WO2012006634A2 - Prostate specific antigen (psa) peptide therapy - Google Patents

Abstract

The present invention is directed to compositions and methods for treating prostate cancer.

Description

PROSTATE SPECIFIC ANTIGEN (PSA) PEPTIDE THERAPY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of US Provisional Patent Application Serial No. 61/363,091, filed July 9, 2010, and US Provisional Patent Application Serial No.

61/426,321, filed December 22, 2010, the disclosures of which are incorporated herein in their entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grant numbers DAMD17-98- 1-8489 awarded by U.S. Army Medical Research and Material Command, and CA088062 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention is directed to compositions and methods for treating prostate cancer.

BACKGROUND OF THE INVENTION

[0004] Prostate cancer (CaP) is the second leading cause of cancer-related mortality in the United States. There were approximately 27,360 deaths caused by CaP in 2009 [Jemal et ah, CA: A Cancer Journal for Clinicians, 59(4): 225-249 (2009)]. Patients who recur after primary ablative therapy respond transiently to androgen deprivation therapy but subsequently progress to hormone-refractory disease for which curative systemic therapies are lacking [Pronzato et al. , Annals of Oncology, 16(Suppl. 4) iv: 80-84 (2005)]. Recent studies have demonstrated that overall survival (OS) of patients with hormone refractory CaP can be modestly extended by vaccination with autologous dendritic cells (DC) loaded with recombinant proteins consisting of granulocyte macrophage colony stimulating factor and prostatic acid phosphatase [Kantoff et ah, Genitourinary Cancers Symposium, American Society of Clinical Oncology, Abstract #8 (2010)]. It is widely assumed that improved outcomes might be achieved by vaccinating patients at earlier points in the development of their disease at a time when host immune effector mechanisms remain robust. [0005] Prostate specific antigen (PSA) contains a Human Leukocyte Antigen (HLA)-A2- restricted epitope, PSA146-154, that is an attractive candidate for specific immunotherapy of HLA-A2+ patients with CaP [Xue et al, Prostate 30: 73-78 (1997); Perambakam et al, Cancer Immunology Immunotherapy 51(5) 263-70 (2002)]. The safety and immunogenicity of PSA146- 154 peptide vaccination in patients with metastatic, hormone-sensitive CaP or disease that is at high risk of recurrence on the basis of tumor stage, serum PSA levels and Gleason score has been previously reported [Perambakam et ah, Cancer Immunology Immunotherapy 55(9): 1033-1042 (2006)]. Herein, we report the clinical outcome of patients up to eight years following vaccination and correlate patients' survival with their immunological responses to the PSA146- 154 vaccine.

[0006] Specific T-cell responses, defined by PSA146-154 peptide :tetramer staining and IFN-γ release assays were quantified in pre- and post- vaccine peripheral blood mononuclear cells (PBMC) and correlated with clinical parameters including, biochemical progression and OS. In addition, microarray whole human gene expression analysis was conducted to identify differentially expressed genes and gene pathways in pre-vaccination PBMC that distinguish strong immune responders from non-responders.

SUMMARY OF THE INVENTION

[0007] The FDA's approval of Sipuleucel-T for the treatment of advanced prostate cancer was a landmark event. The studies leading to the approval of Sipuleucel as well as suggestive results from other prostate cancer vaccine protocols, notably Prostvac® and GVAX, indicates that the clinical course of prostate cancer can be favorably altered by immunotherapeutic manipulation. The present disclosure thus provides compositions/vaccines for the treatment of prostate cancer.

[0008] Accordingly, in one aspect the present disclosure provides a composition comprising a fragment of prostate specific antigen (PSA), said fragment peptide comprising the sequence VISNDVCAQVHPQKVTKFML (SEQ ID NO: 1). In another aspect, a composition is provided comprising a fragment of prostate specific antigen (PSA), said fragment peptide comprising the sequence C ALPERPS LYTKV VH YRKWIK (SEQ ID NO: 2).

[0009] In an embodiment, the disclosure provides a method for treating prostate cancer in a human comprising the step of administering a composition of the disclosure to said human in an amount effective to stabilize or reduce serum PSA levels. In some aspects, methods of the disclosure further comprise administrating granulocyte monocyte colony stimulating factor (GM- CSF). In these aspects, said composition and GM-CSF are co-administered, and in further embodiments said composition and GM-CSF are administered concurrently while in still further embodiments said composition and GM-CSF are administered sequentially. In some aspects, said PSA peptide and GM-CSF are co-administered in a weight-to-weight ratio of about 1:5.

[0010] The disclosure also provides embodiments in which the PSA peptide and GM-CSF are co-administered in multiple injections. In some of these aspects, PSA peptide and GM-CSF are co-administered in up to five injections.

[0011] In some embodiments, the PSA peptide is administered as a composition of dendritic cells pulsed with the PSA peptide. In various aspects, a total of about 100 μg PSA peptide is administered in multiple injections.

[0012] Administration of a composition or vaccine of the disclosure is, in various aspects, intradermal.

[0013] Thus, the disclosure also provides a vaccine comprising: (i) a composition selected from the group consisting of the composition of claim 1, the composition of claim 2 and a composition comprising a prostate specific antigen (PSA) peptide (PSA peptide 146-154; SEQ ID NO: 3), or combinations thereof, and (ii) a pharmaceutically acceptable carrier. In some aspects, the vaccine further comprises granulocyte monocyte colony stimulating factor (GM- CSF). In further aspects, the vaccine further comprises a TLR9 agonist in an amount effective to increase a T cell immune response. In one specific aspect, the TLR9 agonist is a CpG- oligodeoxynucleotide (CpG-ODN).

[0014] In further embodiments, the vaccine further comprises an inhibitor of CTLA4 in an amount effective to increase a T cell immune response, and in a specific aspect the inhibitor of CTLA4 is a monoclonal antibody.

[0015] In additional embodiments, the vaccine further comprises an inhibitor of PD-1 in an amount effective to increase a T cell immune response. In a specific aspect, the inhibitor of PD- 1 is a monoclonal antibody.

[0016] The disclosure also provides a method of vaccinating an individual comprising the step of administering a vaccine of the disclosure to the individual in an amount effective to vaccinate the individual. In some aspects, the PSA peptide is co-administered with GM-CSF, and in further aspects the PSA peptide and GM-CSF are co-administered in multiple injections. In further aspects, the PSA peptide and GM-CSF are administered concurrently while in yet further aspects the PSA peptide and GM-CSF are administered sequentially. In one aspect, the PSA peptide and GM-CSF are co-administered in up to five injections. In a further aspect, a total of about 100 μg PSA peptide is administered in multiple injections.

[0017] In one embodiment, the PSA peptide is administered in weeks 1, 4 and 10, and then every six months up to four years. In another embodiment, the PSA peptide is administered in weeks 1, 4 and 10, and then every six months up to four years, wherein an inhibitor of CTLA4 is administered in weeks 1, 4 and 10, and then every eight weeks up until week 52. In a further embodiment, the PSA peptide is administered in weeks 1, 4 and 10, and then every six months up to four years, wherein an inhibitor of PD-1 is administered in weeks 1, 4 and 10, and then every eight weeks up until week 52.

[0018] The disclosure also provides a method of identifying a patient that is a candidate for prostate cancer therapy comprising the step of: measuring expression level in a sample from a test individual of one or more genes identified in Table 1 or Table 2 relative to a reference expression level, wherein an increase or a decrease in expression of the genes identified in Table 1 or Table 2 relative to the reference expression level is determinative for identifying whether the patient is a candidate for prostate cancer therapy according to a method of the disclosure. In an aspect, the method further comprises collecting the sample from the test individual. In another aspect, the method further comprises comparing the expression level to the reference expression level.

[0019] Accordingly, in some aspects, an increase in expression of a gene selected from the group consisting of 2'-5' oligoadenylate synthetase 1 (OAS1), mitogen- activated protein kinase 1 (MAPK1), Sh2 domain containing IB (SH2D1B), vannin 1 (VNN1), CD58 molecule (CD58), DEAD box polypeptide 58 (DDX58), X-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4) and interferon-induced transmembrane protein-3 (IFITM3) is indicative of the patient being a candidate for prostate cancer therapy according to a method of the disclosure. [0020] In further aspects, a decrease in expression of a gene selected from the group consisting of tumor necrosis factor receptor superfamily-member 25 (TNFRSF-25), chemokine C-C motif receptor 7 (CCR7), and phosphoinositide-3-kinase, regulatory subunit 1 alpha (PIK3R1) and epiregulin (EREG) is indicative of the patient being a candidate for prostate cancer therapy according to a method of the disclosure.

[0021] In another embodiment, the disclosure provides a method of rendering an individual a candidate for prostate cancer therapy comprising the steps of: (i) modulating expression of at least one gene listed in Table 1 to a degree that renders the individual a candidate for prostate cancer therapy; and (ii) administering a therapeutically effective amount of a composition or vaccine of the disclosure to said patient to treat prostate cancer.. In some aspects, modulating increases expression and in one specific aspect the gene is 2'-5' oligoadenylate synthetase 1 (OAS1). In further aspects, modulating decreases expression

Table 1 , Genes that discriminate among immune responders versus non responders,

238577 s teashirt zinc finger

4.12E-04 1 0,0079365 24 39,97 0,6 Info TSHZ2

at homeobox 2

RNF144 ring finger protein

4.42E-04 1 0,015873 71.93 37.56 1.92 235549 at Info

1 144B

MANIC mannosidase, alpha,

4.58E-04 1 0,015873 87,73 130,55 0,67 214180 at Info

i class 1C, member 1

1552634 a zinc finger protein

4.66E-04 1 0.0079365 302.66 513,53 0,59 Info 2NF101

jt 101

220545 s testis-specific serine

4.94E-04 1 0,0079365 29,78 16,73 1,78 Info TSKS

I kinase substrate

211721 s zinc finger protein

5.43E-04 1 0,0079365 81,07 134,14 0,6 Info Z F551

at 551

2'5'-oligoadenvlate

205552 s

5.82E-04 1 0,0079365 836,67 337,13 2,48 Info 0AS1 synthetase E

I 40/46kDa cysteine conjugate-

5.93E-04 1 0,0079365 251,05 404,66 0,62 227748 at info CCBL2

beta lyase 2

6.02E-04 1 0,015873 149,5 96,59 1.55 21S7&9 3t Info u latexin metastasis suppressor

6.17E-04 1 0.015873 : 42,28 28.51 1.48 210359 at !nfo TSS1

1

208078 s

6.32E-04 1 0.0079365 718,82 1067,28 0,67 Wo 1 NA

S 210281 s zinc finger, MYM-

6.44E-04 1 0,0079365 275,9 380,02 0,73 Info ZMYM2

i type 2

6.69E-04 1 0,0079365 374.69 838,19 0,45 235213 at info NA

7.14E-04 1 0,0079365 90,55 167,69 0,54 1S66448 at Info CD6 CD6 molecule

tumor necrosis factor

211282 x TNFRSF

S.87E-04 1 0,015873 222,66 324,87 0,69 receptor supeifamily, at Info

25

member 25

204005 s

9.59E-04 1 0.015873 4035,16 1964,19 2,05 info NA

I

LINE-l type

9.75E-04 1 0,0079365 70,75 23,39 3,02 219955 at Info L1TD1 transposase domain containing 1 mitochondrial

224331 s MRP13

9.95E-04 1 0,0079365 694,23 529,76 1,31 Info ribosomal protein

at

L36

213526 s lin-37 homolog (C.

E03E-03 1 0,015873 236,62 177,09 1,34 _!

at elegans)

1554340 a ClorflS chromosome 1 open

1.07E-03 1 0,015873 53,64 23.45 2,29 Info 7

1 I reading frame 187 2',5'-oligoadenylate

1.09E-O3 1 0.0079365 1408,76 348.23 4,05 202869 at s synthetase 1,

40/46kDa

family with sequence

231396 s FA 12

Ι,ΠΕ-03 1 0.015873 76.34 52,66 1,45 info similarity 126,

1 member A

225884 s GDNF-inducible zinc

1.13E-03 1 0.015873 345,08 538.14 0,64 Info Curl

finger protein 1

POU class 6

1J4E-03 1 0,0079365 45.88 61,72 0,74 229809 at info P0U6F1

homeobox 1 microtubule associated

L20E-03 1 0.0079365 66.51 106.51 0,62 225611 at info AST4 serine/threonine kinase family member 4

protein tyrosine

200732 s

1.28E-03 1 0,015873 848,3 1377,88 0,62 Info PTP4A1 phosphatase type

at

IVA, member 1

CHU C churchill domain

L31E-03 1 0.015873 1259,08 678.25 1,86 223210 at

I continuing 1 1.34E-03 1 0,0079365 301.79 448,79 0,67 2304S9 at Info 1 NA

210360 s metastasis suppressor

1.34E-03 1 0,0238095 91.06 55,47 1,64 Info TSSl

i 1

1.38E-03 1 0.0079365 90,94 205,89 0,44 215592 at info NA

SH2 domain

L39E-03 1 0,0238095 178.76 93.07 1,92 1553176 at If* containing IB low density

221790 s LDLRAP

L42E-03 1 0,0079365 409,74 597.37 0,69 lipoprotein receptor

It i adaptor protein 1 ribosomal protein

L45E-03 1 0,015873 159,23 483,35 0,33 227722 at info PS23

S23

203037 s metastasis suppressor

1.48E-03 1 0,0238095 2738.12 1420,45 1,93 info MTSSl

I 1

1558508 a chromosome 1 open

1.49E-03 1 0,0079365 29.54 18,28 1,62 info Clorf53

1 reading frame 53

L54E-03 1 0,0079365 364.23 133,07 2,74 205844 at iOii! VNN1 vanin 1

zymogen granule

1.54E-03 1 0.015873 107,04 64.92 1,65 228058 at ipfo ZG16B protein 16 homolog B

(rat) ADP-ribosylation

214483 s

1.54E-03 1 0,0079365 117,12 63,86 1.83 Info ARFIPl factor interacting at protein 1

200856 x

1.56E-03 1 0.015873 139.43 103,4 1,35 Info 1 NA

at

1568978 s Cllorf2 chromosome 11 open

1.64E-03 1 0,0079365 128.59 89,15 1,44 info

A I reading frame 21

1.67E-03 1 0,015873 73.74 57,49 1,28 222094 at info 1 NA

1.70E-03 1 0,0079365 7.31 9.22 0.79 239155 at Info M NA

1.73E-03 1 0,0238095 472.99 56,68 8.34 206834 at Info HBD hemoglobin, delta

phosphoribosyl

1.79E-03 1 0,0079365 522,48 324.75 1.61 230352 at info PRPS2 pyrophosphate synthetase 2

GRB10 interacting

1.94E-03 1 0,0079365 15,27 11,03 1,38 1558305 at Info GIGYF2

GYF protein 2 family with sequence

221880 s FA 17

2.03E-03 1 0,0079365 35.45 0,76 info similarity 174,

it 1 member B

2.06E-03 1 0,0079365 9.78 12,39 0,79 243855 at Info N NA

tumor necrosis factor

211841 s T FRSF

2.10E-03 1 0.015873 153,73 236,01 0,65 Info receptor superfamily,

at

member 25

mm transmembrane

2.13E-03 1 0,015873 58,87 40.13 1,47 230647 at Info

I protein 53 leucine rich repeat (in

2.14E-03 1 0.0079365 259,79 189.98 1.37 218364 at info LRRFIP2 FLU) interacting protein 2

C14orf6 chromosome 14 open

2.17E-03 1 0.0079365 43,36 66,18 0,66 1564211 at Info

4 reading frame 64

nuclear receptor

2.18E-03 1 0,0079365 224,82 610,54 0.37 209750 at Info NR1D2 subfamily 1, group D, member 2

1552264 a mitogen-activated

2.45E-03 1 0,015873 902,08 657.71 1,37 info MAPK1

A protein kinase 1 mm s cytochrome b

2.49E-03 1 0.015873 123,26 62,52 1,97 Info CYBRD1

at reductase 1

222157 s WD repeat domain

2.53E-03 1 0.0079365 293,85 198.79 1,48 WDR48

48 serine/threonine-

2.55E-03 1 0.015873 32.07 45.19 0,71 1554929 at QSJ

protein kinase QS chemokine (C-C

2.56E-03 1 0,0238095 750.18 1449.8 0,52 206337 at Info CC 7

motif) receptor 7

222680 s denticleless homolog

2.60E-03 1 0.0079365 23.29 17.81 1,31 Info

at E (Drosophila)

200965 s actin binding L

2.61E-03 1 0,0079365 904,27 1492,2 0,61 info ABLI l

I protein 1

caspase 5, apoptosis-

2.67E-03 1 0,0079365 205.99 99.24 2,08 207500 at Info CASP5 related cysteine peptidase myelodysplasia

1562731 s syndrome 2

76 2.67E-03 1 0,0238095 113,92 188,55 0,6 Info MDS2

J translocation associated

77 2.69E-03 1 0,0079365 169,46 238,58 0.71 239388 at Info 1 NA n 2.70E-03 1 0.015873 19,33 33,29 0,58 230552 at Info 1 NA

Ras protein-specific

RASGRF

79 2.82E-03 1 0,0238095 146,54 249,67 0,59 228109 at |nfo guanine nucleotide- 2 releasing factor 2

glutamate receptor,

80 2.90E-03 1 0,0079365 16.49 12,92 1.28 208552 at Info GRS 4

ionotropic, kainate 4

81 2.90E-03 1 0,0079365 40,81 27.18 1,5 241841 at Μί2 1 NA

PRP40 pre-mRNA

P PF40 processing factor 40

82 2.91E-03 1 0,015873 23.21 17,74 1,31 235477 at Info

A homoloe A (S, cerevisiae) _{1 46} 216842 s„,

2.92E-03 1 0,015873 628,01 430,35 Info CD58 CD58 molecule ii 2.93E-03 1 0,0079365 33,75 46,41 0,73 236260 at info NA

LOC284 hypothetical protein

2.95E-03 1 0,0079365 23,66 17,2 1,38 240661 at jnfo

475 LOC284475 teashirt zinc finger

2.96E-03 1 0,015873 36,58 50,07 0,73 235616 at o TSHZ2

homeobox 2

neuroepithelial cell

1 0,0238095 122,86 182,12 0,67 201829 at Jnfo m transforming 1

METTL7 methyltransferase

2.99E-03 1 0,015873 37,55 27,91 1,35 227055 at Info

1 lie7B

LOC129 hypothetical protein

2.99E-03 1 0,0238095 411,65 662.69 0.62 227867 at info ϋ LOC129293

YODI OTU

deubiquinating

2.99E-03 1 0,0079365 20,5 15,49 1,32 215150 at Info YODl

enzyme 1 horaolog (S, cerevisiae)

3.04E-03 1 0,0079365 119,84 92,21 1,3 236449 at Info 1 NA ankyrin repeat and

AN DD

92 ¹ 3.06E-03 1 0.015873 46,85 75,85 0,62 223497 at info death domain

Ik containing 1A

small nuclear RNA

93 3.07E-03 1 0.0079365 199,75 144,67 1,38 204001 at info SNAPC3 activating complex, polypeptide 3, 50kDa

TMEM1 transmembrane

94 3.09E-03 1 0.0079365 58,07 45,73 1,27 230633 at Info

02 protein 102 unc-50 homolog (C.

95 3.10E-03 1 0.0238095 447,63 317,35 1.41 203583 at tofo UNC50

elegans) zinc finger protein

96 3.13E-03 1 0.0079365 359,02 966,16 0,37 219228 at lofe ZNF331

331

97 3.15E-03 1 0,015873 9,92 13,61 0,73 1570357 at info STX8 syntaxin 8 family with sequence

FA 10

91 3.19E-03 1 0,0238095 1316,13 1969,6 0,67 226905 at s similarity 101,

1 member B

205173 x

99 3.20F.-03 1 0,015873 1270,49 861,04 1,48 Info CD58 CD58 molecule i

G protein-coupled

100 3.21E-03 1 0.015873 30,11 21,04 1,43 221149 at GPR77

receptor 77 glycoprotein V

101 3.22E-03 1 0.015873 37,21 61.72 0,6 207926 at info 6£5

(platelet)

102 sphingosine-1-

3.23E-03 1 0.015873 328,75 608.37 0,54 204642 at o S1P 1

phosphate receptor 1

WD repeat, sterile

WDSUB alpha motif and Li-

103 3.25E-03 1 0,015873 359,63 243.41 1,48 226668 at Info

I box domain containing 1

engulfment and cell

104 3.28E-03 1 0.0079365 796.74 553.15 1,44 55692 at info E1. 02

motility 2 spire homolog 1

105 3.30E-03 1 0.0079365 33,73 26,11 1,29 225018 at info SP!REl

(Drosophila)

SH2 domain

106 3.32E-03 1 0,015873 835,14 564,8 1,48 226673 at Info SH2D3C

containing 3C

107 3.36E-03 1 0,0079365 15,48 10.01 1,55 230399 at Jnfo NA

C14orf6 chromosome 14 open

108 3.36E-03 1 0,015873 48,7 82.54 0,59 1559097 at info

! reading frame 64

109 3.36E-03 1 0.015873 16,35 22.57 0.72 233416 at info 1 NA

110 3.41E-03 1 0.015873 219,39 111.28 1,97 218701 at o LACTB2 lactamase, beta 2 T EM2 transmembrane

111 3.42E-03 1 0,0019365 51.87 29.7 1.75 234994 at Info

OOA protein 200A

112 3.42E-03 1 0,015873 198,89 97.52 2,04 239740 at Info Hi ets variant 6

223007 s chromosome 9 open

113 3.42E-03 1 0,015873 216.78 306,86 0.71 info C9orf5

at reading frame 5

WD repeat domain

114 3.44E-03 1 0,0238095 587,73 389,98 1,51 221735 at Info WDR48

48

115 3.45E-03 1 0,015873 11.01 8,61 1.28 214967 at Info 1 NA

ectonucleotide pyrophosphatase/pho

116 3.48E-03 1 0,0079365 21,49 15,44 1.39 237054 at Info. ENPP5

sphodiesterase 5 (putative function)

spindle and kinetochore

117 3.60E-03 1 0,015873 170,12 126.15 1,35 225686 at Info SKA2

associated complex subunit 2 DEAD (Asp-Giu-

218943 s

118 3.62E-03 1 0,015873 284,46 156,6 1.82 info DDX58 Ala-Asp) box at

polypeptide 58

119 3.65E-03 1 0,0079365 56,45 74,21 0.76 239925 at info 1 NA

212096 s mitochondrial tumor

120 3.73E-03 1 0,015873 26,97 43,06 0,63 Info TUS1

at suppressor 1

208614 s

121 3.74E-03 1 0,0079365 215,46 278.87 0.77 Info FL B filamin B, beta

I

myeloidlymphoid or mixed-lineage leukemia (trithorax

122 3.74E-03 1 0,0079365 74,79 12819 0,58 1569652 at info MLLT3

homolog,

Drosopliila); translocated to, 3

228661 s

123 3.77E-03 1 0.0079365 123,98 172,07 0.72 Info 1 NA

I

X-ray repair complementing

210813 5

124 3.81E-03 1 0,0019365 117,49 85.23 1,38 XRCC4 defective repair in

i Chinese hamster cells

4

pyruvate

125 3.82E-03 1 0,0079365 254,06 177,02 1,44 200979 at Info PDHA1 dehydrogenase

(lipoaraide) alpha 1 mitochondrial

218049 s MRPL1

126 3.85E-03 1 0.015873 353,79 264,67 1,34 Info ribosomal protein at 3

L13 family with sequence

FAM20

127 3.92E-03 1 0,0079365 15.9 10,75 1.48 241981 at Info similarity 20,

A member A

mitogen-activated

128 3.92E-03 1 0,0238095 598,3 391,96 1,53 1552263 at Ik MAPK1

protein kinase 1

LOC650 hypothetical protein

129 4.05E-03 1 0,0238095 51.97 89,58 0,58 227984 at Info

12 ^" LOC650392

G protein-coupled

130 4.06E-03 1 0,0238095 115.3 60,53 1.9 214510 at iOfo GP 20

receptor 20 trinucleotide repeat

131 4.07E-03 1 0.0019365 198.49 252,6 0,79 213254 at Info TNRC6B

containing 6B

203714 s tubulin folding

132 4.09E-03 1 0.015873 426.34 323,59 1.32 |nfo

at 11 cofactor E

215923 s pleckstrin and Sec7

133 4.18E-03 1 0.0079365 217,89 162.17 1,34 l PSD4

at domain containing 4

214414 x

134 4J9E-03 1 0,0238095 19762,36 4524,98 4.37 info

I 1 NA

229571 s C21orf4 chromosome 21 open

135 419E"03 1 0,0079365 42,74 31,8 1.34 o

M 1 reading frame 45 collagen, type IV,

136 4.20E-03 1 0,0079365 7,6 9,73 0.78 213992 at Info COL4A6

alpha 6

137 4.21E-03 1 0,0238095 202,26 320,5 0.63 239122 at 1 NA

TCP11L t-cotnplex ll

138 4.22E-03 1 0,0238095 27,86 43.11 0,65 1559413 at infe

I (mouse)-like 2

207765 5 1AA15

139 4.24E-03 1 0,015873 337,46 236,59 1,43 info IAA1539

at .1

140 4.27E-03 1 0.015873 112,75 83,24 1,35 201714 at Info TUBG1 tubulin, gamma 1 TIP41J0R

22S619 x signaling pathway

141 4.31E-03 1 0.0238095 188,6 107,85 1,75 Info in

i regulator-like (S,

cerevisiae)

TRAP and TNF

142 4.31E-03 1 0,0079365 1241.97 980.99 1.27 202266 at TT AP receptor associated protein

protein tyrosine

217777 s PTPLAD

143 4.35E-03 1 0,015873 78,83 58,94 1,34 Info phosphatase-like A at i domain containing 1

intercellular adhesion

207194 s molecule 4

144 4.36E-03 1 0,015873 182,1 71.78 2,54 lofo ICAM4

li (Landsteiner-Wiener blood group)

cytochrome b

145 4.36E-03 1 0,0238095 515,45 273,23 1,89 222453 at iois CYBRD1

reductase 1 sodium channel,

146 4.37E-03 1 0,0079365 10,32 8,16 1,27 206950 at info SCN9A voltage-gated, type

IX, alpha subunit

solute earner family 11 (proton-coupled

210423 s SLC11A

147 4.38E-03 1 0,0238095 1359,8 732,09 1.86 Info divalent metal ion

at 1

transporters), member 1

nucleosome assembly

4.39E-03 1 0,0238095 55,91 118,03 0.47 204749 at NAP1L3

protein 1 -like 3

NCRNA non-protein coding

149 4.40E-03 1 0.015873 59,77 44_,49 1.34 233812 3t Info

00028 RNA28 tripartite motif-

150 4.43E-03 1 0,015873 9,42 7,55 1,25 223599 at info TRIM6

containing 6

ANAPC anaphase promoting

151 4.44E-03 1 0,015873 97,29 70,87 1,37 225521 at Info 7 complex subunit 7 chromodomam

152 4.48E-03 1 0.015873 430,63 551,21 0.78 225077 at info CHD2 helicase DNA

binding protein 2 zinc finger protein

153 4.51E-03 1 0.015873 256.45 381.11 0,67 1552633 at ZNF101

101

154 4.53E-03 1 0,0079365 692,3 1047.21 0,66 225262 at Info F0SL2 FOS-like antisen 2

211699 x

155 4.54E-03 1 0.031746 14763,3 2292,73 6.44 Info NA

I pleomorphic

156 4.54E-03 1 0,0238095 17,16 23.98 0.72 205372 at !Qfo PLAG1

adenoma gene 1

157 4.58E-03 1 0,015873 10,41 13.45 0,77 236952 at 1 NA

interferon induced

212203 x

158 4.60E-03 1 0,0238095 8355,56 5403,36 1.55 IFIT 3 transmembr it info ane protein 3 (1-8U)

159 4.64E-03 1 0.015873 54.15 70.03 0,77 243111 at ioi!. 1 NA

204143 s enolase superfamily

160 4.66E-03 1 0,0238095 158,5 245.54 0,65 info EN0SF1

at member 1 hematopoietic

161 4.67E-03 1 0.0079365 18.25 14.56 1.25 206726 at info HPGDS prostaglandin D

synthase 162 4.69E-03 1 0.031746 99,31 217,27 0,46 233127 at Info 1 NA

163 4.71E-03 1 0,015873 137,76 196,03 0.7 226528 at Info MTX3 nietaxin 3

201596 x

164 4J9E-03 1 0,0238095 68.88 96,12 0,72 Info RT18 keratin 18

M

165 4.93E-03 1 0,0079365 148,97 113,18 1,32 230279 at iofe 1 NA cell division cycle

166 4.93E-03 1 0,0079365 716,09 572,6 1,25 201725 at info CDC123 123 homolog (S.

cerevisiae)

'Observed v. Expected' table of GO classes and parent classes, in list of 166 genes shown above:

Only GO classes and parent classes with at least 5 obsen'ations in the selected subset and with an Observed vs, Expected¹ ratio of at least 2 are shown.

Molecular Function

|GO:0043565-;sequence-specific DNA binding

Biological Process

21 BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 depicts representative flow cytometric data showing the detection of CD8+ PSA146-154 peptide-tetramer+ cells in patient UPIN28. PBMC were sensitized in vitro with PSA146-154 peptide for 3 cycles and resulting T-cells were doubly stained with PSA146-154 peptide-tetramer-PE (middle panel, FL2) or negative control tetramer-PE (lower panel, FL2) and CD8-FITC (lower panel and middle, FL1). A greater number of CD8+ PSA146-154 peptide- tetramer+ cells (upper right quadrants) were observed on post- vaccine compared to pre-vaccine samples.

[0023] Figure 2 depicts a comparison of tetramer levels based on vaccination methods. The average fold increase in CD8+ PSA146-154peptide:tetramer+ T-cells at week 26 over pre- vaccine levels was 4.4 greater on protocol-1 (PSA146-154 peptide admixed with GM-CSF injected intradermally) as compared to protocol-2 (intravenous administration of peptide-pulsed, autologous DC) particularly in high-risk patients with locally advanced disease (p-value=0.007).

[0024] Figure 3 depicts overall survival for high risk, locally advanced and metastatic hormone- sensitive CaP. The mean OS was 60 months (95% CI 51 to 68 months) for all patients (upper panel). The median OS was greater than 84 months for patients with high risk, locally advanced disease (middle panel), while the median OS was 75 months for patients with metastatic, hormone-sensitive CaP (lower panel) at a median follow-up of 6.30 years since the onset of immunotherapy.

[0025] Figure 4 depicts the correlation between augmented specific tetramer responses and serum PSA status. The average tetramer measurements at week 26 minus pre-vaccine levels (Δ26) inversely correlated with lower risk of serum PSA progression at six months following the onset of immunotherapy(p=0.02). "NP" denotes stable biochemical disease or non progression, while "P" denotes biochemical progression.

[0026] Figure 5 depicts a comparison of overall survival between immune responders versus non responders. There was a trend towards greater OS in men with high-risk, hormone-sensitive CaP who developed strong specific DTH or tetramer response (upper and middle panel, respectively) following vaccination with PSA146-154 peptide.

[0027] Figure 6 shows that CTL-PSA210-230 and CTL-PSA154-173 demonstrated specific release of IFN-gamma cytokine per ELISPOT analysis. [0028] Figure 7 shows that CTL-PSA210-230 demonstrated strong lysis of HLA-A201+ T2 targets per ⁵¹Cr release assay.

[0029] Figure 8 shows that PSA210-230 peptide binds HLA-A0201 with high affinity in a concentration dependent fashion.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0030] The terms "therapeutically effective amount," as used herein, refer to an amount of a compound sufficient to treat, ameliorate, or prevent prostate cancer, or to exhibit a detectable therapeutic, prophylactic, or inhibitory effect. The effect can be detected by, for example, an improvement in clinical prostate cancer, or reduction in symptoms associated with prostate cancer. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Where a drug has been approved by the U.S. Food and Drug Administration (FDA) or a foreign counterpart agency, a "therapeutically effective amount" refers to the dosage approved by the FDA or its counterpart foreign agency for treatment of the identified disease or condition.

[0031] As used herein, a patient "in need thereof" is a patient who would benefit from administration of a composition of the disclosure. The patient may be suffering from any disease or condition for which a composition of the disclosure may be useful in ameliorating symptoms. In various aspects, the patient is a patient diagnosed with prostate cancer.

[0032] As used herein, "co-administration" is understood to include concurrent administration and sequential administration. Thus, the terms are understood to encompass administration simultaneously, or at different times, and by the same route or by different routes, as long as the two agents are given in a manner that allows both agents to be affecting the body at the same time.

[0033] "Sequential administration" as used herein is understood to mean one of the compounds or agents is given first followed by the second. When administered sequentially, the combination may be administered in two or more administrations. [0034] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. , antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, dyes and combinations thereof, as would be known to one of ordinary skill in the art (see, for example and without limitation, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0035] "Vaccine" as used herein can be either a therapeutic vaccine or a prophylactic vaccine. A prophylactic vaccine is understood to be a composition that is administered to a healthy individual to prevent a disease. A therapeutic vaccine is administered to an individual that already has a disease in order to alleviate or eliminate the disease.

Compositions/V accines

[0036] It is an aim of the present disclosure to provide compositions and vaccines for treating or preventing prostate cancer. Thus, PSA peptides are provided herein that are contemplated for administration to a human. In various embodiments, the PSA peptide(s) are administered: (i) alone; (ii) with an additional agent (biologic or chemical); (iii) in a composition of dendritic cells that have been pulsed with the PSA peptide(s); (iv) with a delivery vector; (v) with an

immunomodulating adjuvant; and (vi) as part of a vaccine composition.

[0037] Accordingly, in various embodiments the disclosure provides a composition

comprising a fragment of prostate specific antigen (PSA), said fragment peptide comprising the sequence VIS ND VC AQ VHPQKVTKFML (SEQ ID NO: 1). In various aspects, a composition is provided comprising a fragment of prostate specific antigen (PSA), said fragment peptide consisting of the sequence VIS NDVCAQ VHPQKVTKFML (SEQ ID NO: 1). In various aspects, a composition is provided comprising a fragment of prostate specific antigen (PSA), said fragment peptide consisting essentially of the sequence VIS NDVCAQ VHPQKVTKFML (SEQ ID NO: 1). In further aspects, the PSA peptide(s) are administered: (i) alone; (ii) with a delivery vector; (iii) with an immunomodulating adjuvant; (iv) with additional biologic agent or chemical; (v) in a composition of dendritic cells that have been pulsed with the PSA peptide(s); and (vi) as part of a vaccine composition. [0038] In various embodiments, a composition is provided comprising a fragment of prostate specific antigen (PSA), said fragment peptide comprising the sequence

CALPERPSLYTKVVHYRKWIK (SEQ ID NO: 2). In various aspects, a composition is provided comprising a fragment of prostate specific antigen (PSA), said fragment peptide consisting of the sequence CALPERPSLYTKVVHYRKWIK (SEQ ID NO: 2). In various aspects, a composition is provided comprising a fragment of prostate specific antigen (PSA), said fragment peptide consisting essentially of the sequence CALPERPSLYTKVVHYRKWIK (SEQ ID NO: 2).

[0039] In various embodiments, the disclosure provides a vaccine comprising: (i) a

composition of the disclosure or combinations thereof, and (ii) a pharmaceutically acceptable carrier. In one aspect, the vaccine comprises PSA peptide 146-154 (SEQ ID NO: 3). In various aspects, the vaccine further comprises GM-CSF.

[0040] In various embodiments, the vaccine further comprises a Toll-like receptor 9 (TLR9) agonist. In one aspect, the TLR9 agonist is a CpG-oligodeoxynucleotide (CpG-ODN).

[0041] In various embodiments, the vaccine further comprises an inhibitor of Cytotoxic T- Lymphocyte Antigen 4 (CTLA4) in an amount effective to increase a T cell immune response. In a specific aspect, the inhibitor of CTLA4 is a monoclonal antibody.

[0042] In various embodiments, the disclosure provides a vaccine that further comprises an inhibitor of Programmed Death 1 (PD-1) in an amount effective to increase a T cell immune response. In various embodiments, the inhibitor of PD-1 is a monoclonal antibody.

[0043] PSA peptides disclosed herein are contemplated for use in compositions to be administered according to the methods described below. Also contemplated for use are allelic variants, conservative substitution variants and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined below.

[0044] Conservative amino acid substitutions can frequently be made in a protein or peptide without altering either the conformation or the function of the protein or peptide. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example and without limitation, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant.

[0045] Fusion proteins are made by techniques well known to a person skilled in the art, such as by linking the PSA peptide of the disclosure with other recombinant peptides or proteins. There is no restriction as to the peptides or proteins fused to the peptide of the present disclosure. Non-limiting examples of fusion proteins contemplated by the disclosure include PSA peptide(s) coated onto a microbead or microsphere and PSA peptide(s) incorporated into a liposome.

[0046] Compositions/vaccines, in various embodiments, comprise one or more PSA peptides as disclosed herein formulated, combined, mixed, incorporated into and/or matrixed with one or more adjuvants, diluents, carriers and the like that is administered to a subject by any suitable route to induce protective and/or ameliorative immune responses to the PSA peptide.

"Adjuvant" refers to any substance that is distinct from the PSA peptide which when

incorporated into a composition acts generally to accelerate, prolong, enhance, augment and/or potentiate the host's immune response to the PSA peptide, and includes compositions

encompassed by the terms immunomodulator, immunopotentiator and immunoenhancer. In general, adjuvants comprise a heterogeneous group of compounds broadly classified as oil emulsions, mineral compounds, bacterial products, liposomes and immuno stimulating complexes (ISCOMs).

[0047] Exemplary adjuvants include without limitation, ADJUMER™ (polyphosphazene); aluminum phosphate gel; algal glucans; algammulin; aluminum hydroxide gel (alum); high protein adsorbency aluminum hydroxide gel; low viscosity aluminum hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80(0.2%), Pluronic L121(1.25%), phosphate-buffered saline pH 7.4); AVRIDIE™ (propanediamine); BAY R1005™ ((N-(2-Deoxy-2-L-leucylamino-b-D- glucopyranosyl)-N-octadecyldod- ecanoylarmide hydroacetate); CALCITRIOL™ (la, 25- dihydroxyvitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles);

cholera holotoxin, cholera toxin A 1 -protein A-D fragment fusion protein, cholera toxin B subunit; CRL 1005 (Block Copolymer P1205); cytokine containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC

(dimyristoyl phosphatidylcholine); DMPG (dimyristoyl phosphatidylglycerol); DOC/ Alum Complex (Deoxycholic Acid Sodium Salt); Freund's Complete Adjuvant; Freund's Incomplete Adjuvant; Gamma Inulin; Gerbu Adjuvant (mixture of: i) N-Acetylglucosaminyl-(Pl-4)— N- acetylmuramyl-L-alanyl-D-glutamine (GMDP), ii) Dimethyl dioctadecylammonium. chloride (DDA), iii) Zinc L-proline salt complex (ZnPro-8); GM-CSF; GMDP (N-acetylglucosaminyl- (bl-4)-N-acetylmuramyl-L-al- anyl-D-isoglutamine); IC31™; Imiquimod (l-(2-methypropyl)- IH-imidazo[4,5-c]quinol- in-4-amine); ImmTher™ (N-acetylglucosaminyl-N-acetyhnuramyl-L- Ala-D-iso- Glu-L-Ala-glycerol dipalmitate); DRVs (Immunoliposomes prepared from

Dehydration-Rehyrdation Vesicles); Interferon-. gamma.; Interleukin-l.beta.; Interleukin-2; Interleukin-7; Interleukin-12; ISCOMS™ (Immune Stimulating Complexes); ISCOPREP 7.0.3.™; Liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT Oral Adjuvant™ (E. coli labile enterotoxin protoxin); Microspheres and Microparticles of any composition; MF59™; (squalene.water emulsion); MONTANIDE ISA 51™ (purified Incomplete Freund's Adjuvant); MONTANIDE ISA 720™ (metabolizable oil adjuvant); MPL™ (3-Q-desacyl-4'- monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D- isoglutaminyl- -L-alanine-2-(l,2-dipalmitoyl-sn-glycero-3-(hydroxy-phosphoryloxy)) ethylamide, mono sodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3);

MURAPALMITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGIn-sn-glyc- erol dipalmitoyl); NAGO (Neuraminidase-galactose oxidase); Nanospheres or Nanoparticles of any composition; NISVs (Non-Ionic Surfactant Vesicles); PLEURAN™ (.beta.-glucan); PLGA, PGA and PLA (homo-and co-polymers of lactic and glycolic acid; micro-/nanospheres);

PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (oroteinoid microspheres); Polyethylene carbamate derivatives; Poly rA:Poly rU (Poly- adenylic acid-poly-uridylic acid complex); Polysorbate 80 (Tween 80); Protein Cochleates (Avanti Polar Lipids, Inc., Alabaster, AL); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-Amino-otec,-dimethyl-2- ethox- ymethyl-lH-imidazo[4,5-c]quinoline-l-ethanol); SAF-1™ (Syntex Adjuvant

Formulation); Sendai proteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitan trioleate); Specol (emulstion of Marcol 52, Span 85 and Tween 85); Squalene or Robane® (2,6,10,15, 19,23-hexamethy- ltetracosane and 2,6,10,15,19, 23-hexamethyl-2,6,10,14,18,22 tetracosahexaene); Stearyl Tyrosine (Octadecyl tyrosine hydrochloride); Theramide® (N- acetylglucosaminyl-N-acetylinuramyl-L-Ala-D-isoGlu-L-Al- a-dipalmitoxy propylamide);

Theronyl-MDP (Termurtide™ or [thr 1]-MDP; N-acetyl muramyl-L-threonyl-D-isoglutamine); Ty Particles (Ty-VLPs or virus like particles); Walter Reed Liposomes (Liposomes containing lipid A adsorbed to aluminum hydroxide).

[0048] In further embodiments, PSA peptide(s) are administered with a delivery vector. As used herein, a delivery vector is any vector that comprises a polynucleotide sequence that encodes and is able to direct expression of a PSA peptide of the disclosure in an individual.

Suitable expression vectors are known to those of skill in the art. In various aspects, the delivery vector is a (recombinant) DNA or RNA vector known in the art, or is a plasmid comprising a polynucleotide sequence encoding a PSA peptide of the disclosure that is operably linked to regulatory sequences conferring expression and translation of the encoded messengers. In various aspects, the vector is any DNA or RNA virus, such as, but not limited to Adenovirus, Adeno- Associated Virus (AAV), a retrovirus, a lentivirus, modified Vaccinia Ankara virus (MVA) or Fowl Pox virus, a Herpes virus, or any other viral vector capable of conferring expression of the encoded PSA polypeptide. In various aspects, DNA vectors are non- integrating, such as episomally replicating vectors or are vectors integrating in the host genome by random integration or by homologous recombination. A delivery vector, in various aspects, comprises a liposome that comprises a nucleic acid sequence capable of directing expression of a PSA peptide of the disclosure.

Methods

[0049] As described herein above, the disclosure provides compositions/vaccines for the treatment and/or prevention of prostate cancer. Also provided are methods of their use.

Accordingly, in various embodiments, methods are provided for treating prostate cancer in a human comprising the step of administering a composition of the disclosure in an amount effective to stabilize or reduce serum PSA levels. In various aspects, the serum PSA levels are reduced by at least about 1%. In further aspects, the serum PSA levels are reduced by at least about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 5-fold, about 10-fold, about 100- fold or more relative to a human that was not administered the composition of the disclosure, or to a previously measured serum PSA level in the same human prior to being administered a composition of the disclosure.

[0050] In various embodiments, the disclosure provides a method of vaccinating an individual comprising the step of administering a vaccine of the disclosure to the individual.

[0051] Methods provided contemplate the use of any agent that stimulates, promotes or otherwise augments an immune response. In various embodiments, the agent is an adjuvant and/or a cytokine, and in various aspects, the cytokine is tumor necrosis factor, interleukin-2, interleukin-4, interleukin-12, granulocyte macrophage colony stimulating factor (GM-CSF), γ- interferons and/or combinations thereof.

[0052] In various embodiments, the PSA peptide and GM-CSF are co-administered in a weight- to -weight ratio of at least about 1:5. Other contemplated ratios are at least about 1:6, about 1:7, about 1:8, about 1:9, about 1:10 or higher.

[0053] A PSA peptide of the disclosure is contemplated for use in a composition as described herein in an amount effective to stabilize or reduce serum PSA levels. In various embodiments, a total of about 100 μg PSA peptide is administered to a human. In various embodiments, a total of at least about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 190 μg, about 200 μg, about 210 μg, about 220 μg, about 230 μg, about 240 μg, about 250 μg, about 260 μg, about 270 μg, about 280 μg, about 290 μg, about 300 μg, about 310 μg, about 320 μg, about 330 μg, about 340 μg, about 350 μg, about 360 μg, about 370 μg, about 380 μg, about 390 μg, about 400 μg, about 410 μg, about 420 μg, about 430 μg, about 440 μg, about 450 μg, about 460 μg, about 470 μg, about 480 μg, about 490 μg, about 500 μg or more is administered to a human.

[0054] In various embodiments, the PSA peptide and GM-CSF are co-administered in multiple injections. In various aspects, the PSA peptide and GM-CSF are co-administered in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more injections.

[0055] Routes of administration for the PSA peptide, optionally including an agent that stimulates an immune response, include intravenous, subcutaneous, intramuscular,

intraperitoneal, intradermal, oral, intranasal, intradermal, and intrapulmonary (i.e., by aerosol). The chosen route of administration will dictate the formulation that is administered and would be understood by the clinician of skill in the art.

[0056] In various embodiments, the invention provides administration of dendritic cells pulsed with said PSA peptide. In various aspects, the antigen-presenting cells are autologous to the recipient of the treatment or heterologous to the recipient of the treatment. In various aspects, the antigen-presenting cells are dendritic cells, whether autologous or heterologous, and are expanded in culture prior to being pulsed with the PSA peptide. Culture methods known in the art for expanding antigen-presenting cells are used in the practice of the invention. In varous aspects, the dendritic cells are expanded in culture for at least about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or at least about 21 days prior to being pulsed with PSA antigen. In various aspects, the dendritic cells are pulsed with at least about 25 μg/ml, about 30 μg/ml, about 35 μg/ml, about 40 μg/ml, about 45 μg/ml, about 50 μg/ml, about 100 μg/ml, about 150 μg/ml, about 200 μg/ml, about 300 μg/ml, about 350 μg/ml, about 400 μg/ml, about 450 μg/ml or about 500 μg/ml or more.

[0057] In various embodiments, treatment of CaP with a composition or vaccine of the disclosure results in an increase in PSA peptide-tetramer staining CD8⁺ cells (Kim et al, J Immunology, 2000, 165: 7285-7299) of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold or more.

[0058] In various embodiments, the PSA peptide(s) of the disclosure are administered with an additional agent. In various aspects, the additional agent is PROSTVAC®. In further aspects, the additional agent is GVAX. In further embodiments, methods are contemplated which include combination therapy with a chemotherapeutic agent. Chemotherapy treatment can employ anti-neoplastic agents including, for example, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and

chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine

(methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM),

Methylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5- fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5- azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6- thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine

(EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine

(VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate;

epipodophylotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin

(mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinium coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (ο,ρ'-DDD) and aminoglutethimide; hormones and antagonists including

adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.

Treatment Regimens

[0059] In administering any of the compositions/vaccines of the disclosure, it is contemplated that various treatment regimens are utilized.

[0060] In various embodiments, the composition/vaccine is administered in weeks 1, 4 and 10, and then every six months up to four years. Thus, the composition/vaccine is administered one or more times during weeks 1, 4 and 10 of treatment, and then the composition/vaccine is administered one or more times every six months up to four years. In some embodiments, treatment may continue beyond four years.

[0061] In various embodiments, the composition/vaccine is administered in weeks 1, 4 and 10, and then every six months up to four years, wherein an inhibitor of CTLA4 is administered in weeks 1, 4 and 10, and then every eight weeks up until week 52.

[0062] In various embodiments, the composition/vaccine is administered in weeks 1, 4 and 10, and then every six months up to four years, wherein an inhibitor of PD-1 is administered in weeks 1, 4 and 10, and then every eight weeks up until week 52.

[0063] Combinations of the above treatment regimens are also contemplated by the disclosure. For example and without limitation, administration of both an inhibitor of CTLA4 and an inhibitor of PD-1 in weeks 1, 4 and 10, and then every eight weeks up until week 52 is also contemplated. In various aspects, co-administration of a composition/vaccine of the disclosure with a TLR9 agonist is contemplated. Administration of the TLR9 agonist is contemplated in conjunction with an inhibitor of CTLA4 and/or an inhibitor of PD-1, as well as in the absence of the inhibitor of CTLA4 and/or the inhibitor of PD-1.

[0064] Administration of any of the compositions or combination of compositions of the disclosure is contemplated, in various embodiments, once a week, twice a week, three times a week, four times a week, five times a week, six times a week and seven times a week.

Administration of a composition or combination of compositions of the disclosure that takes place more than once a week is contemplated to occur on either sequential or non- sequential days.

[0065] In various embodiments, administration of any of the compositions or combination of compositions of the disclosure occurs once a day, twice a day, three times a day, four times a day, five times a day or more by any one or more of the routes disclosed herein.

[0066] In various embodiments, administration of any of the compositions or combination of compositions of the disclosure occurs every week, every second week, every third week, every fourth week, every fifth week, every sixth week, every seventh week, every eighth week, every ninth week or every tenth week. Thus, administration of any of the compositions or combination of compositions of the disclosure occurs, in various embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more times per month, or every second month, or every third month, or every fourth month, or every fifth month, or every sixth month, or every seventh month, or every eighth month, or every ninth month, or every tenth month, or every eleventh month or every twelfth month or more.

[0067] Administration of any of the compositions or combination of compositions of the disclosure is contemplated, in various aspects, to occur for a duration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more weeks. In various

embodiments, administration of any of the compositions or combination of compositions of the disclosure is contemplated to occur for a duration of 1, 2, 3, 4, 5 or 6 days.

[0068] In various embodiments, any of the compositions or combination of compositions of the disclosure is contemplated to be administered to a patient for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.

[0069] Administration of any of the compositions or combination of compositions of the disclosure is contemplated, in various aspects, to be discontinuous. In various aspects, discontinuous administration is undertaken to maximize the therapeutic efficacy of the composition or combination of compositions, or in response to one or more adverse events experienced by the patient being treated. Accordingly, administration of any of the compositions or combination of compositions of the disclosure is contemplated to occur for an amount of time and then cease for an amount of time, after which administration may resume. The amount of time that administration of a composition or combination of compositions of the disclosure is ceased is, in various embodiments, at least 1, 2, 3, 4, 5 or 6 days. In further aspects, the amount of time that administration of a composition or combination of compositions of the disclosure is ceased is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more weeks. In various embodiments, and following discontinuation of administration of any of the compositions or combination of compositions of the disclosure, it is contemplated that administration is resumed according to any of the treatment regimens disclosed herein.

Identifying a Candidate for Therapy

[0070] As exemplified below, understanding the molecular intricacies of why some patients respond to a well defined peptide target, while others do not, leads to the application of optimal vaccine strategies for appropriately selected patients and shed light on strategies to make targeted immunotherapy applicable to a wider array of patients.

[0071] The gene expression data shown in Table 1 or Table 2 provides the ability to analyze the expression profile of those patients that are strong immune responders to a therapy of the disclosure. By identifying the genes whose expression is modulated in a strong immune responder versus a non responder, it is contemplated that a further treatment regimen is aimed at either up- or down-regulating the expression of one or more gene products. By way of example, if a particular gene product is down-regulated in a strong immune responder versus a non responder, then a treatment regimen for the non responder will include an additional agent that increases expression of that gene product. In general, the disclosure contemplates modulating the gene expression profile of a non responder so that it more closely matches the gene expression profile of a strong immune responder.

[0072] Thus, in various embodiments the disclosure provides a method of administering a composition/vaccine to a patient in need thereof comprising the steps of: (i) modulating the expression of at least one gene listed in Table 1 or Table 2; and (ii) administering the composition/vaccine of the disclosure. In various aspects, the modulating increases expression and in some aspects, modulating decreases expression. It is contemplated that the modulating results in at least a 1% increase or decrease in expression of a gene. In further aspects, the modulating results in at least a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 50-fold, 100-fold or more increase or decrease in expression of a gene.

[0073] In various aspects of the disclosure, obtaining the gene expression profile information from a patient that is in need of treatment with a composition/vaccine disclosed herein allows a clinician to determine whether that patient is likely to respond favorably to the treatment.

[0074] Thus, the disclosure provides a method of identifying a patient that is a candidate for prostate cancer therapy comprising the step of measuring expression level in a sample from a test individual of one or more genes identified in Table 1 or Table 2 relative to a reference expression level, wherein an increase or a decrease in expression of one or more of the genes identified in Table 1 or Table 2 relative to the reference expression level is determinative for identifying whether the patient is a candidate for prostate cancer therapy according to a method disclosed herein. In various aspects, the method further comprises collecting a sample from the test individual, and in another aspect the method further comprises comparing the expression level to the reference expression level.

Table 2. Differentially expressed genes between immune responders and non responders.

DEAD box polypeptide 58 218943_s_at 1.82 Innate immune response

(DDX58)

Interferon induced 212203_x_at 1.55 Immune response

transmembrane protein 3

1-8U (IFITM3)

Mitogen- activated protein 1552263_at 1.53 1.37 T-cell and B-cell receptor signaling kinase 1 (MAPKl) 1552264_a_at VEGF signaling pathway

TGF-beta signaling

natural killer mediated cytotoxicity

CCR3 signaling in eosinophils

CXCR4 signaling pathway

CD58 molecule (CD58) 216942_s_at 1.46 IL-17 signaling pathway

205173_x_at 1.48

X-ray repair 210813_s_at 1.38 T-cell differentiation in the thymus complementing defective Immunoglobulin V(D)J recombination repair in Chinese hamster

cells 4 (XRCC4)

Tumor necrosis factor 211841_s_at 0.65 Cytokine -cytokine receptor interaction receptor superfamily,

member 25

Chemokine C-C motif 206337_at 0.52 Cytokine -cytokine receptor interaction receptor 7 (CCR7)

Phosphoinositide-3 -kinase , 212249_at 0.62 T-cell activation

regulatory subunit 1 alpha T-cell and B-cell receptor signaling

(PIK3R1) CXCR4 signaling pathway

VEGF signaling pathway

Toll-like receptor signaling pathway

Epiregulin (EREG) 205767_at 0.26 positive regulation of innate immune

response

Gene expression analysis was performed on un-manipulated pre-vaccination PBMC. Of the 166 genes differentially expressed, only genes affecting the immune function associated pathway are shown.

[0075] In various aspects, an increase in expression of a gene selected from the group consisting of 2'-5' oligoadenylate synthetase 1 (OAS1), mitogen- activated protein kinase 1 (MAPKl), Sh2 domain containing IB (SH2D1B), vannin 1 (VNNl), CD58 molecule (CD58), DEAD box polypeptide 58 (DDX58), X-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4) and interferon-induced transmembrane protein-3 (IFITM3) is indicative of the patient being a candidate for prostate cancer therapy according to a method disclosed herein. [0076] In various aspects, a decrease in expression of a gene selected from the group consisting of tumor necrosis factor receptor superfamily-member 25 (TNFRSF-25), chemokine C-C motif receptor 7 (CCR7), and phosphoinositide-3-kinase, regulatory subunit 1 alpha (PIK3R1) and epiregulin (EREG) is indicative of the patient being a candidate for prostate cancer therapy according to a method disclosed herein.

[0077] The invention is illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLES

Example 1

Patient Characteristics

[0078] Long-term follow-up of all patients previously enrolled on a phase IB peptide vaccine protocol was performed with the authorization of the Institutional Review Board of the

University of Illinois at Chicago. Twenty eight HLA-A2+ patients with pathologically confirmed CaP who had completed vaccination with PSA146-154 peptide between July 2002 and September 2004 were included in the study [Perambakam et ah, Cancer Immunology Immunotherapy 55(9): 1033-1042 (2006)]. The clinical characteristics of patients are listed in Table 3, below. All patients had undergone radio-therapy or surgical ablation of the prostate a minimum of 6 weeks prior to initiation of vaccine study. Patients either had advanced local disease with high risk of recurrence based on the presence of T3, T4 disease, a serum PSA level > 10 ng/ml or a Gleason grade > 7 (Group A), or they had confirmed metastatic disease which was associated with declining serum PSA on ADT or a stable or improving bone scan or CT scan in response to hormone therapy (Group B). All patients were immunologically reactive to a panel of mumps, measles, and Candida.

[0079] The Unique Patient Identifying Number (UPIN) assigned in the original report was retained. Relevant information pertinent to morbidity, disease specific mortality and OS was collected from patients and/or family members following appropriate informed consent.

Table 3. Patient baseline characteristics.

All patients had completed primary therapy a minimum of 6 weeks prior to enrollment vaccine study. Vaccine Protocol and Dendritic Cell Culture

[0080] Patients were either treated by intradermal administration of PSA146-154 peptide and GM-CSF (protocol 1, n=14) or by intravenous administration of peptide-pulsed, autologous dendritic cells (DC) (protocol 2, n=14) as previously detailed [Perambakam et ah, Cancer Immunology Immunotherapy 55(9): 1033-1042 (2006)]. Patients were vaccinated on three occasions (week 1, 4 and 10) and monitored. DC was derived from monocyte and cultured in serum free AIM-V (Life Technologies, Grand Island, NY) medium with IL-4 and GM-CSF for a total of 8 days in T-150 flasks in clinical grade sterile laminar airflow hood per the method of Lau et al [Lau et ah, Journal of Immunotherapy 24: 66-78 (2001)]. Release criteria for the final DC product included sterile bacterial, fungal and mycoplasmia cultures, negative endotoxin per Limulus Amoebacyte lysate assay, viability of at least 90% and greater than 50% CD86, CD80, HLA-DR or CDla positive cells and less than 10% CD 14 positive cells by flow cytometric analysis. The final DC product was divided into 3 equal parts. The first infusion included fresh DC while the 2nd and 3rd infusions consisted of frozen DC product. At the time of infusion, DC were rapidly thawed at 37° C, again checked for sterility and viability and administered intravenously to patients.

Results -- Dendritic Cell Product

[0081] Two healthy donors and 14 patients underwent 7-9 liter leukapheresis and DC were cultured for 8 days under identical conditions and phenotyped. The average HLA-DR% was 54.51 (median 52.92), the average CD86% was 58.77 (median 62.56), the average CDla% was 28.17 (median 30.95) and the average CD14% was 1.31 (median 0 or negative expression). DC product was also phenotyped for CD80 and CD83. However, only 2 of 14 patients DC showed CD80 expression, while CD83 was negative in all the patients. The average percent HLA-DR, CD86, CDla and CD14 were 70.03%, 76.6%, 30.58% and 5.94%, respectively in healthy individuals. The yield of total DC from PBMC ranged from 0.94 to 2.02 xlO cells (average 1.499, median 1.555) per vaccine in the 14 patients. Functional activity of DC product also was tested in several patients. DC, cultured in IL-4/GM-CSF for 8 days were able to stimulate significant (> 20-fold) allogeneic T-cell proliferative responses compared to DC pulsed autologous T-cells. Additionally, upon maturation with TNF-oc or LPS for 24 hours, the expression of CD83, a late DC marker, was up-regulated (negative expression to 25% expression).

Delayed Type Hypersensitivity Skin Testing

[0082] Immune responses were monitored by Delayed Type Hypersensitivity (DTH) skin testing on weeks 4, 14, 26, ad 52 by intradermal injection of 0 (carrier only), 1, 10 and 20 microgram of peptide dissolved in 200 microliter of 33% DMSO as previously detailed

[Perambakam et ah, Cancer Immunology Immunotherapy 55(9): 1033-1042 (2006)]. DTH reactions were measured at 48-72 hours following injection. An induration of > 15 mm was considered as a positive reaction.

T-Cell Culture and Peptide-Specific Stimulation Of Peripheral Blood Mononuclear Cells

[0083] Frozen PBMC obtained at various study time points, pre- vaccine (1 to 3 weeks prior to vaccination), week 26 and week 52 were rapidly thawed, washed, checked for viability and re- suspended in RPMI-1640 medium (BioWhittaker, WalkersviUe, MD) containing 10% human AB serum (complete medium). Viability was > 90% (range 90 to 99%, mean 95+1.26). PBMC (2xl0⁶) were plated in 24 well plates (Nunc, Naperville, IL) and cultured in complete medium containing PSA146-154 peptide (SEQ ID NO: 3) (20ug/ml) and IL-2 (20 U/ml) for 7+1 days (1 cycle). PBMC were alternatively stimulated with HLA-A2 binding control peptide, Flu-Mi, in some patients. Spent medium was aspirated and replenished with complete medium plus IL-2 and re-stimulated with irradiated autologous PBMC pulsed with peptide for 2 additional cycles prior to tetramer and cytokine analysis.

Tetramer Analysis

[0084] PSA146-154 peptide stimulated PBMC (lxlO⁶ per tube) were doubly stained with

PSA 146-154 peptide-tetramer-PE (Immunomics, San Diego, CA) and CD8-FITC (BD

Biosciences, San Diego, CA) at room temperature for 30 minutes in phosphate-buffered saline containing 0.5% para-formaldehyde (Sigma, St. Louis, MO). Cells were washed, re-suspended in buffer and analyzed by a Calibur flow cytometer (Becton Dickinson, Mountain View, CA).

Cells also were stained separately with a negative control tetramer-PE, of unknown sequence that does not recognize CD8 + T-cells of any HLA alleletype, to assess the level of background PE fluorescence. As a positive control, tetramer-PE staining for Flu-Mi peptide also was performed in some of the patients. The percentage of CD8+ tetramer+ double positive cells was determined from the quadrant dot plots per Cell Quest software (Becton Dickinson, Mountain View, CA). The results were represented as the number of tetramer+ cells per CD8+ cells and are calculated as the number of tetramer+ CD8+ cells divided by total number of CD8+ cells.

Results -- Immunological Responses

[0085] Three distinct read-outs were used to detect specific immune responses. First via the induction of DTH skin responses to PSA146-154 peptide in vivo, second via detection of CD8+ PSA146-154 peptide-tetramer+ T-cells, and third via PSA146-154 peptide induced release of IFN-γ ίη pre- versus post-vaccine PBMC samples. In vitro sensitization of PBMC with PSA146- 154 peptide was essential prior to tetramer and CBA analysis to detect specific T-cells in peripheral blood. This procedure was applied uniformly to all specimens and was necessary to overcome high background. Similar techniques have been employed in previous cancer vaccine trials [Lau et al., Journal of Immunotherapy 24: 66-78 (2001); Meidenbauer et al., Prostate 43: 88-100 (2000)]. Lau et al. have shown induction of peptide- specific CTL stimulated twice with melanoma-associated peptides over 24 days in IFN-y ELISA [Lau et al., Journal of

Immunotherapy 24: 66-78 (2001)]. Meidenbauer et al. have shown PSA-reactive responses per IFN-y ELISPOT following two stimulations in patients with prostate cancer [Meidenbauer et al., Prostate 43: 88-100 (2000)].

[0086] et al. Tetramer assay denotes a physical measure of the number of CD8+ PSA 146-154 peptide- specific T-cells while the specific release of IFN-γ cytokine following the recognition T2 pulsed targets cells represents a functional readout of T-cells. Although, IFN-γ was the predominant cytokine expressed, specific release of TNF-oc, IL-4 and IL-5 was also observed (Table 4, below).

Table 4. Induction of specific cytokine responses.

UPIN45 30.6 88.8 25.8 3 2.5 7.2 0 8.3

UPIN71 20.9 31 0 0 0 0 0 0

UPIN43 -3.5 -11.2 0 -0.5 1.1 0 -0.2 -0.2

UPIN2 63.9 -50.2 10.7 0 9.3 13.1 21.8 0

UPIN21 1064.5 -9.3 18.7 0 30.4 0 92 0

UPIN27 2236 ND 37 ND 13.8 ND 1.5 ND

UPIN38 112.1 1.3 2.8 0 11.5 0 18.9 0

UPIN82 -3.2 -3.2 -0.2 -0.2 0 0 -2.4 -2.4

UPIN49 0 113.5 0 113.5 0 79.9 0 0

UPIN69 1293.5 133.4 26.7 0 20.4 0 46.7 0.1

UPIN88 -34.3 230.4 0 4.6 0 2.4 -2.9 0.8

UPIN53 25.3 2417.4 0 0.3 0.3 94.3 -1.2 29.8

UPIN81 -2.9 -2.9 0 0 0 0 0 0

UPIN51 0 0 0 0 0 0 0 0.2

UPIN26 255.5 1211.4 -0.3 69.4 10 201 2 875.2

UPIN32 0 0 0 0 0 0 0 0.7

UPIN35 -10.4 24.3 0 0.3 -12.4 -8.5 0 46.4

UPIN37 1.4 0 0 0 0 0 0 0

UPIN85 0 0 0 0.7 0 0 0 0

UPIN89 -46.9 -46.9 -1.2 -1.2 -13 -12.7 -16.8 -16.8

UPIN67 40.3 0 0 0 0 0 -3.5 -2.6

UPIN70 130.9 0 -0.6 -0.6 -4.7 -4.7 -0.6 -2.1

Cytokine responses were evaluated on PBMC at pre-vaccine, week 26 and 52 as detailed in methods section. Value represents absolute changes in post- vaccine cytokine levels minus the pre-vaccine levels are shown. ND denotes not done.

[0087] Overall, fifty percent of patients demonstrated positive DTH skin responses to PSA146-154 peptide (Table 5, below). Baseline (week 4) DTH responses were negative in a majority of patients (13 of 14 patients), however, measurable induration became evident over time and increased with successive DTH testing. Responses were dose-dependent with increasing doses of the PSA146-154 peptide eliciting increasing degrees of induration in responding patients [Perambakam et ah, Cancer Immunology Immunotherapy 55(9): 1033-1042 (2006)]. Injection of carrier only, i.e. 33% DMSO did not cause significant induration. Both CD4+ and CD8+ T-cells were derived from the positive DTH skin biopsy that demonstrated specific cytolytic and cytokine activity as detailed in a previous publication [Perambakam et ah, Cancer Immunology Immunotherapy 55(9): 1033-1042 (2006)].

[0088] Fourteen of 28 patients developed > 4-fold increase in CD8+ PSA146-154-tetramer+ T-cells at week 26 and/or week 52 over baseline levels (Table 5). On average, 3.5 CD8+ PSA146-154-tetramer+ T-cells was observed for every 100 CD8+ T-cells at week 26, while an average of 2.0 CD8+ PSA146-154-tetramer + T-cells were detected for every 100 CD8+ T-cells at week 52. On an average, 1.0 CD8+ PSA146-154-tetramer+ T-cell could be detected per 100 CD8+ T-cells prior to the onset of immunotherapy. Figure 1 is a representative tetramer staining analysis showing increased CD8+ PSA146-154-tetramer+ T-cells post vaccine (week 26) compared to pre-vaccine following in vitro sensitization of PBMC with PSA146-154 peptide. Tetramer responses were not detectable in un-stimulated PBMC population. Comparable results were observed by Lau and co-workers in a peptide-DC based melanoma study [Lau et ah, Journal of Immunotherapy 24: 66-78 (2001)].

[0089] Similarly, 14 of 28 patients demonstrated specific release of IFN-γ (defined as > 100 ng/ml of absolute change) by week 52 from the outset of immunotherapy. Specific release of other cytokines, namely, TNF-oc, IL-4 and IL-5 also was observed (Table 4). The CBA analysis in the current study was performed with unsorted T-cell populations, therefore it was not possible to determine whether IFN-γ was released by CD8+ and/or CD4+ T-cells.

[0090] Eight of 14 (57%) positive tetramer responders also mounted specific DTH responses to PSA146-154 peptide, while only 4/14 (28%) tetramer non-responders were positive for DTH responses to the peptide, indicating concordance between the development of peptide- specific DTH responses in the skin and specific T-cell immune responses in peripheral blood of patients.

Table 5. Immunological outcomes based on specific DTH, tetramer and IFN-γ responses.

UPIN88 + 1.42 0.06 - -34.5 230.4 +

UPIN2 - 3.09 5.83 + 63.9 -50.2 -

UPIN21 - 0.51 5.43 + 1064 -9.3 +

UPIN26 - 0.35 0.81 - 255.5 1211.4 +

UPIN27 - 82.11 ND + 2236 ND +

UPIN32 - 1.13 1.05 - 0 0 -

UPIN35 - 1.31 0.09 - -10.4 24.3 +

UPIN37 - 0.80 0.26 - 1.4 0 -

UPIN38 - 10.79 1.37 + 112.1 1.3 +

UPIN43 - 1.11 5.06 + -3.5 -11.2 -

UPIN67 - 1.72 0.07 - 40.3 0 -

UPIN70 - 0.46 1.85 - 130.9 0 +

UPIN82 - 4.47 0.24 + -3.2 -3.2 -

UPIN85 - 2.50 0.08 - 0 0 -

UPIN89 - 2.93 0.02 - -46.9 -46.9 -

Fourteen of 28 (50%) patients developed positive tetramer, IFN-γ and/or DTH responses to PSA146-154 peptide by week 52. A positive tetramer response is defined as > 4-fold increase in tetramer levels by week 52 over pre- vaccine levels, while positive IFN-γ response was defined as > 100 ng/ml of absolute change in cytokine levels at week 26 or 52 minus pre-vaccine levels. A positive DTH reaction is defined as > 15 mm of induration to PSA146-154 peptide. A stringent cut-off value was taken into consideration to measure true immune responses and to avoid false positives.

[0091] The method of vaccination appeared to impact tetramer response. The average fold increase in CD8+ PSA146-154 peptide :tetramer+ T-cells in patients vaccinated under protocol-1 (intradermal peptide) was 4.4 times higher than achieved with protocol-2 (peptide pulsed DC) in high-risk patients with locally advanced disease at 6 months following vaccination (p- value=0.007) (Figure 2). However, no significant differences remained at week 52, possibly due to declining tetramer responses over time.

Cytokine Bead Array Analysis

[0092] PSA146-154 peptide stimulated PBMC also were evaluated for specific release of cytokines following recognition of peptide-pulsed targets. Cytokines released into the culture supernatant, including, IFN-γ, TNF-oc, IL-4, IL-5 and IL-10, were measured concurrently by cytokine bead array analysis (BD Biosciences, San Diego, CA) as described earlier

[Perambakam et ah, Cancer Immunology Immunotherapy 55(9): 1033-1042 (2006)]. Briefly, the antigen presenting cell line, T2 (ATCC, Manassas VA), was used as a stimulator and was pulsed with 20 of PSA-peptide or control HLA-A2 binding peptide, HIV-RT476-484 or diluent alone (0.4 % volume by volume). T2 cells (25,000/well) were cultured with T-cells (100,000/well) in complete medium containing 30 U/ml of IL-2 in a total volume of 1 ml per well in 48- well plates. This particular stimulator to responder ratio was found to be optimal for culture in 48-well plates. Cells were incubated at 37° C for 24 hours in 5% C0₂ atmosphere. Supernatants were harvested and stored in sterile vials at -80° C. At the time of assay, samples were thawed and cytokines were measured using a CBA kit as per the manufacturer's protocol with a Calibure flow cyto meter (Becton Dickinson, Mountain View, CA). Results were represented as net cytokine levels (pg/ml) which were obtained by subtracting non-specific background responses (T2 cells pulsed with HIV-RT476-484 or diluent).

Microarray and Bioinformatic Analysis

[0093] Total RNA was extracted from pre-vaccine PBMC samples of patients with both strong specific DTH and tetramer responses (UPIN13, UPIN28, UPIN40, UPIN45 and UPIN71) and patients with negative DTH and tetramer responses (UPIN32, UPIN35, UPIN37 and UPIN70) using RNeasy mini kit (Qiagen, Valencia, CA). The quantity and quality of RNA were estimated with a NanoDrop TM 3300 Fluoro spectrometer (Thermo Fisher Scientific, Waltham, MA), and an Agilent bioanalyzer, respectively (Agilent Technologies, Santa Clara, CA). All RNA samples were stored at -80° C. Microarray analysis was performed at the functional Genomics

Laboratory of the University of Illinois at Urbana Champaign, using the human genome U133 plus 2.0 chips (Affymetrix, Santa Clara, CA). Data was extracted from the Affymetrix array and normalized by the Robust Multichip Average (RMA) method [Mzarry et al. , Nucleic Acids Research 31(el5) (2003)]. Class comparison analysis was conducted per the Biometric Research Branch (BRB) array tool (National Cancer Institute, Bethesda, MD). Gene expression data was compared between strong immune responders (UPIN13, UPIN28, UPIN40, UPIN45 and UPIN71- positive DTH and tetramer responses) and non responders (UPIN32, UPIN35, UPIN37 and UPIN70- negative DTH and tetramer responses).

Results -- Gene Expression Profiles of Immune Responders Versus Non Responders

[0094] Affymetrix human genome U133 plus 2.0 chips array analysis was performed on pre- vaccine PBMC, in order to identify genes and gene pathways that are differentially expressed between patients who developed strong immune responses versus patients who did not. Immune responders included patients with strong tetramer (>4.9 fold) responses in conjunction with a positive DTH skin reaction to the PSA146-154 peptide, while non-responders included patients who were negative for both tetramer and DTH responses.

[0095] Class comparison analysis per BRB array tools revealed that 166 of 54,675 genes were differentially expressed at a significance level of p<0.005 (Table 1). Predictably, the gene ontology class belonging to the biological process category of immune system development (GO ID: 0002520) was affected with an observed to expected ratio of 2.1. Of the 166 differentially expressed genes, 12 genes were involved in the immune function associated pathway (Table 2). A 4-fold increase in 2'-5' oligoadenylate synthetase 1 (OAS1) was noted in immune responders versus non-responders. Other genes that were over-expressed included, mitogen- activated protein kinase 1, Sh2 domain containing IB, vannin 1, CD58 molecule and interferon-induced transmembrane protein-3. Tumor necrosis factor receptor superfamily-member 25, chemokine C-C motif receptor 7 and phosphoinositide-3-kinase, regulatory subunit 1 alpha genes and epiregulin showed decreased expression in immune responders versus non-responders.

Clinical Evaluation

[0096] The disease status of patients was monitored by clinical examination and serial serum PSA levels scans on weeks 1, 4, 7, 14, 26, and 52. Biochemical progression (P) was defined as at least a 20 % increase in serum PSA at week 52 over week 1 (study entry) with an absolute PSA value >0.2 ng/ml. Stable biochemical disease or non progression (NP) was defined as less than a 20 % increase in serum PSA over week 1 with an absolute PSA value less than 0.2 ng/ml.

[0097] Survival status was established for all 28 vaccinated patients by review of the Social Security Death Registry Index and by direct contact of patients or their relatives. Time (in months) from the onset of vaccine therapy (week 1) till death or until May 1, 2010 for patients who were deceased or surviving, respectively was calculated followed by computation of OS per Kaplan-Meier analysis (SAS software version 9.2, Cary, NC). The median follow-up period was 6.30 years (mean 5.36 years; range 1.35 to 7.68 years).

Results -- Clinical Outcomes: Toxicity, Serum PSA and Survival Status

[0098] Toxicity: Both methods of vaccination were well tolerated with no treatment related grade 3/4 toxicities, graded according to the NIH Common Terminology Criteria for Adverse Events, version 3.0. Mild pain, itching, and erythema with or without induration were observed at the site of injection for all patients treated under protocol- 1. There were no late safety concerns or deleterious sequelae identified after six to eight years of monitoring.

[0099] PSA progression: Thirteen of 27 (48.1%) patients manifested stable or declining serum PSA, while 14 of 27 (51.6%) patients evidenced PSA progression at one year following the onset of PSA146-154 peptide vaccine therapy. One patient, UPIN27, did not return for follow-up at week 52 and hence, his biochemical status was not evaluable. However, the survival status was determinable in all 28 patients. As of May 1 2010, 15 of 28 (54%) patients were alive while 13 (46%) patients had died. In most patients, death was CaP specific, however, one patient, UPIN16, died of late occurring esophageal cancer.

[0100] Survival: OS is the most definitive standard to assess the outcome of anticancer therapies and was determined per Kaplan-Meier analysis eight years after the initiation of the protocol. The median follow-up period for individual patients was 6.30 years (range 1.35 to 7.68 years) from the onset of immunotherapy. The mean OS was 60 months (95% CI 51 to 68 months) for all patients (Figure 3-upper panel). The median OS has not yet been reached for patients with high risk, locally advanced disease, exceeding 84 months. On the other hand, the median OS was 75 months for patients with metastatic, hormone-sensitive CaP (Figure 3- middle and lower panel).

Correlation of Clinical Outcome With the Induction of Specific Immune Responses

[0101] The development of specific T-cell immune responses was correlated with patients' serum PSA and survival status. The results indicate that the average tetramer measurements at week 26 minus pre-vaccine levels inversely correlated with changes in serum PSA levels (Figure 4, p=0.02). Thus, a decreased risk of biochemical progression was observed in patients who developed augmented tetramer responses at six months compared to pre-vaccination levels. No significant correlation remained at one year, as specific immune responses became attenuated over time.

[0102] OS of patients who developed positive DTH responses, tetramer or IFN-γ responses to PSA146-154 peptide versus patients who did not develop specific immune responses were correlated by log-rank testing. The mean OS was 58 months (95% CI, 50 to 66 months) for strong DTH responders versus 54 months (95% CI, 41 to 68 months) for non-responders (p=0.21). The mean OS was 61 months (95% CI, 50 to 71 months) in patients who showed strong tetramer responses versus 44 months (95% CI, 35 to 52 months) for non-responders (p=0.46). The mean OS was 61 months (95% CI, 50 to 73 months) in patients who showed strong IFN-γ responses versus 55 months (95% CI, 43 to 68 months) for non-responders (p=0.65). Although these findings did not reach statistical significance, the patients who developed strong T-cell immunity in terms of specific DTH and tetramer responses to PSA146- 154 peptide within one year following vaccination, demonstrated a trend towards greater OS (Figure 5).

Statistical Analysis

[0103] A marginal longitudinal model was used to compare tetramer or cytokine

measurements over time within similar groups of patients. The dependent variable was the log of the tetramer values or cytokine measurements. The independent variables included intercept, group, time dummies, and interactions between group and time dummies. Pearson correlation coefficients were used to evaluate correlation between the fold increase in tetramer levels and absolute change IFN-γ cytokine. Spearman analysis was used to evaluate the correlation of tetramer or cytokines values with serum PSA status. The two sample t-test with unequal variance was used to identify genes that were differentially expressed between immune responders and non responders per BRB array tools. OS was evaluated per Kaplan-Meier analysis. The log-rank tests were used to evaluate differences in survival curves.

Conclusions

[0104] In the current study, 15 of 28 (54%) patients were alive at eight years from the initiation of the protocol while 13 (46%) patients had died. Of note, a trend towards greater survival in men with high-risk, hormone-sensitive CaP who developed strong specific DTH or tetramer responses following vaccination with PSA146-154 peptide was observed. Two previous cancer vaccine studies conducted in hormone-refractory CaP patients showed that survival positively correlated with the induction of specific immune responses [Thomas-Kaskel et al., International Journal of Cancer 119(10): 2428-2434 (2006); Gulley et al., Cancer Immunology Immunotherapy 59(5): 663-674 (2010)]. The demonstration of statistically significant survival advantages by immunization of hormone-sensitive CaP patients with longer life expectancies will require extended periods of observation and expanded patient cohorts. Importantly, the availability of quantitative metrics for monitoring the induction of specific T- cell immunity to defined target antigens as in the study presented herein, should provide an important surrogate for gauging vaccine efficacy, if a causal relationship between the induction of specific T cell immunity and survival advantages can be definitively established. This in turn would speed vaccine optimization for early phases of CaP.

[0105] DC are central to successful vaccination and can be directly targeted in vivo with antigen and adjuvants, such as GM-CSF, as demonstrated in early pioneering studies [Palucka et al, Immunological Reviews 220: 129-150 (2007); Disis et al., Blood 88(1): 202-210 (1996); Disis et al, Journal of Clinical Oncology 20(11): 2624-2632 (2002)]. Alternatively, ex vivo generated monocytic or CD34-derived DC loaded with tumor antigen can be utilized for specific active immunotherapy of cancer patients [Nestle et ah, Nature Medicine 4(3): 328-332 (1998); Thurner et ah, Journal of Experimental Medicine 190(11): 1669-1678 (1999); Banchereau et ah, Cancer Research 61(17): 6451-6458 (2001); Timmerman et al, Blood 99(5): 1517-1526 (2002)] . However, DC-based vaccine formulations involve laborious manipulations ex vivo and incur considerable cost. Therefore, the efficacy of PSA146-154 peptide vaccine by both techniques was compared in a randomized fashion. The results revealed that the average fold increase in CD8+ PSA146-154peptide:tetramer+ T-cells was 4.4 times higher in patients vaccinated with PSA146-154 peptide admixed with GM-CSF injected intradermally as compared to intravenous administration of peptide-pulsed, autologous DC. The finding that a simple method of intradermal vaccination is efficacious has important implications for the affordability and applicability of the technique to the general population. These results are corroborated by a similar study, wherein, intradermal injection of E75 HER2/neu peptide GM-CSF was found to be efficacious in high risk node positive breast cancer patients [Peoples et al, Journal of Clinical Oncology 23(30): 7536-7645 (2005)].

[0106] The study presented herein showed that a set of molecular determinants expressed within PBMC distinguish immune responders and non responders undergoing vaccination with a peptide-based cancer vaccine. Genomic and bioinformatics analysis revealed 166 genes that are differentially expressed between strong immune responders versus non responders. In particular, genes associated with innate immune response were over-expressed, including, OAS1, which belongs to a family of IFN-stimulated proteins [Haralambieva et al, Human Immunology 71(4):

383-391 (2010)]. Interestingly, OAS1 also is postulated to be associated with radiation resistance in human breast cancer and CaP cell lines and with the regulation of cell growth in mammary and prostate glands [Tsai et al., Cancer Research 67(8): 3845-52 (2007); Maia et al., Molecular and Cellular Biochemistry 314(1-2): 113-121 (2008)].

[0107] To date, the majority of tumor vaccines have been evaluated in patients with the most advanced forms of disease. In the present disclosure, it was observed that the development of specific T-cell immunity in terms of positive peptide- specific tetramer and IFN-γ responses (> 4- fold increase or > 100 pg/ml fold change, respectively) in 50% of patients vaccinated at points in the spectrum of prostate cancer that precede the development of castrate -resistance. Importantly, patients who developed augmented tetramer responses at six months compared to pre- vaccination levels had a decreased risk of biochemical progression at one year following the onset of immunotherapy. The inclusion of patients with hormone-sensitive disease who are immunologically robust, as reported herein, may be key to harnessing the full potential of novel vaccine regimens.

[0108] In summary, twenty eight HLA-A2+ patients with high-risk, locally advanced or metastatic, hormone-sensitive prostate cancer were immunized with a peptide homologue of prostate specific antigen, PSA146-154, between July 2002 to September 2004 and monitored for clinical and immune responses. Fifty percent of the patients developed strong PSA146-154- peptide specific tetramer and/or IFN-γ responses within one year and were positively correlated (p<0.001). Thirteen patients had stable or declining serum levels of PSA one year post- vaccination. A decreased risk of biochemical progression was observed in patients who developed augmented tetramer responses at six months compared to pre-vaccination levels (p=0.02). Thirteen patients have died while 15 patients remain alive with a mean overall survival of 60 months (95% CI, 51 to 68 months) per Kaplan-Meier analysis. A trend towards greater overall survival was detected in men with high-risk, hormone- sensitive CaP who developed specific T-cell immunity following vaccination with PSA146-154 peptide.

Example 2

[0109] PSA146-154 peptide vaccination by the intradermal route can be readily exported to other study sites and is amenable to multiparametric immunological monitoring that can provide accurate quantification and standardization across a trials network. The target tumor, prostate cancer, is typically indolent and, thus amenable, to immunization schema that may have long latencies to response. Prostate cancers are commonly detected and treated in low tumor burden states, an optimal condition for immunotherapeutic attempts. Relapse and progression of prostate cancer can be readily monitored and quantified by biochemical markers, well before measurable differences in standard Response Evaluation Criteria in Solid Tumors (RECIST) criteria can be observed. In sum, PSA146-154 peptide vaccine provides a promising backbone for testing and analysis of combinatorial vaccination schema envisioned by the Clinical

Immunotherapy Trials Network (CITN).

[0110] Multiple immune inhibitory pathways have been implicated in prostate cancer.

Programmed Death- 1 (PD-1), B7 Homologue 3 (B7-H3) and B7x are especially conspicuous. Release of inhibitory signals with specific antibodies can potentiate cellular immune responses. Ipilimumab, an anti-CTLA4 mAb under development by Medarex and Bristol Myers Squibb, disrupts the interaction of Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) with B7.1 and mediates important anti-tumor effects in clinical trials. Potentiating effects also have been observed in clinical trials with the Medarex monoclonal antibody, MDX1106, which blocks the interaction of PD-1 with PD1-L. In murine models the combination of co-inhibitory blockade with the Toll- Like Receptor 9 (TLR9) agonist, CpG-Oligodeoxynucleotide (CpG-ODN) substantially augments T cell immune responses to specific peptide vaccines. An equivalent compound CPG7909 under development by Pfizer Corporation (PF351276) showed potentiating effects in clinical trials and limited toxicity at the doses used. The compound is well tolerated. We propose to move forward with phase I II studies to evaluate whether similar synergies can be obtained in humans by vaccinating prostate cancer patients with PSA146-154 peptide in combination with a TLR9 agonists and co-inhibitory blockade of both the CTLA-4 and PD-1 pathways. Vialized PSA146-154 peptide is held under IND BB8691. A suggested protocol is outlined below:

[0111] Purpose

[0112] To test the safety and efficacy of combinatorial PSA146-154 peptide vaccination with a TLR-9 agonist and concurrent blockade of the CTLA-4 and PD-1 co-inhibitory pathways.

[0113] Specific Aims

1). To determine the safety of the combination of PSA146-154 peptide/GM-CSF vaccination with adjuvant CpG7909 plus anti-CTLA-4 mAB (ipilimumab) and anti-PDl mAb (MDX1106 ) in patients with recurrent, low burden castration-resistant prostate cancer (cRPC) after primary ablative therapy.

2) To determine the efficacy of the combinatorial vaccine to elicit and sustain PSA146- 154 peptide specific T cell immunity by immunomonitoring of specific Delayed Type

Hypersensitivity (DTH), PSA peptide-tetramer analysis, and multiplex cytokine release assays over time.

3) To determine the correlation between the quantity and function of PSA146-154 peptide specific T cell immunity induced by vaccination at defined intervals with disease course and survival.

[0114] Endpoints

[0115] Phase I: Safety

[0116] Phase II:

1) . Multiparametric quantitation of peptide specific immunity;

2) . Progression Free Survival (PFS) (biochemical); Overall Survival; Quality of Life

Index

3) . Correlative analysis comparing actual survival outcomes to prospectively modeled survival based on Halabi normograms

[0117] Eligibility

Men of the HLA-A2.1 phenotype with asymptomatic castrate resistant PCa and projected survival of >6 mo

[0118] Inclusion

Male

Age 18 and above

Histologically proven PSA+ prostate cancer, status post primary ablative therapy of the prostate

Castrate resistant with biochemical disease recurrence, asymptomatic with no detectable metastases Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) 0-1 Life expectancy > 6 months

HLA-A2.1 phenotype

[0119] Exclusion

Prior immunotherapy

Other malignancies except adequately treated basal cell or squamous cell skin carcinoma Autoimmune disorders

Human Immunodeficiency Virus (HIV), hepatitis B, hepatitis C or other active infections

Corticosteroids or other immunosuppressive therapies

Known immunodeficiency disorders

Pprostate Specific Antigen (PSA) doubling time <2 month

Specified co-morbid conditions or organ dysfunction

White Blood Count (WBC)<2000/microliter

Absolute Neutrophil Count (ANC)<1000/microliter

Platelets< 100 x 103 /microliter

Aspartate Amino Transferase/ Alanine Aminotransferse (AST/ALT)>2.5 x ULN

Bilirubin > 2X Upper Limit of Normal (ULN)

Serum Creat > 2.0 x ULN

[0120] Schema

[0121] Phase I (cohorts of 6-12, total of 24 patients)

Cohort I.

PSA peptide 100 μg + GM-CSF 500 U + CpG-ODN 500 μg i.d. (PGC vaccine) weeks 1, 4 and 10 then every 6 months up to 4 years (PGC vaccine = PSA peptide + GM-CSF + CpG- ODN vaccine)

Cohort II. PGC vaccination i.d. weeks 1, 4, and 10 then every 6 months plus:

Anti-CLTA-4 mAb (Ipilimumab) lOmg/kg week 1, 4 and 10 then every 8 weeks to week

52

Cohort III.

PGC vaccination i.d. week 1, 4, and 10 then every 6 months plus:

Anti-PD-1 mAb (MDX 1106) 10/mg week 1, 4 and 10 then every 8 weeks to week 52

Cohort IV.

PGC vaccination plus anti-CTLA-4 mAb plus anti PD-1 mAb

[0122] Phase II (100 patients, 50 per arm)

Arm 1. PGC vaccination (per Phase 1 -Cohort 1 schedule)

Arm 2. PGC vaccination plus anti-CTLA-4 mAb and anti-PD-1 mAb

[0123] Outcomes

Immunomonitoring (baseline, week 4, 12 and 26 then annually)

Peptide specific DTH testing (biopsy of positive DTH for immunophenotyping and Chromium Release Assay (CRA)):

PSA peptide-tetramer analysis of CD8+ Peripheral Blood Lymphocyte (PBL):

Multiplex cytokine array analysis of CD8+ PBL

Gene array analysis of pre and post vaccination PBMC

Clinical monitoring (baseline, monthly x 6 then every 3months

Clinical assessment and exam

Screen for autoimmune toxicity

Complete Blood Count (CBC) and metabolic panel, Antinuclear Antibody (ANA), Coombs, Thyroid Stimulating Hormone (TSH), c-Reactive Protein (CRP)

Serum PSA (baseline, monthly x 6 then every 3 months)

Bone Metabolites Radiographic studies as clinically indicated

[0124] The individual components of the proposed regimen are prioritized compounds of the 2007 Immunotherapy Agent Workshop and likely to be accessible for incorporation into network studies. The proposed protocol aims to confirm preclinical studies that combined TLR stimulating adjuvants and co-inhibitory blockade can amplify peptide specific immunization to levels needed to achieve significant clinical effect. The results will be applicable to diverse tumor types for which distinguishing tumor associated peptide epitopes are identified. Favorable results will intensify the search for epitopes that will be applied to the treatment of other tumor types in the context of diverse HLA phenotypes.

Example 3

[0125] The expression of PSA is highly restricted to normal and transformed prostatic epithelial tissues. Immunohistochemical staining of prostate cancer revealed PSA-specific staining in 99% of primary and metastatic lesions [Ford et ah, Br J Urol 57: 50-5 (1985)]. Thus PSA is a suitable tumor-associated antigen (TAA) for the induction of specific cytototic T lymphocytes (CTL). CTL recognize processed peptide antigens (approximately 9 to 10 amino acids in length) in association with class I molecules of the major histocompatibility complex (MHC), also called human leukocyte antigen or HLA [Townsend et ah, Annu Rev Immunol 7: 601-24 (1989)].

[0126] Previous work by the inventors identified an HLA-A201 restricted epitope of PSA (PSA146-154 of amino acid sequence KLQCVDLHV (SEQ ID NO: 3)) that induced specific CTL responses in healthy individuals and patients with prostate cancer [Xue et ah, Prostate 30(2): 73-8 (1997); Perambakam et al, Cancer Immunol Immunother 51(5): 263-70 (2002)]. Previous work by the inventors also yielded results of a clinical trial involving PSA146-154 peptide (SEQ ID NO: 3) [Perambakam et al., Cancer Immunol Immunother 55(9): 1033-42 (2006)]. Intradermal administration of peptide (SEQ ID NO: 3) admixed with GM-CSF or intravenous injection of autologous dendritic cell-bound peptide, induced specific T-cell immunity in 50% of patients with prostate cancer of HLA-A2 phenotype. Importantly, patients who demonstrated specific immunity in terms of induction of specific tetramer responses within one year of the first vaccination, showed a lower risk of serum PSA progression, further validating the efficacy of PSA146-154 peptide vaccine [Perambakam et al, Clin Dev Immunol 2010: 473453 (2010)].

[0127] Herein, the induction of specific CTL with cytolytic and cytokine activity by in vitro sensitization is reported using two long-chain synthetic peptides corresponding to human PSA protein residues, 154-173 and 210-230 in healthy individuals. In silico analysis revealed that PSA 154-173 and PSA 210-230 peptides contain multiple putative HLA binding motifs. These results open up new avenues for the PSA-based multi-peptide therapy of diverse array of prostate cancer patients.

Methods

PSA Peptides

[0128] Two long chain synthetic peptides corresponding to PSA residues 154-173 (amino acid sequence VIS ND VC AQ VHPQKVTKFML; SEQ ID NO: 1) and 210-230 (amino acid sequence CALPERPSLYTKVVHYRKWIK; SEQ ID NO: 2) were purchased from Research Genetics Inc (Huntsville, AL) at greater than 95% purity. Peptides were dissolved in dimethyl sulphoxide at a concentration of 5 mg/ml in 2-5 ml aliquots and stored at -80° C for long term storage.

Establishment Of Specific CTL by Sensitization in vitro

[0129] Healthy individuals were recruited in the study following informed consent of the Institutional Review Board of University of Illinois at Chicago. They were typed for HLA- ABDR per manufacturer's instructions (One Lambda Inc, CA) and peripheral blood mononuclear cells (PBMC) were obtained by ficoll-gradient centrifugation as previously described [Xue et ah, Prostate 30(2): 73-8 (1997)].

[0130] CTL-PSA154-173 and CTL-PSA210-230 were induced from PBMC by multiple cycles of in vitro sensitization with respective PSA peptides as previously detailed [Xue et ah, Prostate 30(2): 73-8 (1997)]. Briefly, PBMC (1.25xl0⁶ per well) were stimulated with peptide (20 microgram/well) and cultured in RPMI-1640 medium containing 10% human AB serum (complete media) and rIL-2 (20U/ml) in 24- well plates and cultured at 37° C in 5% C0₂ atmosphere for 7+1 days (1 cycle). In subsequent cycles, T-cell cultures were re-stimulated with autologous irradiated PBMC (lxl0⁵/well) pulsed with respective PSA peptide (SEQ ID NO: 1 or SEQ ID NO: 2). Following 5-8 cycles of stimulation, CTL were pooled and assayed for specificity and functionality as described below.

Enzyme Linked Immune- Spot Assay

[0131] The specificity of the induced CTL-PSA154-173 and CTL-PSA210-230 were evaluated in IFN-gamma enzyme linked immune-spot (ELISPOT) assay as previously detailed [Perambakam et ah, Cancer Immunol Immunother 51(5): 263-70 (2002)]. Briefly, target cells (PWM stimulated autologous lymphoblasts at 10,000 cells/well) were pulsed with PSA-peptide or control HIV-RT 476-484 peptide (at 20μg/ml) or in the absence of peptide and were co- cultured with CTL (5000 cells/well) in complete medium containing 30 U/ml of rIL-2 in 96-well polystyrene plate (Greiner, Germany) followed by incubation at 37° C in 5% C0₂ atmosphere for 24 hours. The number of spot- forming cells was visualized by a two-step, purified mouse anti- human IFN-gamma capture and biotinylated mouse anti-human detection monoclonal antibody system (BD Biosciences, San Diego, CA). Assays were developed with an avidin-biotin complex substrate conjugate system (Santa Cruz Biotechnology, Santa Cruz, CA) followed by tetramethylbenzidine (TMB) liquid color developer (Sigma, St Louis, MO). The blue spots were counted microscopically, and the results were represented as the number of spot forming cells.

Chromium Release Assay

[0132] The cytolytic activity of induced CTL was analyzed by standard 4-hour chromium release assay as previously described [Xue et ah, Prostate 30(2): 73-8 (1997)]. Briefly, targets (T2 cells) were labeled with 100 micro Curies of Na⁵¹Cr0₃ (Amersham Pharmacia Biotech, Piscataway, NJ). Chromium labeled targets (lxl0⁴/well) were then pulsed with PSA-peptide or HIV-RT 476-484 peptide or no peptide and incubated with graded number of CTL for 4 hours in 96-well 'V bottom plate (Nunc, Naperville, IL). Supernatants were harvested and assayed for gamma emission using Top-count NXT scintillation counter (PerkinElmer, Waltham, MA) and percent lysis was calculated as detailed previously [Xue et ah, Prostate 30(2): 73-8 (1997)].

Peptide-Binding Assay

[0133] The antigen-processing defective cell line, T2, was used to assay peptide binding to

HLA-A201 as previously detailed [Xue et al, Prostate 30(2): 73-8 (1997)]. Briefly, T2 cells

(5xl0⁵/well) were pulsed with PSA-peptide or DMSO at the designated concentration and cultured in 24- well plates overnight (18 hours) at 37° C in 5% C0₂ atmosphere. HLA-A2 expression was measured by flow cytometry using FITC conjugated anti HLA-A2 antibody (clone BB7.2) from BD Biosciences (San Diego, CA). in silico analysis for HLA Peptide Binding Motifs

[0134] The 261 amino acid protein sequence (accession number P07288) obtained from national center for biotechnology (www.ncbi.nlm.nih.gov) was utilized as "in-put" sequence for human PSA. Bioinformatics and molecular analysis section (BIMAS, http://www- bimas.cit.nih.gov/molbio/hla_bind/) algorithm of the National Institute of Health, Bethesda, Maryland was utilized to evaluate the HLA peptide binding predictions.

Results

Induction of specific CTL by in vitro sensitization of PBMC with PSA peptides

[0135] CTL-PSA154-173 and CTL-PSA210-230 were induced by multiple cycles of in vitro sensitization of PBMC. Healthy individual was HLA-A2/A2, HLA-B61/B51 and HLA- DR4/DR10 phenotype as evaluated by HLA-ABDR typing per manufacturer's instructions (One Lambda Inc, CA). These CTL lines contained greater than 90% CD8+ T-cells as determined by flow cytometric analysis. Importantly, CTL-PSA154-173 and CTL-PSA210-230 specifically released IFN-gamma cytokine as evaluated by ELISPOT assay (Figure 6). A greater than 3.5 fold-increase in IFN-gamma cytokine was observed against PSA-peptide pulsed autologous lymphoblasts compared to baseline levels (lymphoblasts pulsed with control HIV-RT 476-484 peptide or no peptide) for both CTLs.

[0136] The cytolytic potential of induced CTL-PSA154-173 and CTL-PSA210-230 were evaluated in chromium release assay and results indicated specific killing of PSA-peptide pulsed T2 cells (Figure 7). Additionally, CTL-PSA210-230 was significantly more cytolytic compared to CTL-PSA154-173 (Figure 7). These results validated PSA154-173 and PSA210-230 peptides as potential candidates for inducing specific CTL.

Long- Chain Peptides of PSA Contain Multiple Putative HLA Binding Motifs

[0137] The ability of a peptide to elicit CTL restricted by a particular class I MHC molecule is contingent on the ability of the peptide to bind to class I MHC molecule. Therefore, the bioinformatics and molecular analysis section (BIMAS) algorithm was utilized to identify HLA peptide binding motifs. BIMAS analysis revealed that the 20-mer PSA 154-173 peptide (SEQ ID NO: 1) contained putative binding motifs for HLA-B7 and B51 (Table 6). The 21-mer PSA 210-230 peptide (SEQ ID NO: 2) included putative binding motifs for HLA-A201, A3, Al lOl, B7, B31 and HLA-B61 (Table 6).

[0138] T2 binding assay was conducted to confirm the HLA-A201 binding capacity of PSA210-230 peptide and as observed, PSA210-230 peptide (SEQ ID NO: 2) binds with high affinity in a concentration dependent fashion (Figure 8).

Table 6. PSA 154-173 (SEQ ID NO: 1) and PSA 210-230 (SEQ ID NO: 2) peptides contain multiple putative 9- and 10-mer HLA binding motifs .

HLA peptide binding predictions were evaluated using BIMAS algorithm http://www- bimas.cit.nih.gov/molbio/hla bind/. An estimate of half-time of disassociation of long-chain peptide or protein containing this subsequence. Only the top 6 ranking motifs (out of 20) were included.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a fragment of prostate specific antigen (PSA), said fragment peptide comprising the sequence VISNDVCAQVHPQKVTKFML (SEQ ID NO: 1).

2. A composition comprising a fragment of prostate specific antigen (PSA), said fragment peptide comprising the sequence CALPERPSLYTKVVHYRKWIK (SEQ ID NO: 2).

3. A method for treating prostate cancer in a human comprising the step of administering to said human the composition of claim 1 and/or claim 2 in an amount effective to stabilize or reduce serum PSA levels.

4. The method of claim 3 further comprising the step of administrating granulocyte monocyte colony stimulating factor (GM-CSF).

5. The method of claim 4 wherein said composition and GM-CSF are coadministered.

6. The method of claim 5 wherein said composition and GM-CSF are administered concurrently.

7. The method of claim 5 wherein said composition and GM-CSF are administered sequentially.

8. The method of any one of claims 3 to 7 wherein administering is intradermal.

9. The method of claim 5 wherein said PSA peptide and GM-CSF are coadministered in a weight- to- weight ratio of about 1:5.

10. The method of any one of claims 5 to 9 wherein PSA peptide and GM-CSF are co-administered in multiple injections.

11. The method of claim 10 wherein PSA peptide and GM-CSF are co-administered in up to five injections.

12. The method of claim 3 wherein the PSA peptide is administered as a composition of dendritic cells pulsed with the PSA peptide.

13. The method of any one of claims 3 to 11 wherein a total of about 100 μg PSA peptide is administered in multiple injections.

14. A vaccine comprising: (i) a composition selected from the group consisting of the composition of claim 1, the composition of claim 2 and a composition comprising a prostate specific antigen (PSA) peptide (PSA peptide 146-154; SEQ ID NO: 3), or combinations thereof, and(ii) a pharmaceutically acceptable carrier.

15. The vaccine of claim 14 further comprising granulocyte monocyte colony stimulating factor (GM-CSF).

16. The vaccine of claim 14 or claim 15 further comprising a TLR9 agonist in an amount effective to increase a T cell immune response.

17. The vaccine of claim 16 wherein the TLR9 agonist is a CpG- oligodeoxynucleotide (CpG-ODN).

18. The vaccine of any one of claims 14-17 further comprising an inhibitor of CTLA4 in an amount effective to increase a T cell immune response.

19. The vaccine of claim 18 wherein the inhibitor of CTLA4 is a monoclonal antibody.

20 The vaccine of any one of claims 14-18 further comprising an inhibitor of PD-1 in an amount effective to increase a T cell immune response.

21. The vaccine of claim 20 wherein the inhibitor of PD-1 is a monoclonal antibody.

22. A method of vaccinating an individual comprising the step of administering the vaccine of any one of claims 14 through 21 to the individual in an amount effective to vaccinate the individual.

23. The method of claim 22 wherein the PSA peptide is co-administered with GM- CSF.

24. The method of claim 23 wherein the PSA peptide and GM-CSF are coadministered in multiple injections.

25. The method of claim 23 wherein the PSA peptide and GM-CSF are administered concurrently.

26. The method of claim 23 wherein the PSA peptide and GM-CSF are administered sequentially.

27. The method of claim 24 wherein the PSA peptide and GM-CSF are coadministered in up to five injections.

28. The method of claim 24 wherein a total of about 100 μg PSA peptide is administered in multiple injections.

29. The method of any one of claims 22 to 28 wherein the PSA peptide is administered in weeks 1, 4 and 10, and then every six months up to four years.

30. The method of any one of claims 22 to 28 wherein the PSA peptide is administered in weeks 1, 4 and 10, and then every six months up to four years, wherein an inhibitor of CTLA4 is administered in weeks 1, 4 and 10, and then every eight weeks up until week 52.

31. The method of any one of claims 22 to 28 wherein the PSA peptide is administered in weeks 1, 4 and 10, and then every six months up to four years, wherein an inhibitor of PD-1 is administered in weeks 1, 4 and 10, and then every eight weeks up until week 52.

32. A method of identifying a patient that is a candidate for prostate cancer therapy comprising the step of:

measuring expression level in a sample from a test individual of one or more genes identified in Table 1 relative to a reference expression level,

wherein an increase or a decrease in expression of the one or more genes identified in Table 1 relative to the reference expression level is determinative for identifying whether the patient is a candidate for prostate cancer therapy according to the method of claim 3.

33. The method of claim 32 further comprising collecting the sample from the test individual.

34. The method of claim 32 or claim 33 further comprising comparing the expression level to the reference expression level.

35. The method of any one of claims 32 to 34 wherein an increase in expression of a gene selected from the group consisting of 2'-5' oligoadenylate synthetase 1 (OASl), mitogen- activated protein kinase 1 (MAPKl), Sh2 domain containing IB (SH2D1B), vannin 1 (VNNl), CD58 molecule (CD58), DEAD box polypeptide 58 (DDX58), X-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4) and interferon-induced transmembrane protein-3 (IFITM3) is indicative of the patient being a candidate for prostate cancer therapy according to the method of claim 3.

36. The method of any one of claims 32 to 34 wherein a decrease in expression of a gene selected from the group consisting of tumor necrosis factor receptor superfamily-member 25 (TNFRSF-25), chemokine C-C motif receptor 7 (CCR7), and phosphoinositide-3-kinase, regulatory subunit 1 alpha (PIK3R1) and epiregulin (EREG) is indicative of the patient being a candidate for prostate cancer therapy according to the method of claim 3.

37. A method of rendering an individual a candidate for prostate cancer therapy comprising the steps of:

(i) modulating expression of at least one gene listed in Table 1 to a degree that renders the individual a candidate for prostate cancer therapy; and

(ii) administering a therapeutically effective amount of the composition of claim 1 or claim 2, or the vaccine of claim 14 to said patient to treat prostate cancer.

38. The method of claim 37 wherein modulating increases expression.

39. The method of claim 37 or claim 38 wherein the gene is 2'-5' oligo adenylate synthetase 1 (OAS1).

40. The method of claim 37 wherein modulating decreases expression.