), (I)(1)(b), (I)(1)(c), (I)(1)(d), (I)(1)(e) and (I)(1)(f); or ii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (I)(2)(a), (I)(2)(b) and (I)(2)(c); or iii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (I)(3)(a), (I)(3)(b) and (I)(3)(c); or iv. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (I)(5)(a) and (I)(5)(b); or v. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (I)(6)(a) and (I)(6)(b); or vi. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (I)(7)(a), (I)(7)(b), (I)(7)(c) and (I)(7)(d); or vii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (I)(8)(a), (I)(8)(b) and (I)(8)(c); or viii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (I)(9)(a), (I)(9)(b), (I)(9)(c), (I)(9)(d), (I)(9)(e) and (I)(9)(f); or ix. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (II)(2)(a), (II)(2)(b), (II)(2)(c), (II)(2)(d); or x. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (III)(1)(a), (III)(1)(b), (III)(1)(c), (III)(1)(d); or xi. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (IV)(1)(a), (IV)(1)(b), (IV)(1)(c); or xii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (V)(1)(a), (V)(1)(b), (V)(1)(c), (V)(1)(d), (V)(1)(e), (V)(1)(f), (V)(1)(g), (V)(1)(h), (V)(1)(i); or xiii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (VI)(1)(a), (VI)(1)(b), (VI)(1)(c), (VI)(1)(d), (VI)(1)(e), (VI)(1)(f), (VI)(1)(g), (VI)(1)(h), (VI)(1)(i), (VI)(1)(j); or xiv. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (VI)(2)(a), (VI)(2)(b), (VI)(2)(c); or xv. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (VII)(1)(a), (VII)(1)(b), (VII)(1)(c); or xvi. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (VIII)(1)(a), (VIII)(1)(b), (VIII)(1)(c), (VIII)(1)(d); or xvii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (IX)(1)(a), (IX)(1)(b), (IX)(1)(c), (IX)(1)(d); or xviii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (X)(1)(a), (X)(1)(b), (X)(1)(c), (X)(1)(d); or xix. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XI)(1)(a), (XI)(1)(b), (XI)(1)(c), (XI)(1)(d); or xx. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XIII)(2)(a) and (XIII)(2)(b); or xxi. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XIII)(3)(a) and (XIII)(3)(b); or xxii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XIII)(4)(a) and (XIII)(4)(b); or xxiii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XIII)(5)(a), (XIII)(5)(b), (XIII)(5)(c) and (XIII)(5)(d); or xxiv. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XIII)(6)(a), (XIII)(6)(b), (XIII)(6)(c), (XIII)(6)(d), (XIII)(6)(e) and (XIII)(6)(f); or xxv. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XIII)(7)(a), (XIII)(7)(b), (XIII)(7)(c), (XIII)(7)(d), (XIII)(7)(e) and (XIII)(7)(f); or xxvi. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XIII)(8)(a), (XIII)(8)(b), (XIII)(8)(c), (XIII)(8)(d), (XIII)(8)(e) and (XIII)(8)(f); or xxvii. an enriched population comprising a mix of at least two modified LPS molecular species of Formulas (XIII)(9)(a), (XIII)(9)(b), (XIII)(9)(c), (XIII)(9)(d), (XIII)(9)(e), (XIII)(9)(f), (XIII)(9)(g), (XIII)(9)(h), (XIII)(9)(i) and (XIII)(9)(j).
28. A method of: treating and/or preventing cancer, an inflammatory disease or an infectious disease in a subject in need thereof, stimulating an immune response in a subject in need thereof, or vaccinating a subject in need thereof, comprising administering to said subject a naturally-occurring non-toxic LPS molecular species and/or an enriched population of modified LPS molecular species, or a composition comprising the same, wherein said naturally-occurring non-toxic LPS molecular species and/or enriched population of modified LPS molecular species is selected from: the enriched population of modified LPS molecular species according to claim 21; or a LPS molecular species which does not comprise two secondary C.sub.12 and/or C.sub.14 fatty acid chains simultaneously present at positions C2′ and C3′ of their lipid A domain.
29. The method according to claim 28, wherein said LPS molecular species or composition is selected from the group consisting of: a LPS molecular species selected from the group consisting of LPS molecular species of Formulas (I)(0)(a), (I)(0)(b), (II)(0), (II)(1)(a), (II)(1)(b), (II)(1)(c), (II)(1)(d) as shown in
30. The method according to claim 28, wherein treating and/or preventing cancer, an inflammatory disease or an infectious disease further comprises administering to said subject at least one additional therapeutic agent.
31. The method according to claim 30, wherein the at least one additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, targeted therapy agents, cytotoxic agents, antibiotics, antivirals, cell cycle-synchronizing agents, ligands for cellular receptor(s), immunomodulatory agents, pro-apoptotic agents, anti-angiogenic agents, cytokines, growth factors, antibodies or antigen-binding fragments thereof, cell therapy, antigens, and combinations thereof.
32. The method according to claim 30, wherein the at least one additional therapeutic agent is a targeted therapy agent.
33. The method according to claim 32, wherein the targeted therapy agent is an anti-CD20 antibody or an antigen-binding fragment thereof.
34. The method according to claim 33, wherein the anti-CD20 antibody is rituximab or an antigen-binding fragment thereof.
35. The method according to claim 30, wherein the at least one additional therapeutic agent is an immunostimulatory agent.
36. The method according to claim 35, wherein the immunostimulatory agent is an immune checkpoint inhibitor.
37. The method according to claim 36, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.
38. The method according to claim 28, wherein vaccinating, or stimulating an immune response in, a subject in need thereof further comprises administering to said subject at least one antigen.
39. The method according to claim 38, wherein the at least one antigen is selected from the group consisting of a pathogen-related antigen, a self-antigen, an allergen-related antigen and a neoantigen.
40. The method according to claim 28, wherein the subject in need thereof is an animal other than a human and said naturally-occurring non-toxic LPS molecular species and/or modified LPS molecular species or composition comprising the same is selected from a LPS molecular species of Formula (XII)(1) as shown in
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0820] The present invention is further illustrated by the following examples.
Example 1
[0821] Production of Chemically-Modified LPS Molecular Species
[0822] In the following, the figures “x.Math.y” following the LPS name refer to: [0823] i. first figure (“x”): where 0 means no alkaline treatment, and 1 means one alkaline treatment; [0824] ii. second figure (“y”): where 0 means no acidic hydrolysis treatment, 1 means one acidic hydrolysis treatment with a strong acid (i.e., with pK.sub.a<0) in aqueous solution, 2 means one acidic hydrolysis treatment with a strong acid (i.e., with pK.sub.a<0) in organic solution; 3 means one acidic hydrolysis treatment with a strong acid (i.e., with pK.sub.a<0) in organic solution in presence of ethanol; and 4 means one acidic hydrolysis treatment with a weak acid (i.e., with pK.sub.a>0) in aqueous solution.
[0825] Structures
[0826] “LPS B.sub.2 0.0”, as used herein, refers to a mix of the two naturally-occurring LPS molecular species from Bordetella pertussis with Formulas (I)(0)(a) and (I)(0)(b), as shown in
[0827] “LPS W.sub.1 0.0”, as used herein, refers to the naturally-occurring LPS molecular species from Vitreoscilla filiformis with Formula (II)(0), as shown in
[0828] “LPS SM.sub.1 0.0”, as used herein, refers to the naturally-occurring LPS molecular species from Salmonella enterica enterica serovar Minnesota R595 with Formula (XII)(0), as shown in
[0829] Production & Purification
[0830] LPS B.sub.2 0.0, LPS W.sub.1 0.0 and LPS SM.sub.1 0.0 were produced and purified using conventional methods, in particular the method described in U.S. Pat. No. 8,137,935.
[0831] Other naturally-occurring LPS molecular species have been produced and purified similarly (structures not shown), including:
i. “LPS ST.sub.1 0.0”, from Salmonella enterica enterica serovar Typhimurium Ra;
ii. “LPS E.sub.2 0.0”, from Escherichia coli K12;
iii. “LPS E.sub.1 0.0”, from Escherichia coli J5;
iv. “LPS A.sub.1 0.0”, from Actinobacillus pleuropneumoniae;
v. “LPS K.sub.1 0.0”, from Burkholderia pseudomallei;
vi. “LPS M.sub.1 0.0”, from Moraxella catarrhalis;
vii. “LPS N.sub.1 0.0”, from Neisseria sicca;
viii. “LPS P.sub.1 0.0”, from Pseudomonas aeruginosa;
ix. “LPS P.sub.2 0.0”, from Pseudomonas fluorescens;
x. “LPS I.sub.1 0.0”, from Bordetella hinzii;
xi. “LPS T.sub.1 0.0”, from Bordetella trematum; and
xii. “LPS V.sub.1 0.0”, from Vibrio fischeri.
[0832] Chemical Modification
[0833] After purification, the LPS molecular species were chemically treated by acidic hydrolysis at a temperature below 85° C.; and/or by alkaline hydrolysis, as indicated by the figures “x.Math.y” following the LPS name. If both, alkaline hydrolysis was carried out preferably before acidic hydrolysis.
[0834] The innovative process is that acidic hydrolysis at a temperature below 85° C. targets dephosphorylation of the lipid A glycosidic phosphate, which is removed in situ, in the complete LPS molecular species, i.e., without splitting it in its lipid A and polysaccharide moieties.
[0835] It follows from these treatments several LPS molecular species as defined in Table 1 below:
TABLE-US-00001 TABLE 1 Natural Modified Modified Modified LPS Formulas FIG. LPS Formulas FIG. LPS Formulas FIG. LPS Formulas FIG. B.sub.2 0.0 (I)(0)(a) FIG. 1 B.sub.2 0.1 (I)(1)(a) FIG. 2 B.sub.2 1.1 (I)(2)(a) FIG. 3 B.sub.2 1.2 (I)(3)(a) FIG. 4 (I)(0)(b) (I)(1)(b) (I)(2)(b) (I)(3)(b) (I)(1)(c) (I)(2)(c) (I)(3)(c) (I)(1)(d) (I)(1)(e) (I)(1)(f) W.sub.1 0.0 (II)(0) FIG. 5 W.sub.1 1.0 (II)(1)(a) FIG. 6 W.sub.1 1.2 (II)(2)(a) FIG. 7 (II)(1)(b) (II)(2)(b) (II)(1)(c) (II)(2)(c) (II)(1)(d) (II)(2)(d) SM.sub.1 0.0 (XII)(0) FIG. 27 SM.sub.1 1.2 (III)(1)(a) FIG. 8 (III)(1)(b) (III)(1)(c) (III)(1)(d) ST.sub.1 0.0 n/a n/a ST.sub.1 1.2 (IV)(1)(a) FIG. 9 E.sub.2 0.0 n/a n/a E.sub.2 1.2 (IV)(1)(b) V.sub.1 0.0 n/a n/a V.sub.1 1.2 (IV)(1)(c) E.sub.1 0.0 n/a n/a E.sub.1 0.1 (V)(1)(a) FIG. 10 (V)(1)(b) (V)(1)(c) (V)(1)(d) (V)(1)(e) (V)(1)(f) (V)(1)(g) (V)(1)(h) (V)(1)(i) A.sub.1 0.0 n/a n/a A.sub.1 0.1 (VI)(1)(a) FIG. 11 A.sub.1 1.2 (VI)(2)(a) FIG. 12 (VI)(1)(b) (VI)(2)(b) (VI)(1)(c) (VI)(2)(c) (VI)(1)(d) (VI)(1)(e) (VI)(1)(f) (VI)(1)(g) (VI)(1)(h) (VI)(1)(i) (VI)(1)(j) K.sub.1 0.0 n/a n/a K.sub.1 1.2 (VII)(1)(a) FIG. 13 (VII)(1)(b) (VII)(1)(c) M.sub.1 0.0 n/a n/a M.sub.1 1.2 (VIII)(1)(a) FIG. 14 (VIII)(1)(b) (VIII)(1)(c) (VIII)(1)(d) N.sub.1 0.0 n/a n/a N.sub.1 1.2 (IX)(1)(a) FIG. 15 (IX)(1)(b) (IX)(1)(c) (IX)(1)(d) P.sub.1 0.0 n/a n/a P.sub.1 1.2 (X)(1)(a) FIG. 16 P.sub.2 0.0 n/a n/a P.sub.2 1.2 (X)(1)(b) (X)(1)(c) (X)(1)(d) I.sub.1 0.0 n/a n/a I.sub.1 1.2 (XI)(1)(a) FIG. 17 T.sub.1 0.0 n/a n/a T.sub.1 1.2 (XI)(1)(b) (XI)(1)(c) (XI)(1)(d)
Example 2
[0836] DLS Aggregate Size Analysis
[0837] Materials and Methods
[0838] Determination of aggregates sizes of LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 and of synthetic GLA (PHAD®, from Avanti® Polar Lipids, Inc.) in aqueous and PBS suspensions was determined by Dynamic Light Scattering (DLS) using the Malvern Instruments Zetasizer Nano S90 with a scattering angle Θ=90°, incident laser wavelength λ=633 nm and temperature at 25° C.
[0839] Samples for measurements were prepared as follows: 200 μg of dry samples in 4 mL glass vials were suspended in 1 mL of water or PBS. LONZA LAL Reagent water and Gibco PBS pH 7.4 (without CaCl.sub.2 or MgCl.sub.2) were used. The solvents were added progressively (250 μL plus 250 μL plus 500 μL). Three homogenization cycles were applied after each addition of a solvent, consisting in a 10-second sonication in a Fisher Scientific FB 15050 ultrasonic bath and 5 seconds vigorous vortex agitation.
[0840] The final 0.2 mg/mL suspensions were transparent for all the three LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 samples, whether in water or in PBS, and slightly opaque but homogeneous for GLA.
[0841] 800 μL of each suspension were transferred to a disposable Eppendorf polystyrene semi-micro cuvette and DLS measurements were performed three times.
[0842] Results
[0843] As seen in
[0844] GLA in water also shows a monodisperse profile, but unlike LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1, the average particle size is of about 544 nm (
[0845] The same observations were made in PBS, where LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 have an average particle size of about 15.0 nm, 21.4 nm and 13.5 nm, respectively (
Conclusion
[0846] Previous studies have shown that LPS comprising a full O-antigen domain have lengths ranging from about 17±10 nm to about 37±9 nm. In absence of O-antigen domain, LPS have lengths ranging from about 3±2 nm to about 5±3 nm (Strauss et al., 2009. J Mol Recognit. 22(5):347-55).
[0847] In view of these elements, the data presented herein clearly demonstrate that LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 are soluble or easily dispersible. In water or PBS, they form stable suspensions of monodispersed nano-particles (micelles) whose size is less than 30 nm. No dispersing reagents like detergents, nor special mechanical or thermal treatments (ultrasound, microfluidic or extrusion) are necessary to obtain this result. This is in contrast to synthetic GLA which precipitates (as indicated by the slightly opaque samples) and forms aggregates of >500 nm and more. In fact, GLA aqueous formulations are produced in presence of an additional lipid using heating and microfluidic treatment. The above-mentioned properties of LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 and other similar products make them fairly easy to formulate as drug products. They can also be easily filtered at 200 nm and thus fulfill the major characteristics required for injectable drugs to avoid, e.g., bacterial contamination. These data are summarized in
Example 3
[0848] Biophysical Characterization
[0849] Materials and Methods
[0850] Thin Layer Chromatography (TLC)
[0851] TLC was carried out on glass HPTLC silica gel plates (Merck). 20 μg of LPS were deposited at the origin of the HPTLC plate, and chromatographed in a solvent mixture of isobutyric acid and 1 M ammonium hydroxide (5:3 v:v) as previously described (Caroff & Karibian, 1990. Appl Environ Microbiol. 56(6):1957-9). Products were visualized by charring (in an oven at 150° C. for 5 minutes) after spraying with 10% sulfuric acid in ethanol.
[0852] SDS-Polyacrylamide Gel Analysis of LPS
[0853] 15% poly-acrylamide gels were used, and 0.2 μg of LPS were loaded onto the 4% stacking gel. LPS samples preparation, electrophoresis process and Tsai & Frash Silver Nitrate coloration were performed as previously described (Laemmli, 1970. Nature. 227(5259):680-5; Tsai & Frasch, 1982. Anal Biochem. 119(1):115-9).
[0854] Matrix-Assisted Laser Desorption/Ionization (MALDI)-Time-of-Flight (TOF) Analysis
[0855] MALDI-TOF was performed as previously described (Chafchaouni-Moussaoui et al., 2011. Rapid Commun Mass Spectrom. 25(14):2043-8), in the linear mode with delayed extraction using a Perseptive Voyager STR (PE Biosystem, France) time-of-flight (TOF) mass spectrometer and/or on a Shimadzu Axima Performance system.
[0856] A suspension of lipid A (1 mg/mL) in chloroform:methanol:water (3:1.5:0.25, v:v:v) was desalted with a few grains of Dowex 50W-X8 (H+), and 1 μL was deposited on the target, mixed with 1 μL of gentisic acid (2,5-dihydroxybenzoic acid, DHB) matrix suspended at 10 μg/μL in the same solvent or in 0.1 M aqueous citric acid, and dried.
[0857] Analyte ions were desorbed from the matrix with pulses from a 337 nm nitrogen laser. Spectra were obtained in the negative-ion mode at 20 kV.
[0858] When LPS were analyzed directly, they were suspended in water and decationized in the same way, using the same matrix and conditions.
[0859] Results
[0860] The mass-to-charge ratios (m/z) of the different LPS molecular species described herein are reported in Table 2. They correspond to [M-H].sup.− ions of LPS molecular species, and their core and lipid A moieties fragments. The molecular weight (MW) corresponding to the lipid A moieties (i.e., without their R substituent) are also displayed with the corresponding structures in
TABLE-US-00002 TABLE 2 Calculated Observed masses masses [M − H].sup.− [M − H].sup.− Interpretation LPS B.sub.2 0.0 (Bordetella pertussis) (FIG. 1) 1332.62 1332.8 Lipid A 1558.98 1559.0 Lipid A 2293.04 2292.7 PS 2496.07 2496.9 PS 3853.02 3852.5 LPS 4056.05 4056.4 LPS LPS B.sub.2 0.1 (Bordetella pertussis) (FIG. 2) 872.03 871.7 Lipid A 1042.28 1042.6 Lipid A 1098.39 1098.4 Lipid A 1268.64 1269.1 Lipid A 1308.75 1309.1 Lipid A 1479.00 1479.5 Lipid A 2373.02 2372.4 PS 2496.07 2495.5 PS 3246.05 3246.0 LPS 3369.10 3369.6 LPS 3416.3 3417.4 LPS 3472.41 3472.8 LPS 3595.46 3595.4 LPS 3642.66 3643.3 LPS LPS B.sub.2 1.1 (Bordetella pertussis) (FIG. 3) 872.03 872.2 Lipid A 1098.39 1098.5 Lipid A 1308.75 1308.8 Lipid A 2180.85 2180.3 PS 2373.02 2372.4 PS 2496.07 2496.4 PS 3053.88 3053.6 LPS 3246.05 3246.6 LPS 3369.1 3370.1 LPS LPS B.sub.2 1.2 (Bordetella pertussis) (FIG. 4) 872.03 872.3 Lipid A 1082.03 1082.8 Lipid A 1308.75 1309.2 Lipid A 2293.04 2292.8 PS 2373.02 2373.1 PS 2496.07 2496.4 PS 3166.07 3165.6 LPS 3246.05 3246 LPS 3369.1 3369.7 LPS LPS SM.sub.1 1.2 (Salmonella enterica enterica serovar Minnesota R595) (FIG. 8) 872.03 872.2 Lipid A 1292.75 1292.9 Lipid A 1074.19 1073.3 LPS 1092.21 1092.0 LPS 1214.39 1213.0 LPS 1312.39 1311.2 LPS 1512.93 1512.8 LPS 1715.09 1714.3 LPS 1733.11 1733.1 LPS LPS ST.sub.1 1.2 (Salmonella enterica enterica serovar Typhimurium Ra) (FIG. 9) 872.03 872.1 Lipid A 1054.33 1054.4 Lipid A 1262.68 1262.7 Lipid A 1807.48 1807.8 PS 1930.53 1930.7 PS 2662.49 2662.2 LPS 2680.51 2680.4 LPS 2785.54 2785.4 LPS 2803.56 2803.0 LPS 2882.67 2882.9 LPS 2900.69 2901.5 LPS 3005.72 3006.0 LPS 3023.74 3024.2 LPS LPS E.sub.2 1.2 (Escherichia coli K12) (FIG. 9) 872.03 872.0 Lipid A 1054.33 1054.6 Lipid A 1262.68 1263.0 Lipid A 1796.46 1796.4 PS 1919.51 1919.5 PS 1999.49 1999.5 PS 2651.47 2651.3 LPS 2669.49 2669.4 LPS 2774.52 2774.6 LPS 2792.54 2792.1 LPS 2853.63 2854.8 LPS 2871.65 2872.0 LPS 2994.7 2995.0 LPS 3074.68 3075.0 LPS LPS W.sub.1 0.0 (Vitreoscilla filiformis) 1544.91 1544.8 Lipid A 1667.96 1668.1 Lipid A 1791.00 1791.3 Lipid A 3208.3 LPS 3331.6 LPS 3455.1 LPS 3524.0 LPS LPS W.sub.1 1.0 (Vitreoscilla filiformis) (FIG. 6) 839.79 839.8 Lipid A 1022.10 1022.3 Lipid A 1204.40 1205.1 Lipid A 1262.7 PS 1855.2 PS 2504.1 LPS 2667.7 LPS 2685.8 LPS 2877.9 LPS 3059.3 LPS LPS W.sub.1 1.2 (Vitreoscilla filiformis) (FIG. 7) 759.81 759.6 Lipid A 943.12 942.2 Lipid A 1125.30 1124.7 Lipid A 1262.1 PS 1854.4 PS 2047.6 PS 2405.8 LPS 2423.2 LPS 2568.8 LPS 2587.1 LPS 2606.2 LPS 2798.1 LPS 2943.4 LPS 2961.2 LPS 2980.3 LPS
[0861] MALDI-MS Spectra Analysis of MPL and the Salmonella enterica and Escherichia coli Candidates (
[0862] MPL
[0863] The MALDI-MS spectrum of MPL (monophosphoryl lipid A) is shown for comparison. MPL is a natural LPS derivative, originating from Salmonella Minnesota R595 Re-Type, from which the phosphate group at position C1 of the reducing end of the lipid A domain has been liberated by acidic hydrolysis, together with the core part of the LPS molecular species. It is used in different vaccine preparations by GSK.
[0864] As expected, the spectrum corresponding to MPL only shows peaks in the lower-mass region. A major peak signals at m/z 1729 and corresponds to a molecular species with six fatty acid chains, among which three 3OH—C.sub.14, one C.sub.16, one C.sub.14, one C.sub.12; and only one phosphate at position C4′ of the di-GlcN disaccharide. A peak at m/z 1491 corresponds to a molecular species without C.sub.16, and at m/z 1280 without C.sub.16 and C.sub.14.
[0865] In the present invention, as seen by comparison of the spectra given also in
[0866] LPS SM.sub.1 1.2
[0867] The spectrum corresponds to a Salmonella enterica enterica serovar Minnesota R595 LPS molecular species, modified by removal of the primary ester-linked fatty acids, followed by removal of the glycosidic lipid A phosphate group. Therefore, the main peak at m/z 1292 corresponds to a lipid moiety with one phosphate group and 4 fatty acids, namely two 3-OH—C.sub.14, one C.sub.16 and one C.sub.12 (Formula (III)(1)(d) of
[0868] LPS ST.sub.1 1.2
[0869] The spectrum corresponds to a Salmonella enterica enterica serovar Typhimurium Ra LPS molecular species, modified by removal of all ester-linked fatty acids for a fragment-ion signaling at m/z 872 (Formula (IV)(1)(a) of
[0870] LPS E.sub.2 1.2
[0871] The spectrum corresponds to Escherichia coli K12 LPS molecular species, modified by removal of ester-linked fatty acids and giving signals in the lipid A region corresponding to molecular species described for ST.sub.1 1.2 at m/z 872 (Formula (IV)(1)(a) of
[0872] The core structure was slightly different from ST.sub.1 LPS with one Kdo, four heptoses, four hexoses, and two phosphate groups for a molecular weight of 1797 u signaling at m/z 1796 for the corresponding ion. It comes at m/z 1876 plus phosphate or at m/z 1919 plus PEA and m/z 1999 for PPEA. With an additional lipid A moiety at m/z 872, the LPS molecular species appear at m/z 2669, m/z 2792 plus PEA, 2872 plus PPEA or at 2872 from 2651 (the anydro-core structure) plus Kdo and at m/z 2995 for additional PEA and m/z 3075 for an additional phosphate.
[0873] MALDI-MS Spectra Analysis of the Bordetella pertussis Candidates (
[0874] LPS B.sub.2 0.0
[0875] The negative-ion spectrum of LPS B.sub.2 0.0 shows three zones of signals: [0876] i. in the 3000-4200 Da region, corresponding to LPS molecular ions; [0877] ii. in the 1800-2800 Da region, corresponding to core oligosaccharide-fragment ions; and [0878] iii. in the 1200-1700 Da region, corresponding to lipid A-fragment ions.
[0879] In the 1200-1700 Da region, the two major peaks at m/z 1332 and 1559 correspond respectively to tetra- and penta-acylated species of the lipid A moiety of LPS B.sub.2 0.0, with Formulas (I)(0)(a) and (I)(0)(b) respectively, as shown in
[0880] The 1800-2800 Da region corresponds to the naturally heterogeneous core oligosaccharide moieties. The peak at m/z 2293 corresponds to the well-characterized anhydro-dodecasaccharide described in the literature, followed by its diphosphoryl and diphosphoryl-ethanolamine forms, signaling respectively at m/z 2453 and 2497.
[0881] In the 3000-4200 Da region, corresponding to native LPS molecular species, the peaks at m/z 4056 and 3852 correspond to the penta-acylated LPS with a dodecasaccharide core, with and without PPEA, respectively.
[0882] LPS B.sub.2 0.1
[0883] In the 1200-1700 Da region, peaks correspond to fragment-ions monophosphoryl lipid A moieties obtained by targeted chemical dephosphorylation of the native LPS B.sub.2 0.0 molecules. Dephosphorylation was aimed precisely at the level of the glycosidic phosphate of the lipid moiety, as described above, and correspond to small amounts of mono-phosphoryl penta-acyl lipid A at m/z 1479 (Formula (I)(1)(f) of
[0884] As described above for the native LPS B.sub.2 0.0 molecular species, the core oligosaccharide and LPS molecular species appear respectively in the 2000-2600 Da region, and higher masses up to 3000-3700 Da respectively. Compared to the native LPS B.sub.2 0.0 molecules described above, a decrease in m/z ratio is observed in the higher mass regions, due to the chemical modification (corresponding to the dephosphorylation at position C1 of the reducing end of the lipid A domain, and partial de-O-acylation).
[0885] LPS B.sub.2 1.1
[0886] Peaks in the higher mass region show the dephosphorylation at position C1 of the reducing end of the lipid A domain and additional removal of fatty acid chains, namely a 3-OH—C.sub.14 primary fatty acid chain, a 3-OH—C.sub.10 primary fatty acid chain and a C.sub.14 secondary fatty acid chain. These new species display signals at m/z 3053 and 3370. When the PEA group is removed from m/z 3370, the resulting species displays a signal at m/z 3246.
[0887] Peaks in the lower mass region are mainly represented by the mono-phosphoryl di-acyl lipid A at m/z 872 (Formula (I)(2)(a) of
[0888] LPS B.sub.2 1.2
[0889] Three main molecular species appear in the higher masses' region. The one at m/z 3165 corresponds to a modified LPS with a lipid A moiety at m/z 872 from which the glycosidic phosphate group and all ester-linked fatty acids were removed, plus a dodecasaccharide at m/z 2292 (Formula (I)(3)(a) of
[0890] MALDI-MS Spectra Analysis of the Vitreoscilla filiformis Candidates (
[0891] LPS W.sub.1 0.0
[0892] Two major peaks are observed in the lower mass region of the spectrum, corresponding to fragment-ions generated during the mass spectrometry process. One is observed at m/z 1544, corresponding to a lipid A molecular species containing two glucosamines, two phosphates, four primary hydroxydecanoic acids (3-OH—C.sub.10) and two secondary dodecanoic acids (C.sub.12) (Formula (II)(0) of
[0893] Peaks appearing in the higher mass region correspond to natural heterogeneity, they signal between m/z 3208 and 3577 corresponding to a core polysaccharide of undescribed structure at about 1800 Da, linked at position C6′ of the non-reducing end of the lipid A domain.
[0894] LPS W.sub.1 1.0
[0895] Peaks appearing in the lower mass region of the spectrum correspond to fragment-ions of the Vitreoscilla filiformis lipid A, from which were removed two ester-linked 3-OH—C.sub.10 and signaling at m/z 1205 (Formula (II)(1)(d) of
[0896] LPS W.sub.1 1.2
[0897] When the alkaline treatment is followed by the release of the glycosidic phosphate group by acidic treatment, peaks appear at m/z 1124 (Formula (II)(2)(d) of
Conclusion
[0898] LPS B.sub.2 0.0 corresponds to a native candidate (i.e., before chemical treatment by acidic hydrolysis and optionally by alkaline hydrolysis) displaying some heterogeneity due to the natural heterogeneity of the lipid A domain made of two main molecular species: a tetra- and a penta-acylated molecular species. Other bacterial strains and/or growth conditions can lead to either homogeneous tetra- or penta-acylated LPS molecular species. Heterogeneity at the level of the Bordetella pertussis oligosaccharide core is mainly due to the presence of nona- and dodeca-saccharide cores. Some heterogeneity is also observed in the oligosaccharide core region due to a partial substitution with a PEA group.
[0899] LPS B.sub.2 0.1 was obtained after chemical treatment by acidic hydrolysis of LPS B.sub.2 0.0, thus removing the glycosidic phosphate at position C1 of the reducing end of its lipid A domain, and partial removal of some ester-linked fatty acids.
[0900] LPS B.sub.2 1.1 was obtained after chemical treatment by acidic hydrolysis of LPS B.sub.2 0.0 and further by alkaline hydrolysis, which removed some fatty acids.
[0901] LPS B.sub.2 1.2 was obtained after de-O-acylation by alkaline treatment and acidic hydrolysis of LPS B.sub.2 0.0, thereby removing the glycosidic phosphate at position C1 of the reducing end of the de-O-acylated lipid A domain.
[0902] LPS W.sub.1 0.0, isolated from Vitreoscilla filiformis, contains a hexa-acylated lipid A moiety with mainly short chain fatty acid (four 3-OH—C.sub.10 and two C.sub.12). It is also characterized by the presence of phosphoryl-ethanolamine substituents on both phosphate groups, leading to some heterogeneity in addition to LPS classical heterogeneity due to fatty acid chains. This original structure is expected to react differently from the classical Escherichia coli or Salmonella enterica LPS molecules.
[0903] LPS W.sub.1 1.0 was obtained by alkaline treatment of native LPS W.sub.1 0.0, releasing two 3-OH—C.sub.10 and one C.sub.12. The resulting molecular species correspond to a major tri-acyl molecular LPS and a minor tetra-acyl one.
[0904] LPS W.sub.1 1.2 was obtained by targeted dephosphorylation of the lipid A glycosidic phosphate from the LPS W.sub.1 1.0 de-O-acylated LPS.
[0905] These examples of different LPS structures from different bacterial genera illustrate the capacities of the innovative chemical modifications applied to native LOS and LPS structures, to obtain new in situ dephosphorylated lipid A molecules remaining linked to their core oligosaccharide moieties.
[0906] It should be noted that dephosphorylation at position C1 of the reducing end of the lipid A domain was achieved with a very high specificity and efficacy, with more than 90% and even up to 99% of LPS molecular species being dephosphorylated at position C1 while retaining their core oligosaccharide moiety at position C6′ of the non-reducing end of their lipid A domain.
[0907] The new dephosphorylation process can now be applied to detoxify all LPS structures described so far, and leads to safe and powerful immunostimulant compounds. This process has been applied to a wide panel of different structures from Enterobacterial and non-Enterobacterial LPS, among which those described in Example 1 above.
Example 4
[0908] In Vitro Pyrogenic Activity of LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS W.sub.1 0.0
[0909] The pyrogenic activity of naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0, and of modified LPS B.sub.2 0.1 and LPS B.sub.2 1.1 was evaluated in vitro on human cells in monocytes activation tests (MAT−EEU/μg). The objectives were to demonstrate the relevance of selected LPS, and to position the naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0, and modified LPS B.sub.2 0.1 and LPS B.sub.2 1.1 relative to benchmark natural or synthetic TLR4 agonists used in human vaccines (MPL/GSK) or currently in clinical development (GLA/Merck), in terms of endotoxin activity.
[0910] Materials and Methods
[0911] Monocyte Activation Tests (MAT)
[0912] Standardized Monocyte Activation tests (MAT) based on human cryo-blood and with IL-6 readout (HaemoMAT Kit—Haemochrom Diagnostica GmbH) were performed on a wide range of concentrations (from 400 to 0.04 ng/mL) of the naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0 and modified LPS B.sub.2 0.1 and LPS B.sub.2 1.1, following the European Pharmacopeia (Ph. Eur. 9.8 section 2.6.30).
[0913] Briefly, human PBMC (CryoBlood) were cultured overnight in presence of different concentrations (400 to 0.04 ng/mL) of the tested molecules. The concentration of IL-6 (a pro-inflammatory cytokine) was then quantified in the supernatant by ELISA.
[0914] Equivalent endotoxin level (EEU/μg) was then estimated for each molecule. Samples were tested with a positive control (PPC) of 0.5 EU/mL (interference test). The solution was considered free of interfering factors (i.e., valid) if under the conditions of the test, the measured concentration of the endotoxin added to the test solution was within 50% to 200% of the known added endotoxin concentration, after subtraction of any endotoxin detected in the solution without added endotoxin.
[0915] Rabbit Pyrogen Tests (RPT)
[0916] Standardized Rabbit Pyrogen Test (RPT) were conducted according to European Pharmacopeia (Ph. Eur. 5.1.10). Product tested includes the naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0 and modified LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS B.sub.2 1.2, as well as a non-GMP equivalent of the GLA from Merck (Avanti Polar Lipids).
[0917] Briefly, groups of three rabbits, weighing 2.5 to 3 kg received one intravenous injection of the tested product in 1 mL/kg of body weight.
[0918] For each candidate, several dose ranges were tested from 0.1 to 1000 ng/kg. For each animal, the baseline rectal temperature was recorded. After 30 minutes, the rabbits were inoculated in the ear vein. Temperatures were recorded every 6.5 minutes for 3 hours. The rising temperature of the rabbit body temperature is that the highest body temperature measure within the 3 hours, minus the normal body temperature.
[0919] Result judgment: the dose of a given product was considered not pyrogen when the sum of the maximum temperature rises in the three individual rabbits (ΔT+) does not exceed 1.15° C. Several doses of each candidates were tested to define their MNPD corresponding to the highest dose of a given product that can be administered in rabbits without inducing febrile reaction.
[0920] Results
[0921] Monocyte Activation Tests (MAT)
[0922] Table 3 illustrates the pyrogenic activity of the naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0 and modified LPS B.sub.2 0.1 and LPS B.sub.2 1.1 evaluated in vitro on human cells in MAT assays (EEU/μg). These results are compared to the pyrogenicity of other natural or synthetic LPS as described in the literature, including: [0923] monophosphoryl lipid A from Salmonella enterica enterica serovar Minnesota R595 [MPLA] from InvivoGen (a non-GMP analog of MPL), [0924] glucopyranosyl lipid A [GLA] from Merck (a synthetic analog of MPL), and [0925] a natural non-detoxified LPS from Escherichia coli [O55:B5].
TABLE-US-00003 TABLE 3 LPS B.sub.2 0.0 B.sub.2 0.1 B.sub.2 1.1 W.sub.1 0.0 MPLA GLA O55:B5 MAT 995.5 952.5 891.5 867.0 1000.sup.1 15700.sup.2 5000-10000.sup.3 (EEU/μg) (±6.4) (±102) (±26.2) (±45.3) (±0.43) .sup.1Invivogen, MPLAs technical data sheet, version 19C19-MM, available at https://www.invivogen.com/sites/default/files/invivogen/products/files/niplas_tds.pdf .sup.2Misquith et al., 2014. Colloids Surf B Biointerfaces. 113: 312-319 .sup.3Weary et al., 1980. Appl Environ Microbiol. 40(6): 1148-51
[0926] The outcome of MAT assays demonstrated that the pyrogenic activity of the tested LPS is in the same range, but demonstrated that type B.sub.2 LPS (from Bordetella pertussis) are slightly more pyrogen than W0.1 (from Vitreoscilla filiformis); and that LPS B.sub.2 1.1 (presenting higher level of detoxification) is less pyrogen than LPS B.sub.2 0.1 and LPS B.sub.2 0.0 (Table 3).
[0927] This allows a ranking of the naturally-occurring and modified LPS molecular species based on their estimated pyrogenic activity in vitro: [0928] LPS W.sub.1 0.0>LPS B.sub.2 1.1>LPS B.sub.2 0.1>LPS B.sub.2 0.0.
[0929] Rabbit Pyrogen Tests (RPT)
[0930] Set of Rabbit Pyrogen Tests (RPT) led to estimate the maximum non-pyrogenic dose (MNPD) of all naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0 and modified LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS B.sub.2 1.2 (Table 4).
TABLE-US-00004 TABLE 4 GSK1795- EPS W.sub.1 0.0 B.sub.2 0.0 B.sub.2 0.1 B.sub.2 1.1 B.sub.2 1.2 MPL.sup.1 GLA.sup.2 091.sup.3 055:B.sup.4 MNPD 0.1 5 12.5 300 450 320 34 2.5 1 (ng/Kg) .sup.1Carl R. Alving, Immunobiol. 1993 .sup.2Internally generated data .sup.3GlaxoSmithKline. A 2-Part Randomized, Double-Blind (Sponsor-Unblinded), Placebo-Controlled, Ascending Dose and Parallel Group Study of TLR4 Agonist (GSK1795091) Administered to Healthy Subjects. 2017 .sup.4Greisman SE et al., Proc Soc Exp Biol Med. 1969
[0931] Data demonstrated that among naturally-occurring LPS, the MNPD of the type W.sub.1 candidate is 50 times higher than the MNPD of the type B.sub.2, demonstrating that the naturally-occurring LPS W.sub.1 0.0 is significantly more pyrogen than the naturally-occurring LPS B.sub.2 0.0. Among type B.sub.2 LPS, the MNPD of the naturally-occurring LPS B.sub.2 0.0 was also found to be 2.5×, 60× and 90× higher than those of the modified LPS candidates LPS B.sub.2 0.0, LPS B.sub.2 1.1 and LPS B.sub.2 1.1 respectively.
[0932] This demonstrates an inverse correlation between the detoxification level and the pyrogenic activity of the modified LPS. The MNPD of our native and modified LPS were compared to those evaluated or described in the literature for naturally-occurring (not modified) LPS and LPS derivatives (natural or synthetic) currently used in human vaccines (MPL/GSK) in clinical development (GLA/Merck, and GSK1795091/GSK).
[0933] The naturally-occurring LPS B.sub.2 0.0 was found to present a MNPD 2 to 5 times higher than a benchmark natural LPS from Escherichia coli (O55:B5), or the synthetic LPS from GSK (GSK179509), but 8 times and 64 times lower than a non-GMP equivalent of the GLA and the MPL respectively.
[0934] In contrast, the type B.sub.2 LPS with the highest level of modification (LPS B.sub.2 1.2) was found to present a MNPD 450 times higher than a naturally-occurring LPS from Escherichia coli (O55:B5), 13.2 times and 180 times higher than the GLA and GSK1795091 respectively, and substantially higher (1.12 times) than MPL.
Conclusion
[0935] Results from in vitro pyrogen tests (MAT assays) highlight the relevance of our detoxification process to reduce the pyrogenic activity of native LPS. They also allow to position the naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0 and modified LPS B.sub.2 0.1 and LPS B.sub.2 1.1 relative to commercialized natural or synthetic LPS whose pyrogenic activity, evaluated by in vitro pyrogen tests, have been described in the literature (Endotoxin Level—EU/μg). This includes GLA from Merck (a synthetic TLR4 agonist), MPLA from InvivoGen (an analog of MPL from GSK), and a natural, non-detoxified, LPS from Escherichia coli (O55:B5).
[0936] This demonstrates that in term of pyrogenicity, the naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0 and modified LPS B.sub.2 0.1 and LPS B.sub.2 1.1 are close to MPLA and thus of MPL (GSK), currently used as adjuvant in several commercialized vaccines, but they are significantly less pyrogenic than the synthetic TLR4 agonists GLA (Merck) currently in clinical development, or natural, non-detoxified, LPS from E. Coli O55:B5.
[0937] Data from the in vivo pyrogen test (RPT assay) provide proof-of-concept of the capacity of our detoxification process to significantly reduce the pyrogenic activity of native LPS. They also demonstrated that the type B LPS (B.sub.2 1.2), presenting the highest level of modification, presents a pyrogenic activity significantly reduced compared to a commercialized natural LPS and benchmark LPS derivatives currently on the market (MPL—GSK) or in clinical development (GLA—Merck and GK1795091—GSK).
Example 5
[0938] Pre-Clinical Proof-of-Concept Study: Therapeutic Efficacy of LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS W.sub.1 0.0, in combination with a monoclonal antibody
[0939] Therapeutic efficacy of naturally-occurring LPS B.sub.2 0.0 and LPS W.sub.1 0.0, and of modified LPS B.sub.2 0.1 and LPS B.sub.2 1.1, alone or in combination with rituximab (an anti-CD20 monoclonal antibody), was evaluated in relevant tumor graft animal models. The objectives were to demonstrate the efficiency of selected LPS in a context of pre-existing tumor, to compare it with a synthetic TLR4 agonist (glucopyranosyl lipid A, GLA), and to demonstrate the relevance of their synergistic combination with monoclonal antibodies.
[0940] Materials and Methods
[0941] The therapeutic efficacy of native (LPS B.sub.2 0.0 and LPS W.sub.1 0.0) and modified (LPS B.sub.2 0.1 and LPS B.sub.2 1.1) LPS, alone or in combination with rituximab, was analyzed in vivo against a human B-lymphoma tumor model (RL cells) using tumor rejection assays with a therapeutic setting in mice.
[0942] Cohort of CB17-SCID mice were engrafted subcutaneously in the abdominal right flank with 5×10.sup.6 human B-lymphoma cells (RL cells). When the tumor volume had reached 100 mm.sup.3 (at day 20), mice were randomized (n=6 mice/group), and the first treatment was administered.
[0943] Mice were treated once a week for 3 weeks (at days 20, 27 and 34) with LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 or LPS W.sub.1 0.0, used alone or in combination with rituximab (Genentech), an anti-human CD20 mAb.
[0944] Groups of mice treated with GLA (Merck/Immune Design Corp.) administered either intravenously or intraperitoneally at 0.5 mg/kg, alone or in combination with rituximab, were included for comparison.
[0945] LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 or LPS W.sub.1 0.0 were administered intravenously at a dose of 0.5 mg/kg. Rituximab was administered intraperitoneally at a dose of 30 mg/kg.
[0946] Tumor-bearing mice injected with PBS (control) or rituximab alone were used as controls.
[0947] The tumor volume was measured twice a week (length x width) with a caliper. The tumor volume was determined using the formula:
Tumor volume=4/3×π×r.sup.3
[0948] All mice were raised in specific pathogen-free (SPF) environment with free access to standard food and water.
[0949] Results
[0950] Treatment with rituximab alone was found to significantly impair the growth of established RL tumor cells compared to non-treated mice. Treatment with either of the native (LPS B.sub.2 0.0 or LPS W.sub.1 0.0) LPS molecular species alone, the modified (LPS B.sub.2 0.1 and LPS B.sub.2 1.1) LPS molecular species alone, or GLA alone, did not show any impairment in the growth of RL tumor cells (
[0951] However, the combination of rituximab with either of the native (LPS B.sub.2 0.0 or LPS W.sub.1 0.0) or the modified (LPS B.sub.2 0.1 and LPS B.sub.2 1.1) LPS molecular species was found to lead to a more significant impairment of tumor growth compared to each single modality treatments (
[0952] A better therapeutic efficacy was also observed in the group of mice treated with rituximab in combination with LPS B.sub.2 0.1 or LPS W.sub.1 0.0, than with the synthetic TLR4 agonist GLA injected intraperitoneally (
[0953] Finally, while only 17% (1/6) of tumor regression were observed in the group mice treated with rituximab alone, 50% to 67% of complete and sustained regression of established RL tumor cells were observed in groups of mice treated with the native or modified LPS (50% for LPS B.sub.2 0.0 and LPS B.sub.2 1.1; 67% for LPS B.sub.2 0.1 and LPS W.sub.1 0.0) (
[0954] In contrast, no tumor regression was observed in the groups of mice treated with the native or modified LPS alone, or with PBS (
[0955] Complete tumor regression (50%) was also observed in the group of mice treated with rituximab in combination with GLA when injected intraperitoneally.
[0956] However, all the mice treated with GLA intravenously had to be euthanized following the first injection as they displayed a severely altered aspect and behavior: lethargy, hunched over themselves, bristle hair, and fast breathing. Altogether these symptoms were indicative of a strong and fast toxicity of the GLA when injected in the blood stream.
[0957] These altered aspects and behavior were not observed with LPS B.sub.2 0.0, LPS B.sub.2 0.1, LPS B.sub.2 1.1 or LPS W.sub.1 0.0 administered intravenously.
Conclusion
[0958] These results demonstrate the capacity of the native (LPS B.sub.2 0.0 or LPS W.sub.1 0.0) and modified (LPS B.sub.2 0.1 and LPS B.sub.2 1.1) LPS molecular species to significantly boost the therapeutic efficacy of rituximab against an established human B-lymphoma model.
[0959] Results also demonstrate that, alone or in combination with rituximab, both the native (LPS B.sub.2 0.0 or LPS W.sub.1 0.0) and modified (LPS B.sub.2 0.1 and LPS B.sub.2 1.1) LPS molecular species are as least as effective as GLA against a human B-lymphoma tumor model. However, as demonstrated in Example 2, GLA suffers drawbacks, in particular in terms of precipitation and aggregation, which is not observed with the tested LPS molecular species.
[0960] Finally, these results demonstrate that, in contrast to GLA, the systemic injection of the native (LPS B.sub.2 0.0 or LPS W.sub.1 0.0) and modified (LPS B.sub.2 0.1 and LPS B.sub.2 1.1) LPS molecular species by intravenous route is well-tolerated in mice, tending to demonstrate that these LPS molecular species are less toxic than the synthetic TLR4 agonist GLA.
Example 6
[0961] Adjuvant Activity of LPS A.sub.1 0.1 and LPS B.sub.2 0.1 in a Preclinical Mouse Vaccination Model
[0962] The capacity of the LPS A.sub.1 0.1 and LPS B.sub.2 0.1 to enhance the humoral immune response to the ovalbumin antigen (OVA) was evaluated in a preclinical mouse vaccination model.
[0963] The objectives were to demonstrate the adjuvant activity of modified LPS molecular species, and to compare it with the benchmark adjuvants Alhydrogel® (Alum) and MPLA—an analog of the TLR4 agonist MPL (GSK).
[0964] Materials and Methods
[0965] A cohort of BALB/c mice (n=5 mice/group) was immunized intramuscularly with OVA antigen (Invivogen—10 μg/dose or 2 μg/dose) formulated with either LPS A.sub.1 0.1 (25 μg/dose) or LPS B.sub.2 0.1 (25 μg/dose), and received a boost intramuscularly 14 days later (D14).
[0966] Groups of mice immunized with OVA alone (2 or 10 μg/dose) were used as negative controls.
[0967] Groups of mice immunized (prime/boost) with OVA formulated with MPLA (Invivogen—25 μg/dose) or Alum (100 μg/dose) were included for comparison.
[0968] Serum samples were collected on days 28 and 39 from each mouse, and the levels of anti-OVA IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA).
[0969] Results Serum anti-OVA IgG levels, both at days 28 and 39, were significantly higher in mice treated with LPS A.sub.1 0.1 and LPS B.sub.2 0.1 compared to mice treated with OVA without adjuvant (LPS A.sub.1 0.1 vs No Adjuvant: P=0.008 and P=0.0025; LPS B.sub.2 0.1 vs No Adjuvant: P=6.44×10.sup.−7 and P=0.006) (
[0970] Serum anti-OVA IgG levels, both at days 28 and 39, were also significantly higher in mice treated with LPS A.sub.1 0.1 compared to mice treated with Alum or MPLA (LPS A.sub.1 0.1 vs Alum: P=0.018 and P=0.008; LPS A.sub.1 0.1 vs MPLA: P=0.036 and P=0.017) (
Conclusion
[0971] Results demonstrate the capacity of the LPS A.sub.1 0.1 and LPS B.sub.2 0.1 to boost the humoral immune response to OVA in a preclinical mouse vaccination model, and thus demonstrate their adjuvant activity.
[0972] Results also demonstrate that the LPS A.sub.1 0.1 presents an adjuvant activity higher than Alum and MPLA in this preclinical vaccination model.
Example 7
[0973] Veterinary Applications
[0974] LPS SM.sub.1 0.0, a naturally-occurring LPS molecular species from Salmonella enterica enterica serovar Minnesota R595 (Re chemotype) with Formula (XII)(0), as shown in
[0975] In view of an intended veterinary use, LPS SM.sub.1 0.0 was extracted but not purified upon production. The bacterial lysate, comprising LPS SM.sub.1 0.0 and other co-extracted bacterial components (e.g., proteins and the like), was treated to obtain detoxified LPS SM.sub.1 V1 with Formula (XII)(1), as shown in
[0976] LPS SM.sub.1 V1 has lost several fatty chains, as well as its 4-amino-4-deoxy-L-arabinose group (Ara4pN) at position C4′ of the non-reducing end of its lipid A domain. A majority (≈80-90%) of LPS SM.sub.1 V1 maintained its phosphate group at position C4′ of the non-reducing end of its lipid A domain, while the remaining 10-20% lost this phosphate group. LPS SM.sub.1 V1 thus comprises a mix of LPS molecular species with and without phosphate group at position C4′.
[0977] The bacterial lysate comprising LPS SM.sub.1 V1 was used for DLS aggregate size analysis, biophysical characterization, pyrogenic activity assays, and preliminary in vivo tolerance tests.
[0978] LPS SM.sub.1 V1 was diluted in PBS pH 7.5 at 50 nmol/mL, and filtered through a 200-nm filter to remove large debris and aggregates. No nucleic acid contaminants and 2-4 μg/mL of protein contaminants were measured in the sample.
[0979] DLS measurements (carried out as described in Example 2) showed that the sample was soluble or easily dispersible, forming stable suspensions of monodispersed nano-particles (micelles) whose size ranged from about 6 nm to about 18 nm.
[0980] LC-MS/MS and MALDI-TOF analysis was carried out to confirm the structure of LPS SM.sub.1 V1 as compared to the native species LPS SM.sub.1 0.0 (data not shown).
[0981] Pyrogenic activity assays (carried out as described in Example 4) have shown a MAT (EU/mL) of around 7×10.sup.5; using the well-known limulus amebocyte lysate (LAL) test, a very low activity of around 50 EU/mL was measured (i.e., about 0-1 EU/nmol).
[0982] 100 μL of this sample were administered intravenously to mouse, and their tolerance was followed-up for several days (general condition and weight were observed). On day 1 following administration, the tested mice lost in average 13.4% of their weight, and displayed hair bristling out. Quickly on day 2 after administration, the tested mice starting to get back in shape, until day 3 when the tested mice were healthy. This demonstrates that, in contrast to GLA, the systemic injection of LPS SM.sub.1 V1 by intravenous route is well-tolerated in mice.
Example 8
[0983] Production of Chemically-Modified LPS Molecular Species
[0984] In the following, the figures “x.Math.y” following the LPS name refer to: [0985] i. first figure (“x”): where 0 means no alkaline treatment, and 1 means one alkaline treatment; [0986] ii. second figure (“y”): where 0 means no acidic hydrolysis treatment, 1 means one acidic hydrolysis treatment with a strong acid (i.e., with pK.sub.a<0) in aqueous solution, 2 means one acidic hydrolysis treatment with a strong acid (i.e., with pK.sub.a<0) in organic solution; 3 means one acidic hydrolysis treatment with a strong acid (i.e., with pK.sub.a<0) in organic solution in presence of an alcohol; and 4 means one acidic hydrolysis treatment with a weak acid (i.e., with pK.sub.a>0) in aqueous solution.
[0987] Structures
[0988] “LPS B.sub.2 0.0”, as used herein, refers to a mix of the two naturally-occurring LPS molecular species from Bordetella pertussis with Formulas (I)(0)(a) and (I)(0)(b), as shown in
[0989] “LPS X 0.0”, as used herein, refers to the naturally-occurring LPS molecular species from various Enterobacteria with Formula (XIII)(0)(a) and (XIII)(0)(b), as shown in
[0990] Production & Purification
[0991] LPS B.sub.2 0.0 and LPS X 0.0 were produced and purified using conventional methods, in particular the method described in U.S. Pat. No. 8,137,935.
[0992] Chemical Modification
[0993] After purification, the LPS molecular species were chemically treated by acidic hydrolysis at a temperature below 85° C.; and/or by alkaline hydrolysis, as indicated by the figures “x.Math.y” following the LPS name. If both, alkaline hydrolysis was carried out preferably before acidic hydrolysis.
[0994] The innovative process is that acidic hydrolysis at a temperature below 85° C. targets dephosphorylation of the lipid A glycosidic phosphate, which is removed in situ, in the complete LPS molecular species, i.e., without splitting it in its lipid A and polysaccharide moieties.
[0995] It follows from these treatments several LPS molecular species as defined below:
[0996] From B.sub.2 0.0 (formulas (I)(0)(a) and (I)(0)(b) shown in
[1006] From X 0.0 (formulas (XIII)(0)(a) and (XIII)(0)(b) shown in
Example 9
[1016] Biophysical Characterization
[1017] Materials and Methods
[1018] Thin Layer Chromatography (TLC)
[1019] TLC was carried out on glass HPTLC silica gel plates (Merck). 20 μg of LPS were deposited at the origin of the HPTLC plate, and chromatographed in a solvent mixture of isobutyric acid and 1 M ammonium hydroxide (5:3 v:v) as previously described (Caroff & Karibian, 1990. Appl Environ Microbiol. 56(6):1957-9). Products were visualized by charring (in an oven at 150° C. for 5 minutes) after spraying with 10% sulfuric acid in ethanol.
[1020] SDS-Polyacrylamide Gel Analysis of LPS
[1021] 15% poly-acrylamide gels were used, and 0.2 μg of LPS were loaded onto the 4% stacking gel. LPS samples preparation, electrophoresis process and Tsai & Frash Silver Nitrate coloration were performed as previously described (Laemmli, 1970. Nature. 227(5259):680-5; Tsai & Frasch, 1982. Anal Biochem. 119(1):115-9).
[1022] Matrix-Assisted Laser Desorption/Ionization (MALDI)-Time-of-Flight (TOF) Analysis
[1023] MALDI-TOF was performed as previously described (Chafchaouni-Moussaoui et al., 2011. Rapid Commun Mass Spectrom. 25(14):2043-8), in the linear mode with delayed extraction using a Perseptive Voyager STR (PE Biosystem, France) time-of-flight (TOF) mass spectrometer and/or on a Shimadzu Axima Performance system.
[1024] A suspension of lipid A (1 mg/mL) in chloroform:methanol:water (3:1.5:0.25, v:v:v) was desalted with a few grains of Dowex 50W-X8 (H.sup.+), and 1 L was deposited on the target, mixed with 1 μL of gentisic acid (2,5-dihydroxybenzoic acid, DHB) matrix suspended at 10 μg/μL in the same solvent or in 0.1 M aqueous citric acid, and dried.
[1025] Analyte ions were desorbed from the matrix with pulses from a 337 nm nitrogen laser. Spectra were obtained in the negative-ion mode at 20 k
[1026] When LPS were analyzed directly, they were suspended in water and decationized in the same way, using the same matrix and conditions.
[1027] Results
[1028] The mass-to-charge ratios (m/z) of the different LPS molecular species described herein are reported in Table 5. They correspond to [M-H].sup.− ions of LPS molecular species, and their core and lipid A moieties fragments.
TABLE-US-00005 TABLE 5 Calculated Observed masses masses [M − H].sup.− [M − H].sup.− Interpretation LPS B.sub.2 1.2 (Bordetella pertussis) (FIG. 4) 872.03 872.0 Lipid A1 1082.03 1082.3 Lipid A2 1308.75 1308.7 Lipid A3 2293.04 2292.8 PS1 2373.02 2373.1 PS2 3166.07 3167.5 LPS = Lipid A1 + PS1 3246.05 3247.3 LPS = Lipid A1 + PS2 3376.07 3375.8 LPS = Lipid A2 + PS1 3456.07 3456.2 LPS = Lipid A2 + PS2 3602.79 3600.7 LPS = Lipid A3 + PS1 3682.79 3681.4 LPS = Lipid A3 + PS2 LPS B.sub.2 1.3 (Bordetella pertussis) (FIG. 32) 872.03 Non detected Lipid A1 900.1 Non detected Lipid A1′ = Lipid A1 + Ethyl 1082.03 1082.2 Lipid A2 1110.4 1110.5 Lipid A2′ = Lipid A2 + Ethyl 1308.75 1308.8 Lipid A3 1336.8 1336.7 Lipid A3′ = Lipid A3 + Ethyl 2293.04 2292.9 PS1 2373.02 2373.0 PS2 3166.07 3166.8 LPS = Lipid A1 + PS1 3194.2 3195.4 LPS = Lipid A1′ + PS1 3376.07 3376.5 LPS = Lipid A2 + PS1 3456.07 3456.2 LPS = Lipid A2 + PS2 3484.2 3485.2 LPS = Lipid A2′ + PS2 3630.9 3629.9 LPS = Lipid A3′ + PS1 3682.79 3681.4 LPS = Lipid A3 + PS2 LPS B.sub.2 0.4 (Bordetella pertussis) (FIG. 31) 1479.0 1479.3 Lipid A (1P, 2G1cN, 5FA) 2293.04 2292.7 PS 2908.9 2908.2 LPS = LipA(0P, 1GlcN, 2FA)* + PS 3466.6 3466.6 LPS = LipA(0P, 2GlcN, 4FA)* + PS 3693.0 3692.2 LPS = LipA(0P, 2GlcN, 5FA)* + PS 3773.0 3772.8 LPS = LipA(1P, 2GlcN, 5FA)* + PS LPS B.sub.2 1.4 (Bordetella pertussis) (FIG. 33) 2293.04 2292.7 PS 2908.9 2908.6 LPS = LipA(0P, 1GlcN, 2FA)** + PS 3086.0 3085.8 LPS = LipA(0P, 2GlcN, 2FA)** + PS 3296.0 3296.1 LPS = LipA(0P, 2GlcN, 3FA)** + PS 3522.8 3522.5 LPS = LipA(0P, 2GlcN, 4FA)** + PS *LipA(0P, 1GlcN, 2FA): non-phosphorylated mono-glucosamine di-acyl lipid A *LipA(0P, 2GlcN, 4FA): non-phosphorylated di-glucosamine tetra-acyl lipid A *LipA(0P, 2GlcN, 5FA): non-phosphorylated di-glucosamine penta-acyl lipid A *LipA(1P, 2GlcN, 5FA): mono-phosphoryl di-glucosamine penta-acyl lipid A **LipA(0P, 1GlcN, 2FA): non-phosphorylated mono-glucosamine di-acyl lipid A **LipA(0P, 2GlcN, 2FA): non-phosphorylated di-glucosamine di-acyl lipid A **LipA(0P, 2GlcN, 3FA): non-phosphorylated di-glucosamine tri-acyl lipid A **LipA(0P, 2GlcN, 2FA): non-phosphorylated di-glucosamine di-acyl lipid A
[1029] MALDI-MS Spectra Analysis of LPS B.sub.2 1.2, LPS B.sub.2 1.3, LPS B.sub.2 0.4 and LPS B.sub.2 1.4 (
[1030] LPS B.sub.2 1.2
[1031] Three main lipid A related fragments are observed at m/z: 872, 1082, 1308, corresponding to mono-phosphoryl di-, tri- and tetra-acyl lipid A molecular species. Two major PS related fragments are observed at m/z 2293 (PS1) and 2373 (PS2), corresponding to dodecasaccharide cores with respectively non-substituted Kdo and Kdo substituted with a phosphate group. LPS molecular ions correspond to different combinations of these lipid A and PS structures, they are observed at m/z 3167, 3247, 3376, 3456, 3601 and 3681.
[1032] LPS B.sub.2 1.3
[1033] Several peaks common with the modified LPS B.sub.2 1.2 spectrum are observed, e.g., at m/z 1082 and 1308 for lipid A related fragments, at m/z 2293 (PS1) and 2373 (PS2) for PS related fragments and at m/z 3167, 3376, 3456, 3681 for LPS molecular ions. Additional peaks are observed corresponding to molecular species substituted with an ethyl group at position C1 of the reducing end of their lipid A domain (i.e., at +28 mass units relatively to peaks of non-substituted molecular species). These new peaks are observed at m/z 1110 and 1336 for lipid A related fragments and at m/z 3195, 3485 and 3629 for LPS molecular ions.
[1034] LPS B.sub.2 0.4
[1035] Molecular species detected in modified LPS B.sub.2 0.4 contain either mono-phosphoryl lipid A or non-phosphorylated lipid A (see the phosphate group at position C4 of the non-reducing end of the lipid A domain in parenthesis in
[1036] LPS B.sub.2 1.4
[1037] Molecular species detected in modified LPS B.sub.2 1.4 contain predominantly non-phosphorylated lipid A (see the phosphate group at position C4 of the non-reducing end of the lipid A domain in parenthesis in
Example 10
[1038] In Vivo Pyrogenic Activity of LPS B.sub.2 1.2 Versus LPS B.sub.2 1.3
[1039] The pyrogenic activity of modified LPS B.sub.2 1.2 and LPS B.sub.2 1.3 was evaluated in vivo using the rabbit pyrogen test (RPT), as described in section 5.1.10 of the European Pharmacopeia. Both LPS were administered at the same dose of 450 ng/kg. Two experiments were performed for each modified LPS, with 3 rabbits/experiment (A1, A2 and A3).
[1040] Results
[1041] Table 6 summarizes the temperature increase (individual and sum of three animals) observed for modified LPS B.sub.2 1.2 and LPS B.sub.2 1.3 in two RPT experiments.
TABLE-US-00006 TABLE 6 Temperature increase Modified LPS Experiment 1 Experiment 2 B.sub.2 1.2 A1 0.75° C. 1.1° C. A2 1.05° C. 0.70° C. A3 1.10° C. 0.70° C. Sum 2.7° C. 2.5° C. B.sub.2 1.3 A1 0.50° C. 0.45° C. A2 0.25° C. 0.65° C. A3 0.50° C. 1.00° C. Sum 1.25° C. 2.1° C.
[1042] It follows from these results that the additional substitution with an ethyl group at position C1 of the reducing end of the lipid A domain in LPS B.sub.2 1.3 leads to substantial decrease in pyrogenicity when compared with LPS B.sub.2 1.2 in which position C1 of the reducing end comprises an unsubstituted hydroxyl group (as seen by the reduced increase in temperature in all three rabbits, for each duplicate experiment).
Example 11
[1043] Therapeutic Efficacy of Modified LPS as Monotherapy on a Syngeneic Mouse Model of Colorectal Carcinoma
[1044] Therapeutic efficacy of modified LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS B.sub.2 1.3 was evaluated on a syngeneic mouse model of colorectal carcinoma (MC38). The objectives were to demonstrate and compare the efficacy of selected modified LPS as stand-alone treatment against cancer.
[1045] Materials and Methods
[1046] The therapeutic efficacies of modified LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS B.sub.2 1.3 were analyzed in vivo against a mouse model of colorectal carcinoma (MC38 cells) using tumor rejection assays with a therapeutic setting.
[1047] C57BL/6 mice (4- to 6-week-old) were injected subcutaneously (s.c.) into the right flank with 2×10.sup.6 MC38 tumor cells. When tumor volumes reached 150 mm.sup.3 (day 6), mice were randomized into LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS B.sub.2 1.3 groups (n=7 mice per group).
[1048] A group of mice injected with PBS was used as control.
[1049] Modified LPS were administrated intravenously into the tail vein, once a week at 0.5 mg/kg/dose.
[1050] Tumor growth was monitored twice a week by caliper measurement and determined using the formula:
Tumor volume=4/3×π×r.sup.3
[1051] Results
[1052]
[1053] Treatment with modified LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS B.sub.2 1.3 was found to significantly impair the growth of established MC38 tumor cells compared to control mice (
Conclusion
[1054] These results provide proof-of-concept of the therapeutic efficacy of the modified LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS B.sub.2 1.3 as a monotherapy against a syngeneic mouse model of colorectal carcinoma and suggest that the modified LPS B.sub.2 1.3 is slightly more effective than LPS B.sub.2 0.1 and LPS B.sub.2 1.1.
Example 12
[1055] Therapeutic Efficacy of Modified LPS as Monotherapy on an Orthotopic Rat Model of Osteosarcoma
[1056] Therapeutic efficacy of a modified LPS as monotherapy was evaluated against an orthotopic rat model of osteosarcoma (Osa) and was compared to a chemotherapeutic agent (Doxorubicin) commonly used as standard of care in this indication.
[1057] Materials and Methods
[1058] The therapeutic efficacy of the modified LPS B.sub.2 1.3 was analyzed in vivo against an orthotopic rat model of osteosarcoma (Osa) using tumor rejection assays with a therapeutic setting.
[1059] Sprague-Dawley (SD) rats were engrafted with Osa tumor cells in a paratibial position (left posterior tibia) after a periosteal abrasion. When tumors reached an average tumor volume of 150 mm.sup.3, rats (n=3 to 8/group) were randomized into LPS B.sub.2 1.3, Doxorubicin (Doxo), or PBS (Control) groups.
[1060] LPS B.sub.2 1.3 was administered intravenously into the tail vein, twice a week for 2 weeks at 0.25 mg/kg.
[1061] Doxorubicin was administered intraperitoneally twice a week for 2 weeks at 1 mg/kg.
[1062] Tumor growth and survival were monitored twice a week. Tumor volumes V were assessed according to the following formula:
V=0.5*L*C
with L the longest and C the shortest tumor diameter.
[1063] Results
[1064] Treatment with modified LPS B.sub.2 1.3 as monotherapy was found to significantly impair the growth of established Osa tumor cells compared to rats that received doxorubicin (P=0.03 on day 28 post-tumor challenge) or PBS as a control (P=0.05 on day 31 post-tumor challenge) (
[1065] Treatment with modified LPS B.sub.2 1.3 was also found to induce complete tumor regressions in 37.5% of the treated rats (complete responses (CR) on day 31—
[1066] This was associated with an increased significant improvement in the survival of animals. Approximately 90% of animals that received LPS B.sub.2 1.3 were alive on day 31. By contrast, only 40% of animals that received doxorubicin and 0% of animals that received PBS as a control were alive on day 31 (
[1067] No significant difference in tumor growth and percentages of complete responses were observed among the groups of rats that received doxorubicin or PBS as a control, although percentage of animals alive on day 31 were higher in the doxorubicin group.
Conclusion
[1068] These results provide preclinical proof-of-concept of the high therapeutic efficacy of the modified LPS B.sub.2 1.3 as standalone treatment for osteosarcoma and demonstrate that the modified LPS B.sub.2 1.3 is significantly more effective than doxorubicin, a standard-of-care in this indication.
Example 13
[1069] Synergistic Antitumor Effect of Modified LPS with Immune Checkpoint Inhibitors
[1070] Therapeutic efficacy of the modified LPS B.sub.2 1.3 as monotherapy or in combination with an immune checkpoint inhibitor (ICI) (anti-mouse PD-1 monoclonal antibody, clone RMP1-14) was evaluated on a syngeneic mouse model of colorectal carcinoma (MC38). The objectives were to demonstrate the capacity of the modified LPS B.sub.2 1.3 to boost the therapeutic efficacy of an ICI.
[1071] Materials and Methods
[1072] The therapeutic efficacy of the modified LPS B.sub.2 1.3, alone and in combination with an ICI, was analyzed in vivo against a mouse model of colorectal carcinoma (MC38 cells) using tumor rejection assays with a therapeutic setting.
[1073] C57BL/6 mice (4- to 6-week-old) were injected subcutaneously (s.c.) into the right flank with 2×10.sup.6 MC38 tumor cells. When tumor volumes reached 150 mm.sup.3 (day 6), mice were randomized, and the first treatment was administered.
[1074] Groups of mice (n=7 per group) were treated once a week for 2 weeks with the modified LPS B.sub.2 1.3 (0.5 mg/kg in i.v.) alone or in combination with an anti-mouse PD-1 monoclonal antibody (clone RMP1-14—12.5 mg/kg in i.p.).
[1075] Groups of mice administered with the anti-mouse PD-1 monoclonal antibody alone, or with PBS, were used as controls.
[1076] Tumor growth was monitored twice a week by caliper measurement and determined using the formula:
Tumor volume=4/3×π×r.sup.3
[1077] Mice from anti-PD-1 (n=2) and LPS B.sub.2 1.3+anti-PD-1 (n=2) groups, which totally eradicated MC38 tumor cells in primary responses, were rechallenged with live 2×10.sup.6 MC38 cells.
[1078] Age-matched naive mice (n=3) were used as control.
[1079] Tumor growth was monitored twice a week by caliper measurement.
[1080] Results
[1081]
[1082]
[1083] Treatment with the modified LPS B.sub.2 1.3 and the anti-mouse PD-1 mAb as monotherapies were found to significantly impair the growth of established MC38 tumor cells as compared to control mice (
[1084] The combination of LPS B.sub.2 1.3 with the anti-PD1 mAb was found to be more effective than anti-PD-1 mAb alone (P=0.025 on day 18) and LPS B.sub.2 1.3 alone (P=0.016 on day 13) to impair the growth of established MC38 tumor cells (
[1085] All the mice from the anti-PD-1 and LPS B.sub.2 1.3+anti-PD-1 groups, which totally eradicated MC38 tumor cells in primary responses, were used for a rechallenge study with MC38 tumor cells. Mice treated with LPS B.sub.2 1.3 in combination with anti-PD-1 were all found to be resistant to a secondary challenge with new MC38 tumor cells (
Conclusion
[1086] These results demonstrate the capacity of the modified LPS B.sub.2 1.3 to boost the therapeutic efficacy of ICI against established tumors, associated with increased rates of complete responses and survival. Results also demonstrate the capacity of the modified LPS B.sub.2 1.3 to potentiate the capacity of the anti-PD-1 mAb to induce effective anti-tumor memory responses against MC38 tumor cells for long lasting protection against relapses.
[1087] Altogether, these results provide proof-of-concept of synergistic antitumor effect of the modified LPS B.sub.2 1.3 with ICI to control the growth and eradicate established tumors in vivo, but also to prevent relapse or recurrence of the tumors.
Example 14
[1088] Adjuvant Activity of LPS B.sub.2 0.1 in a Preclinical Mouse Vaccination Model
[1089] The capacity of the modified LPS B.sub.2 0.1 to enhance the humoral immune response to the SARS-CoV-2 spike receptor-binding domain (RBD) was evaluated in a preclinical mouse vaccination model. The objectives were to demonstrate the adjuvant activity of the modified LPS B.sub.2 0.1, and to compare it with a non-GMP equivalent of the MPL (GSK), a benchmark LPS derivative currently used as vaccine adjuvant in human vaccines.
[1090] Materials and Methods
[1091] Three cohorts of BALB/c mice (n=5/group) were immunized intramuscularly three times at 2 weeks interval with a recombinant SARS-CoV-2 Spike RBD domain (RBD—10 μg/dose) alone or formulated with the modified LPS B.sub.2 0.1 or MPLA (Invivogen—non-GMP equivalent of MPL).
[1092] MPLA and LPS B.sub.2 0.1 were administered at 20 μg/dose.
[1093] Anti-RBD IgG, IgGI et IgG2a antibody levels were measured by ELISA on blood samples collected on days 18, 25 and 39.
[1094] Results
[1095]
[1096] Higher serum levels of total anti-RBD IgG antibody were detected in the group of mice that received the recombinant SARS-CoV-2 spike RBD formulated with LPS B.sub.2 0.1 compared to control group of mice that only received the recombinant RBD antigen both on day 18 (P=7×10.sup.−4), and day 25 (P=9×10.sup.−4) and notably on day 39 (P=0.001) (
[1097] The higher increase of total anti-RBD IgG antibody levels were observed after the second boost (day 25 vs day 39: P=3×10.sup.−5). No significant differences were observed between the LPS B.sub.2 0.1 and MPLA groups on days 18 and 25, but significantly higher average levels of total RBD-specific IgG antibody were detected on day 39 in the LPS B.sub.2 0.1 group compared to MPLA group (P=0.005). This was associated with the presence of higher average levels of anti-RBD IgG1 (
Conclusion
[1098] Results of this study demonstrate the capacity of the modified LPS B.sub.2 0.1 to potentiate the induction of specific humoral immune responses in a preclinical mouse vaccination model against a SARS-CoV2 antigen, and thus demonstrate its adjuvant activity.
[1099] Results also demonstrate that the modified LPS B.sub.2 0.1 presents a better adjuvant activity than a non GMP equivalent of the benchmark MPL, associated with a polarization of the immune responses toward a more marked Th.sub.1-like phenotype.
[1100] These results thus confirmed the results of our previous study conduct in a preclinical mouse vaccination model against OVA antigen.
Example 15
[1101] Synergistic Anti-Tumor Effect of Modified LPS in Combination of Chemotherapy
[1102] The efficacy of modified LPS B.sub.2 0.1, LPS B.sub.2 1.1 and LPS B.sub.2 1.3 as monotherapy or in combination with chemotherapy was evaluated on human osteosarcoma cells in 3D-cell culture. The objective was to evaluate the synergistic antitumor effect of the modified LPS with chemotherapy.
[1103] Materials and Methods
[1104] 3D-cultures of human osteosarcoma cells (Saos-2 cells—2500 cells/well in very low attachment 96 well plates) were incubated (ratio target/effector: 1/3; n=6 wells/group) with human monocyte-derived macrophages (THP-1 incubated with PMA—150 nM/days for 48 hours), stimulated or not with one of the modified LPS B.sub.2 0.1, LPS B.sub.2 1.1 or LPS B.sub.2 1.3 (100 ng/ml for 16 hours).
[1105] Cultures were performed in presence or absence of a chemotherapeutic agent (etoposide—5 μM final). Cultures without etoposide and stimulated macrophages are used as control.
[1106] IncuCyte® Green Caspase-3/7 (2.5 μM final) is added in each culture well to monitor tumor cell apoptosis.
[1107] After 24 hours of incubation, number of apoptotic Saos-2 tumor cells is evaluated in each 3D-cell culture by fluorescence microscopy and Incucyte® live-cell analysis.