CA2696068A1 - Compositions and methods for the treatment and prophylaxis of hypertension - Google Patents

Abstract

A self-adjuvanting immunogenic composition comprising an immunogen comprising a lipopeptide cap, a universal T helper sequence and an angiotensin B cell epitope. The immunogen also comprises one or more linker sequences and/or polar charged amino acid sequences. The angiotensin B cell epitope of each immunogen has an amino acid sequence of D-R-V-Y-I-H-P-F
SEQ ID NO: 1 (angiotensin II), or D-R-V-Y-I-H-P-F-H-L SEQ ID NO: 2 (angiotensin I) with a free carboxyl terminus. The lipopeptide is a dipalmitoyl-S-glyceryl-cysteine or a tripalmitoyl-S-glyceryl cysteine or N-acetyl (dipalmitoyl-S-glyceryl cysteine), each with an optional neutral amino acid linker. Optional polar sequences of at least four charged polar amino acids enhance solubility of the immunogen and are located at the carboxy terminal end of the lipopeptide cap, optionally flanked by neutral linker amino acids, or elsewhere in the immunogen. Such compositions, at surprisingly low dosages of less than 10 mg per subject, can induce anti-angiotensin peptide antibodies with GMTs of greater than 100,000 or greater than 1,000,000 when employed to immunize a mammalian subject, without any extrinsic adjuvant. Surprisingly low dosages of this immunogen with or without optional boosters appear to deliver therapeutic/vaccinal titers.

Description

COMPOSITIONS AND METHODS FOR THE
TREATMENT AND PROPHYLAXIS OF HYPERTENSION

This application claims the benefit of priority of United States provisional patent application No. 60/955,695 filed August 14, 2007.

BACKGROUND OF THE INVENTION
Elevated blood pressure, termed hypertension, has a higli prevalence worldwide, with the highest prevalence, approximately 30% in adults, in the USA and Eastern Europe (Kearney et al.
2004 J Hypertens 22:11). The renin-angiotensin-aldosterone system is a key system controlling blood pressure (reviewed in Zicha and Kunes 1999 Physiol Reviews 79:1227; Stroth and Unger 1999 J
Cardiovasc Pharmacol 33:S21; Krum and Gilbert 2007 J Hypertens 25:25).
Blockade of this system by enzyme inhibition (angiotensin converting enzyme (ACE) inhibition) and/or receptor blockade (angiotensin II type 1 receptor blockers) has provided excellent control of blood pressure and minimal side-effects for many hypertensive subjects. However, it is estimated that only 30% of hypertensive patients in the USA have adequate control of blood pressure (Elliot 2003 J
Clin Hypertens (Greenwich) 5:3) and multiple drug modalities are often necessary to gain adequate control.
High affinity antibodies can bind to, and block the biological activity of, pathogenic proteins in the circulation, as exemplified by anti toxin vaccines for tetanus and diphtheria. There has been interest over the years in a vaccine approach to lower free angiotensin levels as a component of treathnent for hypertension (Oates et al. 1974 J Exp Med 139:239; Oster et al.
1975 Circ Res 37:607;
review by Michel et al. 1989 Am Heart J 117:756). Minor or no lowering of blood pressure was found in most studies, although marked elevations of total angiotensin in blood of immunized animals and resistance to the pressor effect of exogenous angiotensin attested to the binding activity of the induced anti-angiotensin antibody. Blood angiotensin was assayed after extraction of angiotensin from blood and the high levels of angiotensin detected in immunized animals were attributed to bound angiotensin carried by the circulating anti-angiotensin antibody.
Significantly, no measurements were attempted to measure levels of free angiotensin in blood and the extent to which they were lowered by vaccination.

A major component of the problem in raising high-titer and high-affinity antibodies to angiotensin is likely related to angiotensin's small size. Although angiotensin does contain a sequence that stimulates a specific antibody response of B cells, it lacks T
helper epitopes that stimulate T helper cells to amplify the B cell antibody response. More recent papers have involved coupling angiotensin I to a protein carrier to supply T cell help, and using aluminum hydroxide adjuvant, a mild stimulator of the innate immune system. Stronger adjuvants have generally not been approved for human use by regulatory bodies. This type of vaccine was studied in rats (Downham et al. 2003 Br J Clin Pharmacol 56:505) and man (Brown et al. 2004 Clinical Sci 107:167). Once again, antibodies titers were low and, despite effects on indirect parameters such as blunting of responses to extrinsic angiotensin, no lowering of elevated blood pressures was observed.
Ambuhl et al. (2007 J Hypertens 25:63) used a newer concept that involves conjugating antigens to the surface of the highly repetitive structure of virus like particles. Such immunogens bypass the need for T cell help and induce improved antibody responses with either no extrinsic adjuvant or a mild aluminum hydroxide adjuvant. Importantly, mean anti-angiotensin II titers of 20,000 were obtained in rats. These titers lowered the blood pressure of spontaneously hypertensive rats to levels comparable with rats treated with the angiotensin converting enzyme (ACE) inhibitor ramipril. Proof of concept had been attained. Another group of immunized, spontaneously hypertensive rats was monitored by telemetry. Angiotensin immunized rats developed median titers of 6,000 at 6 weeks and had reduced blood pressure by comparison with control rats.
A phase 1 clinical study was conducted in normotensive human volunteers with this vaccine (Ambuhl et al. 2007 J Hypertens 25:63). Good tolerability was obtained with no changes in blood pressure in these normotensive volunteers. However, titer after a single injection peaked at 3 weeks at 1,000. Total blood angiotensin was elevated in immunized subjects, but again no attempt was made to distinguish free versus bound angiotensin.
In summary, these recent studies suggest that specific anti-angiotensin antibody control of blood pressure is feasible. A key ingredient will be provision of an acceptable vaccine for human use that is capable of inducing high titer antibodies without the need for powerful adjuvants that are proscribed for human use. It is now recognized that three components are generally involved in the generation of strong specific immune responses: First, dendritic cells of the innate immune system recognize pathogen associated molecular patterns (PAMPs) via toll-like receptors (TLR) and secrete inflammatory cytokines that induce activation of T cells and B cells of the specific immune system (Schejetne et al, 2003 J. Immunol., 171:32). Second, helper T cells are activated by recognition of helper T cell epitopes of the pathogen and, in turn, activate and provide help to B cell production of specific antibody. Third, B cells, bearing antibody recognizing presented B
cell epitopes, multiply and, by clonal expansion, provide progeny secreting antibody specific for the presented antigen.
Despite the foregoing studies and the plethora of patent literature in the field of the treatment of hypertension, there remains a need in the art for new and useful compositions and methods for generating a therapeutic immunogenic composition for hypertension, which does not result in adverse side effects. There remains a need for potent angiotensin immunogens capable of inducing high and persistent antibody titers to angiotensin without the addition of potent adjuvants if a successful vaccine for the treatment of hypertension in humans is to be attained.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. lA is a chemical structure of the lipopeptide cap, dipalmitoyl-S-glyceryl cysteine (Pam2C or Pam2Cys).
FIG. 1B is a chemical structure of the lipopeptide cap, N-acetyl (dipalmitoyl-S-glyceryl cysteine) (NAc (Pam2C)).
FIG. 1 C is a chemical structure of the lipopeptide cap, tripalmitoyl-S-glyceryl cysteine (Pam3C or Panl3Cys).
FIG.2 is a bar graph illustrating the 2 week titers (stippled) and 3 week titers (solid) of anti-angiotensin II antibodies in the blood of rats A, B, and C after a single subcutaneous injection of 10 mg of the TANGI-K4 immunogen described in the examples below. Titers at week 3 following first immunization all exceeded 200,000. Rats are indicated by letter and the week of titer, e.g., 2W or 3W.
FIG. 3 is a graph illustrating depletion of free unbound angiotensin by anti-angiotensin antiserums of rats 3 weeks after a single immunization (see Fig. 2), showing reductions of 53 to 83%
in unbound angiotensin. For technical reasons the assay is performed with 10%
serum so the reductions in undiluted serum, with tenfold higher titers, would likely have approached or exceeded 99%. NRS is normal rat serum control. Five replicates are shown for each determination.

FIG.4 is a graph showing the dose-response characteristics (e.g., a standard curve) of the biotin-angiotensin II sandwich ELISA of Example 6 below.
FIG. 5 is a graph showing the result of the angiotensin depletion assay of Example 6 in a representative serum sample with a titer of one million for undiluted serum.
Dilutions were made with normal serum. The graph shows the concentration of unbound biotin-angiotensin II in the presence of dilution titers from 1000 (essentially non-immune serum) up to 1,000,000 of the anti-angiotensin II antiserum.

SUMMARY OF THE INVENTION
The compositions and methods described herein are useful as prophylactic and/or therapeutic agents to address this need in the art.
In one aspect, a self-adjuvanting immunogenic composition useful in the prophylaxis and/or treatment of hypertension is described. This composition includes an immunogen composed of a lipopeptide cap (R2), a universal T helper sequence (R1), and an angiotensin B
cell epitope, which is angiotensin II and/or angiotensin I. The immunogen has one of several embodiments, as discussed below, such as the formulae:
R2- Rl - angiotensin B cell epitope (Formula I) or, alternatively R2-K(R1)-angiotensin B cell epitope (Formula II), or alternatively Rl-K(R2)-angiotensin B cell epitope (Formula III).
In all formulae of the immunogen components, the B cell epitope must have a free carboxy terminus.
In some embodiments, the R2 lipopeptide cap comprises a linker of one to ten amino acids to link it to the other components of the immunogen. In other embodiments a similar linker is employed to link other components of the immunogen together.
In further embodiments, a sequence of charged, polar amino acids provided with or without the linker sequence is present in various positions in the immunogen. In one embodiment, charged polar sequence is inserted after the R2 cap's Cys, or between the R2 lipopeptide cap's optional linker neutral amino acids and the R1 helper sequence. In other embodiments a linker alone or with such a polar sequence is inserted between R1 helper sequence and the angiotensin B
cell epitope.
In certain embodiments for each immunogen, R2 is dipalmitoyl-S-glyceryl cysteine (Pam2Cys) of FIG. 1 A comprising optionally one or up to ten linker amino acids, as described below.

In certain embodiments for each immunogen, R2 is N-acetyl (dipalmitoyl-S-glyceryl cysteine) ((NAc(Pam2C)) of FIG. 1B, which also can comprise an optional amino acid linker, In certain embodiments for each immunogen, R2 is a lipopeptide tripalmitoyl-S-glyceryl cysteine (Pam3Cys) of FIG. 1 C, which can optionally comprise a linker sequence. The compositions induce anti-angiotensin antibodies with geometric mean titers (GMT) of at least 100,000, at least 300,000 or greater than I
million, when employed to immunize a mammalian subject.
In another aspect, a pharmaceutical composition comprises the self-adjuvanting immunogenic compositions defined herein, and a suitable pllarmaeeutical carrier or excipient. This composition also demonstrates induction of antibodies against angiotensin I and/or II with GMT greater than 100,000, or greater than 300,000 or greater than 1 million when a mammalian subject is immunized therewith. In other embodiments, this composition also demonstrates induction of anti-angiotensin antibodies with GMT greater than 3,000,000 when a mammalian subject is immunized therewith.
In yet another aspect, a method of inducing in vivo the production of anti-angiotensin antibodies having a high GMT by immunizing a subject with an effective antibody-inducing amount of the immunogen or pharmaceutical composition described herein. In one embodiment, the method includes iminunizing with a single dose of immunogen. In anotller embodiment, the method includes administering one or more booster dosages. In certain embodiments, the GMT
resulting from immunization is greater than 100,000. In other embodiments, particularly where the method employs a prime dose and one or more booster dose of the composition, the GMT is considerably higher, e.g., on the order of greater than 300,000 or greater than 1,000,000. In another aspect of the method, the effective amount is a unit dose of about 10 mg or less, alternatively 1 mg or less, alternatively, 0.1 mg or less, or alternatively, 0.01 ing or less.
In another aspect, use of the immunogens described above in the manufacture of a medicament for the treatment and/or prophylaxis of hypertension is provided.
The medicament induces in vivo the production of anti-angiotensin antibodies with high GMT, even at low dosages In yet another aspect, introduction of charged polar residues in at least one position within the immunogen confers aqueous solubility and facilitates an aqueous and/or lyophilized formulation of the immunogen.
Other aspects and advantages of these methods and compositions are described further in the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION
The compositions and metliods described herein address the need in the art for tlierapeutic and prophylactic immunogenic compositions for use in treating, retarding progression of, and preventing, hypertension in human subjects. In one embodiment these compositions involve the formulation and application of therapeutic and prophylactic therapies that are efficacious against hypertension without incurring adverse reactions related to powerful extrinsic adjuvants. To interdict the angiotensin pressor system, the inventor provides an immunogen that (1) incorporates a B cell epitope comprising angiotensin II and/or its precursor angiotensin I; (2) creates a powerful immunogenic moiety that overcomes self-tolerance and is acceptable for use in humans; and (3) creates an immunogen that is soluble in aqueous solvents, and can be presented as a liquid formulation or a lyophilized product easily reconstituted with water for injection. In a single immunogen, selected sequences that simultaneously enhance a helper T cell response with a B cell response directed to angiotensin II and/or its precursor angiotensin I are combined with a lipoprotein cap that activates the innate immune system, creating a particularly desirable self-adjuvanting lipoprotein, which sequences are modified to enhance aqueous solubility. This combination results in a composition that elicits high, persistent levels of anti-angiotensin antibody titers in vivo.

I. Compositions In one embodiment, the self-adjuvanting immunogenic composition comprises a specifically designed immunogen elnploying a B cell peptide of angiotensin II or angiotensin I, which enables the composition to induce anti-angiotensin antibodies with geometric mean titers of greater than 100,000, greater than 300,000, and greater than 1 million. Each "immunogen" as used herein is a composition that does not occur in nature, but can be produced by synthetic technologies, e.g., chemical synthetic techniques for peptides and lipopeptides. This chemical synthesis is completely scalable, allowing for a relatively inexpensive process for producing large quantities of immunogen.
Recombinant DNA
preparation and expression may also be employed to construct some portions of the immunogen, at the selection of the person of skill in the art.
In one embodiment, an immunogenic composition comprises an immunogen including a lipoprotein cap (R2), a universal T helper sequence (Rl) and the angiotensin B-cell epitope. These immunogen components are described in detail below. The immunogens described herein can form a variety of structures, based upon the selection of the formulae below. In certain embodiments, R2 (and optionally R1) individually include a neutral amino acid sequence or linker sequence of from 0 to 10 amino acids in length, which links the lipopeptide of R2 to the other components forming the immunogen, (or links R1 to the B cell epitope) depending upon the formula selected. In other embodiments, a charged polar amino acid sequence is inserted into the immunogen formula with or without flanking neutral linker amino acids, between the components of the formula of the immunogen to enhance solubility. The linker and polar charged sequences are described in detail below.
In one embodiment, an immunogenic composition comprises an immunogen of R2- R1 - angiotensin B cell epitope (Formula I) or, alternatively R2-K(RI)-angiotensin B cell epitope (Formula II), or alternatively R1-K(R2)-angiotensin B cell epitope (Formula III).
In one embodiment of an immunogen of Formula I, the R2 lipopeptide cap which contains a Cys and optionally one or more neutral linker amino acids, is linked to an a-amino group at the amino terminus of the RI T helper sequence. RI is linked to the B cell epitope, thus forming a, linear construct. An optional polar charged sequence is located after the R2 Cys, or between the R2 linker amino acids, thus linking to RI, but may also be located at additional positions between R1 and the B
cell epitope to enhance solubility. Immunogens of this forinula are described in the examples below.
Still other embodiments of immunogens as described herein can take form of Formula II.
According to Formula II, the R2 lipopeptide cap which contains a Cys and optionally one or more neutral linker amino acids, is linked via an inserted lysine residue (indicated in the formula by the single amino acid abbreviation K) directly to the B cell epitope. The T cell helper sequence (R1) within the parentheses of the formula is attached to that inserted lysine via linkage between the R1 carboxy terminus and the s amino group of the lysine. An optional linker amino acid(s) andlor a charged, polar sequence optionally flanked by neutral linker amino acids is optionally interposed between R2 and the B cell epitope on either or both sides of the inserted lysine, or between the RI
carboxy terminus and the P, amino group of the inserted lysine, or is located at the amino terminus of the R1.

In an embodiment of an immunogen of Formula III, an optional lysine residue (K) is inserted between the R1 T helper sequence and the B cell peptide. The R2 within the parentheses is linked to the s-amino group of the inserted K via the R2 Cys or its optional linker amino acid(s) and/or via a charged, polar sequence optionally flanked by neutral linker ainino acids. An optional linker amino acid(s) and/or a charged, polar sequence optionally flanked by neutral linker amino acids is optionally located at the amino terminus of the R1 or interposed between Rl and the B
cell epitope, either before or after the inserted lysine or in both positions.
A. The Angiotensin Epitope Component In one embodiment of an immunogen as described herein the angiotensin B cell epitope of the above immunogen is a peptide sequence of angiotensin II having the sequence D-R-V-Y-I-H-P-F
(SEQ ID NO: 1). In another embodiment the B cell epitope is a peptide sequence of its precursor angiotensin I, i.e., D-R-V-Y-I-H-P-F-H-L (SEQ ID NO: 2).
It is also possible to modify the angiotensin epitopes in the immunogens described above.
For example, other angiotensin epitopes may include homologous or analogous modified epitope sequences, wherein amino acids in either angiotensin peptide above may be conservatively replaced individually by amino acid residues having similar characteristics. For example, the amino acid residue of SEQ ID NO: 1 or SEQ ID NO: 2 may be replaced by other amino acid residues bearing the same charge and/or similar side chain lengths. Similarly the amino acids may be replaced by unnatural alnino acid residues, i.e., an amino acid having a modification in the chemical structure, e.g., a D-amino acid; an amino acid bearing a non-naturally occurring side chains an N-methylated amino acid, etc. See, the cited references relating to N-methylated amino acids, ainong others. See, e.g., L. Aurelio et al, 2002 Organic Letters, 4(21):3767-3769 and references cited therein.
Further, shorter peptides can be used which contain 1 or 2 amino acids deleted from the amino termini of SEQ ID NO: 1 or 2, although such shorter sequences are unlikely to provide any functional difference to the immunogens. In all embodiments of immunogens of Formulae I, II and III, the carboxyl terminus of the angiotensin epitope is free and only the amino terminus is coupled to another component of the immunogen.
B. The Universal T Helper Sequence Rl Another component of the immunogens of the immunogenic compositions described herein is a universal T helper epitope used to enhance the immunogenicity of the B cell epitope in the immunogen, i.e., the angiotensin epitope. The term "T helper epitope" is intended to mean a chain of amino acids which, in the context of one or more class II MHC molecules, activates T helper lymphocytes, which enhances the antibody response to the angiotensin epitope of the immunogen. In certain embodiments, the T helper epitope component of the immunogens is one that is recognized by T helper cells present in the majority of the population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. These are known as "loosely HLA-restricted" or "promiscuous" T helper sequences. In particular, promiscuous T cell epitopes are used as the RI moiety in the formulae above.
Depending upon the formula of the immunogen selected, the linkage between R1 and R2 is one of the following: R2 is linked to the N terminal amino acid of Rl via the R2 Cys or its optional linker ainino acid(s) and/or polar charged sequence as in Formula I.
Alternatively where R2 is linked to the B cell epitope through a lysine residue inserted therebetween, the RI
is linked to the s amino group of the inserted lysine residue, as in Formula II, with optional linkers or polar sequences present in the immunogen. Still alternatively, if R1 is located at the amino terminus of the immunogen, it is linked to the B cell epitope through a lysine residue inserted therebetween.
In this circumstance, R2 is linked via its Cys or its optional linker amino acid(s) and/or polar charged sequence, to the F. amino group of the inserted lysine, as in Formula III. Depending upon the formula of the immunogen selected, the Rl sequence can be linked to the angiotensin B cell epitope either directly or via an optional linker and/or polar charged sequence at the amino terminus of the angiotensin epitope.
Many promiscuous or universal T helper sequences occur naturally in different sources, e.g., microorganisms, or are artificially engineered sequences. Such suitable T cell epitopes are known and may be selected for this use in these immunogens and compositions.
In one embodiment, and as exemplified by the examples below, the R1 T helper sequence in an immunogen as described in Formula I, II, and/or III herein has the sequence, SEQ ID NO: 3:
Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-Xaa, wherein said Xaa is absent or L. This sequence is naturally found in the tetanus toxin at amino acids 830-843(844); see, Panina-Bordignon et al. 1989 Eur J
Immunol 19:2237. Another such tetanus toxin sequence (amino acid 947-967 of tetanus toxin) useful as R1 has the sequence SEQ ID NO: 4:

F-N-N-F-T-V-S-F-W-L-R-V-P-K-V-S-A-S-H-L-E, or a derivative thereof, such as amino acid 950-969 of Tet toxoid. See, Reece JC et al. 1994 J Immunol Methods 172:241-54.
Still other tetanus toxoid T cell helper sequences for use as the R1 of formulae include the sequences SEQ ID NO: 5: I-D-K-I-S-D-V-S-T-I-V-P-Y-I-G-P-A-L-N-I, amino acid residues 632-651 of Tet toxoid, SEQ ID NO: 6: N-S-V-D-D-A-L-I-N-S-T-K-I-Y-S-Y-F-P-S-V, alnino acid residues 580-599 of Tet toxoid, SEQ ID NO: 7: P-G-I-N-G-K-A-I-H-L-V-N-N-E-S-S-E, amino acid residues 916-932 of Tet toxoid, and ] 0 SEQ ID NO: 8: Z-Y-I-K-A-N-S-K-F-I-G-I-T-E, amino acid residues 830-842 of Tet toxoid. For still other universal T cell helper sequences useful as the RI of the immunogens, see, e.g., Ho et al.
1990 Eur J Immuno120:477; Valmori et al 1992 J Immunol 149:717-721; Chin et al. 1994 Immunol 81:428, Vitiello et al 1995 J Clin Invest 95:341; Livingston et al. 1997 J
Immunol 159:1383;
Kaumaya PTP et al. 1993 J Mol Recognition 6:81-94 (1993), and Diethelm-Okita BM et a1.2000 J Inf Dis 175:383-91; all incorporated by reference herein. See also, Raju et al.
1995 Eur J Immunol 25:3207-14 and Diethelm-Okita BM et al. 2000 J Inf Dis 181:1000-9, incorporated by reference herein, which discuss certain diphtheria toxin T cell helper sequences which may be employed as R1 in the immunogens described herein. Still other sequences which may be useful as T helper epitope sequences for R1 of the formulae above are disclosed in Nardin et al. 2001 J
Immunol 166:481-9, incorporated by reference herein.
Examples of other T helper sequences that are promiscuous include sequences from antigens such as Plasmodiumfalciparum circumsporozoite (CS) protein at positions 378-398 (D-I-E-K-K-I-A-K-M-E-K-A-S-S-V-F-N-V-V-N-S; SEQ ID NO: 9), and Streptococcus 18 kD protein at positions 116 (G-A-V-D-S-I-L-G-G-V-A-T-Y-G-A-A; SEQ ID NO: 10). See, e.g., US Patent No.
7,026,443, incorporated herein by reference.
The RI universal T cell helper sequence may also be an artificially engineered sequence, such as the Pan HLA DR-binding (PADRE) molecule (Epimmune, San Diego, Calif.) described, for example, in U.S. Pat. No. 5,736,142 (see, e.g., PCT publication WO 95/07707, incorporated by reference herein). These synthetic compounds are designed to most preferably bind most HLA-DR
(human HLA class II) molecules. Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs. These sequences are recognized by class 2 mixed histocompatibility (MHC) antigens on B cells and macrophages and dendritic cells and enhance B cell production of antigens.
Thus, in one embodiment, the R1 T helper sequence is defined by the formula SEQ ID NO: 11 : Xaa1-K-Xaa2-V-A-A-W-T-L-K-A-A- Xaa3, wherein Xaal and Xaa3 are independently selected from D-Ala or L-Ala, and Xaa2 is L-cyclohexylalanine, Phe or Tyr. These T
helper sequences have been found to bind to most HLA-DR alleles, and to stimulate the response of T
helper lymphocytes from most individuals, regardless of their HLA type. In one embodiment, Rl has the above formula, in which Xaal and Xaa3 are both D-Ala and Xaa2 is cyclohexylalanine. Other PADRE sequences include an alternative of a pan-DR binding epitope that comprises all "L" natural amino acids and can be provided in the form of nucleic acids that encode the epitope. Still other PADRE sequences are disclosed in Vitiello et al. 1995 J Clin Invest 95:341;
Alexander J et al. 1994 Immunity 1:751-61; Del Guercio M-F et al. 1997 Vaccine 15:441-8; Alexander J
et al. 2000 J
Immunol 164:1625-33; Alexander J et al. 2004 Vaccine 22:2362-7; and Agadjanyan MG et al. 2005 J Immunol 174:1580-6, all incorporated by reference herein.
These T helper peptide sequences R1 can also be modified to alter their biological properties.
For example, they can be modified to include D-amino acids or other amino acid modifications to increase their resistance to proteases and thus extend their serum half life.
Further these promiscuous T cell helper sequences or R1 sequences may further include linker and/or polar charged sequences as discussed below.
One of skill in the art is expected to select from among other known promiscuous T cell helper sequences to design other specific immunogens for immunogenic compositions as described herein. A specific embodiment, described below, illustrates the universal T
helper sequence that has been usefi.il within the immunogens at inducing antibodies with high geometric mean titers (GMT) against angiotensin protein.
C. The Lipopeptide Cap Component R2 Another component of the immunogens described herein is a lipopeptide component, preferably a "lipopeptide cap" (R2), that works, in concert with the other components of the immunogens to induce antibodies with GMT of greater than 100,000, or greater than 300,000 or greater than 1,000,000, needed for the angiotensin prophylactic and therapeutic immunogenic compositions as described herein. Lipopeptides have been identified as agents capable of priming CTL against viral antigens and also enhancing humoral antibody responses in vivo against certain antigens. Thus, the R2 moiety of the immunogens is selected from among desirable lipopeptide components having attached thereto a Cys and optionally from one up to ten amino acid linker residues and/or optionally a sequence of charged polar amino acids as described below.
In one embodiment, the R2 lipopeptide is attached to an a-amino group at the amino terminus of the immunogen directly or via its optional linker and/or polar charged amino acid(s), i.e., it is attached to the amino terminus of the R1 T cell helper sequence or directly to the angiotensin epitope, if the RI is in a different position. In another embodiment of an immunogen, the R2 lipopeptide is attached to an s-amino of a lysine residue located between Rl and the first N
terminal amino acid residue of the angiotensin epitope directly via the R2 Cys or via its optional one up to ten amino acid linker residues and/or optionally a sequence of charged polar amino acids. In yet a further embodiment, the immunogen's R2 lipopeptide cap is linked directly via its Cys or via its optional one up to ten amino acid linker residues and/or optionally a sequence of charged polar amino acids to a lysine residue, which is linked directly to the N terminus of the B cell epitope. In this structure, the RI is linked via its carboxy terminus to the s amino group of the lysine.
Specific R2 lipopeptides for such use include, e.g., N-terminal sequences of the E. coli lipoproteins. In one embodiment, R2 is a lipopeptide which is dipalmitoyl-S-glyceryl cysteine (Pam2Cys) of FIG. 1 A with two amino acid linkers and/or a polar charged sequence. In another embodiment, R2 is a lipopeptide which is tripalmitoyl-S-glyceryl cysteine (Pam3Cys) of FIG. 1 C with its amino acid linker and/or polar charged sequence.
Otlier R2 caps include an R-(dipalmitoyl-S-glyceryl) cysteine, wherein the R
is a group consisting of hydrogen, or an alkyl, alkanyl, alkenyl, or alkynl of 1-6 C
atoms. In one embodiment, R2 is a lipopeptide which is N-acetyl (dipalmitoyl-S-glyceryl) cysteine ((NAc(Pam2C)) of FIG. 1B with an optional amino acid linker and/or polar charged sequence (R is N-acetyl).
Other potential R2 moieties are hexadecanoic acid, Hda, and macrophage activating peptide, MALP-2.
Such lipopeptide caps may be selected, synthesized and prepared from those described by Deres, et al., 1998 Nature 342:561; Weismuller et al. 1989 Vaccine 7:29;
Metzger et al. 1991 Int J
Peptide Protein Res 38:545; Martinon et al. 1992 J Immunol 149:3416; Vitiello et al. 1995 J Clin Invest 95:341; Muhlradt et al. 1997 J Exp Med 185:1951; Livingston et al. 1999 J Immunol 162:3088; Zeng et al 2002 J Immunol 169:4905; Borzutsky et al. 2003 Eur J
Immuno133:1548;

Scgjetne et al. 2003 J Immunol 171:32; Jackson et al. 2004 Proc Natl Acad Sci USA 101:15440 Borzutsky et al. 2005 J Immunol 174:6308; Muhlradt PF et al. J Exp Med 11:1951-8 (1997); Obert M et al. Vaccine 16:161-9 (1997); Zeng W et al. Vaccine, 18:1031-9 (2000);
Gras-Masse H Mol Immuno138:423-31 (2001); Zeng W et al J Immunol 169:4905-12 (2002); Schjetne KW et al. J
Immunol 171:32-6 (2003); Spohn R et al. Vaccine 22:2494-9 (2004); Jackson DC
et al Proc Natl Acad Sci USA 101:15440-5 (2004); Zeng W et al Vaccine, 23:4427-35 (2005);
International Patent Application Publication Nos. W02006/026834, W02006/040076, W02004/014956 or W02004/014957, all above-cited documents incorporated by reference herein.
In one embodiment, a particularly effective immunogenic composition comprises the R2 of Pam2CSS, i.e., the dipalmitic acid moiety dipalmitoyl-S-glyceryl- Cys, which is attached via a linker, e.g., Ser-Ser, and/or a polar charged sequence. A Pam2C with a linker is described in PCT
publication WO 2004/014957, incorporated herein by reference. In another embodiment, a particularly effective immunogenic composition comprises the R2 of Pam3Cys-S-S-, i.e., the tripalmitic acid moiety dipalmitoyl-S-glyceryl-Cys, which is attached via a linker, e.g., Ser-Ser, and/or a polar charged sequence. In another embodiment, the R2 is NAc(Pam2C)-S-S-, i.e., the dipalmitic acid moiety N-acetyl (dipalmitoyl-S-glyceryl) eysteine, which is attached via a linker, e.g., Ser-Ser, and/or a polar charged sequence.
As previously described, this R2 lipopeptide is linked directly via its Cys or via its optional one up to ten amino acid linker residues and/or optionally a sequence of charged polar amino acids, to an a-amino group at the amino terminus of the R1 T cell helper sequence, which is in turn linked to the B cell epitope. In another embodiment, the R2 lipopeptide is linked directly via its Cys or via its optional one up to ten amino acid linker residues and/or optionally a sequence of charged polar amino acids, to a lysine inserted between R2 and the amino terminus of the B cell epitope, with Rl linked through the s amino of the inserted lysine. Alternatively R2 is linked via its Cys, its suitable linker amino acid(s) and/or polar charged amino acid sequence, to an s-amino of a lysine residue located between RI and the first amino terminal amino acid residue of the B cell epitope component of the immunogen. In all embodiments, the carboxy terminal end of the B cell epitope is free and not bound to another component of the immunogen.
The R2 cap enhances the antibody response, and proves highly effective in the immunogens of Formula I, II and/or III that form the immunogenic compositions.

D. The Optional Linkers and Polar Sequences Although the R2 cap can be directly linked through its Cys, and the T cell helper sequences R1 can be directly linked to the angiotensin B cell epitope component of the immunogen, a linker is desirably optionally incorporated to link the carboxy terminal end of the R2 lipopeptide cap component to any other component in each immunogen. In other embodiments, linker amino acids or sequences are used also between R1 helper sequence and the B-cell epitope. In another embodiment, an amino acid sequence is used as an optional linker attached to the N- or C-termini of the R1 helper sequence or to the N terminus of the B cell peptide to couple one component to another component of the immunogen, depending upon the formula of the immunogen.
The "linker" located within the R2 cap or positioned elsewhere in the immunogen is typically comprised of from one to ten relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
The linkers are typically selected from, e.g., Gly, Ser, Pro, Thr, or other neutral linkers of nonpolar amino acids or neutral polar amino acids. The optional linker need not be comprised of the same residues and thus may be a heterooligomer, e.g., Gly-Ser- or a homooligomer, e.g., Ser-Ser. When present, the linker in one embodiment is at least one amino acid residues, e.g., Ser or Gly. In another embodiment, the linker is at least two amino acid residues, e.g., Ser-Ser or Gly-Ser. In still other embodiments three to six amino acid residues, and up to 10 or more residues, are used to form the linker. Thus in certain embodiments, the linker sequence includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or mimetics. In another embodiment linkers larger than 10 amino acids may be used in the immunogens.
For example, a linker may be used to couple the lipopeptide cap of R2 to another component of the immunogen. In one embodiment, the linker is the dipeptide Ser-Ser. In another embodiment, the linker is Gly-Gly. In still another embodiment, a heterooligomer, such as Gly-Ser, or Ser-Gly may be used. In another embodiment, a linker such as -Ser- links the T helper sequence R1 to the N-terminal ainino acid of the B cell epitope in the immunogen.
In another embodiment of the immunogens, a sequence of charged, polar amino acids is incorporated within, or replaces, the relatively uncharged linker sequences.
Introduction of a charged, polar sequence has been found to enhance the aqueous solubility of the composition, as demonstrated by the exainples below. For example, these charged polar sequences are employed to enhance the solubility of the immunogens in formulation with water for injection and optionally mannitol for tonicity, without the need for a buffer. These charged polar sequences enable the immunogens to be readily prepared, solubilized and lyophilized. These polar sequences, by enhancing solubility, may also be useful to enhance the immunogenicity of the B cell epitope.
In one embodiment, the polar sequence is composed of 4, 5, 6, 7, or 8 charged polar amino acids. In another embodiment a polar charged sequence of more than 8 amino acids may be employed. In a further embodiment, the polar sequence is composed of 4 amino acids. In yet another embodiment, the polar sequence is composed of 6 amino acids. In one embodiment, the polar sequence is composed of amino acids selected from lysine, arginine, aspartate, and glutamate. In a further embodiment, the polar sequence is composed of amino acids selected from lysine, arginine, and aspartate. In another embodiment, the amino acids in the polar sequence are identical. In further embodiments, 2, 3, or 4 different amino acids are used in the polar sequence.
Thus, in one embodiment, a polar, charged sequence is -Lys-Lys-Lys-Lys- SEQ ID NO: 12, -Lys-Lys-Lys-Lys-Lys-Lys- SEQ ID NO: 13, or -Lys-Glu-Lys-Glu- SEQ ID NO: 14 or -Glu-Glu-Glu-Glu-SEQ ID NO:
or any iteration of from 4 to 8 identical or varying polar, charged amino acids.
15 Optionally, the polar amino acid sequence is flanked on either terminus by an amino acid of the linker to form the sequence -linker amino acid-(polar amino acid)õ-linker amino acid-, wherein n is the number of polar amino acids, e.g., from 4 to 8. Alternatively, the polar amino acid sequence may be used without the flanking linker (neutral, uncharged) amino acids. In one embodiment, the linker with a polar amino acid sequence is composed of Ser-Lys-Lys-Lys-Lys-Ser SEQ ID NO: 16, i.e., a 4 identical amino acid polar sequence within a Ser-Ser linker, or Ser-[Lys]4-Ser SEQ ID NO:
16. In another embodiment, the amino acid linker containing a polar sequence is Ser-[Lys]6-Ser SEQ
ID NO: 17. In other embodiments, the linker with polar sequence is Gly-[Lys]4-Gly SEQ ID NO: 18 or Gly-[Lys]b-Gly SEQ ID NO: 19. In still other embodiments the linker with polar sequence is -Ser-(Lys-Glu-Lys-Glu-)-Ser- SEQ ID NO: 20. As above, any iteration of this sequence that can be assembled by one of skill in the art given the above definition and the -linker amino acid-(polar amino acid)n linker amino acid- formulae.
In one specific embodiment, therefore, an amino acid linker containing a polar, charged sequence, or the linker alone, or the polar charged sequence alone, is located between the immunogen component R2 and any other component of the immunogen with which it is linked.
In another embodiment, the linker and/or polar sequence is located between RI and any other component of the immunogen.
In another embodiment, a polar changed sequence with or without flanking linker amino acids is present only once in the immunogen, e.g., attached only to the carboxy-terminal linker -Ser-of the di- or tripalmitic acid moiety of R2, linking R2 to RI or the B cell epitope or to an inserted lysine in the immunogen. In still another embodiment, linker and/or polar sequences are present in multiple (i.e., 2 or more) positions in the immunogen.
E. Specific Embodiments Specific embodiments of immunogens of Formula I, II or III are employed in the examples below and also include the following immunogens. In one embodiment (using single letter codes for the amino acids), in each immunogen, R2 is formed by two units of palmitic acid linked via a glyceryl group to the sulfur of a cysteine (see FIG. 1 A) and an amino acid linker sequence of -S-S- residues, which links the R2 to the first amino acid of Rl. R1 is Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L SEQ ID
NO: 21 with an optional amino acid linker of -S-, which links to the N
terminal amino acid residue of the angiotensin B cell epitope. While the angiotensin II peptide epitope is used in the embodiments below, it is understood that the angiotensin I B cell epitope may also be used in these embodiments in place of the shorter sequence. Thus one immunogen of Formula I is defined as follows:
SEQ ID NO: 22 :
Pam2C-S-S-Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-S-D-R-V-Y-I-H-P-F.
In a further embodiment, a polar sequence of four lysines (underlined) is incorporated in the linker sequence (-S-S-) connecting R2 to R1. This further immunogen of Formula I is defined as follows:
SEQ ID NO: 23 :
Pam2C-S-K-K-K-K-S-Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-S-D-R-V-Y-I-H-P-F.
In still a further embodiment, a linker containing a polar sequence of four lysines (underlined) is utilized to link R1 helper sequence to the angiotensin B cell epitope component. This further immunogen of Formula I is defined as follows:
SEQ ID NO: 24:
Pam2C-S-S-Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-S-K-K-K-K-S-D-R-V-Y-I-H-P-F.

In yet a further embodiment, a linker containing a polar sequence of four lysines (underlined) is utilized in two places in the immunogen, i.e., both as part of the R2 linker to link the lipopeptide to the remainder of the sequence and to link R1 to the angiotensin epitope component. This further immunogen of Formula I is defined as follows:
SEQ ID NO: 25 :
Pam2C-S-K-K-K-K-S-Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-S-K-K-K-K-S-D-R-V-Y-I-H-P-F.
In one embodiment, in each immunogen, R2 is formed by two units of the palmitic acid linked via a glyceryl group to the sulfur of an N-acetyl cysteine (NAc(Pam2C);
see FIG.1 B) and an amino acid linker sequence of -S-S- residues, which links the R2 to the first amino acid of R1. R1 is Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L SEQ ID NO: 21 with an optional amino acid linker of -S-, which links to the N terminal amino acid residue of the angiotensin peptide. Thus one immunogen of Formula I is defined as follows:
SEQ ID NO: 26 NAc(Pam2C)-S-S-Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-S-D-R-V -Y-I-H-P-F.
In further embodiments, polar sequences are encompassed parallel to the Pam2C
containing embodiments reflected above.
In another embodiment, in each immunogen as defined above, R2 is formed by two units of palmitic acid linked via a glyceryl group to the sulfur of a palmityl-cysteine (i.e., Pam3C in place of Pam2C above; see FIG. 1C) and an alnino acid linker sequence of-S-S- residues.
In further embodiments in which R2 is Pam3C, polar sequences are encompassed parallel to the Pam2C
containing embodiments reflected above.
In an embodiment of Formula II, the Pam3CSS- or Pam2CSS- or NAc(Pam2C)- SS-containing moiety is linked via its Cys and/or an optional linker or polar charged sequence through an inserted lysine to the N-terminal amino acid of the angiotensin epitope. Rl is coupled via the 6 amino group of the same lysine residue, the immunogen being defined as:
(Pam3C-S-S or NAc(Pam2C)-SS or) Pam2C-S-S - K-S-D-R-V-Y-I-H-P-F
I
Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L
In further embodiments of Formula II, the optional linkers and/or polar charged sequences as defined above may be inserted between the two serines of the lipopeptide linkers, between the inserted lysine and the B cell epitope, or between the Rl and the s group of the inserted lysine. Also in further embodiments, multiple linkers comprising polar sequences may be inserted in more than one place parallel to those described with respect to the Formula I
embodiments, above. Similar immunogens of Formula 11 employ the longer angiotensin sequence for the B cell epitope.
In an embodiment of Formula III, in which the RI helper sequence is coupled via an inserted K and an optional linker sequence to the N terminal amino acid of the angiotensin epitope, and the Pam3CSS- or Pam2CSS- or NAc(Pam2C)-SS-containing moiety is coupled via the s amino of the same lysine residue, the immunogen is defined as SEQ ID NO: 27 :
Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-K-S-D-R-V-Y-I-H-P-F
' Pam2C-S-S (or Pam3C-S-S or NAc(Pam2C)-SS) In further embodiments of Formula III, the optional linkers and/or polar charged sequences as defined above may be inserted before or after the inserted lysine or the serine linker linking the T
helper sequence to the angiotensin B cell epitope sequence and/or between the serines (-S-S-) which connect the Pam2C cap to the E amino of the lysine inserted between the universal T helper sequence and the angiotensin epitope. Also in further embodiments, multiple linkers comprising polar sequences may be inserted in more than one place parallel to those described with respect to the Formula I embodiments, above. Similar immunogens of Formula III employ the longer angiotensin sequence for the B cell epitope.
Immunogenic compositions that contain an immunogen as above defined, and as illustrated in the examples below, are characterized by the ability to induce in mammalian animals anti-angiotensin antibodies with a geometric mean titer of greater than 100,000, as discussed in more detail below. In other embodiments the GMTS are greater than 300,000 or greater than 1,000,000, or greater than 3,000,000.
Given the above teachings, one of skill in the art may readily design other immunogens meeting the formulae by selecting from among the components described above.
IL Methods of Making the Immunogens and Immunogenic Compositions Immunogens may be prepared according to the formulae above by carrying out a chemical synthesis in solid phase or in solution. Both synthesis techniques are well known to those skilled in the art. For example, such techniques are described in conventional texts such as Atherton and Shepard in "Solid phase peptide synthesis" (IRT press Oxford, 1989), Stewart &
Young, SOLID
PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co., 1984) and by Houbenweyl in "Methoden der organischen Chemie" [Methods in Organic Chemistry] published by E.Wunsch Vol.
15-I and II, Stuttgart, 1974, and also in the following articles, which are entirely incorporated herein by way of reference: P E Dawson et al. (Science 1994; 266(5186):776 9); G G
Kochendoerfer et al.
(1999; 3(6):665 71); et P E Dawson etal., Annu. Rev. Biochem. 2000; 69:923-60.
Various automated or computer-programmable synthesizers are commercially available and can be used in accordance with known protocols. Further, individual peptide epitopes can be joined using chemical ligation to produce larger peptides that are still within the bounds of the immunogens of Formula I or II as described herein.
Desirably the synthesis involves building the immunogens in direction from the C terminal towards the N-terminal by first immobilizing the C-terminal-most amino acid residue on a solid support, such as by using Fmoc chemistry using HBTU on RAMAGE resin. The lipopeptide cap is then synthesized as a lipoamino acid essentially as described in PCT
publication No.
W02004/014957, incorporated herein by reference. For example, in one embodiment, the Pam2Cys or NAc(Pam2C) or Pam3Cys lipopeptide cap is introduced onto the synthetic angiotensin B cell epitope sequence and T helper sequence by covalently attaching each lipopeptide moiety directly or indirectly via an optional linker and/or polar charged sequence to an alpha-amino group of the N-terminal amino acid of the T helper sequence or to the B cell peptide, or to the epsilon amino group of a lysine introduced between the R1 and the B cell peptide. The resulting immunogen is then cleaved from the resin using standard methods, e.g., trifluoroacetic acid (Reagent K), and optionally converted to a salt, also using conventional methodologies, e.g., a BIO-RAD acetate resin.
The resulting composition contains an immunogen having the lipopeptide cap attached to an a-amino group of the N-terminus of R1 or the B cell epitope, or to an epsilon amino group of a lysine residue inserted between R1 and the B cell epitope. If more than one immunogen is present in a composition, each immunogen can have the same or different R1 helper. A
composition can contain immunogens having the lipopeptide cap at a position different from other immunogens in the composition, such as described in Formula I, II and/or III. In one embodiment the angiotensin II
peptide is sufficient as the B cell epitope in all immunogenic compositions described herein. In another embodiment the angiotensin I peptide is sufficient as the B cell epitope in all immunogenic compositions. In still another embodiment, a mixture of immunogens containing the angiotensin II
epitope and immunogens containing the angiotensin I epitope are employed. The optional linker and/or polar charged sequences can be inserted at various positions in the immunogen as described above using this technique.
An exemplary syntllesis is described in Example 1. The immunogenic composition can be minimally purified to remove solvents and reagents. Rigorous purification is not likely to be necessary for the compositions to pass safety tests. This synthesized mixture can then be tested in animals, or used in humans, in a partially purified form. However, optional conventional purification schemes may be employed, if necessary.
While the above described synthetic method is desirable for its simplicity, an alternative method of preparing the immunogens involves the use of recombinant DNA
technology. As well known in the art, a nucleotide sequence (which encodes the angiotensin peptides, optional linkers (with or without polar sequences, e.g., for solubility), T cell helper sequences, and linkers for the lipopeptide cap) is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., MOLECULAR
CLONING, A
LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
The attachment of the lipopeptide cap involves synthetic methods as described above.
One of skill in the art may readily generate a variety of immunogenic compositions containing an immunogen by following these methods. Similarly, if different immunogens are desired in the composition, the various components of the immunogen of Formula I, II and/or III are modified individually or collectively, as provided above, such as modifications to individual amino acids, uses of optional linker sequences, uses of different T cell helper or lipopeptide caps, uses of angiotensin II
and/or I peptides, modified angiotensin II and/or I peptides, or combinations of different immunogenic compositions made with such modified sequences.
Such immunogenic compositions are able to induce a prophylactic immune response or therapeutic immune response to angiotensin proteins in vivo by inducing anti-angiotensin antibodies with geometric mean titers (GMT) sufficient to prevent or treat, or retard progression of existing hypertension. The titer is the reciprocal of the greatest serum dilution that is still detected at a level of mean + 8 standard deviations (SDs) of control values. The geometric mean titer (GMT) is determined by converting each titer of two or more sera to logio, and averaging these log10 values. The anti-log of this latter value is the GMT. In most embodiments, the GMT is determined from three or more individual titers. The use of the GMT rather than individual titers minimizes extreme outlying results and thus improves accuracy.
In one embodiment, an immunogenic composition as described above induces anti-angiotensin antibodies with GMT of greater than 100,000 or greater. In another embodiment immunogenic compositions induce anti-angiotensin antibodies in vivo with GMT
of 300,000 or greater. In other embodiments, the titers induced are greater than 1,000,000.
The examples below report experiments in laboratory animals that provide evidence of the extraordinary titers induced by single administrations of immunogenic compositions described herein.
Depending upon the selection and composition of other components used in the pharmaceutical compositions and the regimens and routes of administration of these compositions, the induction of such GMT responses is anticipated with compositions other than those specifically exemplified.
X. Pharmaceutical Compositions and Methods of Treatment/Prophylaxis A pharmaceutical composition containing the above-described immunogens is useful for the therapeutic treatment of hypertension and/or as a prophylactic immunogenic composition. In various embodiments, the pharinaceutical compositions employ a self-adjuvanting immunogenic composition which contains an immunogen of the Formula I, II and/or III above, and a pharmaceutically acceptable carrier. Desirably, the mixture of unpurified immunogenic peptide immunogens prepared as described above are dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
As defined herein, pharmaceutically acceptable carriers suitable for use in these immunogenic compositions are well known to those of skill in the art. In one embodiment, a preferred pharmaceutical carrier contains water for injection (WFI) with mannitol added for tonicity at a concentration of about 45 mg/mL. Other possible carriers include, without limitation, and depending upon pH adjustments, buffered water, buffered saline, such as 0.8% saline, phosphate buffer, 0.3%
glycine, hyaluronic acid, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants and excipients, may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusting agents may also be employed.
Buffers include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, trimethamine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like may also be provided in the pharmaceutical carriers. These immunogenic compositions are not limited by the selection of the carrier. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art. See, e.g., texts such as Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, publ., 2000; and The Handbook of Pharmaceutical.
Excipients, 4`h edit., eds. R. C. Rowe et al, APhA Publications, 2003.
Optionally, the pharmaceutical compositions can also contain a mild adjuvant, such as an aluminum salt, e.g., aluminum hydroxide or aluminum phosphate.
The concentration of immunogens of Formula I, II or III in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1 mg/mL, usually at or at least about 2 mg/mL, up to as much as 20 mg/mL, or up to 50 mg/mL or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
A human unit dose form of the immunogenic composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17"' Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985).
These compositions may be sterilized by conventional, well known sterilization techniques, such as sterile filtration for biological substances. Resulting aqueous solutions may be packaged for use as is. In certain embodiments in which at least one polar sequence, a sequence of four lysines, e.g., -K-K-K-K-, is present in the immunogen of the composition, the aqueous solutions are lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
Thus, as yet another aspect is a method of inducing in vivo the production of anti-angiotensin antibodies with GMT of greater than 100,000, or greater than 300,000 or greater than 1,000,000, as provided above. In one embodiment, the pharmaceutical compositions may be therapeutically administered to treat hypertension. In another embodiment, the pharmaceutical composition is administered to a healthy subject as a prophylactic immunogenic composition for prevention of hypertension.
This method involves administering to a subject an effective antibody-inducing amount of the pharmaceutical compositions described herein, so as to induce anti-angiotensin antibody in mammalian subjects having GMT greater than 100,000, greater than 300,000, greater than 1,000,000.
As described above, this method induces antibody with much higher GMT as well.
In one embodiment, the method involves administering one pharmaceutical composition.
In another embodiment, the method involves repeatedly administering the composition but at infrequent intervals, e.g., every 6 months. In healthy patients, the prophylactic immunogenic composition provides the immunized subject with antibodies that block the onset of hypertension.
In one embodiment of this method, the route of administration of these pharmaceutical compositions is subcutaneous injection. Other suitable routes of administration include, but are not limited to, intramucosal, such as intranasal, oral, vaginal, or rectal, and parenteral, intradermal, transdermal, intramuscular, intraperitoneal, intravenous and intraarterial.
The appropriate route is selected depending on a variety of considerations, including the nature of the composition, i.e., as a prophylactic immunogenic composition or as a therapeutic, and an evaluation of the age, weight, sex and general health of the patient and the components present in the immunogenic composition, and similar factors by an attending physician.
Similarly, suitable doses of the self-adjuvanting immunogenic compositions are readily determined by one of skill in the art, whether the patient is already hypertensive and requires therapeutic treatment or prophylactic immunogenic composition treatment, based on the health, age and weight of the patient. The method and routes of administration and the presence of additional components in the compositions may also affect the dosages and amounts of the compositions. Such selection and upward or downward adjustment of the effective dose is within the skill of the art. The amount of composition required to produce a suitable response in the patient without significant adverse side effects varies depending upon these factors. Suitable doses are readily determined by persons skilled in the art. A suitable dose is formulated in a pharmaceutical composition, as described above (e.g., dissolved in about 0.1 mL to about 2 mL of a physiologically compatible carrier) and delivered by any suitable means. Dosages are typically expressed in a "unit dosage", which is defined as dose per subject, e.g., a unit dosage of 1 mg immunogen. Alternatively dosages can be expressed as amount per body weight of the subject or patient, using the norm for therapeutic conversions as 80 kg body weight. For example, a 1 mg unit dose per subject is equivalent to about 12.5 g/kg body weight.

In one embodiment, the intended therapeutic or prophylactic effect is conferred by a single dose, followed by an optional booster dosage given from 3 to 6 months or more following the original dose. According to this regimen, the booster dosages may be lower than the original single dose. In another embodiment, the therapeutic or prophylactic effect is conferred by a priming/boosting dosing regimen, wherein the priming dosages is followed in 3-4 weeks by a boosting dosage, and boosting dosages are repeated at various intervals thereof. In this case also, the boosting dose can be less than the priming dose. For example, the dosage for a single therapeutic administration or for a first priming therapeutic or prophylactic immunogenic composition administration in one embodiment is a "unit dosage" of less than about 0.01 mg to about 100 mg of immunogen. In one embodiment, the unit dosage is 0.01 mg. In another embodiment, the unit dosage is 0.1 mg. In another embodiment, the unit dosage is 1 mg. In still another embodiment, the unit dosage is 10 mg. Thus, the initial single dose or the priming dosage for a human, in certain embodiments, can range from very low unit dosages of at least about 0.01, 0.02, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, to higher dosages of at least I
mg, at least 3 mg, at least 5 mg, at least 7 mg, at least 10 mg, at least 12 mg, at least 15 mg, at least 20 ing. Still other human dosages range from between 21-30 mg, 31-40 mg, 41-50 mg/70-80 kg subject.
These dosages may include fractional dosages between the stated dosages.
Higher or lower dosages may be contemplated.
In one embodiment, the boosting dosages for either therapeutic prophylactic immunogenic composition or prophylactic immunogenic composition use are the same as the above described priming dosage. The saine specific unit dosage or unit dosage ranges as for the priming dosage above may be employed for the boosting dosage. Thus, the boosting dosage for a human, in certain embodiments, can occur in a unit dosage range a "unit dosage" of less than about 0.1 mg to 100 mg of immunogen. In one embodiment, the unit dosage is 0.1 mg. In another embodiment, the unit dosage is 1 mg. In still another embodiment, the unit dosage is 10 mg. Thus, the booster unit dosage for a human, in certain embodiments, can range from very low unit dosages of at least about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, to higher dosages of at least 1 mg, at least 3 mg, at least 5 mg, at least 7 mg, at least 10 mg, at least 12 mg, at least 15 mg, at least 20 mg. Still other human dosages range from between 21-30 mg, 31-40 mg, and 41-50 mg/70-80 kg subject.
These dosages may include fractional dosages between the stated dosages. Even higher dosages may be contemplated. In an alternative embodiment, the boosting dosages are considerably lower than the priming dosages identified above.
In one embodiment, the first "boosting" is administered within weeks of the initial priming dose. In one embodiment, the boosting dose is administered at least 3 weeks after the priming dose, followed by a re-boost administered not earlier than 3 weeks from the preceding boosting dose. In another embodiment, the first boosting dose is administered about 3 to 4 weeks following the priming dose. Additional boosting dosages are administered thereafter at least 3 weeks thereafter, more suitably about 6 months to one or more years, following the first booster dose. In another embodiment of an administration protocol, a priming dosage of a self-adjuvanting immunogenic composition as described herein is administered which is about 10 mg. The subsequent first boosting dosage (e.g., 3-10 mg) is then administered at least three weeks after the priming dosage. Thereafter, additional boosting dosages are administered every 6 months to one year from the preceding boosting dosage.
The timing and dosage of any priming/boosting regimen may be selected by the attending physician depending upon the patient's response and condition as determined by measuring the specific anti-angiotensin antibody titer obtained from the patient's blood, as well as normal considerations related to the physical condition of the patient, e.g., height, weight, age, general physical health, other medications, etc.
In one embodiment of the prophylactic/therapeutic method involves administering an effective amount of the immunogenic composition in a unit dosage of less than or about 10 mg, less than or about 1 mg, less than or about 0.1 mg or less than or about 0.01 mg.
This administration may be followed by a booster administered from 3-12 months following the original administration at the same or lower effective amount. In another embodiment of the prophylactic/therapeutic method, a priming "unit" dosage of less than or about 10 mg, less than or about 1 mg, less than or about 0.1 mg or less than or about 0.01 mg is followed up by the administration by one or two boosters administered at weeks 3 and/or weeks 6 at the same or lower effective unit dosage. These methods induce antibodies of a GMT greater than 100,000, optionally without any extrinsic adjuvant..
In another embodiment of the prophylactic/therapeutic method involves administering a priming effective amount of the immunogenic composition in a unit dosage of less than or about 10 mg, less than or about 1 mg, less than or about 0.1 mg, or less than or about 0.01 mg, and following up the administration optionally by one or two boosters administered at weeks 3 and/or weeks 6 or at month 3 and/or month 6 at the same or lower effective dosage. This method induces antibodies of a GMT greater than 1,000,000 optionally without any extrinsic adjuvant.
In one embodiment, the administration desirably continues until blood pressure has been reduced to normal levels and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
For use as prophylactic immunogenic composition use, the optional boosting dosages are similar to (or lower than) the priming dosages of the therapeutic prophylactic immunogenic composition, but are administered at certain defined intervals from about two weeks to six months after the initial administration of prophylactic immunogenic composition.
Possibly additional prophylactic immunogenic composition administrations may be desirable thereafter.
As indicated in the examples below, the antibody witli high GMT induced by the exemplary pharmaceutical or immunogenic compositions described herein may reduce the need for a high frequency of boosting dosages, or any boosting at all, for either therapeutic or vaccinal use.
In still another embodiment of the methods described herein, the compositions may be used in conjunction with, or sequentially with, other hypertension therapies or pharmaceutical regimens.
The following examples illustrate certain embodiments of the above-discussed compositions and methods. These exainples do not limit the disclosure of the claims and specification.

IV. EXAMPLES

EXAMPLE 1: Generation of an Immunogenic Composition of the Invention Various immunogenic compositions as described above are prepared containing angiotensin peptide components (bolded), a T cell helper sequence (italicized and bolded), linker amino acids (italicized only) and the Pam2C- or Pam3C- lipoprotein cap according to the formulae below (note that the NAc(Pam2C) capped formula is to be prepared in a conventional methodology):
SEQ ID NO: 28 :
(Pam2C or Pam3C or NAc(Pam2C)-S-S-Q-Y-I-K-A-N-S-K-F-I-G-I T-E-L-S-D-R-V-Y-I-II-P-F; or SEQ ID NO: 29 :
(Pam2C or Pam3C or NAc(Pam2C)-S-S-Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-S-D-R-V-Y-I-H-P-F-H-L.
Alternative immunogens are prepared in a similar manner for other similar compositions which are expected to produce similar results. Such similar immunogens include an immunogen of Formula III, e.g., SEQ ID NO: 30:
Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-K-S-D-R-V-Y-I-H-P-F
Pam2C-S-S

and an immunogen of Formula II, Pam2C-S-S-K-S-D-R-V-Y-I-H-P-F
I
Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L
The Pam2C- and Pam3C immunogens prepared according to the preceding formulae are synthesized by Bachem Biosciences, Inc. or Mimotopes, Pty. Ltd. or Anaspec, Inc. using conventional solid phase synthesis techniques and automated synthesizers. Commencing with the appropriate resin that can be cleaved leaving a free hydroxyl group on the last ainino acid, e.g., a free C-terminal Phe-OH, the cycles of synthesis proceed towards the N-terminus of the B cell peptide, through the linker ainino acids, through the helper T cell epitope sequence (which preferably is the tetanus toxoid promiscuous T helper sequence Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L SEQ ID NO: 21) and through Ser-Ser (or optional polar sequence) at the N-terminus for the first immunogen above.
Tripalmitoyl-S-glyceryl-cysteine, and fmoc protected di-palmitoyl-S-glyceryl-cysteine are synthesized by Bachem and are coupled to the N-terminal serine of the nascent peptide chain as indicated. Other synthetic methods known in the art may also be employed.
Trifluoracetic acid is used to cleave the lipopeptide from the resin and deprotect the peptide. The resulting immunogenic product is dried, then taken into aqueous solution, converted to an acetate salt form and dried.
The other immunogens above are prepared in a similar manner, depending upon which amino acid sequence is coupled to the resin. The final products are checked by amino acid analysis for the appropriate content of amino acids, and by mass spectroscopy. Purity is estimated in general to be around 70%, but the resulting lipopeptides are not purified further in the examples provided.
For use in the following experiments, the immunogens referred to as TANGI-K4 and TANGI-K6 each contain a polar charged sequence of four lysine residues (K) or six lysine residues (K) (underlined) inserted between the serines at the carboxy terminal end of the lipopeptide. In these immunogens, angiotensin II has a free carboxy terminus.
TANGI-K4 has the formula:
Pam2C-S-K-K-K-K-S-Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-S-D-R-V-Y-I-H-P-F-SEQ ID NO: 23.
TANGI-K6 has the formula:
Pam2C-S-K-K-K-K-K-K-S-Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L-S-D-R-V-Y-I-H-P-F-SEQ ID NO: 31.

EXAMPLE 2: Immunization Protocol and Titers A. Immunizations Animals were purchased, such as from Harlan Laboratories, and acclimated, such as at Molecular Diagnostic Services, Inc., for at least 1 week before immunization.
Immunogen TANGI-K4 was prepared as described in Example I and taken up in mannito145mg/mL in water for injection (WFI). Rats (n=3), A, B, and C, were immunized by subcutaneous injection with 10 mg/rat with the TANGI-K4 immunogen on day 0. The rats were bled at weeks 2 and week 3, and the anti-angiotensin titer determined, as indicated in FIG. 2. Serums were assayed to determine anti-angiotensin titers against synthetic angiotensin I, angiotensin II and angiotensin III, purchased from Anaspec Inc., by conventional ELISA methods.
As can be observed from the graph of the titer results in FIG. 2, the titers (3 rats) against free angiotensin II at 2 weeks ranged from 70,000 to 500,000 (GMT 190,000) and at three weeks ranged from 250,000 to 900,000 (GMT 500,000). Crossreactivity for antgiotensin I was 8% and for angiotensin III was 70%.
Thereafter a booster administration of 10 mg/rat is administered at weeks 3 and at week 6.
The rats are bled, and titers are determined two weeks after each boost, i.e., at week 5 and week 8. It is anticipated that the geometric mean titers (3 rats) against angiotensin will be greater than about 1,000,000 at 5 weeks and greater than 2,000,000 at 8 weeks after the first injection. The three weekly spacing is expected to greatly enhance the antibody response.
Surprisingly, therapeutic titers were achieved with a single dose of immunogen. Based upon this data, it is anticipated that these immunogens will permit a marked reduction in the vaccine amount required to achieve a successful therapeutic titer. For example, doses as low at 0.1 mg are likely to achieve therapeutically effective titers. Such low therapeutically effective doses will provide dramatic and unexpected therapeutic and cost benefits to the patient population worldwide.
EXAMPLE 3: Reduction of Free Angiotensin Levels By Immune Serum A. Angiotensin Depletion Assay Briefly, this assay employs Protein A-coated beads that are incubated with 10%
serum (immune serum from the immunized rats or control serum from unimmunized rats) to bind serum IgG. The beads are thoroughly washed and exposed to a solution of synthetic angiotensin 11(Anaspec Inc.) and incubated for 30 minutes. The anti-angiotensin antibody binds the synthetic angiotensin, depending upon the amount of captured anti-angiotensin antibody on the beads.
After incubation, the beads are centrifuged and supernatant assayed for free unbound angiotensin II
using a competitive ELISA test kit (Bachem.).
This assay thus measures how well anti-angiotensin antibody in immune serum reduces levels of free angiotensin II. For technical reasons, 10% serum was used so that the Protein A
immunoglobulin binding capacity was not saturated. The titers tested are 1/10 of the titers in undiluted serum.

Quintuple assays were performed for each condition (immune or control) and the concentration of angiotensin II in active supernatant was expressed as percentage of mean level of free angiotensin II in control assays.
B. Results FIG. 3 is a graph showing the results of this assay. Reductions of 53% to 83%
are seen in unbound angiotensin, with each point representing a replicate in normal rat serum control, or in diluted serum with titers of 20,000, 50,000, and 90,000. Reductions in free angiotensin levels in undiluted serum, with tenfold higher titers, would likely have approached or exceeded 99%.

EXAMPLE 4: Aqueous Solubilization by Insertion of Polar Charged Sequence Within the Linker of the Lipopeptide Cap All immunogens designed with Pam2Cys- or Pam3Cys or NAcPam2C with -S-S- linker as described herein were found to be insoluble in aqueous solvents. To achieve solubility of lipopeptides with all three lipopeptides caps in aqueous solvents, immunogens were dissolved in dimetliylsulfoxide (DMSO) and diluted with phosphate buffered saline to 5-10%
DMSO. This yielded an opalescent somewhat turbid solution that was injected into animals.
The TANGI-K4 immunogen described in Example I above has improved solubility, yielding clear solutions at 2 mg/mL in 45 mg/ML mannitol in WFI and at concentrations up to 10 mg/mL.
Solubility in higher concentrations, such as 20 mg/mL, was less complete, but the immunogen at this concentration was highly effective in attaining the titers in Example 2. TANGI-K6 has similar or slightly worse solubility characteristics, as seen in the results of the table below.
A clear solution is anticipated to remain after overnight storage at 4 C. A
summary of solutions and data to be recording is reflected in Table 1, below. The TANGI-K4 immunogen is also expected to be soluble in 25 mg/mL mannitol in water for injection and readily lyophilizable to form a "cake", which is fully soluble in added water for injection.

TABLE 1: ENHANCED SOLUBILITY OF PAM2CSKKKKS- CAPPED IMMUNOGEN

Sample Appearance at Appearance Precipitate after pH
RT after 37 C Centrifugation incubation TANGI-K4 Clear, colorless Clear No 5.5 2 mg/ml in 45 mg/ml mannitol in WFI
TANGI-K4 Slightly cloudy Clear No 5.5 mg/ml in 45 with visible mg/ml mannitol in precipitates WFI
TANGI-K4 Cloudy with large Clear with Not significant 5 mg/ml in 45 visible minor mg/ml mannitol in precipitates precipitate WFI visible TANGI-K4 Not tested Clear with Slight precipitate 5 mg/ml in 45 some small visible mg/ml mannitol in amount of WFI precipitate visible TANGI-K4 Cloudy with large Clear with Some precipitate 5 mg/ml in 45 visible ppt some small visible mg/ml mannitol in amount of WFI precipitate visible TANGI-K6 Clear colorless Clear No 5.5 2 mg/ml in 45 mg/ml mannitol in WFI
TANGI-K6 Cloudier than Cloudy Yes 5 20 mg/ml in 45 Tangi-K4 but mg/ml mannitol in with smaller WFI precipitate 5 EXAMPLE 5: Bioassay of hypertension and inhibition by antibody Efficacy experiments are conducted in spontaneously hypertensive rates monitored by tail-cuff blood pressure measurements, as described substantially in Ambuhl et al, 2007 J. Hypertens., 25(1): 62 at page 65, col. 2 using the TANGI-K4 immunogen of Example 1 above.

Briefly, four groups of rats (10 week old) are used in this experiment: 3 normotensive (normal) rats receiving immunogen, 3 SHR hypertensive rats receiving immunogen, 3 normotensive rats receiving no immunogen (normal control), and 3 SHR hypertensive rats receiving no immunogen (hypertensive control). The two groups of experimental animals are immunized with 10 mg of immunogen TANGI K4 subcutaneously on Day 0. The two control groups receive excipients only.
Arterial blood pressure is measured before immunization and at weeks 3 and 6 by the tail-cuff method. Samples of blood are also titered for levels of anti-angiotensin II
antibody. Angiotensin II
determinations are taken at the same timepoints. During measurements, animals are held in, and acclimated to, restraining devices. Systolic and diastolic blood pressures are determined as a mean of at least 5 determinations at each timepoint. Results are anticipated to show that immunization with the immunogen described herein effectively restores blood pressure to normal levels in hypertensive rats.

EXAMPLE 6: Reduction of Free Angiotensin Levels by Immune Serum using an Alternative ELISA Based Technique A conventional sandwich ELISA assay was performed using non-immune serum of known titer and immune anti-antiogensin II serum of known titer, using a capture antibody recogniziilg the same epitope ( D-R-V-Y-I-H-P-F SEQ ID NO: 1) that is recognized by the antibody developed to the TANGI-K4 immunogen.
Non-immune serum was obtained from unimmunized rats. Immune anti-angiotensin II
serum was generated by injecting 3 rats with the TANGI-K4 immunogen as described above in Example 2. The capture antibody was commercially available from Peninsula Laboratories (T-4004 rabbit IgG a-angiotensin II (human)). The capture reagent is streptavidin/horseradish peroxidase.

A "spike" (approximately 2 ng/mL) of antigen was added to the each assay sample (i.e., containing a titer from a representative sample of serum). The antigen was biotinylated angiotensin II
(i.e., Biotin-Acp-D-R-V-Y-I-H-P-F SEQ ID NO: 1), i.e., angiotensin II
synthesized with a biotin group and an aminocaproic acid (Acp) spacer at the N-terminus. Angiotensin II
has only one B cell epitope at the C-terminus so, for the purpose of this assay, the B cell epitope remained intact.
The capture antibody on the plate can only recognize free antigen, since the angiotensin II
epitope is blocked in the serum sample by the binding of any anti-angiotensin antibody. The assay reveals unbound antigen in each serum sample once the capture reagent is added to the assay samples.
From this, the inhibition of free antigen level for each sample s is calculated.
Using this assay and dilutions from a single representative serum sample, diluted with normal serum, a dose-response curve for biotin-angiotensin II was obtained. The results of this depletion assay are shown in FIGs. 4 and 5. FIG. 4 is a graph showing the dose-response characteristics of the biotin-angiotensin II sandwich ELISA, plotting OD vs. ng/mL of biotinylated angiotensin II. FIG. 5 is a graph showing the concentrations (ng/mL) of free biotin-angiotensin II in the presence of anti-angiotensin II antibodies (serum titers of 1000 to 1,000,000), with 1000 being the titer assigned to non-immune serum and 1,000,000 being the titer of undiluted sample serum).
Reductions of free angiotensin II of 87%, 98% and 99% were present in the presence of anti-angiotensin titers of 30,000, 100,000 and 300,000, respectively. These results are in agreement with the trends found in Example 3 above.
Numerous modifications and variations of the embodiments illustrated above are included in this specification and are expected to be obvious to one of skill in the art.
Such modifications and alterations to the compositions and processes described herein are believed to be encompassed in the scope of the claims appended hereto. All documents listed or referred to above, including the attached Sequence Listing, are incorporated herein by reference.

Claims (40)

1. A self-adjuvanting immunogenic composition comprising an immunogen comprising:
(a) an angiotensin B cell epitope having an amino acid sequence selected from the group consisting of D-R-V-Y-I-H-P-F (SEQ ID NO: 1) and D-R-V-Y-I-H-P-F-H-L (SEQ ID
NO: 2);
(b) a universal T helper sequence (R1); and (c) a lipopeptide cap (R2) selected from the group consisting of a dipalmitoyl-S-glyceryl-cysteine, a dipalmitoyl-S-glyceryl-(N-acetyl-cysteine), and a tripalmitoyl-S-glyceryl cysteine.

2. The composition according to claim 1 having a formula of R2- R1- angiotensin B cell epitope.

3. The composition according to claim 1 having a formula of R2-K(R1)-angiotensin B cell epitope or R1-K(R2)-angiotensin B cell epitope.

4. The composition according to claim 1 further comprising a linker sequence of from one to ten amino acids.

5. The composition according to claim 4, wherein said linker sequence is attached to the Cys of R2 to link R2 to other components or is located between other components of said immunogen.

6. The composition according to claim 3, wherein R1 is linked to an .epsilon.-amino of a K residue located between R2 and the amino terminus of said angiotensin epitope.

7. The composition according to claim 3, wherein R2 is linked to an .epsilon.-amino of a K residue located between R1 and the amino terminus of said angiotensin epitope.

8. The composition according to any of claims 1 to 7, wherein said immunogen further comprises a sequence of charged polar amino acids optionally flanked by one or more neutral linker amino acids, and located at a position selected from the group consisting of (a) as part of the carboxy terminus of R2, (b) between R1 and said B cell epitope, (c) at the free amino terminus of R1, (d) at the free amino terminus of said B cell epitope, (e) interposed between the inserted lysine residue and any other component of the immunogen.

9. The composition according to any one of claims 4-7, wherein said linker is selected from the group consisting of -S-, -S-S-, -G-, and -G-S-.

10. The composition according to claim 8, wherein said polar sequence comprises a sequence of from 4 to 8 amino acids selected individually from the group consisting of K, E, D, and R which is optionally flanked by said linker amino acids.

11. The composition according to claim 10, wherein said polar sequence is selected from the group consisting of -K-K-K-K- SEQ ID NO: 12, -S-K-K-K-K-S- SEQ ID NO: 16, G-K-K-K-K-G
SEQ ID NO: 18, S-K-K-K-K-K-K-S SEQ ID NO: 17, and G-K-K-K-K-K-K-G SEQ ID NO:
19.

12. The composition according to any one of claims 1-11 wherein said R1 sequence is selected from the group consisting of:
(a) Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-Xaa SEQ ID NO: 3, wherein said Xaa is absent or L, with an optional amino acid linker;
(b) Xaa1-K-Xaa2-V-A-A-W-T-L-K-A-A- Xaa3 SEQ ID NO: 11, wherein Xaa1 and Xaa3 are each a D-Alanine and Xaa2 is L-cyclohexylalanine;
and (c) F-N-N-F-T-V-S-F-W-L-R-V-P-K-V-S-A-S-H-L-E- SEQ ID NO: 4.

13. The composition according to claim 1, comprising an angiotensin B cell epitope having the amino acid sequence D-R-V-Y-I-H-P-F SEQ ID NO: 1.

14. The composition according to claim 1, comprising an angiotensin B cell epitope having the amino acid sequence D-R-V-Y-I-H-P-F-H-L SEQ ID NO: 2.

15. The composition according to any of claims 1-14, wherein, in each immunogen, R2 is selected from the group consisting of dipalmitoyl-S-glyceryl-cysteine or N-acetyl (dipalmitoyl-S-glyceryl cysteine) or tripalmitoyl-S-glyceryl cysteine; R2 further comprises an amino acid linker sequence of -S-S- residues; and R1 is Q-Y-I-K-A-N-S-K-F-I-G-I-T-E-L SEQ ID NO:
21 with an optional amino acid linker of -S- linking it to the B cell epitope.

16. The composition according to claim 15, wherein a polar amino acid sequence of -K-K-K-K-SEQ ID NO: 12 is located between the -S-S- of said linker residues.

17. The composition according to any one of claims 1-16, wherein, in each immunogen, R2 is dipalmitoyl-S-glyceryl-cysteine or N-acetyl (dipalmitoyl-S-glyceryl cysteine) or tripalmitoyl-S-glyceryl cysteine, and an amino acid linker sequence of -S-S- residues; and R1 is Xaa1-K-Xaa2-V-A-A-W-T-L-K-A-A- Xaa3 SEQ ID NO: 11, wherein Xaa1 and Xaa3 are each a D-Alanine and Xaa2 is L-cyclohexylalanine, with an optional amino acid linker of -S-.

18. The composition according to any one of claims 1-17, wherein said composition induces in an immunized subject anti-angiotensin antibodies with a geometric mean titer of 100,000 or greater.

19. The composition according to any one of claims 1-18, wherein said composition induces in an immunized subject anti-angiotensin antibodies with a geometric mean titer of greater than 300,000.

20. The composition according to any one of claims 1-18, wherein said composition induces in an immunized subject anti-angiotensin antibodies with a geometric mean titer of greater than 1,000,000.

21. A pharmaceutical composition comprising the self-adjuvanting immunogenic composition of any of claims 1-20, and a suitable pharmaceutical carrier or excipient, wherein said composition induces in an immunized subject anti-angiotensin antibodies with a geometric mean titer of greater than 100,000.

22. The pharmaceutical composition according to claim 21, wherein said geometric mean titer is greater than 1,000,000.

23. A method of inducing in vivo the production of anti-angiotensin peptide antibodies comprising immunizing a subject with an effective antibody-inducing amount of the composition of any one of claims 21 or 22, wherein said effective amount induces anti-angiotensin antibodies with a geometric mean titer of 100,000 or greater.

24. The method according to claim 23, wherein said geometric mean titer is greater than 300,000.

25. The method according to claim 24, wherein said geometric mean titer is greater than 1,000,000.

26. The method according to claim 24, comprising administering to said subject an initial effective amount of said composition, followed by an optional booster effective amount of said composition.

27. The method according to claim 26, comprising administering a first booster effective amount to said subject at least 3 weeks after said priming amount.

28. The method according to claim 27 comprising administering a second booster effective amount to said subject periodically after said priming amount and first booster amount.

29. The method according to claim 26 or 28, wherein said booster administration occurs every 6 months or longer from said prior administration.

30. The method according to any one of claims 23-29, wherein said effective amount is 10 mg or less.

31. The method according to any one of claims 23-29, wherein said effective amount is 1 mg or less.

32. The method according to any one of claims 23-29, wherein said effective amount is 0.1 mg or less.

33. The method according to any one of claims 23-29, wherein said effective amount is 0.01 mg or less.

34. Use of a composition of any of claims 1-22 in the preparation of a medicament for inducing in vivo the production of anti-angiotensin antibodies at a geometric mean titer (GMT) greater than 100,000.

35. Use according to claim 34, wherein the dose of the composition is 10 mg or less.

36. Use according to claim 34, wherein the dose of the composition is 1 mg or less.

37. Use according to claim 34, wherein the dose of the composition is 0.1 mg or less.

38. Use according to claim 34, wherein the dose of the composition is 0.01 mg or less.

39. Use according to claim 34, wherein said GMT is greater than 300,000.

40. Use according to claim 34, wherein said GMT is greater than 1,000,000.