AU2007211899A1 - Biologically active peptides - Google Patents

Description

24-08-2007 03:24PM FROM-A J PARK gton +64 4 4723358 T-714 P.004 F-741 Regulation 3.2 ;Z AUSTRALIA PATENTS ACT, 1990 COMPLETE

SPECIFICATION

FOR A STANDARD

PATENT

ORIGINAL

Name of Applicant CMS PEPTIDES PATENT HOLDING

COMPANY

LIMITED

Actual Inventors; WONG, Wai Ming LAM, Kong Address for service in AJ PARK, Level 11, 60 Marcus Clarke Street, Canberra

ACT

Australia; 2601, Australia Invention Title: Biologically active peptidcs The following statement is a full dsciption of this invention, including the best method of perfonning it known to us.

-(follo -dby page 1- (&llowcdby page Ia) COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-20071 03:24PM FROM A J PARK gton 64 4 4723358 T-714 P.005 F-741 BIOLOGICALLY ACTIVE PEPTIDES This is a divisional application of Australian application 2002319423.

FIELD OF INVENTION The present invention is related to the field of immunology. In particular the present invention is directed to peptides and their pharmaceutical compositions which are capable of modulating an immune responses.

BACKGROUND OF INVENTION 00 Peptidcs are known in the art for treatment of diseases and as pharmaceutical compositions. For example, US Patent No.6,191,1-13 to discloses a peptide that has inhibitory activity for the growth of smooth muscle cells and is therefore useful for preventing and treating pathological conditions associated with Sgrowth of smooth muscle cells such as arteriosclerosis, restenosis after angioplasty, C luminal stenosis after grafting blood vessel and smooth muscle sarcoma. US 6,184,208 discloses another peptide that is found to modulate physiological processes such as weight gain activity of the epithelial growth zone and hair growth.

la COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:25PM FROM-A J PARK gton +64 4 4723358 T-714 P.006 F7T41 Ot. SUMMARY Ofr JyENflON ;Z It is $erefort an bbject of the present inventio~n to identify biologically active polypeptides. Many -peptides were synthesized by standard chemical c-i methods and screened for their biological activity. The peptides are given codes 5 haying the Iqttra CMS followed by a number. A total of 30 peptides have been identified as having in viva -biological activities. The sequences and the coirespndig ED numbers of these biologically active peptides; are shown in Table x 00 Table A Sequence Listing Peotde YpieSqec 71D No. Name 090OQ1 Pro Tbr TbL Tbx Phc FHis rhL 2 CMSOQZ ?a~l FaTTrb he o3 GMSOOS LYSAIA V81 33i HISeA LC S Ml ll u ~,Ir Ala Lou 4 CMSOIO Val Ala Pro flu Gin His Pro Thi Lou Lu a1 fbrlu la Pt',Leu Asu CMS012 Leu I Mtfi~a 0flHis CIn ThiTbTr 6 CM8013 Lou VaiAaPoGlu GlU 53 Pi VlILeU 7 0M3014. Ala Alt His His Pro A A h m r Su 'Vul 8 043015 PoSerIfle Vul GIAr Pro His .9 (31 Val Wt 9CMS01G He~ JAiMet Qlu SsrAlaBUJ lia ULU Lirl2XI -CMSO1IS Va Gi MetI Glyu3L LsAsp 9wer 11 045;T019 Vaul0 Meti ChLyGIn Sot 12 CMSO2O ValtO1 Met GI OlnLy AscSr 7WVZI -13 CMSO21 a l h l l c c c c Lou 14 CM8022 TrSer Phe.

*16 02SO4 TW $orLen 19 CMO2S Ph.s flu Pro Set Ph.a__ CMS029 Gh s lu flu 21 0M5030 Pi0t1 lUflu Met 22 CMSO32 PU. OWu flu Gin 23 CMS033 Pbs Gla Set Phe 24 C43034 Pro1 GlAsnPkhe CMS035 The Vol Assn 26 C 03 kPbeGi Pro Ser Bite 27 CMS003 Pitz Ast Pia Vat Pro 28 CMS0O7 Me A A Aa Pr o AlValfite 29 048009- LenE Val Ala Pro Gin flu His Pro liii Leo (DM5011 A Val Aa Pro Gu Glu hs ProQThrLol .Accordingly, one aspect of the present invention relates to substantiailly pure peptides having sequences identified as sequence ID No.1 to) sequence ID >TC.30. Thus thie present. invention also relates to a substantially pme peptide comprising an ami-no acid sequence selected from the group~co=lsStifls of SEQ ID0 10s. 1-30. It also relates to aL substantially pure peptide consisting essentially of an amino acid sequence selected from the group consisting of SBQ BD NOst 1-30. In a specific embodiment, the peptides, can modulate, but not COMS ID No: ARCS-158250 Received by IP Australia: Time (I-tm) 14:03 Date 2007-08-24 24-08-2007 03:25PM FROM-A J PARK gton +64 4 4723358 T-714 P.007/080 F-741 ;1 limited to modulating, one or more of the following immune activity; hepatitis intction, including but not limited to hepatitis B infection; nephritis; the growth of a oancmr, including but-not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight Another aspect of the present invention relates to substantially pure peptides that are functional derivatives of peptides having sequences identified as sequence ID No.l to 30. Thus thb present invention relates also to a substantially pure peptide comprising an amino acid sequence which is a fbnctioial derivative of an amin acid selected from the group consisting of SEQ ID NOs. 1-30. It also relates tao a substantiall pure peptide consiting essentially of an amino acid sequence which is a'funtional derivative of an amino acid sequence selected from the group consisting of SnEQ D Ns. 1-30. In a specific embodiment, the peptides that are funcitional derivatives can mdulate, but not limited to odulating, one or more of the following immune activity; hepatitis infection, including but not limited to hepatitis B infection; nepitis; the growth of a cancel, including but not limited to sarcoma, liver cancer, leukcmia and melanoma; and body weigh t Another aspect of the present invention relates to nuclei acids that have sequences coding for the peptides identifed above as sequence D No.1 to A frther aspect of the present invention relates to expression vectors that contain the nucleic acid sequences of the peptides shown below as sequence cD No.l to 30. Thus, this aspect of the -prsent invention also relates to a genetic vector comprising a nucleotide sequence encoding a peptide comprsing an aino acid sequence selected from the group comprising of SQ D NOs. 1-30. It also elates to a genetic vector comprising a nnol6oide sequence ecoding a peptide consisting essentially of an amino acid 'sequnce selected from the group consisting eof SEQ IL Os. 1-30. The invention also relates to a genetic vector comprising a nucleotid sequenc encoding a peptide comprising a functional derivative of a biologicaly active ino acid sequence selected from the group consisting of SEQ ID NOs. 1-30. It also relates to a genetic vector comprising a nucleotide sequence ecoding a peptide consisting essentially of a functional amino acid sequence which is a functional derivative of abiologically active amino acid sequence seleced from the group consisting of SEQ ID NOs. 1-30.

Yet another aspedt of the present invention relates to hybrid peptides containing a leader or signal peptide adacent a peptide, the peptide comprising an amino acid sequence selected from the group consising of SEQ ID NOs. 1-30. The present invention also relates to hybrid peptides contaning a leader peptide adjacent a peptide, the peptide comprising a functional derivative of a peptide having an amino acid sequtenc selected from the group consisting of SEQ ID NOs.

The 1 30 preset invention also elates a enetic vector comprising a nucleotide sequence enoding a peptide comprising a leader amino acid sequence adjacent a peptide compising a fnctional amino acid sequence which is a functional derivative of a biologicall active amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30. It also relates to a genetic vector codiprising a nucleotide sequence encoding a peptid comp ng a leader amino ad sequence adjacent a peptide consisting esstially of a functional amino acid sequence which 3 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:26PM FROM-A J PARK gton +64 4 4723358 T-714 P.008/080 F-741

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0 is a functional derivative of a biologically active amino acid sequence selected I from the group consisting of SEQ ID NOs. 1-30.

In a specific embodiment, the peptidcs produced in any of the abovedescribed genetic vectors can modulate, but not limited to modulating one or more of the following immune activity; hepatitis infection, including but not limited to hepatitis 3 infection; nephritis; the growth of A cancer, including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight.

Yet another aspect of the present invention relates to a micro-organism with a genome comprising a nucleotide sequence encoding a peptide comprising an o0 10 amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30. It also relates to a micro-organism with a genome comprising a nucleotide sequence Sencoding a peptide consisting essentially of an amino acid sequence selected from C the group consisting of SEQ ID NOs. 1-30.

SYet another aspect of the present inventioih elates to a micro-organism wit 0 15 genetic material comprising a nucleotide sequence encoding an exogenous peptide C comprising a functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30 -It also relates to a micro-organism with a genetic composition.

comprising a nucleotide sequence encoding an exogenous peptide consisting essentially of a functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30. Exogenous peptide as used herein refers to a peptide having an amino acid sequence that is different from any other peptides normally expressed by the micro-organism in its natural, unmodified form.

Yet another aspect of the present invention relates to a micro-organism with a genetic composition comprising a nucleotide sequence encoding an exogenous hybrid peptide comprising a leader amino acid sequence adjacent a peptide, the peptide comprising an amino acid sequence selected fom the group consisting of SEQ ID NOs. 1-30. It also relates to a micro-organism with a genome compsing a nucleotide sequence encoding a hybrid peptide comprising a leader amino acid sequence adjacent a peptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30.

Yet another aspect of the present invention relates to a micro-organism with a genetic composition comprising a nucleotide sequence encoding an exogenous hybrid peptide comprising a leader amino acid sequence adjacent a peptide, the peptide comprising a functional amino acid sequence which is a fotional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID NOs- 1-30. It also relates to a micro-organism with a genetic composition comprising a nucleotide sequence encoding an exogenous hybrid peptide comprising a leader amino acid sequence adjacent a peptide consisting essentially of a functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30.

In a specific embodiment, the peptides produced in any of the abovedescribed micro-organism can modulate, but not limited to modulating, one or more of the following immune activity; hepatitis infection, including but not limited to iepatitis B infection; nephritis; the growth of a cancer, including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight 4 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:26PM FROM-A J PARK gton +64 4 4723358 T-714 P.00/080 F-T41 0 0 s Yet another aspect of the present invention relates to a pbhamaceutical Scomposition comprising a substantially pure peptide comprising an amino acid sequMcnce seldoted from the group consisting of SEQ ID NOs. 1-30. The invention also relates to pharmaceutical composition comprising a substantially pure peptide consisting essentially of an amino acid sequence selected from the group C consisting of SEQ ID NOs. 1-30.

The present invention ilso relates to a pharmnnaceutical composition comprising a substantially pure peptide comprising a functional derivative of an N amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30. It 00 10 alo relates to a pharmaceutical composition comprising a substantially pure peptide consisting essentially of a functional-derivative of an amino acid sequence cselected from the group consisting of SEQ ID NOs. 1-30. It further relates to pharmaceutical composition consisting of a substantially pure peptid( consisting Sessentially of functional derivative of an amino acid sequence selected from the 0 15 group consisting of SQ-ID NOs- 1-30.

Nc In a specific embodiment, the peptides present in any of the abovedescribed pharmaceutical compositions can modulate, but not limited to modulating, one or more of the following unwe activity, hepatitis infection, including but not limited to hepatitis B infection; nephritis; the growth of a cancer, including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight.

Yet a further aspect of the present invention relates to a method of making a phannaceutical comaposition comprising providing a substantially pure peptide comprissi an amino acid sequence selected from the group consisting of SEQ ID 4Os. 1-30; and mixing said substantially pure peptide with a pharmaceutically acceptable carrier. It also relates to a method of making a pharmacntical composition comprising providing a substatially pure peptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30.

Another aspect of the present invention is a method of making a pharmaceutical composition comprising providing a substantially pure peptide comprising an amino acid sequence which is a functional derivative of an amino acid sequence selected from the group conssting of SEQ ID s 1-30; and mixing said substantially pure peptide *ith a phamaceuticlly acceptabl c-arier.

It further relates to a method of making a phisrmaceutical composition comprising providing a substantially pur peptide consisting essentia4ly of an amino acid sequence which is a functional derivative of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-30.

In connection with any of the above-described method, the peptide can modulate, but not limited to modulating, one or more of the following immune activity; hepatitis infection, including but not limited to hepatitis B infection; nephritis; the growth of a cancer, including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight.

Yet a further aspedt of the present invention relates to a method of treatment of a human comprising administering a pharmaceutically effective dose of a substantially pure peptide comprising an amino acid sequence selected from COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:26PM FROM-A J PARK gton +64 4 4723358 T-714 P.010/080 F-741

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the group consisting of SEQ ID NOs. 1-30 to a human. It also relates to a method of treatment of a human comprising a administering a pharmaceutically effective dose of a substantially pure peptide comprising an amino acid sequence which is a functional derivative of an amino acid sequence selected from the group consisting 7- 5 of SEQ ID NOs. 1-30.

In a specific embodiment, the peptides used for the treatment of human described above may be used to modulate, but not limited to modulating, one or more of the following human conditions: immune activity, hepatitis infection, including but not limited to hepatitis B infection; nephritis; the growth of a cancer, 00 10 including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight SIn connection with any of the above-described nucleic acid sequences, the peptides and/or hybrid peptides expressed from these nucleic acid sequences can 0 modulate, but not limited to modulating, the following immune activity; hepatitis infection, including but not limited to hepatitis B infection; nepbxitis; the growth of N a cancer, including but not limited to sarcoma, liver. cancer, leukemia and melanoma; and body weight.

Another aspect of the present invention relates to the method-of treating diseases comprising administering a pharmaceutically effective dose of a substantially pure peptide having sequence ID No.l to sequence ID No.30. In a specific embodiment, the peptides so administered can modulate, but not limited to modulating, the following immune activity; hepatitis infection, including but not limited to hepatitis B infection; nephritis; the growth of a cancer, including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight.

As described above, another embodiment of the present invention is a peptide or polypeptide consisting essentially of a peptide of the present invention As used herein, the terminology "consisting essentially of' refers to a peptide or polypeptide which includes the amino acid sequence of the peptides of the present invention along with additional amino acids at the carboxyl and/or amino terminal ends and which maintains the activity of the peptides of the present ivention provided herein. Thus, as a non4imiting example, where the activity of the peptide of the present invention is to modulate immune activity, a peptide or polypeptide "consisting essentially of the peptide.ofthe present invention will possess the activity of modulating immune activity as provided herein with respect to that peptide and will not possess any characteristics which materially reduces the ability of the peptide or polypeptide to modulate immune activity or which constitutes a material change the basic and novel characteristics of the peptide as a modulator of immune activity. Thus, in the foregoing example, a full length naturally occurnrig polypoptide which has a primary activity other than modulating immune activity and which contains the amino acid sequence of a peptide of the present invention somewhere therein would not constitute a peptide or polypeptide "consisting essentially of' a peptide of the present invention. Likewise, in the foregoing example, a genetically engineered peptide or polypeptide which has .a primary activity other tan modulating immune activity but includes the amino acid sequence of a peptide of the present invention somewhere therein would not constitute a peptide or polypeptide "consisting. essentially of' a peptide of the present invention, 6 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24

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24-08-2007 03:27PM FROM-A J PARK gton +64 4 4723358 T-714 P.011/080 F-741 0 SBesides the example of immune activity modulation used for illustration Sabove, the foregoing definition also applies to all the peptides of the present Sinvention with respect to the activities provided for such peptides. In particular, the foregoing definition applies to peptides of the invention having activities in 5 modulating the extent of a viral infection, modulating the extent of a hepatitis (infection, modulating the extent ofnephritis, modulating the growth of a cancer, or modulating body weight as set forthin the detailed description below.

Those skilled in the art can readily detennine whether a peptide or polypeptide consists essentially of a peptide of the present invention under the 00 10 foregoing definitions by measuring the activity of the peptide or polypeptide using the assays for modulation of imune activity, modulating the extent of a viral infection, modulating the extent of a hepatitis infection, modulating the extent of nephritis, modulating the growth of a cacer, or modulating body weight which are Sprovided herein with respect to a particuar peptide of the present invention.

O 15 In the preferred embodiment, the terminology "consisting essentially of' Srefers to peptides or polypeptides which have less than 20 amino acid residues in addition to the peptide according to the present invention In a more preferred embodiment, the same terminology refers to a peptides with less than 15 amino acid residues in addition to the peptide according to the instant invention, In an even more prefrred embodiment, the same terminology refers to a peptides with less than 10 amino acid residues in addition to the peptide according to the instant invention. In another preferred embodiment, the same terminology refrs to peptides or polypeptides with less than 6 amino acids in addition to one of the peptide of the prsent invention. In another preferred embodiment the same terminology refes ti peptides or polypeptides with less -han 4 amino acids in addition to one of the peptide of the present invention. In the most preferred embodiment, the same tenmnology refers to peptides or polypeptides with less than 2 amino acids in addition to one of the peptide of the present invention.

7 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:27PM FROM-A J PARK gton +64 4 4723358 T-714 P-012/080 F-T41

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C. DETAILED DESCRIPTION SThe poptides can be readily synthesized by standard synthetic methods from L-amino acids, but may also be synthesized by genetic engineering methods using nucleic acids that have sequences encoding the individual peptides.

tC< 5 1. Biological Activity In order to investigate the possible biological activity of peptides, the immunological effect of the peptides on animal model was examined, with O procedures compliant to the "Principles of Pre-clinical Research of New Drugs" 00 issued by Ministry of Health.ofPeople's Republic of China 10 The T lymphocyte transformation test, NK cll cytotoxicity activity test, and the T lymphocyte IL-2 and IFN-y secretion test were used to detect any possible effect of peptides on specific cellular immune function. The carbon o particle clearace test was used to detect any possible effect of peptides on 1on- Specific cellular immune funtio. The Sheep Red Blood Cell (SRBC) hemolysis test was used to detect any possible effect of peptides on humoral immune function. The lmmunity organ weight test was used to detect any possible effect of peptides at the organ level.

In this study, the saline group was used as negative control, while the IL-2 and IFN-y groups were used as positive controls, since IL-2 and IFN-y are wellstudied immnmostimulants Four arbitrary concentrations of sample peptides were used in this study, to cover a 1000 fold dosage range. Due to the intrinsic complexity of in vivo immunological response, and the lack of prior knowledge on the dosage versus response function, therefore any statistically significant difference over the negative control in any of the dosage groups has been scored as positive biological activity.

Results of this study were as follows: Peptides CMS001, CMS002, CMS003, CMS007, CMS008, CMSO09, CMS010, CMS011, CMS012, CMS015, CMS019, CMS021, CMS029, and CMS034 were found to be able to enhance T lymphocyte transformation, having statistically significant difference from the saline normal control group. Peptides CMSD14 and CMS036 were also founmd to be able to inhibit T lymphocyte transfdirm a t i o n, having statistically significant difference from the saline nomal control group.

2. Peptides CMS001, CMS002, CMS003, CMS008, CMS009, CMS011, CMS012, CMS013, CMS015, CMS016, CMS020, CMS021, CMS022, CMSO23,: CMS024, CMS026, CvIS027, CMSO28, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, and CMS036 were found to be able to increase the cytotaxic activity of NK cells, having statistically significant difference from the saline normal control group.

Peptides CMSOO8 and CMS012, at suitable concentration, were also found to be able to decreasa the cytotoxic activity of NK cells, having statistically significant difference from the saline normal control group.

3. Peptides CMS001, CMS003, CMS007, CMS009, CMSO10, CMS011, CMS012, CMS015, CMS020, CMS022, and CMS034 were found, to be 8 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 gton +64 4 4723358 T-714 P.013/080 F-741 24-08-2007 03:2BPM FROM-A J1 PARK 1 0 0 ci ci able to enhance tl* secretion of interleukin- 2 (IL-2) by T lynq'hocyt-s. having statistically significant differenc fxrm the saline norml control SI'oup- 4. Peptides CMSOOC, CMSOO3, CUSOO9, 0145010, CMS0ll, CMSOI2., GMSOI3, 014016, 0143021, CMSO22, and 0145028 were found to be able to enlhance the secretion of WIN by T lymnphocytes, having statistically ,Significant difeace form the saline normal control group.

PoptideS 0145001, 0148002, CMSOO3, 0143007, 0143008, CMSOO9, 0143010, 0145011, CMS0lZ, CMS0l3, 0143014, 0143015, CMSOI6, 0145018, 0145019, CMSO20, CMSO2t, CMSO2Z, 0143023, 0148024, CM3026, 0145027, CMS02S, 0145029, 0148030, CIVI3032, C14S033, CMS034, 0143035, and CMSO3 6 were found to be able to enhance the synthesis of anti-SRBC antibody uapon the antig3iO challenge. h~avig rtattcally sggificanit difference from the saline nonnil control group.

Peptides OIVSOOZ, 014003, 0145009, 0145010, 0148011, CMSOI3, 01S4304 CMSOIS, CMSOIB, 0A5019, 0143020, CMSO2S, 0143028, dM8029, CWvIS03,0145M034, and 0145036, at sitable couceatatiol1 Were -also found to be able to inhibit the synthesis of anti-SPCC antibody upon the antigenic challenge, having sttistically significant difference from the salie normal control group.

6: Peptides C14S003. 014005,080, CMSO O, CMS UII VE1. UivioDUzz 0148016, 0148018, 014019; 0148020, Q14S02, CMS8024, 0145027, CMSO3O, 0148035, CMS5036 were found to be able to enhance the phagoytOtic activity of xnaoloUClCSY phagocyte. having statistic significant WL.LJ-L'F the saline normal control group.

7. Peptiles 014500', (245002, 01508 M04010, 014802, O4S01, 0145014, 0145015, 0145016, 0148018, 131,0482,0401 014522,014023.0MS24,CM026, 0143027. CMS02S, 0143029, CMS030. 0143032, 0MS033, CMSQ34, CMS035, and CM43036 were found to be able to increase the Weigh Of the thymus glad, having g.11,lt ,Amnificaiit difference from the saline normal control group.

U. etie 81 9 O.lS20 and CMS3030 were found to be able to acrelSCe the weight Of the spleen, having, statistically rngiE~~l difrec from the saline normal r-Ontrol group. MetS 0143001, CMSOO3, CMSOO7, 0143005, 014009, OMSOIO, 0301 9M0, ndCMS31, 014501l5, 0148021, 0145023,0143024, 014302-7, dM302,anCMO6 atsialec3et5t~,wr also found to be able to decrease -the weight of the plehaving statistically significant differene" o tesln normal control .groulp- The materials and methods used to aayse the effect of the peptides n mouse are described bclow.

Ma~teri als A rdrnal txpeiai±enia~ c~LA.Aa BALB/c Mce, 1 8-22g weight, 50% female and 50%o/ matle, provided by Experimental Animal Center, National InstitutO of Medica Science, PR Cbant COMS ID No: ARCS-i 58250 Received by iP Australia: Time 14:03 Date 2007-08-24 24-08-200T1 03:28PM FROM-A J PARK ffton 64 4 4723358 T-714 P.014/080 F-741

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2 Administration recombinant mouse IFN- (rmlFN-r) group: 3x Srecombinant human IL (rhfLt)-2 group: 3xSxIIU/kgday Saline group: 0.Sml/eacb/day peptide dose I group; 500g/kg/day peptide dose II group: 50 g/kg/day peptide dose mI group: Spg/kg/day peptide dose IV group: The above substances were all dissolved in 0.5ml saline and injected 0 0 10 itraperitoneal for 15 continuous days, once per day.

3. Main Reagents c The peptides were custom manufactured by American Peptide Company, Inc., oUSA SFetalbovine-srum, and RPMI-1640 cell culture medium, Gibco, USA C 15 MTT, and ConA, Sigma, USA rmIFN-y, Beijing Biotebh Inc., China rhIL-2, Shanghai Huaxin Biotech Inc., China Lymphocyte separation solution, Research Institute of Heniatologic Disease, National Institute of Medical Science, PR China Vesicular Stomatitis Virus (VSV), IFN-y and IL-2 standard sample, National Institute For The Control Of Pharmaceutical And Biological Products, PR China HT-2 cell and L929 cell, gift from Prof WF Chen of Beijing University Department of Immunology, PR China Method 1. The effect ofpeptides on cellular immunity 1.1 Preparation of spleen cell suspension 2 The BALB/c mice were randomly divided into the peptide, IFN, IL-2, and saline groups. Ten mice per group. The day after the last test substance administration, the mice were sacrificed by cervical dislocation. The spleen was isolated aseptically and manually dispersed in cold D-Hank's solution using an injection needle. The dispersed cell suspension was further sieved through a 100 gauge 150pm diameter stainless steel sieve. After centrifugation at 200g for minutes, the supernatant was discarded. The cell pellet was re-suspended in volume of Tris-NH 4 Cl buffer and then kept standing still for 10 minutes at room tenipeftture. The suspended cells were collected by centifugation at 150g for minutes. The cells were washed 2-4 times with cold D-Hank's solution by resuspending and collecting by centrifugation with condition as decribed above.

The washed cells were then diluted to the desired cell densities by RPMI-1640 culture medium, containing 10% fetal bovine serum.

1.2 The effect ofpeptides on T lymphocyte transformation Spleen cells of density IxlO 6 /ml were placed onto a 96 wells cell culture plate, 100l/well, three parallel wells.each of the assay sample and control sample per mouse. To the assay wells, 100pwell ConA of 100g/ml in RPvI-1640 was COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 T-714 P.015/080 F-741 +fil A 47393~8 24-08-2007 03:28PM FROM-A J PARK on

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added, and 100tl/well plain rPMI-1640 was used for the controls. The clls wert M incubated for 66 bra at 37 0 C, 5% CO. The -cells were then pelleted by cenrifugation at 150g forv10 minutes. The supematant was collected and stored at for cytokies IL2 and IFN detemination.

/well MTT of 1mg/ml in RPMI-1640 was added to the cell pellet and the cells re-suspended by shaking for 2 minutes. The incubation was continued for 4 hours. The supernatatt was' discarded after centrifugation at 150g for O minutes. 12041 40mM HCl-2propanol was added to the cell pellet and shaken for 3 minutes. to obtain ODonm of each well referenced at 630nm. An ELISA 00 10 reader was used Calculation: Each mouse formed three assay wells and three control wells. The o Stimulation Index (SI) of each mouse was obtained by first deriving the average oOD of the three parallel wells, then dividing the value of the assay wells by the control wells.

1.3 The effect of peptids on NK cell activit' Mice spleen cells were prepared to 4x10n/ml as desci'bed in section 1.1 above. Target cells YAC-1 were brought to log phase and adjusted to ixl0s/mL Using a 96 wells cell culture plate, lOp. mouse spleen cells and 100)l culture medium were added to the control well containing only the spleen cells; 100 1 target cells and 100I culture mdium were added to the control well containing only target cells; IOpl mouse spleen cells'and 100l target cells were added to the NK activity assay well. Three parallel sets of the above were prepared per mouse. Samples were centrifuged at 1 50g for 10 minutes to collect the cells. The supernatant was discarded and 50~I/well MTT of lmg/ml was added. The reaction mixture was then shaken for 2 minutes, and incubated at 37C, 5% Cz2 for 4 hours- The supernatant was discarded after centrifugation at 150g for 10 minutes.

120 Il 40mM ICl-2-ppano l was added and shaken for 3 minutes. An ELISA reader was used to obtain ODnonm of each well referenced at 630nm.

Calculation: Each mouse has 9 walls; three 'pleen cells only oontrol, tree target cells only control, and three assay wells with both spleen and trget cells. The INK cell activity index of each mouse was obtained by first deriving the average OD of the three parallel wells of each combination, then applying this average OD to the following formula: NK cell activity index [1-(average OD of spleen and target cell well average OD of spleen cell only well) (average OD of target cell only well)] x 100% IA The effect ofpeptides on the activity T lymphocyte in secreting IL-2 1 -T-2 cells at log phase were collected by centrifgation at 150g for minutes, and washed three times with cold Hank's solution by rsuspension and centrifuga.tion. The collected HT-2 cells were.re-suspended in RPMI-1640 and 11L COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:29PM FROM-A J PARK &ton +64 4 4723358 T-714 P.016/080 F-741

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0 incubated at 370C, 5% C02 for 30 minutes. The cells were further washed twice ;Zwiith RPM-1640 by re-suspension and centrifugation, and re-suspended to final concentration of 2xld0mLwith RPMI-1640.

The supernatant obtained in section 1.2 were diluted to the following C 5 percentage with RPMI-1640: 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%.

rlL-2 was diluted to the- following concentration with RPM-1640: 500LT/n1, 250TU/ml, 125IU/mil, 62.5IU/ml, 31.251U/ml, and 15.51T/l.

A 96 well cell culture plate was set up with three parallel wells per combination: Negative control: 100RPi Wl-1640 100pl HIT-2 cell suspension Cl rlL-2 standard: 100p1rIL-2 solution+ 100pI HT-2 cell suspension Assay well: 100 diluted supernatant 100p1 HT-2 cell suspension The plate was incubated at 370C, 5% COz for 68 hours, then centrifuged at 150g for 15 minutes and the supeinatant removed. 100P 0.5mg/ml TT. in RPMI-1640 without phenolslfonphthalein was added into each well. After shaking for 3-4 minutes to re-suspend-the cells, coninue to incubate for another 4 hours. Samples were then centrifuged at 150g fot 15 minutes and the supernatant was removed. To each well, 120p 40mIM HC1-2-propanol was added, mixed for 3-4 minutes and OD analyzed at 570nm, referenced at 630m with an ELISA plate reade.

Calculation: The average OD of the three parallel Wells of each dilution was taken and plotted against concentration on a semi-log paper, concentration on the X-axis.

The concentration at 50% OD saturation was obtained for both the testing supenatant and rlL-2.

Sample IL-2 activity (sample dilution at 50% maximum action rIL-2 standard dilution at 50% maximum action) x activity of rlL-2 standard at maximum action (IU/ml) 1.5 The effect of peptides on the activity, of T lymphocyte in secreting interferon (FN) The supemrnatant from the section 1.2 was diluted wvith RPMl-1640 culture medium to the following percentages: 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%.

The recombinant interferon (rIFN) standard was diluted with RPMI-1640 to the following concentrations: 500U/ml, 250IU/ml, 125IjAml, 62.5IU/mi, 31,251U/ml, and 15.51U/ml.

Target cells L929 at log phase were adjusted to 2x1&/Iml with RPI-1 640, with treatmnet same as the HT-2 cell described in section 1.4. Stdck VSV was also adjusted to 100 TCIDso with RPMI-1640 The following on a 96 well culture plate was set up, three parallel wells per combination: Negative control well: 150l RPI-1640 100pl L929 12 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007( 03:29PM FROMA J PARK gton +64 4 4723358 T-714 P.017/080 F-741

I

O

C Positive control well: 10091 RPMN-1640 l00il L929 50pl VSV rlFN activity well: 100d rIFN standard 1004 L929 501 VSV Z assay well: 100p diluted supematant 1001 L929 50l VSV Samples were incubated at 37°C, 5% COz for 24 houi.i The positive control wells were observed periodically under inverted microscope to confirm cell lysis, then collected, washed, and OD of all wells was read same as described in section 1.4.

Calculation: 00 Concentrations at 50% maximum action were obtained same way as section 1.4. Calculate IFN activity of sample as following: Sample IFN activity (sample dilution at 50% maximum action rIFN Sstandard dilution at 50% maximum action) x standard UIFN activity at that Smaximum action (lUil) 2. The effect ofpeptides on antibody formation m Sheep red blood cells (SRBC) were prepared by collecting blood from cervical vein and put into a sterile flask with glass beads. The flask was shaken for 3 minutes and the blood then mixed with Alsever solution (glucose 2.05g, NaCI 0.4g, Na lemonade 0.8g, adjust to 100ml with distilled water) and stored at 4 0

C.

Immediately before use, samples were centrifuged at 130g, 5 minutes to collect the SRBC. The cells were washed two times by re-suspension and centrifugation in normal saline. Then the cell pellet was collected by centrifugation at 180g for minutes and re-suspended in saline to make the final wortdng SRBC suspension, 2% Complement was prepared by adding 10 volumes of fresh Cavy serum into one volume centrifuge packed SRBC, and then gently shaking for 30 minutes at 4°C. The SRBC was removed by centrifugation at 200g for 10 minutes. volumes ofnormal saline were added to obtain the worldng complement solution- The BALB/c mice were randomly divided into the peptide group, IFN group, IL-2 group, and saline group, 10 mice per group. The test substances were administered as described in section plus intraperitoneal injection 0.2ml SRBC per mouse on day 12. On the day after the last test substance administration (day 16), blood was collected from the eye canthus and left at room temperature for one hour for serum exudation. After centrifugation at 200g for 10 minutes, the serum was diluted by 500 imes with normal saline.

To iml diluted mouse serum of each mouse, 0.5ml SRBC suspension was added. Ice cold. Then Iml working complement solution was added and incubated at 37C water bath for 10 minutes. Reactions were terminated by ice cold. Samples were then centrifuged at 200g for 10 rinutes to obtain the supernatant To 1ml of this supernatant, 3ml Drabldn solution was added and left at room temperature for 10 minutes, OD0onm was obtained.

Calculation: 13 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:30PM FROM-A J PARK gton +64 4 4723358 T-714 P.018/080 F-741

I

Reference OD540n was obtained by mixing 0.25ml SRBC suspension with SDrabkld solution to 4ml and placed at room temperature for 10 minutes before ODsonmwas taken.

Sample serum index (ODsonm of test sample reference ODs m) X 500 5 3. The effect of peptides on the phagocytosis function of mononuclear phagocyte and the weight of immune organ On the next day after the last test substance administration (day 16), the mice were injected with O.lml/kg body weight India ink (5 times dilution with 00 normal saline) from the tail vein.

10 One minute and five minutes after Indian ink injection, 20pl blood was N obtained from the eye canthus with a heparinized tubing. The blood was mixed with 2ml 0.1% w/v Na 2 CO3 and then OD 6 onm obtained. The outline clear index K O was calculated by the following formula: C K=(lgAt lgA2) (t2-tl) Key: Al: OD680nm at first minute A2: OD68Oan at fifth minute t2: t2:1 minute One day after the last test substance administration (day 16), the liver, spleen, and thymus gland were separated and blotted dry with filter paper aad weighed, The phagocytosis index a was calculated as below: a (4 X)(W Ws) key: W: body weight WLs: weight ofliverplus spleen Thymus gland index (thymus weight body weight) x 100% Spleen index (spleen weight body weight) x 100% Results Due to the large quantity ofraw data involved, only the compiled end result are presented. The groups without statistically significant difference from the saline negative control are also omitted.

1. The effect ofpeptides on T lymphocyte transformation At 500pigg/day, CMS002, CMS007, CMS008, CMS010, CMS012, CMS015, CMS019, CMvS021, and CMS029 were found to be able to enhance

T

lymphocyte transfonation, having statistically sigiicant differenco from the saline group Among these peptides, CMS010 and CMS015 were found to have statistically significant difference from the IFN-y group and IL-2 group (P<0.05) as shown in Table 1 below.

14 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T1 03:3PM FROM-A J PARK gton +64 4 4723358 T-714 P.019/080 F-741 Tabzle I G4-0r-0up. N FMSD (sPKmula 4ind4 3 ;MSOO2 1.0.1* CMS007 9 h.l CMSOOB 9 1.7±0.1P CMS009 10 1.7t0.2* CMSOO 9 2.0-103*@A CMS012 9. 1.6+0.2* CMS015 9 l.9±0.3@A 00 CM$019 9 1.80.3* CMS21oz 10 1.6±0.1* CMSO29 9 1.7±0.3* 10 1.6±02* 0t-2 10' 1.7±0.2* Saline 10 1.3±0.V' comparing to saline group I comparing to IFN-7 group P<0.05 A compuing to TL-2 group P<0.05 At S0gf/kg/day, CMS001, CMS002 nd CMS003 were found to be able to stimulate T lymphocyte transformation, having statistically significant difference from saline group, IFN-y grouip, and IL-2 group CMSOl4 and. CMS036 were found to be able to ihibit T lymphocyte transformation, having statistical significant difference from saline group (PO.05). Data detailed in Table 2 below.

Table 2 Group N X±SD (stimulation index) CMSGOO 10 2.20@ CMS002 10 2-60.3*@A CMS003 8 2.2±0.5*@A CMS014 9 1.0_ a* CMSO36 9 1.0-+0.1* IFN-y 9 1.7±0.2* ]L-2 10 l.8i0-2* Saline 10 1.3±0.1 comparing to saline group P<0.05 compating to IFN-y group A comparig to M1-2 group P<0.05 1 At 5pgglkg/day. CN$001. CMSOS JvS007, and 0MS034 were found to be able to stimulate T lymphocyte transformation, having statistically significant difference from the saline group (P<0.05) as shown in Table 3.

GOMS ID No: ARCS-i58250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:30PM FROM-A J PARK 0 0 ci ci) gton +64 4 4723358 T-714 P.020/080 F-741 Table 3 Group N X±SD (stimulation index) CMS01 10 1.7±0.2* CMS003 10 1.6±0.2* CMS007 8 1.7±0.1* CMS34 9 1.5±0,2* IPN-y 10 1.6i0.2* L2 9 1.6±0.1* Saline 10 1.3±0.1 *comparing to saline group P<0.05 At 0.5 jg/kg/day CMS008, CMSO1O, and CMSO11 were found to be able to stimulate T lymphocyte transformatiou, having statistically significant difference from the saline group (P<0.05) as shown in Table 4.

Table 4 Group N X DS (stimulation index) CMS008 10 1.7±0.3* CMS010 9 1.7±0.3* CMS011 10 1.6±0.4* IFN-y 10 1.60.2* IL-2 10 1.60.1* Saline 10 1.3±0.1 comparing to saline group P<0.05 2. The effect of peptide on NK cell cytotoxic activity At 500gLkg/day, CMSO10, CMS013,- CMS016, CMSO23, CMS024, CMSO26, C S027, 28, CMSO2, 29CMS2 MS030, CMSO32,-CMS033, CMSO34, and CMSO36 were found to be able to increase NK cell cytotoxic activity, with statistically significant difference from the saline group (P<0.05).

Among these peptides, CMS10, CMS016, and CMSO30 were found to have statistically significant difference from the IFNJ group and L-2 group (P<0.05) as shown in Table COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:31PM FROM-A J PARK gton +64 4 4723358 T-714 P.021/080 F-741 N Table O) Gu N XtSD Or)up CM010 9 91t4* CMS013 8 84±9* CMS016 9 9167* CMS023 10 79tl12* CMS024 10 89±8* CMSO26 -10 .89±7* CMSO27 10 88±8* CMSO28 10 90±5 CM8029 10 87±4* 00 .CMSO30 10 915 CM5032 10 87±5* CMSO33 9 89E8* C CMS034 11 85±9' CMS03 8 900*1 CMSO36 10 88±7* IFPN-y 10 77±8* C IL-2 10 77*8* Saline 8 63 ±9 comparing to saline group P<0.05 comparing to FTN-y.group p<0.05 A comparing to IL-2 group P<0.05 At 50jg/kgfday, CMS001, CMS003, CMSO1S5, CMSO21, CMSO26, and CMS035 were found to be able to increase NY, cell cytotoxic activity, having statistically significant difference from the saline group Am6ng these peptides, CMS021 was found to have statistically significant difference from the Dt-y group and L-2 group CMS012 was found to be able to inhibit NK cell cytotoxic activity having statistically significant difference from the saline group Data detailed in Table 6 below.

Table 6 Group N X±.SD CMS01 10 85±10* CMS003 10 85±6* CMS012 .9 40±9* CMS015 8 78*8* CMS021 8 88t12*@^ CMSO26 10 76±9' 10 72£9* IFN-y 10 73±10' 1l-2 10 74±8* Saline 10 56±8 comparinpg to saline group P0.05 comparing to IFN-y group P<0.05 A comparing to IL-2 group P<0.05 At 5pg/kg/day, CMS008, CMS009, CMS01O, CMS011, CMS012, CMS020, CMS024, CMSO34, and CMSO36 were found to be able to increase NK cell cytotoxic activity, having statistically significant difference from the -saline group (p0.05). Among these peptides, CMSOOS and CMSOO9 were found to have 17 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:31PM FROM-A J PARK ton +64 4 4723358 T-714 P.022/080 F-741 statistically significant difference from the IN-y group and IL-2 group (P<0.05) as shown in Table 7.

Table 7 Group N XISD%) CMSU08 10 92±4*I

A

C CMSOO9 8 92,6*D^ -10 82±9* CMS011 10 76±10* CMS012 10 85±7* CMSOZO 9 91±6' 00 CMS024 9 78±3* CMS034 8 90±5* CMS036 10 75±9* C IFN-y 10 80±8* IL-2 10 80I8' Saline 10 60±9 o comparing to saline group P<0.05 C 5 comparing to IFN-y group P<0.05' A compang to L-2 group P<0.05 At 0.5gg/kg/day, CMS002, CMSOII, CMhS012 CMSO22, CMSO28, and were found to be able to increase NK cell cytotoxic activity, having statistically significant difference from the saline group CMS008 was found to be able to inhibit NK cell cytotoxio activity, having statistical significant difference from the saline group Data detailed in Table 8 below.

Table 8 Group N XSD CMS002 8 76±9 CMSOO8 10 46*12*- CMSO11 9 79+3* CMS012 9 77±6* CMSO22 10 '73±11* CMSO28 8 79*3* CMS035 10 76±10* IFN-y 10 72+-9* IL-2 10 74±10* Saline 11 58±7 comparing to saline group P<0.05 3. The effect of peptides on the activity of T lymphocyte in secreting IL-2 At O5pg/kg/day, C(S007, CMS009, CMSO10, and CMS015 were found to be able to promote IL-2 secretion from T lymphocyte, having statistically significant difference from the saline group Among these peptides, CMS007 and CMS015 were found to have statistically significantly difference from the IL-2 group And, CMSOO9 and CMSO1O were also found to have statistically significant difference from both the IFN-y group and IL-2 group (P<0.05) as shown inrf Table 9.

18 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:31PM FROM-A J PARK Kton +64 4 4723358 T-714 P.023/080 F-741 N Table 9 l) Grop N XSD MIU1 CMSQ07 9 86115*A CMS009 10 11413* @A CMS010 .9 125±17 @A 9 85±17*^- IFN-y 10 100±18* IL-2 10 70±13* Saline 10 39±10 comparing to saline group P<0.05 comparing to IFN-y group 00 A comparing to IL-2 group P<0.05 Cl At 50g/kg/day, CMS001 and CMS003 were found to be able to promote activity of T lymphocyte in. secreting IL-2, having statistically significant difference from the saline group Among these peptides, CMS003 was O found to have statistically significant difference from the. IL-2 group (P<0.05) as shown in Table Table Group N X±SD CMS001 10 60±10* CMS003 8 86±9*A IFN-y 9. 99116* IL-2 10 72i112* Saline 10 39±10 comparing to saline group P<0.05 A comparing to L-2 group P<0-05 At 5pg/kg/day, CMSOO7, CMS012, and CMS020 were found to be able to promote T lymphocyte in secreting IL-2, having statistically significant difference from the saline group (F<0.05) as shown in Table 1I.

Table 11 Group N X±SD (IU) CMS0007 8 64±12* CMS012 9 65±16* CMS020 8 63±11* IhN-y 10 96±14* IL-2 10 77±13* Saline 10 .37±9 comparing to saline group P<0.05 At 0.5jg/kg/day, CMS10, CMS011, CMS012, CMS022; and CMSO34 were found to be able to promote T lymphocyte in secreting il-2, having statistically significant difference from the saline group Among these 19 JtAUPo94-,I'PECAPcTsPEC COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:S1PM FROM-A J1 PARK gton +64 4 4723356 T-714 P.024/080 F-741 peptides, CMSQ34 was found to have& statistically sigoificatly difference fromn the 1-_2 group (PcO.05). And, (DM011 and CDMS022 .were also found to have ;Z statistically signtificant difference froma both the WN-y group and IL-2 group <1 (PCQo:5) as, shown in Table 12.

TaleI12 Grup N X±SD (l CMSO)1O 9 6ft11I QA$011 10 i01±9+0^ 00 CMS012 S 59±13" CMS022 9 109l14*@~A CDMS034 10 5U*1 WFN-y 10 7 o 11- 10 73*13* 0Saline 10 38±13 omatg.-to-Slie gro PO00 coinparing to IPN-y group A compazing to IL-2 group NO 4. The e ffe'f of peptides on the activity of T lymphocyte in secreting IFN At so)(jigfkgday, (DM8010, CMvSO13, and CDMS016 wer found to be able to promote T lymphocyte in secreting interferon PMEN, having statistically significant diffrence from the saline group. IFN-y group, and M1-2 group QP<0.05) as shown inlTable 1 3 Table 13 Group N (DM8010 9 6±3A (DM8013 9 154t15*@A (DM5016' 6 162*19*9" IFN-y 10 139±16* 11r2 10 120±13' Saline 10 65±11 %omparhig to saline group NO0.05 comparinlg to IFN-y group -A comparing to IL-2 group P<0.05 At 5OpLSgl~gday, CMSOQ1, (DM5003, and CDM8021 wer found to be-able to promote T lymaphocyte in secreting OFN having statistically significant difference from the saline group Among these peptides, (DMS021 was found to hav&- statistically signilicant difference from the IFN--y group and M1-2 group (J'C0.05) as shown in Table 14.

COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:32PM FROM-A J PARK gton +64 4 4723358 T-714 P.025/080 F-741 0 0 N Table 14 coup N X±SDM CMS001 10 110±15* CMS003 8. 106±16* CMSO21 8 14317*@ pIFN-y 9 125±18* D-2 1To0 113±17* Saline '10 61±+11 comparing to saline group P<0.05 00 comparing to IFN-y group P<0.05 A compaing to IL-2 group P<0.05 At 5pg/kgday, CMSoo9 ond CM5012 were found to be able to promote T o lymphocyte in secreting IYN having statistically significant difference from the Ssaline group, Ang these peptides, CSOO9 was found to have statistically significant difference from the IL-2 group (NO<.05) as shown in Table Table Group N XS CMS009 10 121±15*A CMS012 9 86±9* U,-2 9 105114* Saline 10 66110 comparing to saline group P<0.05 A comparing to I-2 group P<0.05 At 0.5Lgfkg/day, CMSO1O, CMSOI1, CMS022, and CMSO2 were found to be able to promote T lymphocyte in secreting IFN having statistically ignificant difference from the saline group P4?.05). Among these peptides, CMSOIO and 0M8022 were found to have statistically significant difference from the JFNi group and IL-2 group (P<0.05) as shown in Table 16.

Table 16 Group N

XSD

CbASOIO 9 1 4 2 1 S*w CMS011 10 891BH' CMS022 9 145±13* CMSO28 10 96±13* IFN-y 10 124±16* I-2 10 107t13* Saline 10 64±13 *companng to saline group P.0 comparing to IFN-y group NO0.05 A comparing to IL-2 groupP<0.05 5. The effect of peptides on antibodyformation 21 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:32PM FROM-A J PARK gton +64 4 4T23358 T-714 P.026/080 F-l41 At 500Lg/kgday, CMSOO2, CMS003, CMS007, CMvSOOS, CMS009, Ol) CMSOIO, CMSO1I, CMSO12, 0M8013, CMSO14, CMS015, CMS016, CMSOIB, Z CMSo CMSo2O, C18022, CMSO23, CMSO24, CMS029, MS033, and C0S035 were found to be able to promote anti-SRBC aoiibody foumation, having' statistically. sigxdficant cifferenbe from the saline group Among these peptides, CMSOO2, GMSOOS, CMSOO 7, CMS008, CMSO13, CMS019, CMS024, and CMS035 we=- found to have statistically significant difrence from the WN-y group An4 CMS009, CMSO10, CMS011, C1S012, CM014, CMS016,CMS020, CMS023, (DM8029, and CMS033 were also found to have statistically significant difference from both the IFN--y group and IL-2 group (P<0.05) as shown in Table 17.

Table 17 ClGroup N X±SD imt) CMS002 10 8718* 0CM8003 10 96±18'"' CMS007 10 69-17*0 (DMSOO 10 8 215*0 (DM5009 10 11322 *@A CMS010 10 112±30*®A (MSOII 8 i88t16'@" CMS012 8 14 1 2

L*(RA

CMS013 10 80±16*0 CMS8014 10) 13 *@A CMS015 10 136*22@A (MS016 8 14338*19A CMS018 10 66-*16* CMS019 10 91!26*0 CDM5020 6 15535*"& CMS022 8 68:31* (DMS023 9 110±45-@A CMS024. 8 75±29-8 CMS029 8. 115±22*@^ CMS033 10 14327*@A (DM8035 10 88±16*0 IFN-1 9 37±10 1-2 10 71±11P Saline 10 32*7 Comparing to 5line groulp F companag to WN-y group P<0.03 A comparing to IL-2 group At 50pg/g/day, (SO03, CMSOII, CMSOI2, (DM8013, (DM8015, CMS021, (MS022 1 CM023. CM8026, CMS027, (DM8029, CMS030, CMS032, CMS033, CMS034, CMS035,.and CDMS036 Were found to be able to promote anti- SRBC antibody formation, having statistically signifcant difference from the saline group (hO.05). Among these peptides, (MSOL1, CMS013, and CDMS015 were found to have statistically siguificaft difference from the IFNy group And, CMS021, CMS022, CMS023, (DM8026, CMS027, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, and CMS036 were also found to have statistically significant difference from both the IFN-y group and IL-2 group (M8009 was found to be able to inhibit anti-SRBI antibody 22 COMS ID No: ARCS-i58250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:32PM FROM-A J PARK ffton +64 4 4723358 T-714 P.027/080 F-741 fonntion, having statstical significant difference from the saline group (P<0105).

Oil) Data detaied in Table 18 below.

Table 18 ClGroup N X±SD (Unt CMS003 10 .52±11* CMS0O9 8 13±5* CMvS0lI 9 6±* CMOI2 8 S0±l4 t 00 C~MS013 __01 10 54t9*@~ Cl MS021 9 94±2*@A CMASOZ2 9 110±-16,10A oCMS023 8 84±11*@A~ ClCMSOZ.6 9 98±9*@A- CMS027 9 93±11 I*@A CMS029 10 i43±13 *@A CMS030 10 141t33*@A~ CMSO32 -9 131±24*@A CMS1033 8S1±5@ CMSO34 10 136±1 1 *8 0MS035 8 97±10-O^ CMS036 10 ll8±1le8A IFN-Y 9 37±10 IL-2 10 71±1 1 Saline 10 32±7 comparing to salijie group P<0.05 comparing toWICN-y group P<0.05 Acomparing to ZL-2 group PCQ .0 At 54g/kgday, GMSOOI, CM8003, CMSOO7, CMSOOS. a4S0O09, CMSO1I, CMSO12, CMSO13, 0M3015, CMSO1G, CMSOI9, GM8020, 0MS021, CMSOZ3. CM3024, 0M3026: CMSOZ7, CMSO2S, CMS029, 0M8030, CMSOS2, CMSO33, CM5034, CM5035, and CMlVS036 were found to be able to promote anti- MRC antibody formation, having statistically significaint difference from the saline group (Pc0.05). Among these peptides, CMSOO3, CMSOOS, CMSOO9, CMSOI2, CMSO15, 0M3016, CMSO2O, and CMSO2l wer found to have statistically significant differece from the WFN-y group And, CMSOQ1, CMSOO7, 0MS011, CbdS0l9, 0MS023, CMSOZ4, CMSO26, CMSO27, CMSOZS, 0MS029, CMSO3O, CM5032, CMSO3S, 0MS034, CMSOJG, and CM8036 wer also found to have statistically significant difference from both the IEN-y group and IL-2 group (P<0.05) as shown in Table 19.

23 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:33PM FROM-A J PARK gton +64 4 4723358 T-714 P.028/080 F-741 CMS4F319lO±24* CM00O3 9 1±24*@ ClCMS007 9 122±12*@A CMSOOS 9 9-7±26*@ 0MS009 .81 79±18S@ CMSOIl 10 127@ ONCMSOI2 10 81±272*@ ONCMS'013 10 9±2* 00 MS015 8 94±317*@O CMSO16 9 93±32*@ CC MS019 10 1J2*4 0M8020 10 89±24*11 0CMS021 9 82nos@ oCM$023 10 166±27*@A CM02 7 171t-39*@A CM8026* 9 191+17*@OA 0MS027. 9 117±45*OA CMSO2S 10 121I±48*'§tx CMSO29 9 147±3OA CMS03O 9 158±37'4" CMS032 9 l57t37*"' CMS033 7 28t39*s/\ Q MSO34 .8 172tE37*-A CMSg3 176t 391A- ]FN-y 9 37±10O JL-2 10 71±11* Salne 10 32±7 *comparing to saline group pC0.0)5 @comparing to ThN-y group A Companing to Ul.-2 grou O .0 At o-5jgi&g/day. CMSO2I, 0MS023, CMS024, 0MS027, and CMS033 were found to be able to promote anti-SRBC antibody fonnatio-a, having statistically significant difference from the saline grOUP Among these peptides, CMSO21 and CM3033 wete found to have statzsically significant difference from the ThN-y group And, CMS023, qMSO24, and OMS 027 were also found to have statistically -significant difference from both the IFN-y group and -2 group Also, CMSOO2, CMSOO3, CMSOO9, (DM5010, CMSOII, (DM8013, (DM3014, CMSO1S, CMSOLS, CMv.1019, CMSOZOI (DM5026, (DM5028, (DM3029, (DM5030, (DM5034, and CMS036 were found to be able to inhibit anti-SRBC antibody formation, having statistically sioificant difference from the saline group Data detailed in Table 20 below.

24 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:33PM FROM-A J PARK gton +64 4 4723358 T-714 P.029/080 F-741 Table Group N. snnSD(it) CMS02 9 4±1" cMS03 9 2±1* CMSOO9 9 2±1* 10 10±3* CMSO11 '10 5±3* CMS013 10 7±1* CMS014 10 15±6* 00 CMS015 9 13±4* CMS018 9 3±1* CMSO19 9 12t3* CMS020 9 10±3* 0MS021 9 579*@ CM3023 10 108t21*A (N CMS024 10 98±6*§A CMS026 10 19±6* CMS027 10 99±14*@- CMS028 10 18±-5* CMS029 .9 18±7* CMS0SO 9 l9±74 CMS33 9 78±12*@ CMS034 10 20±2* CMSO36 9 20±6* IFN-y 9 37*10 IL-2 10 71±11* Saline- 10 32±77 comparing to saline group P comparing to ]FN-y group A compaing to n-2 group 6. The effect ofpeptides on the phagpoytotic activity of onongeler phagocyte At 500ptg/lcgday, CMS003, GMSOOB, CMS020, CMS02S, and CMS024 were found to be able to enhance the phagocytotic actvitY of mononuclear phagocyte, having statistically signifcant difference from the saline group Among these peptides, CMS022 was found to have statistically: significant difference from the IFN-y group and IL-2 group (FO.05) as shown in Table 21.

COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:33PM FROM-A J PARK

;Z

gton +64 4 4723358 T-714 P.030/080 F-741 Table 21 Gup X±S (h-SO ~Msoo3 10 6.6±0.7* CMS0OS 10 6.5±1l.2* CMS020 10 6.4t0.6* CMS022 10 7.4±O.6*@A CMSO24 10: 6.4±1.0* IFN- 10 6.4±0.9* IL 9 5.7±0.8 Saline .10 5.1±0.6 comparing to saline group p<005 comparing to FN-y group P<0.05 A comparing to II-2 group FC0.05 At 50pgkg/day, CMSO19, CMS024, and CMS030 were found to be able to enhance the phagocytotic activity of mononuclear phaocyte, having statistically signiflcanf difference from the saline group Among these peptides, CMSOI9 was found to have statistically signifcant difference from the M-2 group (P<0.05) as shown in Table 22.

Group N vX±SD (hago-ttic index) GMSO19 9 6.7±0.9" CMS024 8 6.6±0.7* CMS030 10 6.3±0.5* Wkiy 10 6.4±0.9* IL-2 9 5.740.8 Saline 10 5.110.6 *comparing to saline group A compaing to IL2 group P4.05 At 5p/kg/day, CMS03 CMS008, (2009, CMSO10, CMSO11, CMS013,, C3016, CMSOl8, CMS019, and CMS035 were found to be able to enhance the phagocytotic activity of monuclear phagcyte, having statistically significant difference from the saline group Among these peptides, CMS003, CMS009, CMSOiO, C24016, (4S019. and CMSO35 were found to have statistically sjgnificant difference from the 2-2 group (PO.05) as showu in Table 23.

COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:33PM FRO-A J PARK ffton 64 4 4T23358 T-714 P.031/080 F-741 Table 23 Gro N XSDpb&acytotic nd ;MS003 9 6.9±09" CMSOOS 9 6.4i0.5* CMS009 9 Cl CMSOlO 10 7. lt.7*A OMSO11 10 6.4±1.1* CMS013 -10 6.7±0.2* CMS016 9 6.9±0.8*A 00 CVAS018 8 6.7±l.2* CMSO19 -8 6.StO.6*A CMS035 9 6.9t0.9*A 6.44*0.9* 9 5.7±0.8 Saline -10 5.1±0.6 C companng to saline group <O-.05 A comparing to 1L-2 group At .Spg/kg/day, CMS024, 0MS027, and C2S036 were found to be able to enhance the phagocytotic activity of mononuclear phagocyte, having statistically significant difference from the saline group (WC.05). Among these peptides, CMS024 was found to have statistically significant difference from the JL-2 group 0(N.05) as shown in Table 24.

Table 24 Group N c)"c index 0MS024 10 6.7.i. CMS027 10 6.4±0.6* CMS036 9 62-±0.3* IFN-y 10 6.4±0.9* IL-2 9 5.70.8 Saline 10 5.1±0.6 comparing to saline group P<0.05 A comparing toIU-2 group 7. The effect of peptides on the weight f immune organ At 500g/g/day, tMSOOS, CMSOIO, CMS016, CMS019, CMSO22. CMS026, CMS027, GMS028, CM3029, CMS30, CMS0032, (MS033, CMS034, CMS035, and CMSO36 were found to be able to incraee the weight of thymus gland, having statistically significant difference from the saline group (P0,05). Among these peptides,.CMS027 and CMS034. were found to have statistically significant difference from the IL-2 group And, ,1008, CMS022, CMS029, CMSO3. CMSO32, CMS033, and CMSO35 were also found to have statistically significant difference from both the IPN-y group and M1-2 group (P<0.05) as shown in Table 27 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:34PM FROM-A J PARK Vton +64 4 4T23358 T-714 P.032/080 F-741 Table ~h~p N X±SD (0/0, CM08 8 0.21±0.03"A CMS010 10 0,19±0.04* CM8016 9 0.18±0.05* CMS0l9 10 0.19±0.02* 10 0.19±0.04* CMS022 -9 0.26±0.05* 0MS026 10 0.20±0.03* 0MS027 8 0.20_0.03*A CMS028 10 0,19±0.02* 00 CM8029 10 022.04* CMS030 8 0.30,±0.03*@A CMSO32 8 0.25±0.03@ CMS033 9 0.25±0.04A cMS034 9 oiO±+0.05*A CMS03S 10 0.21±0.03*A CMS036 9 0.1 8±0.02* CI IP7N-y 10 0.15±0.04 IL-2 9 0.14±0.03 Saline 9 0,12±0.02 comparing to saline gTop comparing to IFN-y group A comparing to t-2 group P4.05 At 500 Spg//day, CMS019 was found to be able to increase the weight of spleen, vvith statistically significant difference from the saline control groups (P<0.05) as shown in Table 26.. CMSOO1, CMS003, CMS007, aS009, CMSO11, CMSO13, CMS014, CMSO1S, CMS021, CMS023, CMSO24, CMS027, and 4MS036 were fotud to be able to decrease the weight of spleen, with stahstically .igoiflcaiit difference from the saline control group (PCO.05). flata detailed in Table 26 below.

Table 26 Grou N X±+SD CilVS001 10 0.430-O7* CMS003 8 0.40±0.04* cMS007 9 0.32±0.05* CMI009 9 0.41±0.03* CMSO1I 9 0-41±0.04* CMS013 10 0.44±0.07* CMSI4 10 0.400.03* CMS015 9 0.36±0.07* GMOSO9 9. 0.63+0.08* CMSO21 9 0.36±O-0.04*.

CMS023 9 0,36±0.06* CMS024 9 0.34±0.05* CMS027 10 0.37±0.03* CMS036 10 0.40±0.03* Saline 10 0.53±0.05 comparing to saline group P<0.05 28 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T1 03:34PM FROM-A J PARK ffton +64 4 423358 T-714 P.033/080 F-741 At 5OplgSday, CM8002, CMSOO8, CMS012, CMSO14, CMS01O, CMS018, CMOJ9, CMSO2O, CMS022, CMS023, 243024, CMS026, CMSO27, ZCMS028, CM3029. CMSO30, 0MS032, CM5033, CMSO34, and CIS036 were found to be able to increase the weight of thymus gland, having statistically significant differene from the saline group (P0.05). Among these peptides, CMS034 was found to have statistically signifiant difference from the IL-2 group And, CMSOO2, CMS00O, CMSOI2 CMSO14, CMSOI6, CMSO1, CMSOI9, CMS020, CMSO22, CMSO23, CM024, CMSO26. CMSO27, CMS030, (245032, and CMS036 were also found to have statistically sigificant difference 00 10 from both the WN--y group and IL-2 group (P4X 05) as shown in Table 27.

Table 27 GroUP N

X

CMS002 10 .2I±.02 CMSOOB 10 0.2O±0.04*@^n CMS012 10 .26+±O.02*@A CMS014 10 0.210.02*@t (N CMS016 10 0.iO±0.03*@A GMS0IS 10 0.23±.02*@A CMS019 10 0.20±O03*tA Cr48020 10 0.27±O.03*@^ CMS022 10 0.30t0.03*@A CM5023 10 0.20±0.02*@A C108024 10 0.270.02*l@^% CM8026 10 0.27+0.02*§A CMS027 8 021±0.03*@A CMS028 10 0.18±0.04* CMS029. 9 0 18±0.05* Cr4S030 10 0.250.04*@A CMS032 10 0.27±0.03-4@ CMS033 9 0.18±0.03* Cr4034 8 0.19±0.04*A CMSO36 9 0.22±.02-" WFN- 10 0.15±.04 11-2 9 0.14+-0.04 Saline 9 0.12±0.02 *companngto sabine group c comparing to ]FN-y group A comparing to'iL-2 group P<0.05 At i0 1 gqglday, CMS00S, CMSOIO, end CMS029 were found to be able to decrease the weight of spleen, with statistically significant difference from the saline control group Data detailed in Table 28 below.

Table 28 Group N X±D26) CMS008 10 0.390.08* GMS01O 10 0.38±0.05* CMS029 10 0.4210.04* WFN-y 10 0.50±0.04 M1-2 9 0.62±0.07 Saline 9 0.53±0.05 comparing to saline group 29 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T1 03:34PM FROM-A J PARK Vton 64 4 4723358 T-T14 P-034/080 F-41 At Spgtkg/day, MS001, CM3002, CMIS01O, CMSOI1, CMS012, CMS013, CMS014, CMSOlS, CMS016, CMSOlB, cMS0lP, CMSO2O, MS021, CMSO2Z, CMS023, CMS024, 2MS026, CMS2W CMS029, CMS030, CM8032, CMSO33, CMS034, and CM5036 were found to be able to increane'tht weight of thymus gland, having statistically significant difference from the saline group Among these peptides, CMSOO2, CMSO14, CMSO24, and CMS030 were fund to have statistically signifcant difference from the 11-2 .kroup g<0.05). And, CMSOIO, CMS012, CMS0lS, CM8019, GMSO2O, CMS022 1 CMSO26, CMSO2S CM8032, 0MS034, and 0MS036 were also found to have 00 10 statistically significant difference from both the WFN-y group and IL-2 group (P'0.05) as shown in Table 29.

Table 29 Grou N

XNSD

0 00MSO1 10 0.22±0.051 CMS002 9 0.24±0.0*A CNMOI 0 9 0. CMSO11 10 0.21±0.06* MS012 10 0110.06*@A C S013 10 0.21±0.05* CMSO14 10 0.23t0.6td CMS015 9 0.20*0.08* CMS016 10 0.22±0.06* CMSO1B 10 0.24±0.4*§A CMS019 10 0.24±.02*0A CMSOZO 10 0.241t0.07@A CMS021 9 0.20tO.06* CM3022. 9 0.25±0.04*@^ CMSO23 10 0.2340.06* CMS024 9 0.23t0.06*A CMS026 10 0.310.05 *O CZMS2S 10 o.28±0.06*@4 CMS029 10 0.21±0.03* GMS030 10 013t07*A CMS032 10 0.29tO.04OA'In CMS033 10 0.20*0.02* CMS034 9 0.270.06s@^ CMS035 10 0.21±0.04" CMS036 10 0.25±-0.04*/ IFN-y 10 0.15;0,04 IL-2 9 0.14±0.04 Saline 9 0.12±0.02 comp gtos sp<0.0F comparing to IF-y group P<0.05 conpainng to 11-2 groupPO.S At 5]g/kg/day, CMS0O3 was found to be able to increase the weight of spleen, having statistically significant difference from the saline group CMSO1S was found to be able to decrease the weight of spleen, having statistically significant difference from the saline group (NO.05). Data detailed.in Table below.- COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:35PM FROM-A J PARK Ston +64 4 4723358 T-714 P.035/080 F-741 N Table 9 0.380.15* 10 0.64±0o.09* C Saline 10 0.53+0.05 comparing to saline group F<0.05 At 0.5tg/kcglday, CMS002, CMSOS, CMS010, CMS012, CMS014, CMSO18, CMS020, CMS022, CMS026, CMSO28, CMS030, and CMS032 were 00 5 found to be able to .increase the weight of the thymus gland, having statistically significant difference from the saline group Among these peptides, CMS008 and CMS012 were found to have statistically significant difference from Sthe IL-2 group And, CMS002, CM1020, and CMSO30 were also found to have statistically significant difference from both the IFN-7 group and I1-2 o 10 group as shown in Table 31.

Cl Table 31 Group N X M002 8 0.260.06*^ CMS008 10 0.22±0.07*^ CMS010 9 0.21±0.03* CMS012 10 0.22±0.06*t CMS014 10 0.20±0.04* CMS018 10 0.20±0.03* 9 0.23±0.05*

A

CMSO22 10 0.1±0.06* CMS026 9 0.211±0.05* CMSO28 10 0.20±0.06* CMS030 9 0.24±Q.05*@A CMS032 10 0.21±0.06* IFN- 10- 0.150.04 I-2 9 0.14±0.04 Saline 9 0.12±0.02 comparing to saline group comparing to IFN-y group P<0.05 A comparing to ITL-2 At 0.5gkg/day, CMSO20 was found to be able to increase the weight of spleen, having statistically significant difference from the saline group, IFN-y group, and IL-2 group CMS001 was found to be able to decrease the weigh of spleen, having statistically significant difference from the saline group Data detailed in Table 32 below.

31 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:35PM FROM-A J PARK Kton +64 4 4T23358 T-714 P-036/080 F-741 (N Table 32 to Group N X+SD (%y CMS001 10 0.40±0.05* CMS020 8 0.68+0.09*"^ 'Saline 10 0.53±0.05' SIFN-y 10 0.50±0.04 IL-2 10 0.62+0.07 comparing to saline group P<0.05 comparing to-IFN-y group P<0.05 00 A comparing to L-2 group P<0.05 c In summary, we found that peptides CMS001, CMS002, CMS003, CMSDO7, CMS008, CMS009, CMSO10, CMS011, CMS012, CMS013, CMS014, O CMS015, CMS016, CMS018, CMS019, CMS020, CMS021, CMS022, CMS023, C CMS024, CMS026, CMS027, CMS028, CMS029, CMS0 CMSCMS032, CMS033, CMS034, CMS035, and CMS036 have in vivo biological activities in the animal model tested.

I. THE IN VVO ANTIVRAL EFFECT OF PEPTIDES In order to find out whether these peptides have possible therapeutic effect on viral infections, we used the animal model duck hepatitis B infection -in this study to test out the in vivo effects of the above peptides on diseased animals.

Chongqing duck hepatitis B model was set up and treated with peptides by intraperitoneal injection (50pg/kg/day, once per day) for 4 weeks. The serum level of DHBV DNA was analyzed by serum dot-blot hybridization. Lamivudine and normal saline treatment was used as positive and negative control respectively.

The peptide CMS001 was found to be able to reduce the serumlevel of DHBV DNA at the 4th week of the treatment, having statistically significant difference from the saline control group It is concluded that CMS001 at suitable dosage level can be used as a part, or on its own, for viral hepatitis infection management.

Materials and methods 1. Peptides were custom synthesized by American Peptide Company, Inc., USA, from L-amino acids.

2, Animal model I] One-day old Chongqing ducks were inoculated with 0.lml stock serum positive of Duck Hepatitis B Virus (DHIBV) DNA (5X i0 copy/ml) by intraperitoneal injection. One week later, blood samples were collected from the external jugular vein arid infection was confinmed by dot-blot hybridization with DEBV DNA probe labeled with digoxin r. The ducks were breed to 2 weeks old for entrance into the study.

32 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-~007 03:35PM FROM-A J PARK ffton +64 4 4T23358 T-714 P.037/080 F-741

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0 0 S3. Grouping and treatment DHBV infected ducks were randomized into the following groups: a) Negative control group Normal saline was given 1ml per duck 'N 5 k by intraperitoneal injection, once per day.

ONb) Lamivudine group As positive control group. Lamivudine [41 was given 50mg/kg/day by oral 00 administration, once per day.

c)Peptide group 50tg/kg/day peptide (adjusted to final Svavouime of 0.5 to iml with normal saline) o was given once per day by 0 intraperitoneal injection.

Treatmrent lasted for 4 weeks and observation continued for another one week after termination of treatmeht 1 ml blood samples were drawn from the external jugular vein of the ducks on days 0, 7, 14, 21, 28, and 35 when treatment started.

Sera of the blood samples were isolated immediately P1 and stored at -20C until analysis.

4. Serna DIBV DNA level determinations DIHBV DNA probe was fluorescent labeled according to the labeling kit protocol from the kit manufacturer (Amersham Pharmacia Biotech duck sera were dot-blotted (1 duplicated dot per sample) onto nitro-cellulose membrane and hybridized with flnorescent-labeled DHBV DNA probe for quantitation After completion ofhybridation, the blots were developed in.CDP- Star fluorimetry reagent RPN3690 and scanned with Vaego Scan (Brisa-620st) scanner ImageMaster TotaILab vl.ll.Ink software was used.for quantitative analysis of the blott. Statistical analysis.was carried out accbrding to the pair t-test with SPSS software.

Results Table .1 Seram DHBV DNA titer before and after treatment DHBV DNA level (Mean Standard Deviation, l0'units) DDay 0 7 Day 14 Day 21 Day 28 Day Normal 26±12 45±31 49±23 102±66 60±3.8 50±43 saline Lamivudine 21±9 6±4* 7±6* 8±7* 8+5* 20±19 CMS001 21±18. 11113 20±18 14±14 5±3* 18±16 Pair t-test, comparing with day 0 of the same animals: *P<0.05 The negative (nonal saline) and positive (lamivudine) control group 33 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T1 03:36PM FROMPA J PARK ffton +64 4 4T23358 T-714 P.038/080 F-741

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proved successful establi sment of the hepatitis animal model. At the peptide CMS001 was found to be able to decrease the serum DHBV DNA titre Safter 4 weeks of treatment having statistically significant difference (p<0.05) from the before treatment value of the same animals. Upon termination of treatment, the serum DHBV DNA titre rebouced to a value with no statistical difference from S-that before treatment, showing that the effect of peptide CMS001 may be reversible and/or need longer treatment period for eradication of the virus.

Discussion Duck hepatitis animal model 0 is an established experimental model for human hepatitis B pathogenesis studies and for the screening of hepatitis B C therapeutic agents. In this study, CMS001 was found to be able to decrease the o serum titre of DIBV DNA after 4 weeks of treatment, indicating that the peptide o CMS001 at suitable dosage level and with suitable application scheme, can be C 15 useful on its own or in combination with other substances as an agent for human hepatits B management.

In the present study, administration by intraperitoneal injection was tested, but this does not exclude the possible effectiveness of the peptide if administered via other alternative routes. The peptides may also be administered via intravenous injection, intramuscular injection, subcutaneous injection, and subcutaneous implantation, with.or, without delivery facilitating device such as liposome, sustain release protection etc. The peptide may also be administeed in any form of oral administration like tablet, capsule, suspension, solution etc, in the usual form without modification or in slow release form, or with or without gastroenteric protection. The peptide may further be applied in any form of topic application like ointment, cream, gel etc with or without transdernal facilitating device, or as inhalant of powder, dissolved, or as liposome protected form. The peptide may also be interpreted into its genetic sequence and cloned into -an expression system, on its own or in combination with other peptide sequences, to generate a resulting peptide molecule to make use of the activity of the peptide as described in this report, with or without purification of the resulting peptide.

Table 11.2 Peptides Effective for Hepatitis B CMScode SEQID No.

CMS001 1 References 1. Chen Yaxi, Guo shuhua, Zhang Dingfeng, et al. Foundation and application of Chongqing duck hepatitis B model. Chinese Journal of Hepatology. 1993; 89-91 2. Chen Yaxi, Guo shuhua, Chen Xuehua. Preparation and application of DHBV DNA probe labeled with digoxin. Journal of Chongqing University of Medical 34 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:3EPM FROM-A J1 PARK ston +64 4 4723358 T-714 P.039/080 F-741 0 0 Sciences. 1994; 19(4): 295-297 3. Tang Ni, Huang Ailong, Gue shuhus, et al SystefO fUndationd application of seroloical parameters of humoral to k hepatiti8 virus. Chinese Journmal of Hepatology. 2001;9(1): 13-15 Ci 5 4. Che Yaxi, Quo shuu, Qi Zhenyuda et '4i experimental study of lamivudixi against duck hepatitis B virus in combinatLO with famciclOviL Chinese Journal of Hepatology. 2001; 209-211 00 EFFE FPFIDES ON PITIS In order to find out whether these peptides have possible therapeutic effect on nephritis, we used the animal model rat Masugi nephritis in this study to test out o~ the in vivo effects of the above peptides on diseased animals 4] The objective of this study is to investigate the in vivo therapeutic effect of peptides on chicuic glomenilonephritis. Masugi nephritis rat model was catucted by injecting rabbit anti-rat-renal-cortex IgO into heathy Sprague Dawley rats, and then treated immediately with intrapertoaeal injection of peptides at 50gkg/day, once per day for 3 weeks. Hydrocotisoue was used as positive control. It was found that proteinurca, serum orcatiine level, and splexic index of the rats treated with CMS014, CMS018, CMS30, and CMS036 wre lowered compared with that of the control group, with statistical, signifcance Postnoram microscopic examination of the kidney showed that the therapeutic effect of these ptptides was similar to hydocotisone treatment It is concluded that CMS014, -CMSOI8, CMSO30, and CMSO36 may be used as a means for chronic nephritis management.

Materials Sprague Dawley (SD) rats, male, weighing 120dz20g, were from Center of Experinental Animal, Guanghou -University of Traditional Chinese Medicine, and also from First Military Medical Unirsity. Ciabilll rabbits weighing 3g were from Center of Experimental Animal, Guangzho University of Traditional Chinese Medicine.

Peptides, of L-amino acids orin were custom synthesized by American Peptide Company, Inc, USA, and were diluted to l1pg/wl in sterile normal saline.

Hydrocortisone was from Yangzhou Pharmnnaceutical Factory, China.

Serum reatinine level determination kit from Shanghai Rongsheng Biological Technique Company, PR China.

Methods 1- Preparation ofrabbit anti-rat-renal-cortex anti-serum N 20 healthy SD rats were anesthetized with intravenous injection of 3% sodium pentobarbital, COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:36PM FROO-A J PARK ton +64 4 4T23358 T-T14 P.040/080 F-741

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The aorta abdominalis was exposed and the kidneys were perfused with normal saline until clean of blood. The renal cortex was isolated, homogenized in 5 volumes of 0.01M Tis.-HCI buffer, pH 8.1, and filtered through a 140-gauge tsialcss steel mesh. The filtrate was collected and mixed with eitherGIBCO.R

E

complete or incomplete Freunds adjuvant in 1:1 and emulsified to obtain the C working immunization solution.

ON 10 healthy rabbits were immunized with the immunization solutions, first 00 1 time with complete Freunds adjuvant and subsequently with the incomplete one.

The antigen was injected hypodermally through 6 random points, 0.1 ml per point, once per 10 days. From 6" week onwards, blood was collected from the auricular CN vein and the antibody titer was determined by the double diffusion method On the 8h week after starting of immunization, the rabbits were anesthetized with O intravenous injection of 3% sodium pentobarbital, 20mg/kg, and.blood was 0 15 collected from the carotid artery. The anti-serum was then ended and isolateA.

Whole blood was collected from 15 healthy SD rats. The red blood cell was isolated and washed clean thrice with normal saline. The clean red blood cell was sLed with 250 ml rabbit anti-sorum and incubated at 1 0 C overight. After incubation, the blood cell was removed by ceatrifugation and the supernatent was further inactivated at 56°C for 30 mintes, Aftr centrifugation to remove any precipitate, crude IgG was isolated and partially purified from the supernatent by precipitation with (NfH)2S04 taiee 33%, ad then 33%) L 1 The arido IgG was redissolved in 125ml double'distilled water and dialysed clean of ammonium sulphate. The anti-rat-renal-cortex antibody titer was determined by the double diffusion method 1.

2. Induction ofrat glomerulonephritis 0.25ml priming solution (containing about 4mg non-specific rabbit IgG with complete Freumds adjuvant) was injected intraperitoneally into healthy SD rats to prime the rats five days before induction.

With the exception of the normal healthy control group that received normal saline, all groups were subsequently induced,by intraperitoneal injection of Iml rabbit anti-rat-renal-cortex IgG prepared as described in section Method 1.

3. Grouping and administration: male SD rats were randomized into groups of normal healthy control (nornal), nephritis placebo treatment control (placebo), nephritis peptide treatment, and the nephritis hydrocortisone treatment control (hydrocortisone), 12 per group.. Treatment started on the day of induction.

Peptides 50Ag/tkgday, hydrocortisone 3.3mg/kg/day, and normal saline was used as placebo, all administered intraperitoneally once per day and lasted for 3 weeks.

4. Parameters monitored 1 41 urine protein level: each rat was kept individually in one cage.

Adequate drinking water was supplied. Urine was collected once COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:37PM FROMA J PARK ton +64 4 4723358 T-714 P.041/080 F-741 per week, 24 hours per time, Protein content of the urine was determined by the Coomassie blue methodserum creatinine level: blood was collected after three weeks of treatment and serum creatinine level determinration. The creatinine kit was provided .by Shanghai Rongsheng Biological Technique Company.., S(c) Spleen weight index: spleen weight index was determined by the following formula: Spleen weight index mean spleen weight e mean body weight 0 Paithological microscopic examination of kidney: 6 heaviest kiddneys were chosen from each group for pathological examination- Statistical analysis Used t-test for inter-group comparisons, cut off act at p<0.05. For all groups, the two rats with lowest urinary protein levels were excluded from the statisticoal analysis.

Result 1. The effect ofpeptide treatment on the urine protein level Table IIL1 The effect of peptide treatment on urin otein levelunitg) Groups n Week I Week 2 Week 3.

CM8014 10 5.5±4.0* 6.316.8* 2.8*1.9* CMS018 10 7.03.7* 3.33.4* 10 5.4+3.4* 9.5+16.2 2.4*1.5* CMSO36 10 7.9J5.8 5.0A7.1' 1.3*0.9' hydrocortisone 10 3.102.0* 7.57.7 7-67,1* placebo 10 22.9L22 '17.214.5 20.2*29.0 normal 9 2.31.1* 0.40.2 Comparing with placebo, p<0.05 It was found that peptides CMS014, CMS018, CMSO30O, and CMSO36 at once per day can lower the urine protein level of Masugi nephritis rat model, with statistically significant difference from the placebo treated. control group, pc.05,. It was also noted that the growth rate of groups CMS014, CMSO3O, CMSO36, and hydrocortisone have slower growth rate than nornal. The growth rate of the hydrocortisone group decreased to such an extent that after the first week, the treatment dose has to be halved to avoid severs intolerance.

3.7 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:37PM FROM-A J PARK gtcn +64 4 4723358 T-714 P.042/080 F-T4l 1 2. The effect of peptide treatment On the s 'erumn rreathine level Table M1.2 The influence of peptides on the serm cratinine level GrouS ciSrum ratiien el (FKoII--L CMS014 10 1591 CMS01B 10 67*1 00CMS030 10 9341* __CMS036 10 0 Comparing with nephriis rats receiving placebo treatment. (placebo)," p"CQ.OS The seru creatinine level of Masugi neCphiitis rats receiving. placebo treatmnent was much highr than. that of the normnal contrOl. group, showing that the 16 i~ tmamLlun.4'11 a. LL u1huuuusu1- It uOO dimud flint the paptirlit CMSO14. CMSDIlS, CMSO3O, and CM§036 at 5O~g/kgday once per day wore able to lower the level of seru creatinine, wit statistically significant difference from the nephrtitis rats'with placebo teatmnent 3. The effect of peptides on the splee indexes- Table 111.3 The effect of peptides; on the speen idxi' a up spaleen index -CMS014 10 3.&LI.3* CMSO3G 10 3.3+-0.8* plac eb o 10. 4.8*13~ hydrocortisone 1 .1.

Comparing with nephritis rats rcei-ving placebo treatment (placebo), P<0.05 The ipleens of all induced rats were enlaged compared with norma rats, showing that the induction of nepbritis was related to immune response. It wsB found that the peptides CMSO14, CMSOI 8, CMSD3O. and CMSO36 at once per day wer able to decrease the spleen index, "with statistically significant difference from the group of nephlis rats receiving placebo treatment, p< 0.05.

This suggested that the peptides; might exert their corrective effect agis nephrntis through hnanunosuppresslol mechsns.

38 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:38PM FROM-A J PARK gton 164 4 4723358 T-714 P.043/080 F-41

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4. The effects of peptides on the renal pathological microscopic examination Comparing with normal rats, the nephritis rats receiving placebo treatment (placebo group) showed signs of fibrous tissue formation in the glomerular capsule, hyperplasia of the glomeular epithelium, formation of crescents, dilation and congestion of gIomenlar capillaries, edema ofthe proximal tubule epithelium, and cast formation in the distal tubule and collecting duct. These pathological changes confirmed the successful induction of nephritis. It was observed that in -the group CMS014, there was only one rat showed signs of fibrous tissue formation in the glomerular capsule and hyperplasia of the glomerular epithelium.

00 10 Other parameters of the same rat, and other rats of the same group have essentially Sthe same renal histology as normal rats. In the groups CMS018, CMS030, and SCMS036, all pathological parameters of all rats were normal.

SConclusion It is concluded that the peptides CMS014, CMS018, CMS030, and CMS036 at 50pg/kg/day, once per day, administered intrapritoneally can have therapeutic effect on experimental Masugi nephritis rat model. Proteinurea, creatinine excretion, and renal histology of the treated rats were corrected by these peptides, with statistically significant difference from the control group receiving placebo treatment. These peptides may act through immunological mechanisms as indicated by their influence on the spleen weight index, but does not exclude the possibility of their actions via other mechanisms.

Discussion The peptides CMS014, CMS018, CMS030, and CMS036 may be useful as a part, or on its own, in nephritis management in human. For example, the peptides maybe used to correct proteinurea or to restore the excretary fmuntions of nephritis patients. The peptides may also be used to prevent father kidney function impairment in nephritis patients. The peptides may be used on its own, i combinations of two or more peptides, or in combination with other phannaceuticals or food supplements as a total course for nepbritis management.

Table 111.4 Peptides Effective for Nephritis CMS code SEQ D No.

CMS014 7 CMS018 CMS030 21 CMS036 26 References 1. SDA(State Drug Administration, P.,.China). The guideline of preclinioal researches of new drugs. 1994, the 1st edition, Page 96 39 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:38PM FROM-A J PARK ston +64 4 4723358 T-714 P.044/080 F-741 1 2. Xu Shuyun, et al. The methodology of pharmacological expedment, the 4 Peoples Sanitation Publishing Company, Beijing the 2nd edition, S1991:1071.

3. Chen Qi, et al. The .methodology of pharmacological researches of traditional Chinese medicine; the People's Sanitation Publishing Company, Beijing, the 1st edition. 1993:390.

4. Du Guanhua The guideline for pharmacological experiment the Sdiscovery and phanracological evaluation of new drugs, Science SPublishing CompanyBeijing, the 1st edition, 2001:598.

00 5. Wang Shuxian. Nephology, the People's Sanitation Publishing Company, Beijing, the 11* edition, 1987:244.

o IV. EFFECT OF CMS PEPTID ON CANCER In order to find out whether these peptides have possible therapeutic on oancer, we used various standard animal cancer models in this study to test out the biological effects of the above peptides on diseased animals.

Materials 1. Experimental animals BALB/c mice, Cs 7 B/6 mice, and DBA/2 mice, weighing,18-22g, from China Medical Science Institute, PR China.

2. Cell lines Mouse sarcoma Siso cells, Bi6 cells, and Lulo cells, from Cancer Research Department, China Medical Science Institute.

YAC-1 cells, as gift from Prof Yao Zhi, Tianjin Medical University.

3. Main drug and reagent The peptides used in this study were custom manufactured by American Peptide Company, Inc., USA.

Fetal bovine senim, RPMI-1640 cell culture medium, from Gibco, USA.

MTT, ConA, from Sigma, USA.

Recombinant mouse interferon-y (rmlFN-y), from Beijing Biotech Inc., PR China.

Recombinant human interleuldn-2 (rhL-2), from Shanghai Huaxin Biotech Inc., PR China.

Lymphocyte separation solution, from Reserch Institute of Henatologic Disease, National Institute ofMedical Science, PR China COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:39PM FROM-A J PARK ton +64 4 4723358 T-714 P-045/00 F-741

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0 f Cyclophosphamide, from The 12th pharmaceutical factory of Shanghai, PR China.

Methods 1. Administration of test substance oO Intraperitoneal injection, once per day. With the exception of cyolophosphamide group, all groups started treatment 5 days before transplanting C 10 the cancer cells. The cyclophosphamide groups were treated on the next day after Stransplanting the cancer cells. Treatment of all groups with testing substance 0 lasted for 30 days or until the animal died unless otherwise specified.

2. The effect ofpeptides on the growth rate of transplanted Siso sarcoma cells in BALB/c mice, and that on the immunological function of the host BALB/o mice were randomized into peptide group, cyclophosphamide group, lFN-group rhN-y2 group r2 and saline group, 20 animals per group.

Stock Sao sarcoma cells were incubated in DMEMIF12 medium supplemented with 10% fetal bovineserum, 37°C, 5%CO2 for 72 brs, then'washed 3-4 times with Hank's solution at room temperature. Adjust the cell concentration to 1-2 x 10( per litre with Hank's solution. 0.2ml of cell suspension was implanted to several BALB/c mice through the ampit for 10-12 days. The mice were killed by cervical vertebra dislocation. Vigorously growing and non-disrupted tumor masses were harvested and washed clean with sterile saline. The tissue was dispersed in saline to a homogenous cell suspension, in a ratio of Ig tissue to 4 ml saline. The sarcoma bearing mice model was prepared with an injection of 0.2ml cell suspension through the amipit Administration of test substance treatment started as described in section Method 1.

2.1 The effect of peptides on the phagocytotic function of-mononuclear phagocyte in mice with Stso sarcoma was analysed by injecting mice from the tail vein with 0.lml/10g body weight of Indian ink (1:5 dilution with normal saline) on the second day after the last test substance administration. At one minute and five minutes after the injection, 20pl blood was obtained from the eye canthus with hepariised tubing. The blood was mixed with 2ml 0.1% w/v Na2CO3 and then ODssom obtained- The outline clear index K was calculated by the following formula: K= (g A, Ig Az) (t2 tl) Key: Al: ODasam at first minute A2: OD 6 8 orn at fifth minute 41 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:39PM FROM-A J PARK ffton +64 4 4T233586 T-714 P-046/080 F-41

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t2: 5 minutes t2: 1 minute After. the phagocytosis index study, the mice were killed by cervical cA 5 vertebra dislocation. The liver, spleen, and cancer tissue were dissected, blotted dry, and weighed.

The phagocytosis index a was calculated as below: 00 a (3 K) x(W Wis) key: W: body weight WLs: weight of liver plus spleen 2.2 The tumor growth inhibition index was calculated according to the following formula: Tumor growth inhibition index (mean tumor weight of control group mean tumor weight of treatment group) mean tumor weight of control group 3. The effect ofpeptides on the survival of BALB/c mice with transplanted ascitic fluid type liver cancer Hz BALB/c mice were randomly grouped into peptide group, cyclophosphamide group, zmFN-y group, rxIL-2 group and saline group, animals per group.

Stock 22 cells were incubated in DMEM/F12 medium supplemented with fetal bovine senrm, 37rC/5%CO2 for 72 hrs, then washed 3-4 times with Hank's solution at room temperature. Adjust cell concentration to 1-2 x 10 per litre with Hank's solution. 0.2ml of the cell suspension was implanted to the abdominal cavity of several BALB/c mice for 6-8 days The mice were killed by cervical vertebra dislocation. Ascitic fluid of the mice was collected asceptically and the cell concentration adjusted to 1 x 106 per ml with Hank's solution. 0.2ml of the cell suspension was implanted into the abdominal cavity of healthy mice to generate the H= carrying mouse model for ascitic fluid type liver cancer. Administration of test substance started as described in section Method 1.

Survival data of the mice was recorded. If the animal survived longer than the experiment, the survival days was recorded as the duration of the experiment Mean survival day was obtained by the. Kaplan-meier method in the Survival option of the SPSS software. The survival index was calculated according to the following formula: Survival index (mean survival days in treatment group -mean survival days of control group) -+mean survival days of control group x 100% 42 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:40PM FROM-A J PARK ton 64 4 4723358 T-714 P.04T/00 F-741 tb 4. The effect of peptides on the cellular immunity of BALB/c mice with transplanted asciti fluid type liver cancer H122 4.1 Preparation of spleen cell suspension[1'4 Healthy BALB/c mice were randomised into peptide group, rmIFN-y group, rhIL-2 group, and saline group, 15 mice per group, and prepared into Ha carrying mice model as described in. section Method 3. After implantation of the Scancer cells, the test substances were administered for 15 days, the mice were c killed by cervical dislocation. The spleen was isolated aseptically and manually dispersed in cold D-Hank's solutioi using an injection needle. The dispersed cell suspension was further sieved through a 100-gauge 150pm diameter stainless steel C sieve. After centrifugation at 200g for 10 minutes, the supenatant was discarded.

The cell pellet was re-suspended in 10 volume of Tris-NH 4 Cl buffer and then kept O standing still for 10 minutes at room temperature. The suspended cells were 0 15 collected by centrifugation at 150g for 10 minutes. The cells were washed 2-4 times with cold D-Hank's solution by re-sispending and collecting by oentrifugation with condition as described above. The washed cells were then diluted to the detired cell densities by.RF I-1640 culture medium, containing fetal bovine serum.

42 The effect of peptides on the T lymphocyte transformation in mice with ascitic fluid type liver cancer H

A

2 Spleen cells of density 1x10 6 /ml were placed onto a 96 wells cell culture plate, 100pl/well, three prarallel wells each of the assay sample and control sample" per mouse. To the assay wells, 100.I/well ConA of 100g/ml in RPMI-1640 was added, and 100piwell plain RPMI-1640 was used for the controls. The cells were incubated for 66 hra at 37C, 5% CO 2 The cells were then pelleted by centrifugation at 150g for 10 minutes. The supernatant was collected and stored at 0 C for cytokines IL-2 and IFN determination.

MTT of 1mg/ml in RPMI-1640 was added to the cell pellet and the cells re-suspended by shaking for 2 minutes. The incubation was continued for 4 hours. The supernatant was discarded after centrifugation at 150g for minutes. 120 1 40mM HC1-2-propano'was added to the cell pellet and shaken for 3 minutes. Use an ELISA reader to obtain ODnonm of each well referenced at 630mn.

Each mouse formed three assay wells and three control wells. The Stimulation Index (SI) of each mouse was obtained by first deriving the average OD of the three parallel wells, then dividing the value of the assay wells by the control wells, 4-3 The effect of peptides on the NK cell activity in mice with ascitic fluid type liver cancer H22 6 Mice spleen cells were prepared to 4x10 6 /ml as described in section 4.1 above. Target cells YAC-1 were brought to log phase and adjusted to lxl0'/ml.

Using a 96 wells cell culture plate, 100l mouse spleen cells and 100l culture medium were added to the control well containing only the spleen cells; 100j1 43 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T1 03:40PM FROM-A J PARK glton +64 4 4723358 T-T14 P-048/080 F-41

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target cells and 100 culture medium were added to the control well containing P only target cells; 100p mouse spleen cells and 1001l target cells were added to the ;Z NK activity assay well. Three parallel sets of the above were prepared per mouse.

After that the 96 wells cell culture plates were incubated for 4hbr at 370C, 5 C02.

Samples were centrifuged at 150g for 10 minutes to collect the cells. The supematant was discarded and 50pwell MIT of lingml was added. The reaction Smixture was then shaken for 2 minutes, and incubated at 37 0 C, 5% CO 2 for 4 Shours. The supernatant was discarded after centrifugation at 150g for 10 minutes.

00 0 10 120 l 40mMI HC1-2-propanol was added and shaken for 3 minutes. An BLISA Sreader was used to obtain ODson n of each well referenced at 630nm.

C'1 Each mouse.has 9 wells: three spleen cells only control, tree target cells o only control, and three assay wells with both spleen and target cells. The NK cell Sactivity index of each mouse was obtained by frst deriving the average OD of the C 15 three parallel wells of each combination, then applying-this average OD to the following formula: NK cell activity index [l-(average OD of spleen and target cell well average OD of spleen cell only well). (average OD of target cell only well)] x 100% The effect of peptides on the survival of DBA/2 mice with transplanted Lilo leukemia DBA/2 mice, 6-8 weeks old, were randomized into peptide group, cyclophosphamide group, rmlN-y group, rbIL-2 group, and saline group, animals per group.

Stock Lio1 cells were incubated- in DMEM/F12 medium supplemented with 10% fetal bovine serum, 370C/5%C02 for 72 hrs, then washed 3-4 times with Hank's solution, and adjusted to I x 10s cells per litre. 0.lml of the cell suspension was implanted into the abdominal cavity of several healthy DBA mice for 6-8 days. The mice were then killed by cervical vertebra dislocation and their ascitic fluid collected aseptically. The cell concentration of the collected ascitic fluid was adjusted to 1 x 10 per ml with Hank's solution. O.lml of.the cell suspension was inplanted into each of the testing animal and the survival data of the animals recorded. Treatment started in the testing animals as described in section Method 1. Mean survival day was obtained by the Kaplan-meier method in the Survival option of the SPSS software. If the animal survived longer than the experiment, the survival days was entered as the duration of the experiment. The survival index was calculated according to the following formula: Survival index (mean survival days of treatment group mean survival days of control group) mean survival days of control group 44 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:41PM FROM-A J PARK ffton +64 4 4723358 T-14 P.049/080 F-741

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6. The effect of peptides on the humoral immunity of C 57 BL/6 mice bearing transplanted Bi melanoma, and that on the metastatic potential of the inoculated melanoma cells C5BL/6 mice, 6-8 weeks old, body weight 18-22g, were randomized into peptide group, cyclophosphamide, group, rmiN-y group, rbL-2 group, and saline group, 20 animals per group.

0 0 Stock B 16 mouse nelanoma cells were incubated in DMEM/F12 medium supplemented with 10% fetal bovine serum, 37C/5%COz for 72 hrs, then washed NCl 3-4 times with Hank's solution- The cell concentration was adjusted to I x 10 s per ml with Hank's solution and 0.lml of the cell suspension was injected via the tail O vein to the testing mice to generate the B16 melanoma bearing animal model 0 Treatment with test substance started as described in section Method 1.

6.1 The effect of peptides on the humoral immunity of C 57 BL/6 mice bearing transplanted B 6 melanoma t Sheep red blood cells (SRBC) were prepared by collecting blood from the cervical vein and put into a sterile flask with glass beads. The flask was shaken for 3 minutes and the blood then mixed with Alsever solution (glucose 2.05g, NaCI 0.4g, Na lemonade 0.8g, adjust to 100ml with distilled water) and stored at Immediately before use, samples were centrifuged at 130g, 5 minutes to collect the SRBC. The cells were washed two times by re-suspension and centrifugation in normal saline. Then the cell pellet was collected by centrifugation at 180g for minutes and re-suspended in saline to make the final working-SRBC suspension, 2% Complement was prepared by adding 10 volumes of fresh Cavy serum into one volume centrifuge packed SRBC, and then gently shaked for 30 minutes at 4°C. The SRBC was removed by centrifugation at 200g for 10 minutes. volumes of normal saline were added to obtain the working complement solution.

On the testing animals, on the 27 date of test substance treatment, 0.2ml of the working SRBC cell suspension was injected into each aimal to raise antibody.

On the day after the last test substance administration, blodd was collected from the .eye canthus and left at room temperature for one hour for serum exudation.

After centrifugation at 200g for 10 minutes, the collected serum was diluted by 500 times with normal saline.

To lml diluted mouse serum of each mouse, 0.5ml SRBC suspension was added. Ice cold. Then iml working complement solution was added and incubated at 37 0 C water bath for 10 minutes. Reactions were terminated by ice cold. Samples were then centrifuged at 200g for 10 minutes to obtain the supernatant.

To Iml of this supernatant, 3ml Drabkin solution was added and left at room temperature for 10 minutes. ODs4onm was obtained.

COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:41PM FROM-A J PARK go 6 735 -1 .5/6 -4 gton +64 4 4723358 T-714 P.050/080 F-741

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Reference OD4obm was obtaiaed by mixtag 0.25m1 SRBC suspension With Drablin solution to 4nil and placed at room temperature for 10 minutes before

WOD,

0 was taken.

Ilemolysis index (ODs 4 onm Of test sample t reiference ODS40") x 500 6.2 After the humors] immunity study, the mice were killed by oervical vertebra dislocation. Postmorbnrn examination of the animals was perforned. The pathological changes were recorded, and the nm~bers of melanoma lung metastasis foci were counted.

Results 1. The effect of peptide on the cellular phagocytosias in BALBic mice with transplanted Sitoo sarcoma (Method. 2.1)- Table I.1I. The effect of peptide on the phagocytosis index of BALE/c- -mice with transplanted Si13o sarcoma Group Dose N Phagocytosis index CMSOOI S0jig/kg 20 '6SVdu0.33*A CMSOO1 SpLgtkg 19 6.57+OAS""* CMSOS34 spgfkg 19 6.201-0.44*A CMS0S4 O.Sp1gulc 20 6.35;61.02* fl.-2 3x10 5 ILT/kg 19 6.96ff1.37*

IFN-

1 r. 3xlO 3 I1Utkg 17 5.45+0.71 Cyulo-phos 20mglkg 19 5.-92±2.47 pharnide saline 0.5=1 19 5.38+0.85 *compaing to saline, P<0-05 e: omparintg to rmIFN-y, P'C0.05 CMISOOT at 5Oigkg/day and 5Lgt/day, and CMS034 at 5p~g/kg/day and jig/kg/day were found to be able to increase the phagocytosis index, having statistically significant difference from the saline group.

2. The effect of peptide on the growth rate of transplanted Sigo sarcoma in 203 BALB/c mice (Method 2.2) Table IV2. The effect of peptde on trasplanted 51 80 tumor growth Group Dose N Truer weightg) Tumer inhibition index (Y.

CMSO1O SOlpgfkg 20 0.67*0O.35* 48.4 CMS034 0.5pjg/kg 20 0.8-3*0.48* .35.9 CMS035 Zp~g/kg 20 0.71-+0.37* 44.6 It-2 3x<1O 5 TU/kg 20 0.6910.37* 46.2 7NY3xlO 5 Iwlkg 18 0.96+0O.45 25.3 cyolo-phoi 2Omg'kg 20 0.68+0.32* 47.3 pharnide Saline 0.inil 20 1.29+0O.50 *comparing to saline group, ThC0.05 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:41PM FROM-A J1 PARK sa 6 735 -1 .5/8 -4 gton +64 4 4723358 T-714 P.051/080 F-T41

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CMSO1Oo at sO0[gkg/day. CMS034 at 0.Sgglday, and CMSO35 at Sjtg/kg/day were found to be able to reduce the grorwth of the transplanted S 1 80 sarcoma, having statistically significant diffbrene from the saline -group (P2<0.05).

3. The effect of peptide on the survival of BALB/o mice with transplanted ascittc; fluid type liver eanvr fl2 (Method 3) Table IV3 The effect of peptide on the survival index of BALB/c mice .with transplanted ascitic fluid type liver cancr M Group -Dose CMSOOS0 GMSI11 CMS024 CMS 024 CbMSO24 CMS032 rbMf-2 rmIPN-y Cyclo-phos SpLg/kg 5.Sg/kg 3xIOiTU/kg 3x10S1I/lg 20mg/kg 20 50.74b20.9*& 67.8 20 36:44222*& 602 20 36.3t12.7*8' 38.4 19 40.6h14.6* 's 54.8 19 46.4*4.p,%a&5 76.9 20 42.8t12.2*-a& 63.3 20 13.6-L0.5 27.8:7.5 20 24,71±10-2 pbamide Saline 0.Sml 19 comparing to saline, P<0.05 A:I comparingtonnWEN-P<O.OS comparing to rhIL-2, P<0.05 c ompar[ing to oyctophosphanide, PN0.05 26,2-16.8 CMS008 at 5p1gfg/day, CMS041 at Sjigkday, CM-SO24 at CMSD24 at 0.Slg/g/day, and CMSOS2 at 0.5gjg/kg/day Were found to be able to prolong the survival of BALB/c mice with transplanted H12 as-citic fluid type liver cancer, having statistically significant difference from the saline grop It was also observed that in the group CMS024 0.Sjgfkg/day, more than 30% (n76) of the mice could survive longer than 90 days (two months after the experiment ended).

Postmortumn examination of the mice did not show sign of tumor establishment.

CMS 024 at 0.5 jig!kg/day therefore may interfere with the growth of the transplanted 1-1 by interfering with its establishment or inducing the complete healing of the established cancer.

47 COMS ID No: ARCS-i 58250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:42PM FROM-A J PARK gton +64 4 4T23358 T-714 P-052/080 F-741 4. The effect of peptide on T lymphocyte transfoimatiODn m AJ~I/c mice wit trdnsplanted =sitic f luid type liver canner H-L- (Mothod 4.2) ;Z ~Table I4A Tbfe effect of peptide on T lymphocyte tranSfOnnadonm Group DoeN stimulation index CMSO 10 SOO[tg/kg 20 1.45.+.21S GMS019 .igfkg 19 1.50±0.19*5I CMSO2.4 O.Spg/Ig 19 1.4&JJ,19*S 000S04$jgk 20 1.45*0.21?' (N GS034 0.5V.g~g 20 1.37±+0.10*'.

oCMS035 0.Sggkg' 20 1,440+.13--" oCMS035 SjPzglk 20 1.46+R.16*' rhIL-2 3x10 5 1tJ/lcg 19 1.461-021* rMNY.3x10IU/kg 18 1.27+0.14 Cyclo-pjosphamide 2Omglicg 19 1*Ql+QJ3 t 20 1.25+0O.07 *:comparing to Saline, $:comparing to cyclophosphamide, CMSOIO at SO0pgfkg/day, CMS019 at O.5pRgg/day, CMS024 at and 5g/jkgday, and CMvSO35 at 0.5j~ggday and 5pgg/dday were found to be able to increase the atimilation index of T-lyxnphocytc. having statistically significant difference from the saline group (P<0,05)- The effect of peptide on the NK cell activity in BAL~c mice With transplanted ascitic fluid type liver cancer H= (Method 4.3) 48 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:42PM FROM-A J PARK gton +64 4 4723358 T-T14 P.053/080 F-T41 Table 1V5. The effect ofpeptide on the K cell cytotoxic activity index Group Dose N NK activity index(%Yo) CMSO3 500 L/g 17 37.9*14.5 CMS014 O.Sjg/Irg 17 40.7;19 7.* CMS024 0.5iLg/kg 17 39.3+1.7-" 00 CMS024 Sgg/k-g 20 34.9+121A CM3024 50g/kg 20 43.6:k3.9 *AS 0M8032 S tg/Ig 20 52.6412.5*AS& o CM5032 5OVS/kg 19 4l.0t18.7A- CMS034 50g/kg 20 57,±17-9 *AS IL-2 3xlO 5 J/kg 1 9 26.0W9.0 IFN-y SxlOIJ/k 18 20.9+3-3 Cyclo-phos 20mgkg 19 l6.5t7.2* Phamide Salim 0.5ml 20 24.M+8.2 S as compared with saline, P<0.0S.

A as compared with IFNy, P<0.05.

as compared with IL-2, S3: as compared with cyclophosphmido, P<0.05 CMSOO3 at SOOpg/kg/day, CMS014 at 0.5.g/kgfday, CMS024 at 0.Spg/kgday, and 50gg/k,-/day, and CMS034 at SOpg/kg/day were found to be able to increase the NK cell cytotoxie activity in the testing animal model, having statistioally significant differee from the saline group 49 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:42PM FROM-A J PARK Vton +64 4 4723358 T-714 P.054/080 F-41 6. The effect of peptie on the survival of DEBA2 mie with transplanted L 210 leukemia (Method Table IVM6. The effect ofpeptide on the survival index of testing animals Group Dose N Survival days Survival index (O) CMS019 0.5qglkg 20 21.115.8* 26.8 C45035 0.Spgfkg 20 29.3*15.4* 76.1 IL-2 3xlO 5 1U/kg 20 32.0113.7 92.3 ]FN-y 3xl0 5 UT/kg 20 15.6+2.2 Cyclaphos 20ngkg 20 24.0*5.3* 44.2 CA phamide Saline 0.5=1. 21 16. 6+Z.

compaing to saline group, F<005 CM8019 at 0.5jglkg/day and CMS035 at 0.5p4gcgday were found to be able to prolong the survival of DBAI-2 mice with transplanted L1210 leukemia, having statistically signficant difference from the saline group 7. The effect of peptide on the humoral immmnity of C5 7 B/6 mice with transplanted B16 melanoma (Method 6.1) Table IV.7. The effect of peptide on the heznolysis index of C578TJ6 mice with transplanted B16 nelanoma- Group Dose N Heniolysia index CMS001 0.5jig/kg.20 5.+625- CMSOOl sjg/kg 20 4.3+l-7.7*' CMS001 5OItg/kg 19 -45-0431.90 CMSOO1 500±glkg 20 36.0410.2*' CMS003 0.5 g/kg 20 61.6±26.9'* OMS003 Spt/kg 20 37.2ll5.9*$ CMS003 SOj.g/kg 20 38.7*2I3.9*r CMS003 500ig/lcg 20 35.9tL3-0*' CMS008 O.Spg/kg 19 42.7+18.4* CMS008 20 38.912.0*' JLUAP"oP,ISPECCT-SPEC COMB ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:4SPM FROM-A J PARK io 5 735 -1 .5/8 -4 ffton +64 4 4723358 T-714 P.055/080 F-741

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CMSOOS 501pg/kg 2' 0M8008 500j±glkg 2' CMSOIO 0.514g/kg I1 QMSO1O Sptgfkg 2 CMSO1O 50jtg/kj 2 CDUIU -5VUUpIg/Rg 2 CMSO1I O.Spg/kg 2 CMSO1I SO01gg/kg 2 CMS016 500pg/kg 2 CM.S019 0.5 pg/kg 2 045S019' 5A&8k 2 0M5019 50pg/cg 2 CMS024 0,Sj~g/kg 2 CMS 024 Spg/k 2 CMS024 50 jkg 2 CM8024 SOg/kg I CMS034 O.5Sgg/kg I GMS0S4 SpLg/kg 1 CMS034 50pig/kg 2 CMSO3S SpLg/kg 2 CM8035 50pLglkg 2 GMMS SO0jsg/kg 1 rhIL-2 3x10WT/k 1 nnIFN-y 3xlObJJ/kg 1 Cyclo-phos 20rng/lcg 1 phauiide Saline O.Sxnl 2 *:coino~aring to saline, PC0.05 A~comparing to rmWN-y, F<0.05 &:comparing to rhIL-2, P<0.05 $:comaparing to cyclopboaphamide, N0.05 0 0 8 0 0 0.

0 0 0 0' 0' 0.

0 ~0 0 ~0 .0 9 9 9 37.1+16.7* 50 .l 1 M-8' 34,.9±105" 39.617.7-" 32.0114.7* 34.4=L-j9.4* 43.3129,9* 42.0±12.0"' 35A415.l*S 28.3--k7,6 4 43.0+1 030 27.8;19.I* 43.0+11.7*' 30.2*10.9* 38.9=L-21.2*' 44.71*22.74" 40.5z:25.8w' 49.3+24.7* 60.5*17.4* 20.7kL19.1 .0 19.0*19.1 fLUA PJ094-1 IEPELIF C-SPEC COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-M824 24-08-200T 03:43PM FROM-A J PARK gton +64 4 423358 T-714 P.056/080 F-41 0 CCMS001, CMS003, CMS008, CMS0, C CMS011, CMS016, CMS019, CMS024, CMS034, and CMS035 were found to be able to enhance the humoral Sresponse (increase in hemolysis index) of Cs7BL/6 mice with transplanted B 16 melanoma at dosages as shown on Table IV.7, having statistically significant difference from the saline group 8. The effect of peptide on the survival of inoculated B16 melamona cells in ON CsrBIJ6 mice (Method 6.2) 0 Postmortum examination of animals after the ending of test substance treatment did not show any sign of existance of B16 metastatic foci in the lung of mice treated with CMS008 at 0.5ggg/kg/day, 5ang/kgday, and 50pgkg/day, and CMS016 C at Spg/kg/day and 500pg/kg/day.

Ci 'Conclusion In compliance with the Preclinical New Drug Research Guidelines issued by Branch of Drug Administration, Department of Health, PR China in 1993, the effects of peptide on mice with transplanted cancer cells was studied. It was concluded that 1. CMS010, CMS034, and CMSO35 at suitable dosage could significantly 'inhibit the development of transplanted Stoo sarcoma in mice; 2. CMS001 and CMS034 at suitable dosage could enhance the phagocytotic immune activities of mice transplanted with Siso sarcoma; 3. CMS08, CMSO11, CMS024, and CMS032 at suitable dosage could Sprolong the survival of mice transplanted with ascitic fluid type liver cancer H22; 4. CMS010, CMS0 CMS024, CMS034, and CMS035 at suitable dosage could enhance T lymphocyte transformation in mice transplanted with ascitic fluid type liver cancer H22; CMS003, CMS014, CMS024, CMS032, and CMS034 at suitable dosage could increase the NI cell cytotoxic activity of mice transplanted with ascitic fluid type liver cancer 122; 6. CMS019 and CMS035 at suitable dosage could prolong the survival of mice transplanted with L1210 leukemia; 7. CMS008 and CMS016 at suitable dosage could inhibit the development of transplanted B16 melanoma in mice; 8. CMS001, CMS003, CMS008, CMS010, CMS011, CMS016, CMS019, CMS024, CMS034, and CMS035 at suitable dosage could enhance the humoral immune response of mice transplanted with B16 melanoma.

Discussion 52 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:43PM FROM-A J PARK gton +64 4 4723358 T-714 P.057/080 F-741 CMS001, CMSOO3, CMSOO8, CMSO10, CMS11, CMS014, .CMS016, ;ZCMS019, CMSO24, CMS032, CMS034, CMS035 may be useful as apart, or on its own, in cancer management in human. For example, CMS001, CMS003, CMS008, CMSU1O, CMSO1I CMSO14, CMS016, CMS019, CMS024, CMS032, CMS034, and CMS035 may be used to increase the immunity of cancer patients.

Cl CMSOO8, CMSOlO, CMS016, CMSO34, and CMSO35 may be used to interfer with the growth of cancer cells in patients. CMS008, CMS011, CMS019, CMSO24, CMS032, and CMSO35 may be used to prolong the life expectancy of cancer patients. The peptides may be used on its own, in combinations of two or more 00 10 peptides, or in combination with other phannrmaceuticals or food supplements as a total course for cancer management.

t\ Table IV.8 Peptides Effective for Cancer 0 0 Cl CMS code SE ID No.

CMS001 1 CMS003 27 CMS008 3 CMSOIO 4 CMSO11, CMS014 7 CMS016 9 CMS019 11 CMSO24 16 CMS032 22 CMSO34 24 Reference 1. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7:137-143 2. Principles of Pre-clinical Research of New Drugs, People's Republic of China 1993, 7:128-129 3. Yuanpei Zhang, Huaide Su. Pharmacological experiment (second edition).

People's Hegth Publishing House. 1998, 137-138 4. Shuyun Xu, Rulian Bian, Xiu Chen. Methodology of pharmacological experiment. People's Health Publishing House. 1991, 1221-1234 Principles of Pre-clinical kesearch of New Drugs, People's Republic of China. 1993, 7:140 6. Jinsheng He, Ruizhu Li, Tingyi Zong. The study on MTT reduction method of testing NK cell activity. China Immunology Journal. 1996, 356-358 7. Yaoqin Yang, Huchuan Yang, Huihong Tao, et al. The synergic effect of on the antitumor of hyperthermia--Experimental studies of 53 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:44PM FROM-A J PARK gton 64 4 4T23358 T-714 P.058/080 F-T41

I

rmouse melanoma. Cancer Research on Prevention and Treatment.

S1999,26(4): 8-12 8. Jian Fu, Jie Zhen, Weigang Fag, et al. Interleuin-12 gene transfectim into muine B16 melanoma cells suppresses tumorigenicity and decreases metastatic potential. National Medical Journal of China. 1998,78(8): 627- 629 9. Qian Wang. Modem medical experiment method. People's Health Publishing House. 1998,482-483 00 10. Qicbao Pan, Bip Xu. Cancer pharmacology and chemotherapy Henan medical university Publishing House. 2000,66-69 Cl 11. Yuanpei Zhang, Huaide Su. Pharmacological experiment (second edition).

People's Health Publishing House. 1998, 131

O

N V. THE EFFECT ON THE BODY WEIGHT Healthy rats were feeded with high nutrition diet for 5 weeks, with or without simultaneous peptide treatment (intramuscular 300pg/kg/day). Rats on an ordinary diet with saline injection were used as negative control. After 5 weeks of treatment, injection was stopped and the same diet was maintained for another three weeks. Body weight data was collected at once per week interval.

Behaviour of the rats was also observed. Rats receiving peptide CMS015 were found to have statistically significant lower body weight increase compared with control during the course of peptide treatment. This trend of decreased body weight gradually reduced after ceasing the CMS015 treatment. It is concluded that CMS015 at -suitable dosage level can reversibly control obesity development induced by over-feeding.

Materials Sprague-Dawley (SD) rats, weighing 145±10g, were supplied by the Center of Experimental Animal of Guangzhou University of Traditional Chinese Medicine, PR China (certificate No.: 2000A019). Peptides were custom synthesized (of L-amino acids origin) by American Peptide Company, Inc., USA,.

and were diluted to lO)g/nI in normal saline. The high andnormal nutrition diets were prepared in compliance with the guideline for pre-clinical research of antiobesity drug, issued by SDA (State Drug Administration), PR China Methods Healthy rats were randomized into the experiment, positive control, and negative control groups. 10 rats per group, half male and half female. The experiment group of rats was feeded with high nutrition diet for 5 weeks with simultaneous intramuscular injection of 300g/kg/day peptide, once per day.

Positive control group received the same high nutrition diet but placebo saline injection, while negative control group, used to proof successful establishment of obesity model, received normal nutrition diet and placebo saline injection. After weeks treatment, injection was stopped and the same diet was maintained for 5.4 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:44PM FROM-A J PARK +64 4 4723358 T-714 P.059/080 F-41 0 0

(N

notller weeks. Thi Eits were weihed at once per week interval. Behaviour of the rats was also observed.

Statistics The data were presented as mean standard deviation1 Paired t test or single-factor ANOVA was used for inter and intra group comparison. Statistical significance cut-off was at P<0.05.

Results 1, Effect of peptide on the body weight of SD rat Table V.1 Pre-treatment Week 1 SWeek 2 Effect ofpeptide onfthe body weight of SD rats Positive Control Group CMS015 Treatment Group (g) Male n=5 Female Male n=5 145.6 13.6 194.4 14.5 239.6 A 13.4 133:6 4.6 164.4* 8.7 145.6 8.5 183.8 10.6 129.2 3.3 157.8 8.3 I I 11.

118.01 6.4 I a.

I

Week 3 Week 4 265.0h 11.8 287.4 17.7 208.8 ±8.2 227.2 ±8.2 220.6 12.2 239.0 16.0' 258.27 18.1 176.0 11.2 196.0-± 10.5 212.0 13.5 Week 5 299.4 21.2 236.6 10.9 268.8 17.7 221.4 132 Week 6 333.4 27.1 249.4 16.3 299.4 21.2 235.6 16.3 Week 7 349.2 28.9 261.2 13.4 310.4±25.9" 242.2 ±18.8 Week 8 374.4 37.2 255.6 11.5 337.4+ 3.u.o 3 L.I In comparison with positive control group: p<0.05 At the dose of 300 pg/kg/day, CMS015 was found to be able to limit the weight gain of obesity rats induced by over-feeding, having statistically significant difference from the control group The difference of the treatment and control groups increased with the length of treatment. Upon termination of peptide CMS015 treatment in the treatment group, the difference of the treatment and control groups gradually reduced and became statistically insignificant after three weeks, showing that the effect of the peptide on SD rat body weight is rversible.

During the whole course of the experiment, it was observed that the appetite and activity of all groups of rats remained normal Discussion COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:44PM FROM-A J PARK ton 64 4 4723358 T-T14 P-060/080 F-41 It is concluded that CMS015 at suitable dosage level can limit the development of obesity induced by over-feeding. The peptide may put into human use for control of obesity, The peptides may be used on its own, in combinations Sof two or more peptides, or in combination with other phannaceuticals or food supplements as a total course for obesity management.

In the present study, administration by intramuscular injection was tested, but this does not exclude the possible effectiveness of the peptide if administered via other alternative routes. The peptides may be adxministered via intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, Sand subcutaneous implantation, with or without delivery facilitating device such as Sliposome, sustain release protection etc. The peptide may also be administered in any form of oral administration like tablet, capsule, suspension, solution etc, in the Susual form without modification or in slow release form, or with or without gastroo 15 enteric protection. The peptide may further be applied in-any form of topic Capplication like ointment, cream, gel etc with or without transdermal faclitating device, or as inhalant of powder, dissolved, or as liposome protected form. The peptide may also be interpreted into its genetic sequence and cloned into an expression system, on its own or in combination with other peptide sequences, to generate a resulting peptide molecule to make use of the activity of the peptide as described in tlis report, with or without purificationof the resulting peptide.

Table V.2 Peptides Effective for Obesity CMScode SEQ ID No.

CMS01S5 8 References 1. SDA, PR China. The guideline for pre-clinical research of new drugs.

1993 It is understood that it may be possible to add additional amino acids to the amino or carboxyl tennini of the aboye disclosed peptides as another method of practising the present invention. For example, one or two amino acids may be added to the disclosed peptide without affecting its biological function. It may also be possible to add three or four amino acids and still maintain the function of the peptides. These are all referred fo as variants of the same peptide. Alternatively, one or two amino acids may be deleted from the peptide without affecting its biological activity. It may further be possible for three or four amino acids to be deleted without affecting the biological function of the peptides. These are referred to as fragments of the instant peptide.. Furthermore, derivatives of the peptide such as conservative replacement of one amino acid for another within the same functional class may be used to practise another aspect of the present invention. For example, peptides having non-polar or hydrophobic side chains may be possible to substitute one side group for another without reducing biological activity. As a further example, linker/spacer may be inserted into the peptide to form variants, but the variants still retaining its active moiety as the 56 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:45PM FROM-A J PARK fftorl +64 4 4723358 T-714 P-061/080 F-41 0 original peptide used in this study These are also considered variants of the Speptides. A peptide analogue as used herein, includes peptides that have amino acid molecules which mimic the structure of the natural amino acid e.g. an analog with a different backbone structure, or D-amino acid substitution: As a further c" 5 example, although the amino acids used for synthesizing the peptides are in their L optical isomeric form, peptides with one or more of the amino acids in the sequence substituted with the D-fonn may have similar biologidal activities. The term "ftnctional derivative" as used in the claims is meant to include fragments, variants, analogues or chemical derivatives of the peptide..

00 10 As used herein, the term "hybrid peptide" is used to refer to peptides that Scontain additional peptides inserted into the original biologically active peptides having SEQ. ID no. 1-30 or their functional derivatives, but still retain substantially similar activity. The additional peptides include leader peptides that Scontain, for example, an amino acid sequence that is recognized by one or more prokaryotic or eukaryotic cell as a signal for secretion of the hybrid protein into the C -exterior or the cell. The secretion may be a direct secretion, or indirectly through secretory vesicles.

"Substantially pure peptide" refers to peptides that are at least 10%w/w in purity, more preferably 20%, even more preferably 40% and much more preferably 60% and far more preferably larger than 90% pure. In the most preferred embodiment, the purity is larger than 99%. The substantially pure peptide can be used to prepare pharmaceutical and nutritional formulations that may be complex mixtures as described below.

The use of the above-identified peptides in pharmaceutical formulations may be employed as possible treatment for immunological disorders or disease having secondary effect on immunity e.g. cancer or infections or any of the conditions mentioned above. The formulations may contain one of identified peptides mixed with other active or inactive constituents, including other peptides.

E.g. two to several 3-5) of the listed peptides may be added to the same formulation with or without other ingredients. Alternatively, one of the listed peptides may be used to prepare the formulation together with peptides not listed here. They can be administered in the form of intravenous, intramuscular, intracutaneous, subcutaneous or intradermal. The mode of administration may also be intra-arterial injection that leads directly to the organ of problem.. Other modes of administration are tansdermal, inhalation as powder or spray, and other forms of delivery known by one in the art. The formulation may also be orally taken, and may contain carriers that can be used to preventgastric digestion of the peptide after oral intake or any other caniers known in the art (for transdenmal such as liposome).

The pharmaceutical formulation may include any of the known pharmaceutical carriers. Examples of suitable carriers include any of the standard pharmaceutically accepted carrier known to those skilled in the art. These include but are not limited to, physiological saline solution, water, emulsions including oil and water mixtures or triglyceride emulsions, and other types of agents, fillers, coated tablets and capsules. The appropriate carrier may be selected based on the mode of administration of the pharmaceutical composition.

57 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:45PM FROM-A J PARK grton 64 4 4723358 T-714 P-062/080 F-T41 0 The peptides may be administered via intravenous injection, intramuscular Sinjection, intraperitoneal injection, subcutaneous injection, and subcutaneous implantation. -The peptide may also be administered in any form of oral administration like tablet, capule, suspension, solution etc, in the usual form without modification 'or in slow release form, or with or without gastro-enteric C' protection. The peptide may further be applied in any form of topic application like ointment, cream, gel etc with or without transdermal facilitating device. The peptide may also be interpreted into its genetic sequence and cloned into an Sexpression system, on its own or in combination with other peptide sequences, to o 10 generate a resulting peptide molecule to make use of the activity of the peptide as described in this report.

The dose of each peptide may be Ing 10g per kg body weight A preferred dose is O1ng 10mg per kg, and more preferably lIpg Img per kg for an o injection mode of administration. However, the effective dose can be as low as Ing per kg body weight, since one or more of the peptides may operate through CN receptors that will induce a cascade of normal physiological response.

Alternatively, one or more of the peptides can just be an initiator for a whole cascade of reaction. For an oral intake, the amount may be Ing 10g per day per kg body weight, more preferably 0.1lLg Ig per day per kg body weight and even more preferably 1 Lg 10mg per day.

VI. Gene Therapy and Method of Treatment Gene therapy based on the discovered peptide sequences is performed by designing.a nucleic acid sequence that code for one of these peptides. The nucleic acid may be synthesized chemically and operably ligated to a promoter, and cloned into an expression vector. The expression vector is then administered into the human body as the form of gene therapy for expression in the human cell, The term "genetic vectors" as used herein includes these expression vectors. Vectors that can be used for gene therapy includes. adeno-associated virus (Mizuno, M. et aL (1998). Jpn J Cancer Res 89, 76-80), LNSX vectors (Miller, A.D. et al. (1993) Methods Enzymol 217, 581-599) and lentivirus (Goldman, MJ. et al. (1997) Hum Gene Ther 8, 2261-2268).

Other vehicles for peptide delivery include expression vectors encoding the desired peptide that can be transferred into an organism which can replicate in the host organism to which it is desired to administer the peptide without significant detrimental effects on the health of the host organism. For example, the expression vectors may be transferred into an organism which is not pathogenic to the host organism to which it is desired to administer the peptide. In some embodiments the expression vector produces the desired peptide in a bacterial or fungal organism which does not have significant detrimental effects on the health of the host organism to which the peptide is to be administered. For example, the expression vector encoding the desired peptide may be an expression vector which produces the desired peptide in an organism such as lactic acid bacteria, E. coli, or yeast. In one embodiment, the expression vector produces the desired peptide in a microbe normally found in the mammalian gut or a microbe tolerated by the mammalian.digestive tract Some of the microbial species in which the desired peptide can be expressed include, but are not limited to, Lactobacillus species, such as L. acidophilus, L. amylovoru, L. casei, L. crispatus, L. gallinarum. L.

58 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:4PM FROM-A J PARK Clon +64 4 4723358 T-714 P.063/080 F-41 0 gasseri, L. johnsonii, L. paracasei, L. plantarum, L. Luteri, L. rhamnosus or C) others; Bifidobacterumn species, such as B. adolescentis.,B. aninalus, B. bifidum, B. breve, B. infantis, B. lactis, B. longum or others; Enterococcus faecalis or Ent.

facium; Sporolactobacillus inulinus; Bacillus subtilis or Bacillus cereus; 5 Escherichia col; Propionibacterium freudenreichii; or Saccharonoces cerevisiae C or Saccharomyces boulardii Nucleic acid sequences that encode the peptides of the present invention, chemically synthesized or produced by other means, including but not limited to the reverse transcription of mRNA to produce cDNA molecules, are incorporated 00 10 into expression vectors for gene transfer into the desired organisms by methods of genetic engeering familiar to those of skill in the art. The expression vectors may be DNA vectors or RNA vectors. For example, the expression vectors may be c based on plasmid or viral genetic elements. The expression vectors may be vectors Swhich replicate extra-chromosomally or vectors which integrate into the chromosome.

SThe expression vectors comprise-a promoter operably linked to a nucleic acid encoding a peptide of the present invention. The promoter may be a S regulatable promoter, such as.an induoible promoter, or a constitutive promoter In some embodiments, the promoter may be selected to provide a desired level, of peptide expression. In addition, if desired, the expression vectors may comprise other sequences to promote the production, presentation and/or secretion of peptides. In some embodiments a nucleic acid encoding a peptide of the present invention is operably linked to a nucleic acid sequence which directs the secretion of the peptide. 'For example, the nucleic acid encodixig the peptide of the present invention may be operably linked to a nucleic acid enoding a signal peptide.

In some embodiments, the expression vectors which are engineered to encode the peptides of the present invention may be expression vectors which are adapted for expressing the peptide of the present invention in a bacterial species that makes up the normal gut flora of mammals, such as Lactobacillus species and Bacillus subtilis Examples of such expression vectors can be found in US Patents No. 6,100,388, to Casas, and No. .5,728,571, to Bellini, respectively. These documents are hereby expressly incorporated by reference in their entireties. It will be appreciated that any expression vector which facilitates the expression of a peptide of the present invetion in an" organism which is not detrimental to the health of the host organism to which the peptide is to be administered may be used.

In some embodiments, the expression vectors which are engineered to encode the peptides of the present invention may be expression vectors which are adapted for expressing the peptide of the present invention in a yeast species that is well tolerated by the mammaliam gut such as Saccharomyces cerevisiae; or, preferably, Saccharomyces bouladii, which can colonize the human gut and is used to treat certain forms of diarrhea. Yeast expression vectors can be used that constitutively express heterologous proteins and peptides, are highly stable, thus are well transmitted to progeny cells during mitosis and meiosis and may comprise coding st.pnqrnr.n for a signal prptide nr npptidfif- that direct high levels of recombinant protein secretion. An example of such a yeast vector is given in TUS Patent No. 6,391,585, to Jang et al., which is hereby expressly incorporated by reference in its entirety.

59 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:47PM FROM-A J PARK Vton +64 4 4723358 T-T14 P-064/080 F-41 C'I The expression vectors encoding the peptides of the present invention may Z be introduced into the organism in which it is intended to .express the.peptides through techniques Imown in the art. These techniques include traditional methods 1 of transforming bacteria, yeast, or other microbes, through the use of chemically competent bacterial cells,electroporation or lithium acetate transfomation (for yeast), for example, as well as recent advances in the transformation of bacterial species recalcitrant to these procedures. In some embodiments, the expression vectors are introduced into lactic acid bacteria known to be recalcitrat to transfonnation using the method disclosed by Leer et at (WO 95/35389), the 0 10 disclosure of which is incorporated herein by reference in its entirety.. The 00 introduced sequences may be incorporated into microbial chromosomal DNA or may remain as xtrachromosomal DNA elements.

This genetically engineered microbe containing the expression vector can o then be inoculated into the alimentary canal, vagina, trachea etc. to achieve sustained immuno-therapy. In some embodiments, the organisms expressing the C' peptides of the present invention are ingested in an inactive form or, preferably, in live form. In the gut these microorganisms produce said peptides, release them into the lumen by secretion or by lysis of the microorganism or otherwise present the peptides to the host, whereby the peptides produce their intended effect upon the host organism. In other embodiments, peptides are presented to the host at the mucous membrane of the nasal passages, vagina or the small intestine.

Another method of the treatment is the use of liposomes as a means for delivering the specific nucleic acid to the cells in the human body. The nucleic acid (suih as an expression vector containing A nucleic sequence that encodes peptides of sequence ID No.1 to ID No.30) is delivered in an environment that encourages cellular uptake and chromosomal incorporation as described in Gao, X.

and Huang, L. (1995) Gene Ther 2, 710-722 and US 6,207,456. Alternatively, the peptide itself can be encapsulated in the liposome and delivered directly, using a method described in US6,245,427. All the scientific publications and patents indicated above are incorporated herein by reference.

The nucleic acid sequences useful for the above-mentioned gene therapy and method of treatment include sequences that code for these peptides and functional derivatives thereof. Any one of the numerous nucleic acid sequences may be used to code for these peptides and their derivatives based on the degenerate codon system.

The following references are incorporated herein by reference in their entirety.: 1. Principles of Pre-clinical Research of New Drugs, People's Republic of China.

1993,7:134-135 2. Shuyun Xu, Rulian Bian, Xiu Chen. Methodology of pharmacological upcinimeut. People's Health Publishing House. 1991, 1221-1234 3. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7:140 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:47PM FROM-iA J PARK ffton +64 4 4723358 T-714 P.065/080 F-41 c 4. Jinsheng He, Ruizhu Li, Tingyi Zong. 'The study on MTI reduction method of bl) testing NK cell activity. China Immunology Journal 1996, 356-358 Qian Wang. Moderm nedical experiment method. People's Health Publishing Hupl. 1990, 42-483 6. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China 1993, 7:141 7. Principle of now drug research in pre-clinic issued by Ministry of Health, People's Republic of China 1993, 7: 132-133 00 8. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7:128-129 C 9. Yuanpei Zhang, Huaide Su. Phamalogical experiment (secohd edition).

o People's Health Publishing House. 1998, 137-138 Jiatai Li, clinical pharmacology(second edition). People's Health Publishing c House. 1998, 1338-1339.

EXAMPLE 1 Delivery of pentides through enetically engineered Lactobacillus bacterial species The following is provided as one exemplary method to deliver peptides of this invention to a host as described above. A DNA sequence that encodes one of the peptides listed ii table A above is synthesized by chemical means and this DNA sequence is inserted into an expression vector using standard techniques of genetic engineering familiar to those skilled in the art. The expression vector selected contains a- constitutive promoter functional in Lactobacilli, a multiple cloning site for the introduction of DNA sequences in a specific 5' to 3' orientation as well as a selectable marker gene that confers resistance to an antibiotic (to aid in cloning procedures) and may comprise other sequences to assist in the production and/or secretion of the peptides, such as signal peptide sequences. An example of such a vector is provided by US Patent No. 5,592,908, to Favla, which is hereby incorporated by reference in its entirety.. Briefly, this patent discusses several known promoters that function in Lactbbacillus species, as well as a method for discovering novel promoters in said bacteria, any of which may be operably linked to a nucleio acid encoding a peptide of the present invention to express the peptide in Lactobacilli. A nucleic acid encoding -a signal, peptide, such as leptides comprising of 16 to 35 -mostly hydrophobic amino acids that are active in Lactobacillus lactis described in US Patent No. 5,529,908, cited above, is interposed between the promoter and the nucleic acid encoding the peptide of the present invention such that the the nucleic acid encoding the signal peptide is in frame vwith the nuoloio aoid.enooding the paptids of the present invention.

In addition to the coding sequence of, the peptide, the DNA sequence synthesized may comprise sequences to aid in the ligation and cloning of said DNA into the expression vector. For example, restriction enzyme recognition sites that correspond to ones found in the multiple cloning site of the vector can be incorporated into the synthesized DNA at the 5' and 3' ends of the sequence, so 61 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:48PM FROM-A J PARK gton +64 4 423358, T-T14 P.066/080 F-741 CI that the sequence can be cloned in proper orientation within the vector. Both the bO) vector and the synthesi2ed DNA are digested with the particular restriction enzymes, then purified. Ligation reactions with the vector and the synthesized DNA are followed by transformation into a suitable strain of B. Coli. The transformed bacteria are plated .on media containing the antibiotic to which the i C vector confers resistance. A colony of transformed bacteria is selected for growth cultures and plasmid preparation procedures; the presence of the synthesized DNA in the correct orientation is confirmed.

0This expression vector is then transformed into a bacterial host cell of a 03 10 Lactobacillus species, such as L. acidophilus. Transformed cells are selected for by virtue of the selectable marker found within the vector sequence and the secretion of the peptide may be verified by performing a western blot, performing C gel electrophoresis of peptides present in the growth medium or other standard 0techniques. A transformed colony of bacteria is chosen and used to prepare largescale cultures of the genetically engineered bacteria. A culture of the genetically C engineered bacteria expressing the desired peptide is grown up and at least a portion-thereof is administered to the alimentary canal, vagina, trachea or other area of the host organism in which the bacteria are able to replicate. If desired, the bacterial cultures can be treated in a variety of ways to produce a supplement for enteric consumption by the host These treatments include lyopbilization or other methods of preserving the bacteria, in addition to combining the bacteria with carrier agents, .such as solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of these agents to prepare supplements is well known in the art. For example, the bacteria can be used to make cultured milk products or other foodstuffs for human consumption, such that the organisin expressing the peptide colonizes the gut of the host organism. A number of different methods for incorporating specific strains of lactic acid bacteria into foodstuffs such as yogurt, kimchee, cheese and butter are disclosed in US Patent No. 6,036,952, to Oh, which is hereby incorporated by reference in its entirety.

Upon consuming the bacteria through one of any number of routes, the engineered organisms can colonize the gut and allow the presentation and/or absorption of the peptides of this invention via the mucosal layer of the gut.

EXAMPLE 2 Delivery of peptides through a genetically engineered form of Bacillus subtilis The following is provided as another exemplary method to deliver peptides of this invention to a host as described above. A DNA sequence that encodes one of the peptides listed in table A above.is synthesized by chemical means and this DNA sequence is inserted into an expression vector via techniques of genetic engineering, all techniques being known in the art. The.expression vector selected comprises a shuttle vector, such as pTZ18R (Pharmacia, Piscataway, NJ), capable of being propagated in both E. Coli and B. Subtilis and containing an antibiotic resistance gene for selecting colonies of transformed bacteria. This vector can contain a constitutive promoter active in B. subtilis, such as a promoter derived from the Sac B gene of B. subtilis as well as a nucleotide 62 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:48PM FROM-A J PARK Vton +64 4 4723358 T-714 P-06T/80 F-41

O

Ssequence encoding a signal or leader peptide active in B. subtilis that directs bJ) efficient export of expressed heterologous proteins from the bacterial cell. An example of such a vector is -disclosed in US Patent No. 6,268,169, to Fahnestock, the disclosure of which is incorporated herein by reference in its entirety. Briefly, as detailed above, the DNA encoding a peptide of this invention will be (C synthesized with restriction enzymes sites and/or other sequences to facilitate cloning of the DNA through techniques familiar to those with skill in the art. After transformation into E. Colt, plating, selection and propagation of the plasmid to create a plasmid stock, the plasmid is then be transfomned into B, subtilis and transfonnants are selected by virtue of resistance to an antibiotic in the plating 00 media.

Peptide production in and secretion from the genetically engineered B.

C subtilis is verified using techniques well known to those with skill in the art, such as radiolabeling of peptides for autoradiographic detection after SDS-PAGE 0 15 anaylsis or Western blotting.

NC A culture of genetically engineered bacteria is grown up and at least a portion thereof is administered to the alimentary canal, vagina, trachea or other area of the host organism in which the bacteria are able to replicate.

EXAMPLE 3 Delivery of peptides through genetically engineered Saccharomyces yeast species The foll6wing is provided as another exemplary method to deliver peptides of this invention to a host as described above. A DNA sequence that encodes one of the peptides listed in table A above is synthesized by chemical means and this DNA sequence is inserted into an expression vector via techniques of genetic engineering, all techniques being known in the art. The expression vector selected comprises a stably maintained yeast protein expression vector, comprising a constitutive yeast promoter such as pADH1, sites for replication of the vector in both yeast and Coli, a gene or genes that confer'prototrophy to ain auxotrophic yeast mutant for selection purposes, a multiple cloning site (MCS) and, if desired, sequences that code for a signal peptide. Vectors such as this are commercially available and well known in the art or can be readily constructed using standard techniques After insertion of the synthesized DNA into the yeast vector, transformation into E. Coli, plating of transformed E. Cdli onto selective media,' selection of a transfonred bacterial colony and preparation ofplasmid DNA from a growth culture of bacteria from said colony, the vector is transformed into Saccharomyces cerevisiae via well-known techniques such as lithium acetate transformation or electroporation. The strainof Saccharomyces cerevisiae selected for transformation is a mutant auxotrophic strain that will require a gene on the plasmid in order to grow on minimal media plates. Transformed yeast colonies are isolated by plating the yeast on growth media lacking the gene provided on the vector. Only those yeast that have received the vector and its selective gene and are expressing that gene product will be able to grow into colonies on the minimal media. Verification of peptide secretion can be obtained by performing a Western blot, performing gel electrophoresis of peptides present in the growth medium or other standard techniques.

63 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:49PM FROM-A J PARK gton +64 4 4723358 T-714 P.068/080 F-741 0 C A transformed colony of yeast is chosen and used to prepare large scale OS) cultures. A culture of the genetically engineered yeast expressing the desired Speptide is grown up and at least a portion thereof is administered to the alimentary canal, vagina, trachea or other area of the host organism in which the bacteria are able to replicate. If desired, the yeast cultures can be treated in a variety of ways to ri, produce a supplement for enteric consumption by the host These treatments include lyophilization or other methods of preserving yeast, in addition to combining the bacteria with carrier agents, such as solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of these agents to prepare supplements is well known in the art. In another embodiment, the transformed 00 yeast are used in the creation of food products, such as fermented milk products like yogurt and kefir, by techniques known to those skidlled in the art As with live lactic acid bacterial cultures in these foodstuffs, the transformed yeast colonize the gut at least transiently and serve to present peptides to the host via the gat lumen.

0 64 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24

Claims (17)

1. 24-08-2007 03:49PM FROM-A J PARK Klton +64 4 4723358 T-714 P.06/080 F-41 0 WHAT WE CLAIM IS: ci S1. A substantially pure peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30. C, 2. A substantially pure peptide according to claim 1 wherein said peptide modulates at least one of the following conditions: immune activity, hepatitis infection, hepatitis B infection, the extent of nephritis and the growth of a cancer. 00 3. A substantially pure peptide according to claim 1 wherein said peptide modulates at least one of the following activities: T lymphocyte transformation, cytotoxic activity of NK C- cells, secretion of IL-2 by T lymphocytes, secretion of IFN by T lymphocytes, synthesis o of anti-SRBC antibody upon antigenic challenge, phagocytotic activity of mononuclear Ophagocyte, the weight of thymus gland, and the weight of spleen. 4. A substantially pure peptide consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30 A substantially pure peptide according to claim 4 wherein said peptide modulates at least one of the following conditions: immune activity, hepatitis infection, hepatitis B infection, the extent of nephritis and the growth of a cancer. 6- A substantially pure peptide according to claim 4 wherein said peptide modulates at least one of the following activities: T lymphocyte transformation, cytotoxic activity of NK cells, secretion of IL-2 by T lymphocytes, secretion of IFN by T lymphocytes, synthesis of anti-SRBC antibody upon antigenic challenge, phagocytotic activity of mononaclear phagocyte, the weight of thymus gland, and the weight of spleen. 7. A substantially pure peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30. 8- A substantially pure peptide according to claim 7 wherein said peptide modulates at least one of the following conditions: immune activity, hepatitis infection, hepatitis B infection, the extent of nephritis and the growth of a cancer. 9. A substantially pure peptide according to claim 7 wherein said peptide modulates at least one of the following activities: T lymphocyte transformation, cytotoxic activity of NK cells, secretion of IL-2 by T lymphocytes, secretion of IFN by T lymphocytes, synthesis of anti-SRBC antibody upon antigenic challenge, phagocytotic activity of mononuclear phagocyte, the weight of thymus gland, and the weight of spleen. A substantially pure peptide comprising a functional derivative of a biologically active peptide, said biologically active peptide having the amino acid sequence of selected from the group consisting of SEQ ID NOs. 1-15 and 17-30. COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:49PM FROM-A J PARK ffton +64 4 4T23358 T-14 P.070/080 F-741 o 11. A substantially pure peptide according to claim 10 wherein said peptide modulates at least Cl one of the following conditions: immune activity, hepatitis infection, hepatitis B infection, 0.0 the extent of nephritis, and the growth of a cancer. <4 12. A substantially pure peptide consisting essentially of a functional derivative of a biologically active peptide, said biologically active peptide having the amino acid sequence C' 1 selected from the group consisting of SEQ ID NOs. 1-15 and 17-30. 13. A substantially pure peptide according to claim 12 wherein said peptide modulates at least one of the following conditions: immune activity, hepatitis infection, hepatitis B infection, 00 the extent of nephritis, and the growth of a cancer. 14. A substantially pure peptide consisting of a functional derivative of a biologically active peptide, said biologically active peptide having the amino acid sequence selected from the Sgroup consisting of SEQ ID NOs. 1-15 and 17-30. CN 15. A substantially pure peptide according to claim 14 wherein said peptide modulates at least one of the following conditions: immune activity, hepatitis infection, hepatitis B infection, the extent of nephritis, and the growth of a cancer. 16. A genetic vector comprising a nucleotide sequence encoding a peptide consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30. 17. A vector according to claim 16.wherein said peptide modulates at least one ofthe following conditions: immune activity, hepatitis infection, hepatitis B infection, the extent of nephritis, and the growth of a cancer- 18. A genetic vector comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30. 19. A genetic vector comprising a nucleotide sequence encoding a peptide consisting essentially of a functional derivative of a biologically active peptide, said biologically active peptide having the amino acid sequence selected from the group consisting of SEQ D NOs. 1-15 and 17-30. A genetic vector comprising a nucleotide sequence encoding a peptide consisting of a functional derivative of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30. 21. A micro-organism having a genetic composition comprising a nucleic acid sequence encoding a peptide, said peptide comprising a functional derivative of a biologically active peptide, said biologically active peptide having the amino acid sequence selected from the. group consisting of SEQ ID NOs. 1-15 and 17-30. 22. A pharmaceutical composition comprising a substantially pure peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30 admixed with a pharmaceutically acceptable carrier. 66 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:50PM FROM-A J PARK gton +64 4 4T23358 T-714 P.071/080 F-741 o 23. A pharmaceutical composition comprising a substantially Pure peptide consisting Cl essentially of the amino acid sequence selected from the group consisting of SEQ ID NOs- 0.0 1-15 and 17-30 admixed with a pharmaceutically acceptable carier. 24. A pharmaceutical composition comprising a substantially pure peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs- 1-15 and 17-30 Cl admixed with a pharmaceutically acceptable carrier. A pharnaceutical composition comprising a substantially pure peptide comprising a a>-functional derivative of a biologically active peptide, said biologically active peptide 00 having the amino acid sequence selected from the group consisting of SEQ IDI NO0& 1-15 and 17-30 admixed with a pharmaceutically acceptable carrier.
2. 26. A pharmaceutical composition comprising a substantially pure peptide consisting essentially of a functional derivative of a biologically active peptide, said biologically Cl active peptide having the amino acid sequence selected from the group consisting of SEQ ID) NOs. 1-15 and 17-30 admixed with a pharmaceutically acceptable canier.
3. 27. A pharmaceutical composition comprising a substantially pure peptide consisting of a functional derivative of a biologically active peptide, said biologically active peptide having the amino acid sequence selected from the group consisting of SEQ ID) NOs. 1-15 and 17-30 admixed with a pharmaceutically acceptable carrier.
4. 28. A method of making a pharmtaceutical composition comprising providing a substantially pure peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs- 1-15 and 17-30; and mixing said substantially pure peptide with a pharmaceutically acceptable carrier.
5. 29. A method of making a pharmaceutical composition comprising providing a substantially pure peptide consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30; and mixing said substantially pure peptide with a pharmaceutically acceptable carrier. A method of making a phannaceutical composition comprising providing a substantially pure peptide consisting of the amino acid sequence selected from the group consisting of SEQ IEl 140s. 1-15 and 17-30; and mixing said substantially pure peptide with a pharmaceutically acceptable carrier. 3 1. A method of making a pharmaceutical composition comprising providing a substantially pure peptide comprising the amino acid sequence selected from the group consisting of SEQ MD NOs. 1-15 and 17-30.
6. 32. A method of making a pharnaceutical composition comprising providing a substantially pure peptide consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-3 0. 67 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:50PM FROM-A J PARK ffton +64 4 4723358 T-T14 P.072/080 F-741 o 33- A method of making a pharmaceutical composition comprising providing a substantially C'l pure peptide consisting of the amino acid sequence selected from the group consisting of SEQ IDNOs. 1-15 and 17-30. <1 34. A method of making a pharmaceutical composition comprising providing a substantially pure peptide comprising an amino acid sequence which is a functional derivative of a C biologically active peptide, said biologically active peptide having the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30; and mixing said substantially pure peptide with a pharmaceutically acceptable carrier. 00 35. A method of making a pharmaceutical composition comprising providing a substantially pure peptide consisting essentially of an amino acid sequence which is a functional derivative of a biologically active peptide, said biologically active peptide having the Oamino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30. C' 36- Use of a substantially pure peptide comprising the amino acid sequence selected from the group consisting of SEQ I) NOs. 1-15 and 17-30 in the manufacture of a medicament for the modulation of at least one of the following conditions: immune activity, hepatitis infection, hepatitis B infection, the extent of nephritis, and the growth of a cancer.
7. 37. Use of a substantially pure peptide comprising the amino acid sequence selected from the group consisting of SEQ TID NOs. 1-15 and 17-30 in the manufacture of a medicament for the modulation of at least one of the following activities: T lymphocyte transformation, cytotoxic activity of IK cells, secretion of IL-2 by T lymphocytes, secretion of EFN by T lymphocytes, synthesis of anti-SRBC antibody upon antigenic challenge, phagocytotic activity of mononuclear phagocyte, the weight of thymus gland, and the weight of spleen.
8. 38. Use of a substantially pure peptide consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30 in the manufacture of a medicament for the modulation of at least one of the following conditions: immune activity, hepatitis infection, hepatitis B infection, the extent of nephritis, and the growth of a cancer.
9. 39. Use of a substantially pure peptide consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID NOs. l-15 and 17-30 in the manufacture of a medicament for the modulation of at least one of the following activities: T lymphocyte transformation, cytotoxic activity of NKl cells, secretion of IL-2 by T lymphocytes, secretion of IFN by T lymphocytes, synthesis of anti-SRBC antibody upon antigenic challenge, phagoeytotic activity of mononuclear phagocyte, the weight of thymus gland, and the weight of spleen- Use of a substantially pure peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30 in the manufacture of a medicament 68 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:SPM FROM-A J PARK Vton +64 4 4T23358 T-714 P.073/080 F-41 o for the modulation of at least one of the following conditions: immune activity, hepatitis CN infection, hepatitis B infection, the extent of nephritis, and the growth of a cancer.
10. 41. Use of a substantially pure peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30 in the manufacture of a medicament for the modulation of at least one of the following activities: T lymphocyte transformation, Cl cytotoxic activity of NK cells, secretion of IL-2 by T lymphocytes, secretion of IFN by T lymphocytes, synthesis of anti-SRBC antibody upon antigenic challenge, phagocyrotic 0activity of mononuclear phagocyte, the weight of thymus gland, and the weight of spleen. 00 42. Use of a substantially pure peptide comprising a functional derivative of a biologically Sactive peptide, said biologically active peptide having the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-15 and 17-30 in the manufacture of a 0 medicament for the modulation of at least one of the following conditions: immune S activity, hepatitis infection, hepatitis B infection, the extent of nephritis, and the growth of a cancer.
11. 43. A substantially pure peptide according to any of claims 1, 4, 7, 10, 12, 14, substantially as herein described with reference to any example thereof.
12. 44. A genetic vector according to any of claims 16, 18, 19, 20, substantially as herein described with reference to any example thereof. A micro-organism according to claim 21, substantially as herein described with reference to any example thereof.
13. 46. A pharmaceutical composition according to any of claims 22-27, substantially as herein described with reference to any example thereof-
14. 47. A method according to any of claims 28-35, substantially as herein described with reference to any example thereof.
15. 48. A use according to any of claims 36-42, substantially as herein described with reference to any example thereof.
16. 49. A pharmaceutical composition when made by a method according to any of claims 28-35 or 47. 69 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:51PM FROM-A J PARK gton +64 4 4723358 T-714 P.074/080 F-741 PCT/GB02/03203 WO 03/006492 ^to ;Zl WILKG3.001CP1 SEQUENCE LISTING <110> Wong, Wai Ming Lin, Gang <120> BIOLOGICALLY ACTIVE PEPTIDES <130> WILKG3.001CP1 <140> unknown <141> 2002-06-20 <150> 09/904,492 <151> 2001-07-13 <160> <170> FastSEQ for Windows Version <210> 1 <211> <212> PRT <213> Sus scrofa <400> 1 Pro Thr Thr Lys Thr 1 5 Tyr Phe Pro His Phe <210> 2 <211> 9 <212> PRT <213> Sus scrofa <400> 2 Val Val Tyr Pro Trp Thr Gin Arg Phe 1 <210> 3 <211> 13 <212> PRT <213> Sus scrofa <400> 3 Lys Ala Val Gly His Leu Asp Asp Leu 1 5 Pro Gly Ala Leu <210> 4 Page 1 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:51PM FROM-A J PARK ton +64 4 4723358 T-714 P.075/080 F-T41 o WLKG3.001CP1 C( <211> 18 o <212> PRT <213> Sus scrofa <400> 4 Val Ala Pro Glu Glu His Pro Thr Leu Leu Thr Glu Ala Pro Leu Asn S 1 5 10 Pro Lys 00 <210> <211> 13 C <212> PRT <213> Sus scrofa C <400> C( Leu Gly Met Glu Ala Cys Gly Ile His Glu Thr Thr Tyr 1 5 <210> 6 <211> 11 <212> PRT <213> Sus scrofa S<400> 6- Leu Arg Val Ala Pro Glu Glu His Pro Val Leu 1 5 <210> 7 <211> 12 <212> PRT <213> Sus scrofa <400> 7 Ala Ala His His Pro Asp Asp Phe Asn Pro Ser Val, 1 5 <210> 8 <211> 13 <212> PRT <213> Sus scrofa <400> 8 Pro Ser Ile Val Gly Arg Pro Arg His Gin Gly Val Met 1 5 <210> 9 Page 2 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:51PM FROM-A J PARK ton +64 4 4723358 T-714 P.076/080 F-741 0 o WILKG3.001CP1 CI <211> 13 O <212> PRT S<213> Sus scrofa <400> 9 Ci Ile Gly Met Glu Ser Ala Gly Ile His Glu Thr Thr Tyr 1 5 <210> 00 <211> 9 <212> PRT <213> Sus scrofa <400> o Val Gly Met Gly Glu Lys Asp Ser Tyr 1 <210> 11 <211> 9 <212> PRT <213> Sus scrofa <400> 11 Val Gly Met Gly Gin Lys Asp Ser Tyr 1 <210> 12 <211> <212> PRT <213> Sus scrofa <400> 12 Val Gly Met Gly Gin Lys Asp Ser Tyr Val 1 5 <210> 13 <211> <212> PRT <213> Sus scrofa <400> 13 Met Ala Thr Ala Ala Ser Ser Ser Ser Leu 1 5 <210> 14 <211> 3 <212> PRT Page 3 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:52PM FROM-A J PARK gton +64 4 4723358 T-714 P.077/080 F-741 O WILKG3.001CP1 CI <213> Sus scrofa <400> 14 Tyr Ser Phe <210> <211> 3 <212> PRT <213> Sus scrofa 00 <400> Ala Ala Phe C<210> 16 <211> 3 <212> FRT <213> Sus scrofa <400> 16 Tyr Ser Leu 1 <210> 17 <211> 7 <212> PRT <213> Sus scrofa <400> 17 Thr Thr Tyr Asn Set Ile Met 1 <210> 18 <211> <212> 2RT <213> Sus scrofa <400> 18 Phe Glu Glu Asn Met 1 <210> 19 <211> <212> PRT <213> Sus scrofa Page 4 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date
17. 2007-08-24 24-08-2007 03:52PM FROM-A J PARK gton +64 4 4723358 T-714 P.078/080 F-741 0 o WILKG3.001CP1 S <400> 19 Phe Glu Pro Ser Phe 1 C <210> <211> 4 <212> PRT <213> Sus scrofa 00 <400> Phe Asn Glu Glu 1 o <210> 21 <211> 4 Cl <212> PRT <213> Sus scrofa <400> 21 Phe Glu Glu Met 1 <210> 22 <211> 4 <212> PRT <213> Sus scrofa <400> 22 Phe Glu Glu Glu 1 <210> 23 <211> 4 <212> PRT <213> Sus scrofa <400> 23 Phe Glu Ser Phe 1 <210> 24 <211> 4 <212> PRT <213> Sus scrofa <400> 24 Pro Glu Asn Phe Page COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-2007 03:52PM FROM-A J PARK gton +64 4 4723358 T-714 P.079/080 F-741 1 0 o WILKG3-001CFI <210> <211> 4 C <212> PRT <213> Sus scrofa <400> Phe Val Asn Asp. 00 1 C' <210> 26 <211> 0 <212> PRT 0 <213> Sus scrofa <400> 26 Phe Gin Pro Ser Phe 1 <210> 27 <211> 6 <212> PRT <213> Sus scrofa <400> 27 Phe Asn Phe Val Pro Pro 1 <210> 28 <211> <212> PRT <213> Sus scrofa <400> 28 Ala Gly Asp Asp Ala Pro Arg Ala Val Phe 1 5 <210> 29 <211> 11 <212> PRT <213> Sus scrofa <400> 29 Leu Arg Val Ala Pro Glu Glu His Pro Thr Leu 1 5 Page 6 COMS ID No: ARCS-158250 Received by IP Australia: Time 14:03 Date 2007-08-24 24-08-200T 03:52PM FROM-A J PARK ga 8 735 gton +64 4 4T23358 T-714 P.080/080 F-741 o WILKG3.- D1lPi <210> ;Z <211> <212> PRT <213> Sus scrot& <400> Arg Val Ala Pro Gin Glu His Pro Thr Leu 00 Page 7 C-OMS ID No: ARCS-i 58250 Received by IP Australia: Time 14:03 Date 2007-08-24