# Patent

## Title
Methods for treating kidney disease with fragments of ApoB-100

## Abstract

The invention provides compositions comprising immunogenic fragments of ApoB-100 for eliciting an immune response in a subject or vaccinating a subject, so as to treat, prevent, 5 inhibit and/or reduce symptoms of kidney diseases in the subject. The compositions include immunogenic fragments of ApoB-100, CD8+ T cells activated with immunogenic fragments of ApoB-100 or a combination thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Phase of International Application PCT/US2012/063705, filed Nov. 6, 2012, which designated the U.S. and that International Application was published under PCT Article 21(2) in English. The present application also claims the benefit of the filing date of U.S. Provisional Application No. 61/558,908 filed Nov. 11, 2011, the disclosures of each of which are incorporated herein by reference in their entirety.


FIELD OF INVENTION
The invention provides compositions and methods for treating kidney diseases in a subject in need thereof. The compositions include one or more peptides of ApoB-100 as set forth in Table 1 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof.
BACKGROUND OF THE INVENTION
All publications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
The renin-angiotensin system (RAS) or the renin-angiotensin-aldosterone system (RAAS) is a hormone system that regulates blood pressure and fluid balance and is activated when there is a drop in blood volume or blood pressure. When the blood volume is low, renin is secreted into the plasma by the juxtaglomerular cells of the kidneys. The plasma renin carries out the conversion of angiotensinogen released by the liver to angiotensin I (Kumar et al., 2010 “11”. Pathologic Basis of Disease (Eighth ed.). Philadelphia: Saunders Elsevier. p. 493. ISBN 978-1-4160-3121-5). Angiotensin I (AngI) is then converted to angiotensin II (AngII) by the angiotensin converting enzyme (ACE) found in the lungs. AngII is a potent vaso-active peptide which causes blood vessels to constrict, resulting in increased blood pressure. AngII also stimulates the secretion of the hormone aldosterone from the adrenal cortex. Aldosterone causes the tubules of the kidneys to increase the reabsorption of sodium and water into the blood. This increases the volume of fluid in the body, which also increases blood pressure.
AngII activates at least two receptors, namely, angiotensin II type 1 (AT1) and angiotensin II type II (AT2). Majority of the effects of AngII, such as vasoconstriction, proteinuria, fibrosis and inflammation are mediated by the AT1 receptor. Hypertension and proteinuria are the most important risk factors for the progression of renal disease. Haemodynamic and non-haemodynamic effects of angiotensin II are critically involved in the development and maintenance of hypertension and proteinuria. Therefore, suppression of angiotensin II formation by angiotensin-converting enzyme (ACE) inhibitors and blockade of AT1 receptor by angiotensin II receptor blockers (ARB) are powerful therapeutic strategies that effectively slow the progression of renal disease by lowering blood pressure and proteinuria (Wenzel et al., J Renin Angiotensin Aldosterone Syst. 2010 March; 11(1):37-41. Epub 2009 Oct. 27; Wenzel U O, Contrib Nephrol 2001; 135:200-11). It has been speculated that the AT2 receptor may exhibit beneficial effects (such as anti-proteinuric, anti-fibrotic and anti-inflammatory) and that the blockade of the AT1 receptor results in increased synthesis of AngII which in turn stimulates the AT2 receptor (Wenzel et al., J Renin Angiotensin Aldosterone Syst. 2010 March; 11(1):37-41. Epub 2009 Oct. 27).
The inventors observed that peptides of ApoB-100 reduce the levels of the AT1 receptor and therefore these peptides serve as therapeutics in kidney diseases.
SUMMARY OF THE INVENTION
The invention provides composition comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof.
In one embodiment, the composition comprises one or more peptides of ApoB-100 and/or immunogenically active portions, derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and a pharmaceutically acceptable carrier that induces and/or enhances an immune response. The composition elicits an immune response and/or used for immunization. The composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the carriers induce and/or enhance an immune response. In an embodiment, the peptide of ApoB-100 is any one or more of peptides P1 to P302 as set forth in Table 1. In a further embodiment, one or more of the peptides of ApoB-100 are immunogenic peptides of ApoB-100.
In another embodiment, the composition includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and CD8+ T cells. The CD8+ T cells are activated by one or more immunogenic peptides of ApoB-100. The composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the carrier induces and/or enhances an immune response. The CD8+ T cells may be autologous. In an embodiment, the peptide of ApoB-100 is any one or more of peptides P1 to P302 as set forth in Table 1. In a further embodiment, one or more of the peptides of ApoB-100 are immunogenic peptides of ApoB-100.
The compositions of invention may be used for treating kidney disease, inhibiting kidney disease, preventing kidney disease and/or promoting prophylaxis of kidney disease in a subject in need thereof. The methods for these indications include providing compositions that include one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof, and administering an effective amount of the composition so as to treat, inhibit, prevent and/or promote prophylaxis of kidney disease in the subject.



BRIEF DESCRIPTION OF FIGURES
Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
FIG. 1 depicts, in accordance with an embodiment of the invention, (A) a schematic showing the immunization schedule, and (B) that p210 significantly decreased the expression of AT1R in aorta, as assessed by Western blot analysis (I), from mice immunized with p210, adjuvant/carrier control or PBS after 4-weeks of angiotensin II infusion delivered by a subcutaneously implanted pump; (II) Densitometric analysis. *p<0.05 vs. p210 by ANOVA following post-hoc test. N=6 in each group. p210: immunized with p210/cBSA/Alum+AngII; cBSA: immunized with cBSA/Alum+AngII; PBS: immunized with PBS+AngII.
FIG. 2 depicts, in accordance with an embodiment of the invention that the p210 vaccine significantly reduced IL-6 (N=8 each), MCP-1 (N=8 each), and TNF-α (N=10 each) mRNA expression in kidney. *p<0.05 vs. p210. p210: immunized with p210/cB SA/Alum+AngII; cBSA: immunized with cBSA/Alum+AngII; PBS: immunized with PBS+AngII.
FIG. 3 depicts, in accordance with an embodiment of the invention that the p210 vaccine significantly reduced glomerular ROS production. (A) Representative figure of in situ DHE labeling. (B) Densitometric analysis of DHE fluorescence. *p<0.05 vs. p210. N=4 each. p210: immunized with p210/cBSA/Alum+AngII; cBSA: immunized with cBSA/Alum+AngII; PBS: immunized with PBS+AngII.
FIG. 4 depicts, in accordance with an embodiment of the invention that (A) p210 vaccine significantly reduced NOX1, a component of NADPH oxidase, expression in kidney. *p<0.05 vs. p210. N=8 each, and (B) p210 vaccine significantly reduced PAI-1 and TGF-β mRNA expression in kidney. (Left): PAI-1. N=5 each, (Right): TGF-β. N=9 each. *p<0.05 vs. p210. p210: immunized with p210/cBSA/Alum+AngII; cBSA: immunized with cBSA/Alum+AngII; PBS: immunized with PBS+AngII.



DETAILED DESCRIPTION OF THE INVENTION
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.
Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
“Peptidomimetic” as used herein is a small protein-like chain designed to mimic a peptide. They may be modifications of an existing peptide or newly designed to mimic known peptides. They may be, for example peptoids and/or β-peptides and/or D-peptides.
“Beneficial results” may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy.
“Conditions” and “disease conditions,” as used herein may include, but are in no way limited to any form of kidney diseases.
“Kidney disease” as used herein refers to renal inflammatory response. In one embodiment, the renal inflammatory response is angiotensin II-induced renal inflammatory response. In various embodiments, kidney disease may be atherosclerosis-related, hypertension-related, diabetes and/or autoimmune diseases-related. Glomerular and renal dysfunction with resultant clinical or subclinical renal dysfunction is included in kidney diseases as used herein.
“Immunogenic fragment”, “antigenic fragment” or “immunogenic peptides” as used herein indicates a portion of a polypeptide of any length capable of generating an immune response, such as an antigen. An antigen is a molecule recognized by the immune system. An antigenic fragment of ApoB100 is accordingly a portion of ApoB-100 that presents antigenic properties (e.g. a specific humoral or cellular response). “Immunogenic fragments” also refer to derivatives of any fragment of ApoB100, such as mutated fragments (including fragments with replaced, added or deleted residues), oxidative derivatives and/or peptides treated with MDA or copper, which maintain a detectable antigenic property of the original fragment.
“Immune response” as used herein refers to immunities including but not limited to innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and/or overactive immunity.
“T cells” as used herein refers to T lymphocytes belonging to a group of white blood cells (lymphocytes), and participate in humoral or cell-mediated immunity. T cells can be distinguished from other lymphocyte types, such as B cells and natural killer cells (NK cells) by the presence of specific markers on their cell surface such as T cell receptors (TCR). Additional markers identifying T cell include but are not limited to any one or more of CD1a, CD3, CD4, CD8, or a combination thereof and additional markers possibly associated to a T cell state and/or functionality as will be understood by a person of ordinary skill in the art.
“CD8(+) T cells” or “CD8+ T cells” as used herein refer to T cells expressing the CD8 glycoprotein on their cell surface, wherein the CD8 (cluster of differentiation 8) glycoprotein is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Similar to the TCR, CD8 binds to a major histocompatibility complex (MHC) molecule, but is specific for the class I MHC protein. Exemplary CD8+ T cells comprise cytotoxic memory CD8+ T cells, regulatory CD8+ T cells, cytotoxic effector CD8+ T-cells and additional cells identifiable by a person of ordinary skill in the art.
The terms “enhancer” and “enhance” as it pertains to a molecule in connection with CD8(+) T cell refers to the ability of a molecule to modify the immune response by promoting the activation of cells of the immune system. The choice of appropriate enhancer can allow control of activation of the immune response. Exemplary enhancers include cytokines such as IL-10, IL-2, IL-12, IL-4 and IL-16. The term “cytokine” as used herein refers cell signaling molecules that act as has immunomodulating agents, and comprise proteins such as interleukins and interferons as would be identifiable to a skilled person. Selection of a suitable cytokine can result under appropriate conditions in the preferential induction of a humoral or cellular immune response.
The terms “activated” and “activation” as used herein refers to the process by which a T cells interact with an antigen presenting cells which presents a specific antigen for a time and under condition resulting in a T cell having a pre-assigned immunological role (e.g. cytotoxicity) within the immune system. The term “antigen-presenting cell” (APC) indicates a cell that displays antigen complex with major histocompatibility complex (MHC) on its surface. T-cells recognize this complex using their T-cell receptor (TCR). Exemplary APCs comprise dendritic cells (DCs) which are known to play an important role in linking innate and acquired immunity (Chyu, K. et al., 2005. Biochem. Biophys. Res. Commun. 338:1982-1989; Fredrikson, G. N., et al., 2003. Arterioscler. Thromb. Vasc. Biol. 23:879-884) and both immune responses participate in atherogenesis (Fredrikson, G. N., L. et al., 2005 Autoimmunity 38:171-179; Fredrikson, G. N., et al. 2008. J. Intern. Med. 1-8). In various embodiments, activated CD8(+) T cells according to the present disclosure are activated with one or more immunogenic fragment of ApoB100 or an immunogenically active portion thereof and are typically specific for the immunogenic fragment or the immunogenically active portion used for the activation.
“Fragment of ApoB100” as used herein refers not only to fragments/peptides of any length from ApoB100, but also peptides produced by genetic recombination or chemical synthesis, comprising sequences from ApoB100.
“Derivative” as used herein with reference to a first peptide (e.g., an immunogenic fragment), indicates a second peptide that is structurally related to the first peptide and is derivable from the first peptide by a modification that introduces a feature that is not present in the first peptide while retaining functional properties of the first peptide. A derivative peptide of an immunogenic fragment or of any portion thereof retains one or more of the immunogenic activities that are herein described in connection with an ApoB100 immunogenic fragment or portion thereof. The antigenic properties can be verified with methods and systems such as the ones already described for the immunogenic fragments and additional methods and systems identifiable to a skilled person. In an embodiment, a derivative of a peptide of ApoB-100 includes immunogenically active fragments of the peptides of ApoB-100 described herein.
“Immunogenically active portion” as used herein refers to any part of a reference antigen that can elicit a specific immune response. Exemplary immunogenically active portions are the epitopes typically formed by 5 or more residues included within an immunogenic fragment. In some embodiments, epitopes within one or more fragments can overlap. Immunogenic fragments can be expressed by recombinant technology, such as a fusion with an affinity or epitope tag, chemical synthesis of an oligopeptide, either free or conjugated to carrier proteins, or any other methods known in the art to express the ApoB-100 peptides.
Exemplary fragments of ApoB100 are peptides each comprising one of the sequences listed in the Sequence Listing as SEQ ID NO: 1 to SEQ ID NO: 302 described in further detail in the Examples section. Methods and systems suitable to identify an immunogenic fragment in the sense of the present are described in WO 02/080954, hereby incorporated by reference. In an embodiment, the one or more immunogenic fragments of ApoB100 suitable to treat, prevent and/or reduce kidney disease are associated to atherosclerosis reduction.
“Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
“Treatment” and “treating,” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.
As discussed above, Applicants observe that peptides of ApoB-100 reduce the levels of the AT1 receptor and therefore these peptides serve as therapeutics in kidney diseases.
Peptides of the Invention
Specific immunogenic epitopes of ApoB-100 were characterized and a peptide library including 302 peptides, each about 20 amino acid residues in length, covering the complete 4563 amino acid sequence of human ApoB-100 was produced. The peptides were produced with a 5 amino acid overlap to cover all sequences at break points. Peptides were numbered 1-302 starting at the N-terminal of ApoB-100 as indicated in Table 1 below.










TABLE 1







Apolipoprotein 



Peptide
Sequence
B aa
SEQ ID NO







P1:
EEEML ENVSL VCPKD ATRFK
aa 1-20
SEQ ID NO: 1


 


P2:
ATRFK HLRKY TYNYE AESSS
aa 16-35
SEQ ID NO: 2


 


P3:
AESSS GVPGT ADSRS ATRIN
aa 31-50
SEQ ID NO: 3


 


P4:
ATRIN CKVEL EVPQL CSFIL
aa 46-65
SEQ ID NO: 4


 


P5:
CSFIL KTSQC TLKEV YGFNP
aa 61-80
SEQ ID NO: 5


 


P6:
YGFNP EGKAL LKKTK NSEEF
aa 76-95
SEQ ID NO: 6


 


P7:
NSEEF AAAMS RYELK LAIPE
aa 91-110
SEQ ID NO: 7


 


P8:
LAIPE GKQVF LYPEK DEPTY
aa 106-125
SEQ ID NO: 8


 


P9:
DEPTY ILNIK RGIIS ALLVP
aa 121-140
SEQ ID NO: 9


 


P10:
ALLVP PETEE AKQVL FLDTV
aa 136-155
SEQ ID NO: 10


 


P11:
FLDTV YGNCS THFTV KTRKG
aa 151-170
SEQ ID NO: 11


 


P12:
KTRKG NVATE ISTER DLGQC
aa 166-185
SEQ ID NO: 12


 


P13:
DLGQC DRFKP IRTGI SPLAL
aa 181-200
SEQ ID NO: 13


 


P14:
SPLAL IKGMT RPLST LISSS
aa 196-215
SEQ ID NO: 14


 


P15:
LISSS QSCQY TLDAK RKHVA
aa 211-230
SEQ ID NO: 15


 


P16:
RKHVA EAICK EQHLF LPFSY
aa 226-245
SEQ ID NO: 16


 


P17:
LPFSY NNKYG MVAQV TQTLK
aa 241-260
SEQ ID NO: 17


 


P18:
TQTLK LEDTP KINSR FFGEG
aa 256-275
SEQ ID NO: 18


 


P19:
FFGEG TKKMG LAFES TKSTS
aa 271-290
SEQ ID NO: 19


 


P20:
TKSTS PPKQA EAVLK TLQEL
aa 286-305
SEQ ID NO: 20


 


P21:
TLQEL KKLTI SEQNI QRANL
aa 301-320
SEQ ID NO: 21


 


P22:
QRANL FNKLV TELRG LSDEA
aa 316-335
SEQ ID NO: 22


 


P23:
LSDEA VTSLL PQLIE VSSPI
aa 331-350
SEQ ID NO: 23


 


P24:
VSSPI TLQAL VQCGQ PQCST
aa 346-365
SEQ ID NO: 24


 


P25:
PQCST HILQW LKRVH ANPLL
aa 361-380
SEQ ID NO: 25


 


P26:
ANPLL IDVVT YLVAL IPEPS
aa 376-395
SEQ ID NO: 26


 


P27:
IPEPS AQQLR EIFNM ARDQR
aa 391-410
SEQ ID NO: 27


 


P28:
ARDQR SRATL YALSH AVNNY
aa 406-425
SEQ ID NO: 28


 


P29:
AVNNY HKTNP TGTQE LLDIA
aa 421-440
SEQ ID NO: 29


 


P30:
LLDIA NYLME QIQDD CTGDE
aa 436-455
SEQ ID NO: 30


 


P31:
CTGDE DYTYL ILRVI GNMGQ
aa 451-470
SEQ ID NO: 31


 


P32:
GNMGQ TMEQL TPELK SSILK
aa 466-485
SEQ ID NO: 32


 


P33:
SSILK CVQST KPSLM IQKAA
aa 481-500
SEQ ID NO: 33


 


P34:
IQKAA IQALR KMEPK DKDQE
aa 496-515
SEQ ID NO: 34


 


P35:
DKDQE VLLQT FLDDA SPGDK
aa 511-530
SEQ ID NO: 35


 


P36:
SPGDK RLAAY LMLMR SPSQA
aa 526-545
SEQ ID NO: 36


 


P37:
SPSQA DINKI VQILP WEQNE
aa 541-560
SEQ ID NO: 37


 


P38:
WEQNE QVKNF VASHI ANILN
aa 556-575
SEQ ID NO: 38


 


P39:
ANILN SEELD IQDLK KLVKE
aa 571-590
SEQ ID NO: 39


 


P40:
KLVKE ALKES QLPTV MDFRK
aa 586-605
SEQ ID NO: 40


 


P41:
MDFRK FSRNY QLYKS VSLPS
aa 601-620
SEQ ID NO: 41


 


P42:
VSLPS LDPAS AKIEG NLIFD
aa 616-635
SEQ ID NO: 42


 


P43:
NLIFD PNNYL PKESM LKTTL
aa 631-650
SEQ ID NO: 43


 


P44:
LKTTL TAFGF ASADL IEIGL
aa 646-665
SEQ ID NO: 44


 


P45:
IEIGL EGKGF EPTLE ALFGK
aa 661-680
SEQ ID NO: 45


 


P46:
ALFGK QGFFP DSVNK ALYWV
aa 676-695
SEQ ID NO: 46


 


P47:
ALYWV NGQVP DGVSK VLVDH
aa 691-710
SEQ ID NO: 47


 


P48:
VLVDH FGYTK DDKHE QDMVN
aa 706-725
SEQ ID NO: 48


 


P49:
QDMVN GIMLS VEKLI KDLKS
aa 721-740
SEQ ID NO: 49


 


P50:
KDLKS KEVPE ARAYL RILGE
aa 736-755
SEQ ID NO: 50


 


P51:
RILGE ELGFA SLHDL QLLGK
aa 751-770
SEQ ID NO: 51


 


P52:
QLLGK LLLMG ARTLQ GIPQM
aa 766-785
SEQ ID NO: 52


 


P53:
GIPQM IGEVI RKGSK NDFFL
aa 781-800
SEQ ID NO: 53


 


P54:
NDFFL HYIFM ENAFE LPTGA
aa 796-815
SEQ ID NO: 54


 


P55:
LPTGA GLQLQ ISSSG VIAPG
aa 811-830
SEQ ID NO: 55


 


P56:
VIAPG AKAGV KLEVA NMQAE
aa 826-845
SEQ ID NO: 56


 


P57:
NMQAE LVAKP SVSVE FVTNM
aa 841-860
SEQ ID NO: 57


 


P58:
FVTNM GIIIP DFARS GVQMN
aa 856-875
SEQ ID NO: 58


 


P59:
GVQMN TNFFH ESGLE AHVAL
aa 871-890
SEQ ID NO: 59


 


P60:
AHVAL KAGKL KFIIP SPKRP
aa 886-905
SEQ ID NO: 60


 


P61:
SPKRP VKLLS GGNTL HLVST
aa 901-920
SEQ ID NO: 61


 


P62:
HLVST TKTEV IPPLI ENRQS
aa 916-935
SEQ ID NO: 62


 


P63:
ENRQS WSVCK QVFPG LNYCT
aa 931-950
SEQ ID NO: 63


 


P64:
LNYCT SGAYS NASST DSASY
aa 946-965
SEQ ID NO: 64


 


P65:
DSASY YPLTG DTRLE LELRP
aa 961-980
SEQ ID NO: 65


 


P66:
LELRP TGEIE QYSVS ATYEL
aa 976-995
SEQ ID NO: 66


 


P67:
ATYEL QREDR ALVDT LKFVT
aa 991-1010
SEQ ID NO: 67


 


P68:
LKFVT QAEGA KQTEA TMTFK
aa 1006-1025
SEQ ID NO: 68


 


P69:
TMTFK YNRQS MTLSS EVQIP
aa 1021-1040
SEQ ID NO: 69


 


P70:
EVQIP DFDVD LGTIL RVNDE
aa 1036-1055
SEQ ID NO: 70


 


P71:
RVNDE STEGK TSYRL TLDIQ
aa 1051-1070
SEQ ID NO: 71


 


P72:
TLDIQ NKKIT EVALM GHLSC
aa 1066-1085
SEQ ID NO: 72


 


P73:
GHLSC DTKEE RKIKG VISIP
aa 1081-1100
SEQ ID NO: 73


 


P74:
VISIP RLQAE ARSEI LAHWS
aa 1096-1115
SEQ ID NO: 74


 


P75:
LAHWS PAKLL LQMDS SATAY
aa 1111-1130
SEQ ID NO: 75


 


P76:
SATAY GSTVS KRVAW HYDEE
aa 1126-1145
SEQ ID NO: 76


 


P77:
HYDEE KIEFE WNTGT NVDTK
aa 1141-1160
SEQ ID NO: 77


 


P78:
NVDTK KMTSN FPVDL SDYPK
aa 1156-1175
SEQ ID NO: 78


 


P79:
SDYPK SLHMY ANRLL DHRVP
aa 1171-1190
SEQ ID NO: 79


 


P80:
DHRVP ETDMT FRHVG SKLIV
aa 1186-1205
SEQ ID NO: 80


 


P81:
SKLIV AMSSW LQKAS GSLPY
aa 1201-1220
SEQ ID NO: 81


 


P82:
GSLPY TQTLQ DHLNS LKEFN
aa 1216-1235
SEQ ID NO: 82


 


P83:
LKEFN LQNMG LPDFH IPENL
aa 1231-1250
SEQ ID NO: 83


 


P84:
IPENL FLKSD GRVKY TLNKN
aa 1246-1260
SEQ ID NO: 84


 


P85:
TLNKN SLKIE IPLPF GGKSS
aa 1261-1280
SEQ ID NO: 85


 


P86:
GGKSS RDLKM LETVR TPALH
aa 1276-1295
SEQ ID NO: 86


 


P87:
TPALH FKSVG FHLPS REFQV
aa 1291-1310
SEQ ID NO: 87


 


P88:
REFQV PTFTI PKLYQ LQVPL
aa 1306-1325
SEQ ID NO: 88


 


P89:
LQVPL LGVLD LSTNV YSNLY
aa 1321-1340
SEQ ID NO: 89


 


P90:
YSNLY NWSAS YSGGN TSTDH
aa 1336-1355
SEQ ID NO: 90


 


P91:
TSTDH FSLRA RYHMK ADSVV
aa 1351-1370
SEQ ID NO: 91


 


P92:
ADSVV DLLSY NVQGS GETTY
aa 1366-1385
SEQ ID NO: 92


 


P93:
GETTY DHKNT FTLSC DGSLR
aa 1381-1400
SEQ ID NO: 93


 


P94:
DGSLR HKFLD SNIKF SHVEK
aa 1396-1415
SEQ ID NO: 94


 


P95:
SHVEK LGNNP VSKGL LIFDA
aa 1411-1430
SEQ ID NO: 95


 


P96:
LIFDA SSSWG PQMSA SVHLD
aa 1426-1445
SEQ ID NO: 96


 


P97:
SVHLD SKKKQ HLFVK EVKID
aa 1441-1460
SEQ ID NO: 97


 


P98:
EVKID GQFRV SSFYA KGTYG
aa 1456-1475
SEQ ID NO: 98


 


P99:
KGTYG LSCQR DPNTG RLNGE
aa 1471-1490
SEQ ID NO: 99


 


P100:
RLNGE SNLRF NSSYL QGTNQ
aa 1486-1505
SEQ ID NO: 100


 


P101:
QGTNQ ITGRY EDGTL SLTST
aa 1501-1520
SEQ ID NO: 101


 


P102:
SLTST SDLQS GIIKN TASLK
aa 1516-1535
SEQ ID NO: 102


 


P103:
TASLK YENYE LTLKS DTNGK
aa 1531-1550
SEQ ID NO: 103


 


P104:
DTNGK YKNFA TSNKM DMTFS
aa 1546-1565
SEQ ID NO: 104


 


P105:
DMTFS KQNAL LRSEY QADYE
aa 1561-1580
SEQ ID NO: 105


 


P106:
QADYE SLRFF SLLSG SLNSH
aa 1576-1595
SEQ ID NO: 106


 


P107:
SLNSH GLELN ADILG TDKIN
aa 1591-1610
SEQ ID NO: 107


 


P108:
TDKIN SGAHK ATLRI GQDGI
aa 1606-1625
SEQ ID NO: 108


 


P109:
GQDGI STSAT TNLKC SLLVL
aa 1621-1640
SEQ ID NO: 109


 


P110:
SLLVL ENELN AELGL SGASM
aa 1636-1655
SEQ ID NO: 110


 


P111:
SGASM KLTTN GRFRE HNAKF
aa 1651-1670
SEQ ID NO: 111


 


P112:
HNAKF SLDGK AALTE LSLGS
aa 1666-1685
SEQ ID NO: 112


 


P113:
LSLGS AYQAM ILGVD SKNIF
aa 1681-1700
SEQ ID NO: 113


 


P114:
SKNIF NFKVS QEGLK LSNDM
aa 1696-1715
SEQ ID NO: 114


 


P115:
LSNDM MGSYA EMKFD HTNSL
aa 1711-1730
SEQ ID NO: 115


 


P116:
HTNSL NIAGL SLDFS SKLDN
aa 1726-1745
SEQ ID NO: 116


 


P117:
SKLDN IYSSD KFYKQ TVNLQ
aa 1741-1760
SEQ ID NO: 117


 


P118:
TVNLQ LQPYS LVTTL NSDLK
aa 1756-1775
SEQ ID NO: 118


 


P119:
NSDLK YNALD LTNNG KLRLE
aa 1771-1790
SEQ ID NO: 119


 


P120:
KLRLE PLKLH VAGNL KGAYQ
aa 1786-1805
SEQ ID NO: 120


 


P121:
KGAYQ NNEIK HIYAI SSAAL
aa 1801-1820
SEQ ID NO: 121


 


P122:
SSAAL SASYK ADTVA KVQGV
aa 1816-1835
SEQ ID NO: 122


 


P123:
KVQGV EFSHR LNTDI AGLAS
aa 1831-1850
SEQ ID NO: 123


 


P124:
AGLAS AIDMS TNYNS DSLHF
aa 1846-1865
SEQ ID NO: 124


 


P125:
DSLHF SNVFR SVMAP FTMTI
aa 1861-1880
SEQ ID NO: 125


 


P126:
FTMTI DAHTN GNGKL ALWGE
aa 1876-1895
SEQ ID NO: 126


 


P127:
ALWGE HTGQL YSKFL LKAEP
aa 1891-1910
SEQ ID NO: 127


 


P128:
LKAEP LAFTF SHDYK GSTSH
aa 1906-1925
SEQ ID NO: 128


 


P129:
GSTSH HLVSR KSISA ALEHK
aa 1921-1940
SEQ ID NO: 129


 


P130:
ALEHK VSALL TPAEQ TGTWK
aa 1936-1955
SEQ ID NO: 130


 


P131:
TGTWK LKTQF NNNEY SQDLD
aa 1951-1970
SEQ ID NO: 131


 


P132:
SQDLD AYNTK DKIGV ELTGR
aa 1966-1985
SEQ ID NO: 132


 


P133:
ELTGR TLADL TLLDS PIKVP
aa 1981-2000
SEQ ID NO: 133


 


P134:
PIKVP LLLSE PINII DALEM
aa 1996-2015
SEQ ID NO: 134


 


P135:
DALEM RDAVE KPQEF TIVAF
aa 2011-2030
SEQ ID NO: 135


 


P136:
TIVAF VKYDK NQDVH SINLP
aa 2026-2045
SEQ ID NO: 136


 


P137:
SINLP FFETL QEYFE RNRQT
aa 2041-2060
SEQ ID NO: 137


 


P138:
RNRQT IIVVV ENVQR NLKHI
aa 2056-2075
SEQ ID NO: 138


 


P139:
NLKHI NIDQF VRKYR AALGK
aa 2071-2090
SEQ ID NO: 139


 


P140:
AALGK LPQQA NDYLN SFNWE
aa 2086-2105
SEQ ID NO: 140


 


P141:
SFNWE RQVSH AKEKL TALTK
aa 2101-2120
SEQ ID NO: 141


 


P142:
TALTK KYRIT ENDIQ IALDD
aa 2116-2135
SEQ ID NO: 142


 


P143:
IALDD AKINF NEKLS QLQTY
aa 2131-2150
SEQ ID NO: 143


 


P144:
QLQTY MIQFD QYIKD SYDLH
aa 2146-2165
SEQ ID NO: 144


 


P145:
SYDLH DLKIA IANII DEIIE
aa 2161-2180
SEQ ID NO: 145


 


P146:
DEIIE KLKSL DEHYH IRVNL
aa 2176-2195
SEQ ID NO: 146


 


P147:
IRVNL VKTIH DLHLF IENID
aa 2191-2210
SEQ ID NO: 147


 


P148:
IENID FNKSG SSTAS WIQNV
aa 2206-2225
SEQ ID NO: 148


 


P149:
WIQNV DTKYQ IRIQI QEKLQ
aa 2221-2240
SEQ ID NO: 149


 


P150:
QEKLQ QLKRH IQNID IQHLA
aa 2236-2255
SEQ ID NO: 150


 


P151:
IQHLA GKLKQ HIEAI DVRVL
aa 2251-2270
SEQ ID NO: 151


 


P152:
DVRVL LDQLG TTISF ERIND
aa 2266-2285
SEQ ID NO: 152


 


P153:
ERIND VLEHV KHFVI NLIGD
aa 2281-2300
SEQ ID NO: 153


 


P154:
NLIGD FEVAE KINAF RAKVH
aa 2296-2315
SEQ ID NO: 154


 


P155:
RAKVH ELIER YEVDQ QIQVL
aa 2311-2330
SEQ ID NO: 155


 


P156:
QIQVL MDKLV ELTHQ YKLKE
aa 2326-2345
SEQ ID NO: 156


 


P157:
YKLKE TIQKL SNVLQ QVKIK
aa 2341-2360
SEQ ID NO: 157


 


P158:
QVKIK DYFEK LVGFI DDAVK
aa 2356-2375
SEQ ID NO: 158


 


P159:
DDAVK KLNEL SFKTF IEDVN
aa 2371-2390
SEQ ID NO: 159


 


P160:
IEDVN KFLDM LIKKL KSFDY
aa 2386-2405
SEQ ID NO: 160


 


P161:
KSFDY HQFVD ETNDK IREVT
aa 2401-2420
SEQ ID NO: 161


 


P162:
IREVT QRLNG EIQAL ELPQK
aa 2416-2435
SEQ ID NO: 162


 


P163:
ELPQK AEALK LFLEE TKATV
aa 2431-2450
SEQ ID NO: 163


 


P164:
TKATV AVYLE SLQDT KITLI
aa 2446-2465
SEQ ID NO: 164


 


P165:
KITLI INWLQ EALSS ASLAH
aa 2461-2480
SEQ ID NO: 165


 


P166:
ASLAH MKAKF RETLE DTRDR
aa 2476-2495
SEQ ID NO: 166


 


P167:
DTRDR MYQMD IQQEL QRYLS
aa 2491-2510
SEQ ID NO: 167


 


P168:
QRYLS LVGQV YSTLV TYISD
aa 2506-2515
SEQ ID NO: 168


 


P169:
TYISD WWTLA AKNLT DFAEQ
aa 2521-2540
SEQ ID NO: 169


 


P170:
DFAEQ YSIQD WAKRM KALVE
aa 2536-2555
SEQ ID NO: 170


 


P171:
KALVE QGFTV PEIKT ILGTM
aa 2551-2570
SEQ ID NO: 171


 


P172:
ILGTM PAFEV SLQAL QKATF
aa 2566-2585
SEQ ID NO: 172


 


P173:
QKATF QTPDF IVPLT DLRIP
aa 2581-2600
SEQ ID NO: 173


 


P174:
DLRIP SVQIN FKDLK NIKIP
aa 2596-2615
SEQ ID NO: 174


 


P175:
NIKIP SRFST PEFTI LNTFH
aa 2611-2630
SEQ ID NO: 175


 


P176:
LNTFH IPSFT IDFVE MKVKI
aa 2626-2645
SEQ ID NO: 176


 


P177:
MKVKI IRTID QMQNS ELQWP
aa 2641-2660
SEQ ID NO: 177


 


P178:
ELQWP VPDIY LRDLK VEDIP
aa 2656-2675
SEQ ID NO: 178


 


P179:
VEDIP LARIT LPDFR LPEIA
aa 2671-2690
SEQ ID NO: 179


 


P180:
LPEIA IPEFI IPTLN LNDFQ
aa 2686-2705
SEQ ID NO: 180


 


P181:
LNDFQ VPDLH IPEFQ LPHIS
aa 2701-2720
SEQ ID NO: 181


 


P182:
LPHIS HTIEV PTFGK LYSIL
aa 2716-2735
SEQ ID NO: 182


 


P183:
LYSIL KIQSP LFTLD ANADI
aa 2731-2750
SEQ ID NO: 183


 


P184:
ANADI GNGTT SANEA GIAAS
aa 2746-2765
SEQ ID NO: 184


 


P185:
GIAAS ITAKG ESKLE VLNFD
aa 2761-2780
SEQ ID NO: 185


 


P186:
VLNFD FQANA QLSNP KINPL
aa 2776-2795
SEQ ID NO: 186


 


P187:
KINPL ALKES VKFSS KYLRT
aa 2791-2810
SEQ ID NO: 187


 


P188:
KYLRT EHGSE MLFFG NAIEG
aa 2806-2825
SEQ ID NO: 188


 


P189:
NAIEG KSNTV ASLHT EKNTL
aa 2821-2840
SEQ ID NO: 189


 


P190:
EKNTL ELSNG VIVKI NNQLT
aa 2836-2855
SEQ ID NO: 190


 


P191:
NNQLT LDSNT KYFHK LNIPK
aa 2851-2870
SEQ ID NO: 191


 


P192:
LNIPK LDFSS QADLR NEIKT
aa 2866-2885
SEQ ID NO: 192


 


P193:
NEIKT LLKAG HIAWT SSGKG
aa 2881-2900
SEQ ID NO: 193


 


P194:
SSGKG SWKWA CPRFS DEGTH
aa 2896-2915
SEQ ID NO: 194


 


P195:
DEGTH ESQIS FTIEG PLTSF
aa 2911-2930
SEQ ID NO: 195


 


P196:
PLTSF GLSNK INSKH LRVNQ
aa 2926-2945
SEQ ID NO: 196


 


P197:
LRVNQ NLVYE SGSLN FSKLE
aa 2941-2960
SEQ ID NO: 197


 


P198:
FSKLE IQSQV DSQHV GHSVL
aa 2956-2975
SEQ ID NO: 198


 


P199:
GHSVL TAKGM ALFGE GKAEF
aa 2971-2990
SEQ ID NO: 199


 


P200:
GKAEF TGRHD AHLNG KVIGT
aa 2986-3005
SEQ ID NO: 200


 


P201:
KVIGT LKNSL FFSAQ PFEIT
aa 3001-3020
SEQ ID NO: 201


 


P202:
PFEIT ASTNN EGNLK VRFPL
aa 3016-3035
SEQ ID NO: 202


 


P203:
VRFPL RLTGK IDFLN NYALF
aa 3031-3050
SEQ ID NO: 203


 


P204:
NYALF LSPSA QQASW QVSAR
aa 3046-3065
SEQ ID NO: 204


 


P205:
QVSAR FNQYK YNQNF SAGNN
aa 3061-3080
SEQ ID NO: 205


 


P206:
SAGNN ENIME AHVGI NGEAN
aa 3076-3095
SEQ ID NO: 206


 


P207:
NGEAN LDFLN IPLTI PEMRL
aa 3091-3110
SEQ ID NO: 207


 


P208:
PEMRL PYTII TTPPL KDFSL
aa 3106-3125
SEQ ID NO: 208


 


P209:
KDFSL WEKTG LKEFL KTTKQ
aa 3121-3140
SEQ ID NO: 209


 


P210:
KTTKQ SFDLS VKAQY KKNKH
aa 3136-3155
SEQ ID NO: 210


 


P211:
KKNKH RHSIT NPLAV LCEFI
aa 3151-3170
SEQ ID NO: 211


 


P212:
LCEFI SQSIK SFDRH FEKNR
aa 3166-3185
SEQ ID NO: 212


 


P213:
FEKNR NNALD FVTKS YNETK
aa 3181-3200
SEQ ID NO: 213


 


P214:
YNETK IKFDK YKAEK SHDEL
aa 3196-3215
SEQ ID NO: 214


 


P215:
SHDEL PRTFQ IPGYT VPVVN
aa 3211-3230
SEQ ID NO: 215


 


P216:
VPVVN VEVSP FTIEM SAFGY
aa 3226-3245
SEQ ID NO: 216


 


P217:
SAFGY VFPKA VSMPS FSILG
aa 3241-3260
SEQ ID NO: 217


 


P218:
FSILG SDVRV PSYTL ILPSL
aa 3256-3275
SEQ ID NO: 218


 


P219:
ILPSL ELPVL HVPRN LKLSL
aa 3271-3290
SEQ ID NO: 219


 


P220:
LKLSL PHFKE LCTIS HIFIP
aa 3286-3305
SEQ ID NO: 220


 


P221:
HIFIP AMGNI TYDFS FKSSV
aa 3301-3320
SEQ ID NO: 221


 


P222:
FKSSV ITLNT NAELF NQSDI
aa 3316-3335
SEQ ID NO: 222


 


P223:
NQSDI VAHLL SSSSS VIDAL
aa 3331-3350
SEQ ID NO: 223


 


P224:
VIDAL QYKLE GTTRL TRKRG
aa 3346-3365
SEQ ID NO: 224


 


P225:
TRKRG LKLAT ALSLS NKFVE
aa 3361-3380
SEQ ID NO: 225


 


P226:
NKFVE GSHNS TVSLT TKNME
aa 3376-3395
SEQ ID NO: 226


 


P227:
TKNME VSVAK TTKAE IPILR
aa 3391-3410
SEQ ID NO: 227


 


P228:
IPILR MNFKQ ELNGN TKSKP
aa 3406-3425
SEQ ID NO: 228


 


P229:
TKSKP TVSSS MEFKY DFNSS
aa 3421-3440
SEQ ID NO: 229


 


P230:
DFNSS MLYST AKGAV DHKLS
aa 3436-3455
SEQ ID NO: 230


 


P231:
DHKLS LESLT SYFSI ESSTK
aa 3451-3470
SEQ ID NO: 231


 


P232:
ESSTK GDVKG SVLSR EYSGT
aa 3466-3485
SEQ ID NO: 232


 


P233:
EYSGT IASEA NTYLN SKSTR
aa 3481-3500
SEQ ID NO: 233


 


P234:
SKSTR SSVKL QGTSK IDDIW
aa 3496-3515
SEQ ID NO: 234


 


P235:
IDDIW NLEVK ENFAG EATLQ
aa 3511-3530
SEQ ID NO: 235


 


P236:
EATLQ RIYSL WEHST KNHLQ
aa 3526-3545
SEQ ID NO: 236


 


P237:
KNHLQ LEGLF FTNGE HTSKA
aa 3541-3560
SEQ ID NO: 237


 


P238:
HTSKA TLELS PWQMS ALVQV
aa 3556-3575
SEQ ID NO: 238


 


P239:
ALVQV HASQP SSFHD FPDLG
aa 3571-3590
SEQ ID NO: 239


 


P240:
FPDLG QEVAL NANTK NQKIR
aa 3586-3605
SEQ ID NO: 240


 


P241:
NQKIR WKNEV RIHSG SFQSQ
aa 3601-3620
SEQ ID NO: 241


 


P242:
SFQSQ VELSN DQEKA HLDIA
aa 3616-3635
SEQ ID NO: 242


 


P243:
HLDIA GSLEG HLRFL KNIIL
aa 3631-3650
SEQ ID NO: 243


 


P244:
KNIIL PVYDK SLWDF LKLDV
aa 3646-3665
SEQ ID NO: 244


 


P245:
LKLDV TTSIG RRQHL RVSTA
aa 3661-3680
SEQ ID NO: 245


 


P246:
RVSTA FVYTK NPNGY SFSIP
aa 3676-3695
SEQ ID NO: 246


 


P247:
SFSIP VKVLA DKFIT PGLKL
aa 3691-3710
SEQ ID NO: 247


 


P248:
PGLKL NDLNS VLVMP TFHVP
aa 3706-3725
SEQ ID NO: 248


 


P249:
TFHVP FTDLQ VPSCK LDFRE
aa 3721-3740
SEQ ID NO: 249


 


P250:
LDFRE IQIYK KLRTS SFALN
aa 3736-3755
SEQ ID NO: 250


 


P251:
SFALN LPTLP EVKFP EVDVL
aa 3751-3770
SEQ ID NO: 251


 


P252:
EVDVL TKYSQ PEDSL IPFFE
aa 3766-3785
SEQ ID NO: 252


 


P253:
IPFFE ITVPE SQLTV SQFTL
aa 3781-3800
SEQ ID NO: 253


 


P254:
SQFTL PKSVS DGIAA LDLNA
aa 3796-3815
SEQ ID NO: 254


 


P255:
LDLNA VANKI ADFEL PTIIV
aa 3811-3830
SEQ ID NO: 255


 


P256:
PTIIV PEQTI EIPSI KFSVP
aa 3826-3845
SEQ ID NO: 256


 


P257:
KFSVP AGIVI PSFQA LTARF
aa 3841-3860
SEQ ID NO: 257


 


P258:
LTARF EVDSP VYNAT WSASL
aa 3856-3875
SEQ ID NO: 258


 


P259:
WSASL KNKAD YVETV LDSTC
aa 3871-3890
SEQ ID NO: 259


 


P260:
LDSTC SSTVQ FLEYE LNVLG
aa 3886-3905
SEQ ID NO: 260


 


P261:
LNVLG THKIE DGTLA SKTKG
aa 3901-3920
SEQ ID NO: 261


 


P262:
SKTKG TLAHR DFSAE YEEDG
aa 3916-3935
SEQ ID NO: 262


 


P263:
YEEDG KFEGL QEWEG KAHLN
aa 3931-3950
SEQ ID NO: 263


 


P264:
KAHLN IKSPA FTDLH LRYQK
aa 3946-3965
SEQ ID NO: 264


 


P265:
LRYQK DKKGI STSAA SPAVG
aa 3961-3980
SEQ ID NO: 265


 


P266:
SPAVG TVGMD MDEDD DFSKW
aa 3976-3995
SEQ ID NO: 266


 


P267:
DFSKW NFYYS PQSSP DKKLT
aa 3991-4010
SEQ ID NO: 267


 


P268:
DKKLT IFKTE LRVRE SDEET
aa 4006-4025
SEQ ID NO: 268


 


P269:
SDEET QIKVN WEEEA ASGLL
aa 4021-4040
SEQ ID NO: 269


 


P270:
ASGLL TSLKD NVPKA TGVLY
aa 4036-4055
SEQ ID NO: 270


 


P271:
TGVLY DYVNK YHWEH TGLTL
aa 4051-4070
SEQ ID NO: 271


 


P272:
TGLTL REVSS KLRRN LQNNA
aa 4066-4085
SEQ ID NO: 272


 


P273:
LQNNA EWVYQ GAIRQ IDDID
aa 4081-4100
SEQ ID NO: 273


 


P274:
IDDID VRFQK AASGT TGTYQ
aa 4096-4115
SEQ ID NO: 274


 


P275:
TGTYQ EWKDK AQNLY QELLT
aa 4111-4130
SEQ ID NO: 275


 


P276:
QELLT QEGQA SFQGL KDNVF
aa 4126-4145
SEQ ID NO: 276


 


P277:
KDNVF DGLVR VTQKF HMKVK
aa 4141-4160
SEQ ID NO: 277


 


P278:
HMKVK HLIDS LIDFL NFPRF
aa 4156-4175
SEQ ID NO: 278


 


P279:
NFPRF QFPGK PGIYT REELC
aa 4171-4190
SEQ ID NO: 279


 


P280:
REELC TMFIR EVGTV LSQVY
aa 4186-4205
SEQ ID NO: 280


 


P281:
LSQVY SKVHN GSEIL FSYFQ
aa 4201-4220
SEQ ID NO: 281


 


P282:
FSYFQ DLVIT LPFEL RKHKL
aa 4216-4235
SEQ ID NO: 282


 


P283:
RKHKL IDVIS MYREL LKDLS
aa 4231-4250
SEQ ID NO: 283


 


P284:
LKDLS KEAQE VFKAI QSLKT
aa 4246-4265
SEQ ID NO: 284


 


P285:
QSLKT TEVLR NLQDL LQFIF
aa 4261-4280
SEQ ID NO: 285


 


P286:
LQFIF QLIED NIKQL KEMKF
aa 4276-4295
SEQ ID NO: 286


 


P287:
KEMKF TYLIN YIQDE INTIF
aa 4291-4310
SEQ ID NO: 287


 


P288:
INTIF NDYIP YVFKL LKENL
aa 4306-4325
SEQ ID NO: 288


 


P289:
LKENL CLNLH KFNEF IQNEL
aa 4321-4340
SEQ ID NO: 289


 


P290:
IQNEL QEASQ ELQQI HQYIM
aa 4336-4355
SEQ ID NO: 290


 


P291:
HQYIM ALREE YFDPS IVGWT
aa 4351-4370
SEQ ID NO: 291


 


P292:
IVGWT VKYYE LEEKI VSLIK
aa 4366-4385
SEQ ID NO: 292


 


P293:
VSLIK NLLVA LKDFH SEYIV
aa 4381-4400
SEQ ID NO: 293


 


P294:
SEYIV SASNF TSQLS SQVEQ
aa 4396-4415
SEQ ID NO: 294


 


P295:
SQVEQ FLHRN IQEYL SILTD
aa 4411-4430
SEQ ID NO: 295


 


P296:
SILTD PDGKG KEKIA ELSAT
aa 4426-4445
SEQ ID NO: 296


 


P297:
ELSAT AQEII KSQAI ATKKI
aa 4441-4460
SEQ ID NO: 297


 


P298:
TKKII SDYHQ QFRYK LQDFS
aa 4457-4476
SEQ ID NO: 298


 


P299:
LQDFS DQLSD YYEKF IAESK
aa 4472-4491
SEQ ID NO: 299


 


P300:
IAESK RLIDL SIQNY HTFLI
aa 4487-4506
SEQ ID NO: 300


 


P301:
HTFLI YITEL LKKLQ STTVM
aa 4502-4521
SEQ ID NO: 301


 


P302:
STTVM NPYMK LAPGE LTIIL
aa 4517-4536
SEQ ID NO: 302









The invention provides compositions comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof. In one embodiment, the compositions elicit an immune response and comprise one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier. In another embodiment, the composition is for immunization and comprises one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier that induces and/or enhances an immune response. In a further embodiment, the compositions for eliciting an immune response include one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and CD8(+) T cells. The CD8+ T cells (such as autologous CD8+ T cells) are activated by one or more immunogenic peptides of ApoB-100. In an embodiment, the peptide of ApoB-100 is any one or more of peptides P1 to P302 as set forth in Table 1. In a further embodiment, one or more of the peptides of ApoB-100 are immunogenic peptides of ApoB-100. In an additional embodiment, the compositions for immunization include one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and CD8(+) T cells. The CD8+ T cells (such as autologous CD8+ T cells) are activated by one or more immunogenic peptides of ApoB-100. In an embodiment, the peptide of ApoB-100 is any one or more of peptides P1 to P302 as set forth in Table 1. In a further embodiment, one or more of the peptides of ApoB-100 are immunogenic peptides of ApoB-100.
The compositions for eliciting an immune response and/or for immunizations/vaccinations comprising one or more of the immunogenic peptides of ApoB-100 described herein may be used to treat, inhibit, prevent and/or promotes prophylaxis of kidney diseases in subjects. The compositions eliciting an immune response and/or for immunizations/vaccinations comprising one or more of the immunogenic peptides of ApoB-100 described herein may also be used to mitigate the effects of kidney diseases, reduce the severity of kidney diseases, reduce the likelihood of developing kidney disease and/or slow the progression of kidney disease.
In some embodiments, a percentage of kidney disease reduction after administration of the compositions described herein is at least about 20%, at least about 30%, from about 40% to about 60% and/or about 50% to about 80%. In a further embodiment, the expected improvement in kidney disease after immunizations with a composition comprising one or more immunogenic peptides of ApoB-100 and/or administration with of activated CD8+ T cells is at least about 20% and/or about 20-80% relative to control subjects. In an embodiment, administration of the compositions described herein reduces mortality due to kidney disease, and/or reduce incidence of kidney disease.
Peptide Compositions for Vaccination
The invention provides composition comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof. In one embodiment, the composition elicits an immune response and comprises one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier. In another embodiment, the composition is a vaccine and comprises one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier that induces and/or enhances an immune response.
In some embodiments, the compositions (for eliciting an immune response and/or for vaccination) comprising fragments of ApoB-100, such as the immunogenic fragments of ApoB-100 associated with treating kidney disease, inhibiting kidney disease, preventing kidney disease, promoting prophylaxis of kidney disease, mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease, include at least one or more of peptides p1 (SEQ ID NO: 1), p2 (SEQ ID NO: 2), p10 (SEQ ID NO: 10), p11 (SEQ ID NO:11), p25 (SEQ ID NO:25), p30-p34 (SEQ ID NOs:30-34), p40 (SEQ ID NO:40), p40 (SEQ ID NO:40), p45 (SEQ ID NO:45), p68 (SEQ ID NO:68), p74 (SEQ ID NO:74), p94 (SEQ ID NO:94), p99 (SEQ ID NO:99), p100 (SEQ ID NO:100), p102 (SEQ ID NO:102), p103 (SEQ ID NO: 103), p105 (SEQ ID NO:105), p107 (SEQ ID NO:107), p111 (SEQ ID NO:111), p129 (SEQ ID NO:129), p143 (SEQ ID NO:143), p148 (SEQ ID NO:148), p149 (SEQ ID NO:149), p154 (SEQ ID NO:154), p162 (SEQ ID NO:162), p169 (SEQ ID NO:169), p177 (SEQ ID NO:177), p199 (SEQ ID NO:199), p210 (SEQ ID NO:210), p222 (SEQ ID NO:222), p236 (SEQ ID NO:236), p252 (SEQ ID NO:252), or p301 (SEQ ID NO:301), or immunologically active fragments thereof and/or combinations thereof.
In further embodiments, the compositions (for eliciting an immune response and/or for vaccination) comprising the fragments of ApoB-100, such as the immunogenic fragments of ApoB-100 associated with treating kidney disease, inhibiting kidney disease, preventing kidney disease, promoting prophylaxis of kidney disease, mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease, include one or more of peptides p2 (SEQ ID NO:2), p11 (SEQ ID NO:11), p32 (SEQ ID NO:32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO:148), p162 (SEQ ID NO:162), or p210 (SEQ ID NO:210), or immunologically active fragments thereof and/or combinations thereof.
In additional embodiments, the compositions (for eliciting an immune response and/or for vaccination) comprising the fragments of ApoB-100 such as the immunogenic fragments of ApoB-100 associated with treating kidney disease, inhibiting kidney disease, preventing kidney disease, promoting prophylaxis of kidney disease, mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease, include one or more of peptides p2 (SEQ ID NO:2), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102) and/or p210 (SEQ ID NO:210) or immunologically active fragments thereof and/or combinations thereof.
In a further embodiment, the compositions (for eliciting an immune response and/or for vaccination) comprising the fragment of ApoB-100 associated with treating kidney disease, inhibiting kidney disease, preventing kidney disease, promoting prophylaxis of kidney disease, mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease includes amino acids 3136-3155 of human ApoB-100 (P210; SEQ ID NO: 210) or an immunogenically active portion thereof.
T Cells and Peptide Compositions for Vaccination
The composition for eliciting an immune response and/or for vaccination to treat kidney disease, inhibit kidney disease, prevent kidney disease, promote prophylaxis of kidney disease, mitigate the effects of kidney disease, reduce the severity of kidney disease, reduce the likelihood of developing kidney disease and/or slow the progression of kidney disease, comprise activated CD8+ T cells (for example, autologous T cells) wherein the CD8+ T cells are activated by contacting CD8+ T cells with one or more immunogenic fragments of ApoB100. In an embodiment, activated CD8+ T cells specific for an immunogenic fragment of ApoB100 may be obtained by contacting a CD8+ T cells (for example, autologous CD8+ T cells) with any one or more of peptides p1 to p302 of ApoB-100 or an immunogenically active portion thereof for a time and under condition to activate the CD8+ T cell.
In some embodiments, activated CD8+ T cells specific for an immunogenic fragment of ApoB100 may be obtained by contacting a CD8+ T cells (for example, autologous CD8+ T cells) with any one or more of peptides p1 (SEQ ID NO: 1), p2 (SEQ ID NO: 2), p10 (SEQ ID NO: 10), p11 (SEQ ID NO:11), p25 (SEQ ID NO:25), p30-p34 (SEQ ID NOs:30-34), p40 (SEQ ID NO:40), p40 (SEQ ID NO:40), p45 (SEQ ID NO:45), p68 (SEQ ID NO:68), p74 (SEQ ID NO:74), p94 (SEQ ID NO:94), p99 (SEQ ID NO:99), p100 (SEQ ID NO:100), p102 (SEQ ID NO:102), p103 (SEQ ID NO: 103), p105 (SEQ ID NO:105), p107 (SEQ ID NO:107), pill (SEQ ID NO:111), p129 (SEQ ID NO:129), p143 (SEQ ID NO:143), p148 (SEQ ID NO:148), p149 (SEQ ID NO:149), p154 (SEQ ID NO:154), p162 (SEQ ID NO:162), p169 (SEQ ID NO:169), p177 (SEQ ID NO:177), p199 (SEQ ID NO:199), p210 (SEQ ID NO:210), p222 (SEQ ID NO:222), p236 (SEQ ID NO:236), p252 (SEQ ID NO:252), or p301 (SEQ ID NO:301), or immunologically active fragments thereof and/or combinations thereof. In various embodiments, the above activated CD8+ T cells are administered as immunizations and/or to elicit an immune response.
In further embodiments, activated CD8+ T cells specific for an immunogenic fragment of ApoB100 may be obtained by contacting a CD8+ T cells (for example, autologous CD8+ T cells) with any one or more of peptides p2 (SEQ ID NO:2), p11 (SEQ ID NO:11), p32 (SEQ ID NO:32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO:148), p162 (SEQ ID NO:162), or p210 (SEQ ID NO:210), or immunologically active fragments thereof and/or combinations thereof. In various embodiments, the above activated CD8+ T cells are administered as immunizations and/or to elicit an immune response.
In additional embodiments, activated CD8+ T cells specific for an immunogenic fragment of ApoB100 may be obtained by contacting a CD8+ T cells (for example, autologous CD8+ T cells) with any one or more of peptides SEQ ID NO:2, SEQ ID NO: 45, SEQ ID NO: 74, SEQ ID NO: 102, SEQ ID NO:210, or immunologically active fragments thereof and/or combinations thereof. In various embodiments, the above activated CD8+ T cells are administered as immunizations and/or to elicit an immune response.
In a further embodiment, activated CD8(+) T cells specific for an immunogenic fragment of ApoB100 may be obtained by contacting a CD8(+) T cells (for example, autologous CD8+ T cells) with an immunogenic fragment which includes the amino acids 3136-3155 of human ApoB-100 (for example P210; SEQ ID NO: 210) or an immunogenically active portion thereof. In various embodiments, the above activated CD8+ T cells are administered as immunizations and/or to elicit an immune response.
In general, the same combination of immunogenic fragments proven or expected to be associated with treatment and/or prevention of kidney disease in an individual are also expected to be able to activate CD8(+) T cells to be used in treatment and/or prevention of kidney disease in the individual. In particular, T cell activation can be performed using any of the molecules herein described administered in vivo in an amount suitable to be used with the methods of the invention (for example, to treat or prevent kidney disease). Activation of T cell can also be performed in vitro using methods and procedures such as the ones described in (R. Wu, et al., 1996 Scand. J. Immunol. 43, 381-384) as well as additional procedures identifiable by a skilled person.
In some embodiments, compositions comprising peptides of ApoB-100 (such as immunogenic peptides of ApoB-100) and CD8(+) T cells (for example, activated CD8+ T cells and/or autologous CD8+ T cells) further include an enhancer of CD8(+) T cell activation.
In an embodiment, the enhancer can be interleukin 2 (IL2), interleukin 10 (IL10), Interleukin 15 (IL-15), TGF-beta (TGF-β), IL2-antiIL-2 antibody complex and/or additional enhancer identifiable by a skilled person upon reading of the present disclosure. Reference is made to the references Mitchell et al 2010 (J Immunol. 2010 Jun. 15; 184(12):6719-30. Epub 2010 May 14), Perret et al 2008 (Eur J Immunol. 2008 October; 38(10):2886-95) and Kamimura et al 2007 (J Exp Med. 2007 Aug. 6; 204(8):1803-12. Epub 2007 Jul. 30). In some embodiments, the enhancing is performed by reducing CD86 expression and/or IL12 secretion by dendritic cells in the individual.
As disclosed herein, the immunogenic fragments of ApoB-100 or immunogenically active portion thereof, CD8 (+) T cell, and enhancers described herein can be provided as a part of systems to treat and/or prevent kidney disease or of a condition associated thereto. In an embodiment, the system comprises CD8(+) T cell activated with at least two ApoB-100 fragments or immunogenically active portions thereof, and one or more cytokine able to enhance the activated CD8(+) T cell.
In an embodiment, the system comprises at least two or more immunogenic fragments of ApoB-100 or immunogenically active portion thereof and one or more of an activated CD8(+) T cell specific for an immunogenic fragment of ApoB-100.
Methods of the Invention
The invention provides methods for treating or inhibiting kidney disease in a subject in need thereof. The method comprises providing a composition comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and administering an effective amount of the composition to the subject so as to treat or inhibit kidney disease in the subject.
In one embodiment, the composition for treating or inhibiting kidney diseases includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier that may induce and/or enhance an immune response.
In another embodiment, the composition for treating or inhibiting kidney diseases includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and CD8(+) T cells, wherein the CD8+ T cells are activated by one or more immunogenic peptides of ApoB-100. The CD8+ T cells may be autologous. The composition may further comprise enhancers.
In a further embodiment, the compositions for treating or inhibiting kidney diseases include a first composition comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier, and a second composition comprising activated CD8+ T cells and optionally, enhancers. In one embodiment, the first and second compositions are administered concurrently. In another embodiment, the first and second compositions are administered sequentially.
In some embodiments, the peptide of ApoB-100 in the compositions for treating or inhibiting kidney diseases is any one or more of peptides P1 to P302 as set forth in Table 1, or immunologically active fragments thereof and/or a combination thereof. In a further embodiment, the peptide of ApoB-100 is any one or more of peptides p2 (SEQ ID NO:2), p11 (SEQ ID NO:11), p32 (SEQ ID NO:32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO:148), p162 (SEQ ID NO:162), or p210 (SEQ ID NO:210), or immunologically active fragments thereof and/or a combination thereof. In an embodiment, the peptide is p210 (SEQ ID NO:210).
The invention further provides methods for preventing kidney diseases or promoting prophylaxis of kidney diseases in a subject in need thereof. The method comprises providing a composition comprising one or more peptides of ApoB-100 and/or immunogenically active portions, derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and administering an effective amount of the composition to the subject so as to prevent or promote prophylaxis of kidney disease in the subject.
In one embodiment, the composition for preventing kidney diseases or promoting prophylaxis of kidney diseases includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier that may induce and/or enhance an immune response.
In another embodiment, the composition for preventing kidney diseases or promoting prophylaxis of kidney diseases includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and CD8(+) T cells, wherein the CD8+ T cells are activated by one or more immunogenic peptides of ApoB-100. The CD8+ T cells may be autologous. The composition may further comprise enhancers.
In a further embodiment, the compositions for preventing kidney diseases or promoting prophylaxis of kidney diseases include a first composition comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier, and a second composition comprising activated CD8+ T cells and optionally, enhancers. In one embodiment, the first and second compositions are administered concurrently. In another embodiment, the first and second compositions are administered sequentially.
In some embodiments, the peptide of ApoB-100 in the composition for preventing kidney diseases or promoting prophylaxis of kidney diseases is any one or more of peptides P1 to P302 as set forth in Table 1 or immunologically active fragments thereof and/or a combination thereof. In a further embodiment, the peptide of ApoB-100 is any one or more of peptides p2 (SEQ ID NO:2), p11 (SEQ ID NO:11), p32 (SEQ ID NO:32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO:148), p162 (SEQ ID NO:162), or p210 (SEQ ID NO:210), or immunologically active fragments thereof and/or a combination thereof. In an embodiment, the peptide is p210 (SEQ ID NO:210).
The invention also provides methods for mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease in a subject in need thereof. The methods comprise providing a composition comprising one or more peptides of ApoB-100 and/or immunogenically active portions, derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and administering an effective amount of the composition to the subject so as to mitigate the effects of kidney disease, reduce the severity of kidney disease, reduce the likelihood of developing kidney disease and/or slow the progression of kidney disease in the subject.
In one embodiment, the composition for mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier that may induce and/or enhance an immune response.
In another embodiment, the composition for mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and CD8(+) T cells, wherein the CD8+ T cells are activated by one or more immunogenic peptides of ApoB-100. The CD8+ T cells may be autologous. The composition may further comprise enhancers.
In a further embodiment, the compositions mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease include a first composition comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier, and a second composition comprising activated CD8+ T cells and optionally, enhancers. In one embodiment, the first and second compositions are administered concurrently. In another embodiment, the first and second compositions are administered sequentially.
In some embodiments, the peptide of ApoB-100 in composition for mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease is any one or more of peptides P1 to P302 as set forth in Table 1 or immunologically active fragments thereof and/or a combination thereof. In a further embodiment, the peptide of ApoB-100 is any one or more of peptides p2 (SEQ ID NO:2), p11 (SEQ ID NO:11), p32 (SEQ ID NO:32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO:148), p162 (SEQ ID NO:162), or p210 (SEQ ID NO:210), or immunologically active fragments thereof and/or a combination thereof. In an embodiment, the peptide is p210 (SEQ ID NO:210).
The invention also provides methods for decreasing the expression of Angiotensin II type I receptor (AT1R) in a subject in need thereof. The methods comprise providing a composition comprising one or more peptides of ApoB-100 and/or immunogenically active portions, derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and administering an effective amount of the composition to the subject so as to decrease the expression of AT1R.
In one embodiment, the composition for reducing the expression of AT1R includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier that may induce and/or enhance an immune response.
In another embodiment, the composition for reducing the expression of AT1R includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and CD8(+) T cells, wherein the CD8+ T cells are activated by one or more immunogenic peptides of ApoB-100. The CD8+ T cells may be autologous. The composition may further comprise enhancers.
In a further embodiment, the compositions for reducing the expression of AT1R include a first composition comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier, and a second composition comprising activated CD8+ T cells and optionally, enhancers. In one embodiment, the first and second compositions are administered concurrently. In another embodiment, the first and second compositions are administered sequentially.
In some embodiments, the peptide of ApoB-100 in composition for reducing the expression of AT1R is any one or more of peptides P1 to P302 as set forth in Table 1 or immunologically active fragments thereof and/or a combination thereof. In a further embodiment, the peptide of ApoB-100 is any one or more of peptides p2 (SEQ ID NO:2), p11 (SEQ ID NO:11), p32 (SEQ ID NO:32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO:148), p162 (SEQ ID NO:162), or p210 (SEQ ID NO:210), or immunologically active fragments thereof and/or a combination thereof. In an embodiment, the peptide is p210 (SEQ ID NO:210).
In one embodiment, kidney disease may be atherosclerosis-related. In another embodiment, the kidney disease may be hypertension-related. In a further embodiment, the kidney disease may be diabetes. In an additional embodiment, the kidney disease may be autoimmune diseases-related.
In an embodiment, the effective amount of activated CD8(+) T cells is about 500,000 to about 2,000,000 cells, about 500,000 to about 1,500,000 cells, about 500,000 to about 1,000,000 cells, about 750,000 to about 2,000,000 cells, about 750,000 to about 1,500,000 cells, or about 750,000 to about 1,000,000 cells. In an embodiment, administration of about 1,000,000 activated CD8(+) T cells is expected to result in both treatment and prevention of kidney diseases.
Administration is expected to be performed in accordance with dosages and schedules which will be identified based on the condition of the subject to be treated and the desired effect. For example if administration directed to prevention, administering an effective amount of activated CD8(+) T cell can performed by performing either a single administration, or a plurality of administrations (e.g. 3 administrations or more, 6 administrations or more) of activated CD8(+) T cell herein described in intervals to obtain a desired immunization based on the condition of the individual. In particular, a plurality of administrations can be performed whenever a prolonged immunizing effect is desired.
Administration of CD8(+) T cell herein described can be performed according to methods to immunize an individual identifiable to a skilled person. In an embodiment, administration can be performed by parenteral administration. Parenteral administration is a systemic route of administration where the substance is given by route other than the digestive tract and includes but is not limited to intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal, administration, intraperitoneal administration, and intravesical infusion. The activated CD8(+) T cells may be administered one time, or multiple times, depending on the desired duration of the immunization effect.
Dosages of the Invention
In some embodiments of the invention, the effective amount of one or more peptide of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof in the compositions for eliciting an immune response and/or for immunization for treating kidney disease, inhibiting kidney disease, preventing kidney disease, promoting prophylaxis of kidney disease, mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease can be in the range of about 0.01-0.05 mg/day, 0.05-0.1 mg/day, 0.1-0.5 mg/day, 0.5-1 mg/day, 1-5 mg/day, 5-10 mg/day, 10-50 mg/day, 50-100 mg/day, 100-150 mg/day, 150-200 mg/day, 100-200 mg/day, 200-300 mg/day, 300-400 mg/day, 400-500 mg/day, 500-600 mg/day, 600-700 mg/day, 700-800 mg/day, 800-900 mg/day, 900-1000 mg/day, 1000-1100 mg/day, 1100-1200 mg/day, 1200-1300 mg/day, 1300-1400 mg/day, 1400-1500 mg/day, 1500-1600 mg/day, 1600-1700 mg/day, 1700-1800 mg/day, 1800-1900 mg/day, 1900-2000 mg/day, 2000-2100 mg/day, 2100-2200 mg/day, 2200-2300 mg/day, 2300-2400 mg/day, 2400-2500 mg/day, 2500-2600 mg/day, 2600-2700 mg/day, 2700-2800 mg/day, 2800-2900 mg/day or 2900-3000 mg/day. The ApoB-100 peptides may be any one or more of peptides P1 to P302 as set forth in Table 1, or immunologically active fragments thereof and/or combinations thereof. In some embodiments, the compositions comprising the ApoB-100 peptides or immunologically active fragments thereof and/or combinations thereof comprise peptides in the above dosage ranges. In other embodiments, compositions comprising the ApoB-100 peptides or immunologically active fragments thereof and/or combinations thereof and activated CD8+ T cells comprise peptides in the above dosage ranges.
In other embodiments of the invention, the effective amount of one or more peptide of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof in the compositions for eliciting an immune response and/or for immunization for treating kidney disease, inhibiting kidney disease, preventing kidney disease, promoting prophylaxis of kidney disease, mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease can be in the range of about 0.01-0.05 mg/immunization, 0.05-0.1 mg/immunization, 0.1-0.5 mg/immunization, 0.5-1 mg/immunization, 1-5 mg/immunization, 5-10 mg/immunization, 10-50 mg/immunization, 50-100 mg/immunization, 100-150 mg/immunization, 150-200 mg/immunization, 100-200 mg/immunization, 200-300 mg/immunization, 300-400 mg/immunization, 400-500 mg/immunization, 500-600 mg/immunization, 600-700 mg/immunization, 700-800 mg/immunization, 800-900 mg/immunization, 900-1000 mg/immunization, 1000-1100 mg/immunization, 1100-1200 mg/immunization, 1200-1300 mg/immunization, 1300-1400 mg/immunization, 1400-1500 mg/immunization, 1500-1600 mg/immunization, 1600-1700 mg/immunization, 1700-1800 mg/immunization, 1800-1900 mg/immunization, 1900-2000 mg/immunization, 2000-2100 mg/immunization, 2100-2200 mg/immunization, 2200-2300 mg/immunization, 2300-2400 mg/immunization, 2400-2500 mg/immunization, 2500-2600 mg/immunization, 2600-2700 mg/immunization, 2700-2800 mg/immunization, 2800-2900 mg/immunization or 2900-3000 mg/immunization. The ApoB-100 peptides may be any one or more of peptides P1 to P302 as set forth in Table 1. The ApoB-100 peptides may be any one or more of peptides P1 to P302 as set forth in Table 1, or immunologically active fragments thereof and/or combinations thereof. In some embodiments, the compositions comprising the ApoB-100 peptides or immunologically active fragments thereof and/or combinations thereof comprise peptides in the above dosage ranges. In other embodiments, compositions comprising the ApoB-100 peptides or immunologically active fragments thereof and/or combinations thereof and activated CD8+ T cells comprise peptides in the above dosage ranges. In an additional embodiment, an effective amount of p210 is about 100 μg of p210/immunization. In an alternative embodiment, an effective amount of p210 is about 250 μg to about 500 μg.
In further embodiments of the invention, the effective amount of one or more peptide of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof in the compositions for eliciting an immune response and/or for immunization for treating kidney disease, inhibiting kidney disease, preventing kidney disease, promoting prophylaxis of kidney disease, mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease can be in the range of about 0.01-0.05 mg/kg, 0.05-0.1 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, 1-5 mg/kg, 5-10 mg/kg, 10-50 mg/kg, 50-100 mg/kg, 100-150 mg/kg, 150-200 mg/kg, 100-200 mg/kg, 200-300 mg/kg, 300-400 mg/kg, 400-500 mg/kg, 500-600 mg/kg, 600-700 mg/kg, 700-800 mg/kg, 800-900 mg/kg, 900-1000 mg/kg, 1000-1100 mg/kg, 1100-1200 mg/kg, 1200-1300 mg/kg, 1300-1400 mg/kg, 1400-1500 mg/kg, 1500-1600 mg/kg, 1600-1700 mg/kg, 1700-1800 mg/kg, 1800-1900 mg/kg, 1900-2000 mg/kg, 2000-2100 mg/kg, 2100-2200 mg/kg, 2200-2300 mg/kg, 2300-2400 mg/kg, 2400-2500 mg/kg, 2500-2600 mg/kg, 2600-2700 mg/kg, 2700-2800 mg/kg, 2800-2900 mg/kg or 2900-3000 mg/kg. The ApoB-100 peptides may be any one or more of peptides P1 to P302 as set forth in Table 1, or immunologically active fragments thereof and/or combinations thereof. In some embodiments, the compositions comprising the ApoB-100 peptides or immunologically active fragments thereof and/or combinations thereof comprise peptides in the above dosage ranges. In other embodiments, compositions comprising the ApoB-100 peptides or immunologically active fragments thereof and/or combinations thereof and activated CD8+ T cells comprise peptides in the above dosage ranges.
In an embodiment, the effective amount of immunogenic peptide of ApoB-100 may vary depending on the number and combination of peptides utilized for each immunization, and specific characteristic and conditions of the individual treated (e.g. immune system, diet and general health and additional factors identifiable by a skilled person). Additionally, lower or higher amounts within the defined range are expected to be effective in an individual depending on factors such as weight, age, gender of the individual as well as additional factors identifiable by a skilled person.
In some embodiments, the immunogenic peptides herein described or related immunogenically active portions can be administered in combination with an adjuvant or other carrier suitable to affect and in particular increase immunogenicity of the peptide o active portion thereof. In some embodiments, the immunogenic peptide or active portion thereof can be conjugated to the adjuvant or carrier according to procedures identifiable to a skilled person. Suitable carriers comprise BSA, and in particular, cationized BSA, Human Serum Albumin (HSA) and in particular cationized HSA, aluminum salts such as aluminum phosphate and aluminum hydroxide and additional carriers identifiable by a skilled person.
In an embodiment, the administering is performed according to a schedule of administration to be determined in view of the desired effect. In particular, administration is expected to be performed in accordance with dosages and schedule which will be identified based on the condition of the individual to be treated and the desired effect. For example, administration can be performed by performing either a single administration, or a plurality of administrations (e.g. 2 administrations or more, in particular up to 6 administrations) of immunogenic fragments or immunogenically active portion thereof herein described in intervals to obtain a desired immunization based on the condition of the individual.
The route of immunization can vary depending on the purposes of immunization described herein. Successful prevention and treatment of kidney diseases in mice occurred by subcutaneous osmotic pump injections. The type of immune response triggered is largely determined by the route of immunization. Various routes can be used comprising subcutaneous, parenteral, and systemic among the others. In particular, the mucosal linings of airways and intestines contain lymphatic tissue that, when exposed to antigen, elicits anti-inflammatory, immunosuppressive responses. Distinct immunological features of the respiratory and intestinal mucosa lead to partly different types of protective immunity upon antigen exposure by the nasal or oral route.
As described above, administering an effective amount of CD8+ T cells activated with any one or more of peptides (for example immunogenic peptides) of ApoB-100 is associated with treating kidney disease, inhibiting kidney disease, preventing kidney disease, promoting prophylaxis of kidney disease, mitigating the effects of kidney disease, reducing the severity of kidney disease, reducing the likelihood of developing kidney disease and/or slowing the progression of kidney disease. In an embodiment the effective amount of activated CD8+ T cells may be between about 500,000 and 2,000,000 cells. In another embodiment the effective amount of activated CD8+ T cells may be between about 750,000 and about 1,500,000 cells. In an additional embodiment, the effective amount of activated CD8+ T cells may be about 1,000,000 cells.
Typical dosages of an effective amount of one or more peptide of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof in the composition can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. The same or similar dosing can be used in accordance with various embodiments of the present invention, or an alternate dosage may be used in connection with alternate embodiments of the invention. The actual dosage can depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of relevant cultured cells or histocultured tissue sample, or the responses observed in the appropriate animal models.
In one embodiment, administration of activated CD8+ T cell herein described can be performed according to methods used in the art to immunize an individual. In another embodiment, the administering can be performed by parenteral administration. Parenteral administration is a systemic route of administration where the substance is given by route other than the digestive tract and includes but is not limited to intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal, administration, intraperitoneal administration, and intravesical infusion. In an embodiment the administering can be performed by intravenous administration.
The invention also provides that the activated CD8+ T cells, obtained by contacting a CD8+ T cells (for example, autologous CD8+ T cells) with any one or more of peptides P1 to P302 as set forth in Table 1, may be administered alone or in combination with an effective amount of any one or more peptides (for example immunogenic peptides) P1 to P302 of ApoB-100 or immunogenic portions thereof.
In an embodiment, compositions comprising peptides of ApoB-100 including peptides P1 to P302 and/or immunogenic fragments thereof may be administered once daily or multiple times daily. Similarly, activated CD8+ T cells, obtained by contacting a CD8+ T cells (for example, autologous CD8+ T cells) with any one or more of peptides P1 to P302 as set forth in Table 1, may be administered once daily or multiple times daily depending on the desired duration of the immunization effect.
In some embodiments, administering of an immunogenic fragment and/or a CD8(+) T cell can be performed in combination with an enhancer of CD8(+) T cell activation including but not limited to Interleukin 2 (IL2), Interleukin 15 (IL-15), TGFbeta(TGF-β), IL2-antiIL-2 antibody complex and/or additional enhancer identifiable by a skilled person upon reading of the present disclosure. Reference is made to the references Mitchell et al (J Immunol. 2010 Jun. 15; 184(12):6719-30. Epub 2010 May 14), Perret et al (Eur J Immunol. 2008 October; 38(10):2886-95) and Kamimura et al (J Exp Med. 2007 Aug. 6; 204(8):1803-12. Epub 2007 Jul. 30), each incorporated by reference in its entirety, which describe exemplary use of enhancer in connection with T cell activation. In additional embodiments, the enhancing is performed by reducing CD86 expression and/or IL12 secretion by dendritic cells in the individual.
The subjects in the claimed invention may be any one or more of human, non-human primate, monkey, ape, dog, cat, cow, horse, rabbit, mouse and rat.
Pharmaceutical Compositions and Kits
As described above, the invention provides compositions (for eliciting an immune response and/or for immunizations) comprising one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof. In one embodiment, the composition comprises one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or analogs thereof and a pharmaceutically acceptable carrier that induces and/or enhances an immune response. In another embodiment, the composition includes one or more peptides of ApoB-100 and/or derivatives, peptidomimetics, pharmaceutical equivalents and/or an analogs thereof and CD8(+) T cells. The CD8+ T cells are activated by one or more immunogenic peptides of ApoB-100. In an embodiment, the peptide of ApoB-100 is any one or more of peptides P1 to P302 as set forth in Table 1. In a further embodiment, one or more of the peptides of ApoB-100 are immunogenic peptides of ApoB-100.
The compositions (for eliciting an immune response and/or for immunizations) can be formulated as freeze-dried or liquid preparations according to any means suitable in the art. Non-limiting examples of liquid form preparations include solutions, suspensions, syrups, slurries, and emulsions. Suitable liquid carriers include any suitable organic or inorganic solvent, for example, water, alcohol, saline solution, buffered saline solution, physiological saline solution, dextrose solution, water propylene glycol solutions, and the like, preferably in sterile form.
The compositions (for eliciting an immune response and/or for immunizations) can be formulated in either neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
In various embodiments, the compositions are administered intradermally, subcutaneously, intramuscularly, or intravenously. The compositions may be formulated for inoculation or injection into the subject. For injection, the compositions of the invention can be formulated in aqueous solutions such as water or alcohol, or in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution can contain formulatory agents such as suspending, preserving, stabilizing and/or dispersing agents. Injection formulations can also be prepared as solid form preparations which are intended to be converted, shortly before use, to liquid form preparations suitable for injection, for example, by constitution with a suitable vehicle, such as sterile water, saline solution, or alcohol, before use.
The compositions (for eliciting an immune response and/or for immunizations) can also be formulated in sustained release vehicles or depot preparations. Such long acting formulations can be administered by inoculation or implantation (for example subcutaneously or intramuscularly) or by injection. Thus, for example, the vaccine compositions can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well-known examples of delivery vehicles suitable for use as carriers.
The compositions (for eliciting an immune response and/or for immunizations) can comprise agents that enhance the efficacy of the composition, such as adjuvants. Adjuvants include any compound or compounds that act to increase an immune response to the ApoB100 peptides or immunogenically active portions thereof, thereby reducing the quantity of antigen necessary in the vaccine, and/or the frequency of administration necessary to generate a protective immune response. Adjuvants can include for example, emulsifiers, muramyl dipeptides, pyridine, aqueous adjuvants such as aluminum hydroxide, chitosan-based adjuvants, and any of the various saponins, oils, and other substances known in the art, such as Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, CpG sequences, synthetic oligonucleotides and combinations thereof (Schijns et al. (2000) Curr. Opin. Immunol. 12:456), Mycobacterialphlei (M. phlei) cell wall extract (MCWE) (U.S. Pat. No. 4,744,984), M. phlei DNA (M-DNA), and M-DNA-M. phlei cell wall complex (MCC).
Compounds which can serve as emulsifiers include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds. Among the synthetic compounds, anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of lauric and oleic acid, the calcium, magnesium and aluminum salts of fatty acids, and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltrhethylammonlum bromide, while synthetic nonionic agents are exemplified by glycerylesters (e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers, and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan monopalmitate). Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol.
Other suitable adjuvants can be formed with an oil component, such as single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, or animal oil. Mineral oils are liquid hydrocarbons obtained from petrolatum via a distillation technique, and are also referred to in the art as liquid paraffin, liquid petrolatum, or white mineral oil. Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange roughy oil and shark liver oil, all of which are available commercially. Suitable vegetable oils include, for example, canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like. Freund's Complete Adjuvant (FCA) and Freund's Incomplete Adjuvant (FIA) are two common adjuvants that are commonly used in vaccine preparations, and are also suitable for use in the present invention. Both FCA and FIA are water-in-mineral oil emulsions; however, FCA also contains a killed Mycobacterium sp.
Immunomodulatory cytokines can also be used in the compositions to enhance the efficacy of the composition, for example, as an adjuvant. Non-limiting examples of such cytokines include interferon alpha (IFN-α), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor (GM-CSF), or combinations thereof. GM-CSF is highly preferred.
Compositions comprising ApoB100 peptides or immunogenically active portions and further comprising adjuvants can be prepared using techniques well known to those skilled in the art including, but not limited to, mixing, sonication and microfluidation. The adjuvant can comprise from about 10% to about 50% (v/v) of the vaccine composition, about 20% to about 40% (v/v), about 20% to about 30% (v/v), or any integer within these ranges.
Administration of the compositions can be by infusion or injection (e.g., intravenously, intramuscularly, intracutaneously, subcutaneously, intrathecal, intraduodenally, intraperitoneally, and the like). The vaccine compositions can also be administered intranasally, vaginally, rectally, orally, or transdermally. Additionally, vaccine compositions can be administered by “needle-free” delivery systems. In an embodiment, the compositions are administered by intradermal injection. Administration can be at the direction of a physician or physician assistant.
The injections can be split into multiple injections, with such split inoculations administered preferably substantially concurrently. When administered as a split inoculation, in an embodiment, the dose of the immunogen may be proportioned equally in each separate injection. If an adjuvant is present in the vaccine composition, the dose of the adjuvant may be proportioned equally in each separate injection. The separate injections for the split inoculation may be administered substantially proximal to each other on the subject's body. In some aspects, the injections are administered at least about 1 cm apart from each other on the body, at least about 2.5 cm apart from each other on the body, at least about 5 cm apart from each other on the body, at least about 10 cm apart from each other on the body or more than 10 cm apart from each other on the body, for example, at least about 12.5, 15, 17.5, 20, or more cm apart from each other on the body. Primary immunization injections and booster injections can be administered as a split inoculation.
Various alternative pharmaceutical delivery systems can be employed. Non-limiting examples of such systems include liposomes and emulsions. Certain organic solvents such as dimethylsulfoxide also can be employed. Additionally, the vaccine compositions can be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. The various sustained-release materials available are well known by those skilled in the art. Sustained-release capsules can, depending on their chemical nature, release the vaccine compositions over a range of several days to several weeks to several months.
Effective amounts of an immunogenic fragment to treat and/or prevent kidney disease will depend on the individual wherein the activation is performed and will be identifiable by a skilled person. For example in an embodiment the T cell activation can be performed with an effective amount of from about 1 to about 100 μg immunogenic fragment or immunogenically active portion thereof. In an embodiment, treatment and/or prevention of kidney disease can be performed with an effective amount of from about 1 to about 100 mg ApoB fragment or immunogenically active portion thereof. Additional effective amounts are identifiable by a skilled person in view of the individual where activation is performed and the desired activation. In an embodiment, an effective amount for the treatment or prevention can be about 100 μg or more. A greater concentration can be used in some embodiments depending on the desired effect as illustrated in the present disclosure. For example, in embodiments wherein treatment of kidney disease is expected to be performed with an effective amount be 250 μg or more. In another example, wherein the kidney disease is less severe an effective amount to treat the kidney disease is expected to be 25 μg or 50 μg.
The effective amount of the composition can be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the patient, the type of formulation, the mode or manner or administration, or the presence or absence of risk factors that significantly increase the likelihood that kidney disease may occur. The effective amount is also expected to vary depending on the number and combination of peptides utilized for each particular composition, and specific characteristic and conditions of the individual treated (e.g. immune system diet and general health and additional factors identifiable by a skilled person).
Toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in patients. The dosage of such compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
Toxicity information can be used to more accurately determine useful doses in a specified subject such as a human. The treating physician can terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions, and can adjust treatment as necessary if the clinical response is not adequate, to improve the response. The magnitude of an administrated dose in the prevention of recurrent kidney disease will vary with the severity of the patient's condition, relative risk for recurrence, or the route of administration, among other factors. The severity of the patient's condition can, for example, be evaluated, in part, by standard prognostic evaluation methods.
The compositions can be administered to a patient on any schedule appropriate to induce and/or sustain protective immunity against kidney disease relapse and more specifically to induce and/or sustain a cytotoxic T lymphocyte response to ApoB fragment or immunogenically active portion thereof. For example, patients can be administered a composition as a primary immunization as described and exemplified herein, followed by administration of a booster to bolster and/or maintain the protective immunity.
In some aspects, patients can be administered the vaccine compositions 1, 2 or more times per month. In an embodiment, once per month for six consecutive months may establish the protective immune response, particularly with respect to the primary immunization schedule. In some aspects, boosters can be administered at regular intervals such as every 6 or more months after completion of the primary immunization schedule. In an embodiment, administration of the booster is every 6 months. Boosters can also be administered on an as-needed basis.
The administration schedule for the composition including primary immunization and booster administration can continue as long as needed for the patient, for example, over the course of several years, to over the lifetime of the patient. In some aspects, the vaccine schedule includes more frequent administration at the beginning of the vaccine regimen, and includes less frequent administration (e.g., boosters) over time to maintain the protective immunity.
The composition can be administered at lower doses at the beginning of the regimen, with higher doses administered over time. The composition can also be administered at higher doses at the beginning of the regimen, with lower doses administered over time. The frequency of primary administration and booster administration and dose of ApoB fragments or immunogenically active portions thereof administered can be tailored and/or adjusted to meet the particular needs of individual patients, as determined by the administering physician according to any means suitable in the art.
The present invention is also directed to kits to treat, inhibit, reduce severity of, mitigate the effects of and/or promote prophylaxis of kidney disease in a subject in need thereof. The kit comprises ApoB-100 peptides or immunogenic fragments thereof, for example peptides set forth in Table 1 or a derivative, variant, pharmaceutical equivalent, peptidomimetic and/or analog thereof. Alternatively, the kits comprise CD8+ T cells (for example, activated CD8+ T cells) and pharmaceutically acceptable carriers. The kits may also comprise ApoB-100 peptides or immunogenically active fragments thereof and activated CD8+ T cells. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Additional components of the kit may include enhancer molecules.
The exact nature of the components configured in the inventive kit depends on its intended purpose. In one embodiment, the kit is configured particularly for human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.
Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to treat, prevent, inhibit, prevent metastasis of and/or promote prophylaxis of cancer (for example leukemia) in a subject. Optionally, the kit also contains other useful components, such as, measuring tools, diluents, buffers, pharmaceutically acceptable carriers, syringes or other useful paraphernalia as will be readily recognized by those of skill in the art.
The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a bottle used to contain suitable quantities of the compositions of the invention. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
EXAMPLES
The following example is provided to better illustrate the claimed invention and is not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
The inventors assessed the effect of p210 immunization on AngII-induced renal inflammatory responses in apoE (−/−) male mice. For the experiments described herein, native p210 peptide (KTTKQ SFDLS VKAQY KKNKH) was conjugated to cationic bovine serum albumin (cBSA) as carrier. Alum was used as an adjuvant. At 7, 10, and 12 weeks of age, male apoE (−/−) mice were subcutaneously injected with 100 μg of p210/cBSA/Alum. As immunization controls, 100 μg of cBSA/Alum, or PBS were injected. At 10 weeks of age after first immunization booster, 1000 ng/Kg/min of AngII was delivered by subcutaneously implanted osmotic pump for 4 weeks (FIG. 1A).
As shown in FIG. 1B, administration of p210, an immunogenic peptide of ApoB-100 reduces the levels of the AT1 receptor. p210 immunization significantly decreased the expression of Angiotensin II type 1 receptor (AT1R) in aorta.
Serum creatinine (Cr.) (an indicator of renal function) level at 14 weeks of age was assessed by commercially available creatinine assay kit following the manufacture's instruction. As shown in Table 2, p210 immunization protected against AngII-induced renal damage. Serum creatinine level was significantly lower in p210 group. *p<0.05 vs. p210. N: p210=12, cBSA=11, PBS=9.











TABLE 2







P210
cBSA
PBS





















Serum Cr. (mg/dl)
0.6 ± 0.1
1.1 ± 0.2*
1.3 ± 0.1*










At euthanasia, kidney was harvested for quantitative PCR analysis or histological analysis. Data are presented as mean±SEM. Data were analyzed by ANOVA following post-hoc test for multiple group comparison. Immunization with p210 vaccine significantly attenuated AngII-induced renal damage. As shown in FIG. 2, the p210 immunization down-regulated pro-inflammatory gene expressions (IL-6, MCP-1, and TNF-α) in kidney.
Superoxide production in kidney was measured by in situ dihydroethidium (DHE) methods with freshly cut frozen sections. As shown in FIG. 3, the p210 immunization significantly reduced glomerular ROS production.
As shown in FIG. 4A, p210 immunization significantly reduced NOX1, a component of NADPH oxidase. As shown in FIG. 4B, the p210 vaccine significantly downregulated profibrotic gene expression (PAI-1 and TGF-β) in kidney. These anti-hypertensive and renal protective effects are associated with significant reduction of inflammatory cytokines and chemokine gene expression, ROS production mediated by NADPH oxidase, and profibrotic gene expression in kidney. Accordingly, in an exemplary embodiment, immunogenic peptide p210 and/or immunogenic fragments thereof may be good therapeutic agents for Angiotensin associated or related diseases such as kidney diseases and kidney malfunction.
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).
The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.
While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).

## Claims

1. A method of treating kidney disease in a subject in need thereof, wherein the subject has been diagnosed with kidney disease, comprising:
(a) providing a composition comprising one or more peptides of ApoB-100 or immunogenically active portions of said peptides, wherein the one or more peptides of ApoB-100 is any one or more of peptides p1 (SEQ ID NO: 1), p2 (SEQ ID NO: 2), p10 (SEQ ID NO: 10), p11 (SEQ ID NO: 11), p25 (SEQ ID NO: 25), p30-p34 (SEQ ID NOs: 30-34), p40 (SEQ ID NO: 40), p45 (SEQ ID NO: 45), p68 (SEQ ID NO: 68), p74 (SEQ ID NO: 74), p94 (SEQ ID NO: 94), p99 (SEQ ID NO: 99), p100 (SEQ ID NO: 100), p102 (SEQ ID NO: 102), p103 (SEQ ID NO: 103), p105 (SEQ ID NO: 105), p107 (SE ID NO: 107), p111 (SEQ ID NO: 111), p129 (SEQ ID NO: 129), p143 (SEQ ID NO: 143), p148 (SEQ ID NO: 148), p149 (SEQ ID NO: 149), p154 (SEQ ID NO: 154), p162 (SEQ ID NO: 162), p169 (SEQ ID NO: 169), p177 (SEQ ID NO: 177), p199 (SEQ ID NO: 199), p210 (SEQ ID NO: 210), p222 (SEQ ID NO: 222), p236 (SEQ ID NO: 236), p252 (SEQ ID NO: 252), or p301 (SEQ ID NO: 301), or a combination thereof; and
(b) administering an effective amount of the composition to the subject, so as to treat kidney disease in the subject.

2. The method of claim 1, wherein the peptide of ApoB-100 is any one or more of peptides p2 (SEQ ID NO: 2), p11 (SEQ ID NO: 11), p32 (SEQ ID NO: 32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO: 148), p162 (SEQ ID NO: 162), or p210 (SEQ ID NO: 210), or a combination thereof.

3. The method of claim 1, wherein the peptide of ApoB-100 is any one or more of peptides p2 (SEQ ID NO: 2), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p210 (SEQ ID NO: 210), or a combination thereof.

4. The method of claim 1, wherein the peptide of ApoB-100 is p210 (SEQ ID NO: 210).

5. The method of claim 1, wherein the effective amount is about 100 to about 500 μg.

6. The method of claim 1, wherein the kidney disease is atherosclerosis-related kidney disease, hypertension-related kidney disease or diabetes.

7. A method of treating kidney disease in a subject in need thereof comprising:
(a) providing a composition comprising CD8+T cells activated with one or more peptides of ApoB-100 or immunogenically active portions of said peptides, wherein the one or more peptides of ApoB-100 is any one or more of peptides p1 (SEQ ID NO: 1), p2 (SEQ ID NO: 2), p10 (SEQ ID NO: 10), p11 (SEQ ID NO: 11), p25 (SEQ ID NO: 25), p30-34 (SEQ ID NOs: 30-34), p40 (SEQ ID NO: 40), p45 (SEQ ID NO: 45), p68 (SEQ ID NO: 68), p74 (SEQ ID NO: 74), p94 (SEQ ID NO: 94), p99 (SEQ ID NO: 99), p100 (SEQ ID NO: 100), p102 (SEQ ID NO: 102), p103 (SEQ ID NO: 103), p105 (SEQ ID NO: 105), p107 (SEQ ID NO: 107), p111 (SEQ ID NO: 111), p129 (SEQ ID NO: 129), p143 (SEQ ID NO: 143), p148 (SEQ ID NO: 148), p149 (SEQ ID NO: 149), p154 (SEQ ID NO: 154), p162 (SEQ ID NO: 162), p169 (SEQ ID NO: 169), p177 (SEQ ID NO: 177), p199 (SEQ ID NO: 199), p210 (SEQ ID NO: 210), p222 (SEQ ID NO: 222), p236 (SEQ ID NO: 236), p252 (SEQ ID NO: 252), or p301 (SEQ ID NO: 301), or a combination thereof; and
(b) administering an effective amount of the composition to the subject so as to treat kidney disease in the subject.

8. The method of claim 7, wherein the peptide of ApoB-100 is any one or more of peptides p2 (SEQ ID NO: 2), p11 (SEQ ID NO: 11), p32 (SEQ ID NO: 32), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO: 148), p162 (SEQ ID NO: 162), or p210 (SEQ ID NO: 210), or a combination thereof.

9. The method of claim 7, wherein the peptide of ApoB-100 is any one or more of peptides p2 (SEQ ID NO: 2), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p210 (SEQ ID NO: 210), or a combination thereof.

10. The method of claim 7, wherein the peptide of ApoB-100 is p210 (SEQ ID NO: 210).

11. The method of claim 7, wherein the effective amount is between about 500,000 and about 2,000,000 activated CD8+ T cells.

12. The method of claim 7, wherein the effective amount is about 1,000,000 activated CD8+ T cells.

13. The method of claim 7, wherein the method further comprises administering an effective amount of one or more enhancers.

14. The method of claim 7, further comprising administering a composition comprising an immunogenic peptide of ApoB-100 or an immunogenically active portion thereof.

15. The method of claim 14, wherein the composition comprising activated CD8+ T cells and the composition comprising an immunogenic peptide of ApoB-100 or an immunogenically active portion thereof, are administered sequentially or concurrently.