WO1997034921A1

WO1997034921A1 - Conjugated mucin peptide vaccines

Info

Publication number: WO1997034921A1
Application number: PCT/US1997/004493
Authority: WO
Inventors: Philip O. Livingston; Shengle Zhang
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 1996-03-20
Filing date: 1997-03-20
Publication date: 1997-09-25
Also published as: CA2249395A1; AU2536997A; JP2001510440A; EP0923605A4; AU734253B2; EP0923605A1

Abstract

This invention provides a vaccine capable of producing an immune response which recognizes a mucin, comprising an amount of mucin peptide conjugated to an immunogenic protein effective to stimulate or enhance immune response in the subject, an effective amount of an adjuvant and a pharmaceutically acceptable vehicle. This invention further provides a method for stimulating or enhancing production of an immune response which recognizes a mucin, comprising administering to the subject the above-described vaccine.

Description

CONJUGATED MUCIN PEPTIDE VACCINES

The invention disclosed herein was made with government support under NIH Grant No. CA 61422 from the Department of Health and Human Services. Accordingly, the U.S. Government has certain rights in this invention.

Background of the Invention

Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.

Mucins, such as the mucin MUC1, are extensively glycosylated high molecular weight (> 200 kD) proteins abundantly expressed in many human cancers of epithelial origin (1-5) . Recent studies have demonstrated that MUC1 contains a variable number of tandem repeats of a 20 amino^' acid peptide (PDTRPAPGSTAPPAHGVTSA) (SEQ ID. 7) in the extracellular portion of the molecule and that the most antigenic epitome recognized by anti-mucin mAbs and cytotoxic T cells is the APDTR segment within the repeats (6-8) . Expression of MUC1 on normal tissues is largely restricted to the apical surface of secretory cells (1, 4) , a site with minimal access to the immune system. In addition, the extensive glycosylation of MUC1 expressed on normal tissues may further limit the immune system exposure to the peptide backbone. It has been suggested that the peptide backbones of mucins are not fully glycosylated in carcinomas, resulting in exposure to the immune system of peptide sequences which are not normally exposed (6) . Consequently, MUC1 peptide specific monoclonal antibodies show specificity for ca rcinoma-associated mucins from cancers of breast, pancreatic and ovary origin though the amino acid sequences in both normal and carcinoma mucins are probably the same (9) . Immunization of rats with a vaccinia recombinant expressing MUCl has resulted in protection from challenge with MUCl-expressing tumor cells (5) . These findings have suggested that immunization against MUCl may be possible and that this immunization might prevent tumor regrowth in patients with breast or pancreatic cancer. In this study, applicants' synthesized MUCl peptides of different lengths and sequences and prepared vaccines containing these peptides mixed with various adjuvants or covalently attached them using different linkers to the protein carrier keyhole limpet hemocyanin (KLH) . In preparation for clinical trials, the impact of the different vaccines on humoral and cellular immunological responses and protection from tumor challenge was compared in mice.

Human mucin MUCl is abundantly expressed in some cancers of epithelial origin and is largely restricted to the apical surface of secretory cells in normal tissues. It is therefore a potential target for cancer i munotherapy. In preparation for clinical trials, vaccines containing synthetic MUCl peptides of different lengths and sequences mixed with various adjuvants or covalently attached, using different linker methods, to protein carrier keyhole limpet hemocyanin (KLH) were studied in mice. MUCl peptides

(containing 30 amino acids) , plus adjuvants QS-21 or BCG, were incapable of inducing antibody. However, MUCl peptide conjugated to KLH (MUCl-KLH) , plus QS-21, induced high titer antibody against the immunizing peptides and against MUCl-expressing tumor cells. Although T cell responses including delayed type hypersensitivity, lymphocyte proliferation and cytotoxic T lymphocyte were not observed in mice immunized with any of these vaccines, significant protection from MUCl-expressing tumor cell challenge in mice immunized with MUCl-KLH was observed. Based on these studies, a vaccine containing MUCl-KLH conjugate prepared with m-Maleimidobenzoyl-N-hydroxysuccinimide ester linker, plus QS-21, has been constructed for testing in clinical trials.

Summary of the Invention

This invention provides a vaccine capable of producing an immune response which recognizes a mucine, comprising an amount of mucin peptide conjugated to an immunogenic protein effective to stimulate or enhance immune response in the subject, an effective amount of an adjuvant and a pharmaceutically acceptable vehicle.

In an embodiment, the subject is a human.

In another embodiment, the immunogenic protein is Keyhole Limpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin.

In a separate embodiment, the mucin is MUCl. The mucin may include other mucins such as MUC 2-5. A person of ordinally skill in art would be able to apply this invention in other mucins.

In a further embodiment, the MUCl peptide ranges from ten amino acids to three hundred amino acids in length.

In an embodiment, the effective amount of conjugated MUCl peptide is an amount between about 1 μg and about lmg. In another embodiment, the adjuvant is QS-21.

In an embodiment, the effective amount of QS-21 is an amount between about lOμg and about 200μg. In an separate embodiment, the effective amount of QS-21 is about 100 μg.

In another embodiment, the subject is afflicted with cancer and the immune response produced in the subject upon administration of the vaccine effectively treats the cancer. In a further embodiment, the subject is susceptible to cancer and the immune response produced in the subject upon administration of the vaccine effectively prevents the cancer. In an embodiment, cells of the cancer have the mucin on their surface.

In a further embodiment, the cancer is a breast cancer, prostate cancer, colon cancer, lung cancer or pancreas cancer. This invention is applicable to other cancers of epithelial origin.

This invention also provides a method of producing an immune response which recognizes the mucin comprising administering to the subject an effective dose of the above-described vaccine.

This invention proviαes a method for treating cancer in a subject afflicted with cancer comprising administering to the subject an effective dose of the above-described vaccine.

This invention provides a method for preventing cancer in a subject susceptible to cancer comprising administering to the subject an effective dose of the above-described vaccine .

In the above methods, the immunogenic protein may be Keyhole Limpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin. In an embodiment of the above methods the adjuvant is QS-21.

Brief Description of the Ficrures

Figure 1 Conjugation of MUCl to KLH.

Detailed Description of the Invention

This invention provides a vaccine capable of producing an immune response which recognizes a mucin, comprising an amount of mucin peptide conjugated to an immunogenic protein effective to stimulate or enhance immune response in the subject, an effective amount of an adjuvant and a pharmaceutically acceptable vehicle. In an embodiment, the subject is a human.

The effective amount of the conjugated mucin peptide may easily be determined by simple titration experiment. Animals may be immunized with different amounts of the conjugated peptide and tested with the immune response generated. The effective amount will generate an appropriate immune response.

In another embodiment, the immunogenic protein is Keyhole Limpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin. Other appropriate immunogenic proteins may also be used in this invention. An ordinary skilled artisan may test the appropriateness of an immunogenic protein by conjugating the tested immunogenic protein with a mucin peptide known to be capable of illiciting an immune response. The mucin peptide conjugated may be administrated in animals to test whether it can generate good immune responses. Proteins with good immune response are the appropriate immunogenic proteins.

In a separate embodiment, the mucin is MUCl. The mucin may include other mucins such as MUC 2-5. A person of ordinally skill in the art would be able to apply this invention in other mucins.

In a further embodiment, MUCl peptide ranges from thirty amino acids to three hundred amino acids in length. In a specific embodiment, the mucin peptide is selected from a group consisting of APDTRPAPGSTAPPAHGVTS , TAPPAHGVTSAPDTRPAPGS, APDTRPAPGSTAPPAHGVTSAPDTRPAPGS, VTSAPDTRPAPGS TAP PAHGVTSAPDTRPA , a nd (VTSAPDTRPAPGSTAPPAHG)₂VTSAPDTRPA. In a further specific embodiment , the mucin peptide is VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA.

In a preferred embodiment, the effective amount of conjugated MUCl peptide is an amount between about 1 μg and about lmg.

In another embodiment, the adjuvant is QS-21. As it can be readily appreciated by persons of ordinary skill in the art, other appropriate adjuvants may be similarly used.

In a preferred embodiment, the effective amount of QS-21 is an amount between about lOμg and about 200μg. In a further preferred separate embodiment, the effective amount of QS- 21 is about 100 μg.

In another embodiment, the subject is afflicted with cancer and the immune response produced in the subject upon administration of the vaccine effectively treats the cancer.

In a further embodiment, the subject is susceptible to cancer and the immune response produced in the subject upon administration of the vaccine effectively prevents the cancer. In an embodiment, cells of the cancer have the mucin on their surface.

This invention also provides a method for stimulating or enhancing in a subject production of an immune response which recognizes the mucin comprising administering to the subject an effective dose of the above-described vaccine.

This invention provides a method for treating cancer in a subject afflicted with cancer comprising administering to the subject an effective dose of the above-described vaccine.

In the above methods, the immunogenic protein includes, but is not limited to Keyhole Limpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin.

In an embodiment of the above methods, the adjuvant is QS- 21.

In a separate embodiment, the mucin is MUCl. The mucin may include other mucins such as MUC 2-5. A person of ordinary- skill in the art would be able to apply this invention in other mucins.

The mucin may include other mucins such as MUC 2-5. A person of ordinally skill in art would be able to apply this invention in other mucins.

In a further embodiment, MUCl peptide ranges from thirty amino acids to three hundred amino acids in length. In a specific embodiment, the mucin peptide is selected from a group consisting of APDTRPAPGSTAPPAHGVTS, TAPPAHGVTSAPDTRPAPGS, APDTRPAPGSTAPPAHGVTSAPDTRPAPGS,

VTSAP DTR PAPGS TAP PAHGVTSAPDTR PA , a n d

(VTSAPDTRPAPGSTAPPAHG)₂VTSAPDTRPA. In a further specific embodiment , the mucin peptide is VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA.

In a preferred embodiment, the effective amount of QS-21 is an amount betv/een about lOμg and about 200μg. In a further preferred separate embodiment, the effective amount of QS- 21 is about 100 μg.

In an embodiment, cells of the cancer have the mucin on their surface.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter. Experimental Details

FIRST SERIES OF EXPERIMENTS

MATERIALS AND METHODS

Materials. Peptides. MUCl peptides containing 20, 30 and 50 amino acids with different sequences were synthesized using an Applied Biosystems Model 431A automated peptide synthesizer in the Core Facilities of Memorial Sloan- Kettering Cancer Center. A cysteine was added as indicated to the original sequence at the C- or N-terminal of the synthetic peptides to facilitate conjugation with protein carriers (Table 1) .

Table 1 Sequences of Synthetic MUCl Peptides Used For Vaccine Preparation and Testing Peptide Amino Acid Sequence

MUCK20A) APDTRPAPGSTAPPAHGVTS (C) (SQ ID 2)

MUCl (20) TAPPAHGVTSAPDTRPAPGS (C) (SQ ID 3)

MUCK30A) APDTRPAPGSTAPPAHGVTSAPDTRPAPGS (C) (SQ ID 4)

MUCl (30) (C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA (SQ ID 5) MUCl (50) (C) (VTSAPDTRPAPGSTAPPAHG) ₂VTSAPDTRPA (SQ ID 6)

MAbs and cell lineε. HMFG-2 is a MUCl-reactive mouselgG mAb (10) . 410.4 is a murine (BALB/c) mammary epithelial cancer cell line (11) and E4 is derived from a clone of 410.4 cells transfected with the MUCl gene (12) . HMFG-2, 410.4 and E4 were kindly provided by Dr. Joyce Taylor- Papadimitriou (Imperial Cancer Research Fund, London, U. K.) . MCF 7 is a human breast carcinoma cell line (13) .

Animals and adjuvants. Female BALB/c x C57BL/6 Fl mice, BALB/c x C3H Fl mice , or BALB/c mice, 6 weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, Maine) . Adjuvant QS-21, a purified saponin fraction (16) , was obtained from Cambridge Biotech, Inc. (Worcester, MA) . Bacille Calmette-Guerin (BCG) was purchased from Connaught Laboratories (Ontario, Canada) .

Conjugation of MUCl peptides to keyhole limpet hemocyanin (KLH) . KLH (Perlmmune Inc., RockviUe, MD) was used as carrier protein for MUCl peptide conjugates. m- Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS, Pierce Co., Rockford, IL) , N-Succinimidyl 3- (2-pyridyldithio) propionate (SPDP, Pierce Co., Rockford, IL) and glutaraldehyde (Aldrich Chemical Co., Milwaukee, WI) were used as linkers for making MUCl peptide conjugates (Fig.l) .

MBS conjugation method (14) . One mg MBS in 70μl dimethylformamide (Sigma Chemical Co., St. Louis, MO) was added to 5 mg KLH in 1 ml 0.01 M phosphate buffer (PB) pH 7.0. One hour later, MBS/KLH solution was applied on Sephadex G-15 column equilibrated with 0.1 M PB pH 6.0. The first peak at OD280 (MBS-KLH) was collected and mixed with 5 mg MUCl peptide and stirred at room temperature for 2 • hours. The unconjugated peptide was separated from MUCl-KLH conjugate using a Centriprep-30 concentrator (Amicon Inc., Beverly, MA) . The MUCl/KLH conjugation ratio (500/1 - 600/1) was calculated based on the starting amount of peptide and KLH and the amount of unconjugated peptide in the filtrate by spectrophotometer.

SPDP conjugation method (15) . Conjugation by the SPDP method was similar to that by the MBS method except that MBS was replaced by SPDP. The conjugate ratio of MUCl/KLH resulting from the SPDP method was calculated in 2 different assays: 1) based on the amount of unconjugated peptide, and 2) based on the SPDP by-product (pyridine-2- thione) produced by the conjugation reaction. The MUCl/KLH ratio was 400/1 -500/1 by both assays.

Glutaraldehyde conjugation method (14) . Five mg KLH in 1 ml borate buffer pH 10 was mixed with 5 mg MUCl peptide. One ml of 0.3% glutaraldehyde was added and stirred at room temperature for 2 hours . Unreacted glutaraldehyde was blocked by adding 0.25 ml 1 M glycine for 30 min. The MUCl- KLH solution was dialysed against PBS overnight. Because glutaraldehyde interferred with the absorbance of unconjugated peptide at OD215, the ratio of MUCl/KLH was assumed based on the starting ratio, 500/1.

Vaccine preparation and administration. Mice were immunized with 8-15 μg MUCl peptide alone or conjugated to KLH plus 8-10 μg QS-21 or 5 x 10⁵ BCG, 2-3 times at one week intervals. Eight to ten days after the 2nd or 3rd immunization, mice were bled and the sera separated for testing with ELISA and flow cytometry assays.

Serological assays. ELISA. ELISAs were performed as previously described (17) . MUCl peptide in 0.1 M carbonate buffer pH 9.6 were coated on ELISA plates at 0.1 μg/well. A series of antiserum dilutions were incubated with the • coated peptide for 1 hour. Secondary antibodies were alkaline phosphatase-con ugated goat anti-mouse IgG or IgM at a dilution of 1/200 (Southern Biotechnology Associates, Inc., Birmingham, AL) . ELISA titer is defined as the highest dilution yielding an absorbance of 0.1 or greater over that of normal mouse sera. MAb HMFG-2 was used as positive control in each assay.

Flow cytometry. Tumor cells (2 x 10⁵) were incubated with 40 1 of 1/30 diluted antisera or 1/2 diluted mAb supernatant for 30 min on ice. After washing with 3% fetal calf serum/phosphate buffered saline, the cells were incubated with 20_/ l of 1/15 diluted fluorescein-isothiocyanate- labeled goat anti-mouse IgM or IgG (Southern Biotechnology Associates, Inc., Birmingham, AL) . The positive population of the stained cells were quantitated by flow cytometry

(EPICS-Profile II, Coulter Co., Hialeah, FL) , as previously described (18) .

T lymphocyte assays. Proliferative assay (19) . Lymphocytes

(2 x loVwell) were prepared from the spleens of mice seven days after the 2nd immunization, and incubated with MUCl peptide (0.1-10 μg/ml) or KLH (1-20 μg/ml) in 37°C/5% C02 for 3-5 days. Eighteen hours after adding 0.5 μCi ³H- Thymidine (ICN, Irvine, CA) per well, the cells were processed and analyzed with a 1204 Betaplate (Wallac Oy, Finland) .

Delayed type hypersensitivity (DTK) (20) . Two weeks after the 2nd immunization, 5 μg MUCl peptide or KLH were injected in 20 μl PBS into the hind foodpad. Footpad thickness was measured at 24 and 48 hours.

Cytotoxic T lymphocyte (CTD assay (21) . Seven days after the 2nd immunization, splenic lymphocytes were sensitized in vitro with 1-8 μg/ml MUCl peptide and 10 unit/ml IL-2 (Boehringer Mannheim, Germany) for 7-10 days. The sensitized lymphocytes were then incubated with europium- labeled E4 or 410.4 cells at ratios of 10:1-100:1 for 4 hours. Percent release of europium from target cells were measured with a time-resolved 1232 Delfia fluorometer (Wallac Oy, Finland) (18) .

Tumor challenge in mice. Three days after the 2nd immunization, mice were injected i.v. with 2 x 10⁵ E4 cells. Twenty five days later, the mice were sacrificed, the lungs fixed with 10% formaldehyde, and the number of tumor colonies in the lungs were counted, as previously described (22) . EXPERIMENTAL RESULTS

Effect of MUCl-KLH Vaccine Construction on Serological Response. Comparison of MUCl peptides and MUCl conjugates (Table 2) . After 3 vaccinations, sera from 6 mice immunized with 15 μg peptide MUCl (30) plus 10 μg QS-21 or 5 x 10^s BCG per vaccination failed to show significant titers of IgM or IgG antibody against MUCl, while sera from mice immunized with 15 μg MUCl (30) conjugated with KLH plus 10 μg QS-21 showed high titer IgG and modest titer IgM (median titer 1:409600 and 1:800, respectively) . Sera from mice immunized with MUCl (30) -KLH, but not those immunized with unconjugated MUCl, also showed strong reactivity with MUCl- expressing E4 murine breast cancer cells and MCF 7 human breast cancer cells.

Table 2. Serological Response with Anitisera from Mice Immunized with MUCl Peptide or MUCl-KLH Conjugates*

ELISA Titers On Flow Cytometry (IgG) *

Immunizing MUCl positive ce ills peplιde

Antisera IgM IgG E4 cells MCF7 cells

-

Controls unprimed mice 1 blank^d blank 4 5 unprimed mice 2 blank blank 2 4 anti-GD3-KLH 1 < 1:20 < 1:25 2 5 anti-GD3-KLH 2 < 1:20 < 1:25 4 7 mAb HMFG-2 - 1:320 94 57

Mucl(30) + BCG

G2-1 < 1:20 < 1:25 3 -

G2-2 < 1:20 < 1:25 2 -

G2-3 < 1:20 < 1:25 4 -

G2-4 < 1:20 < 1:25 3 -

G2-5 < 1:20 < 1:25 1 -

G2-6 < 1:20 < 1:25 1 - median < 1:20 < 1:25 1 4^b

Mucl(30) + QS21

G3-1 < 1:20 < 1:25 1 - G3-2 < 1:20 < 1:25 1

G3-3 < 1:20 < 1:25 1

G3-4 < 1:20 < 1:25 1

G3-5 < 1:20 < 1:25 1

G3-6 < 1:20 < 1:25 1 median < 1:20 < 1:25 1

Mucl(30)- LH +

QS21

G5-1 1:400 1:819200 93 69

G5-2 1:800 1:204800 66 25

G5-3 1:800 1:819200 86 56

G5-4 1:1600 1:819200 97 79

G5-5 1:800 1:204800 72 40

G5-6 1:3200 1:204800 94 67 median 1:800 1:409600 90 62

Mucl (30A) -KLH +

OS21

G4-1 1:800 1:409600 83 83

G4-2 1:800 1:819200 92 58

G4-3 1:800 1:204800 95 45

G4-4 1:400 1:204800 81 20

G4-5 1:1600 1:819200 93 57

G4-6 1:400 1:204800 85 48 median 1:800 1:307200 89 53

Mucl (50) -KLH +

QS21

G6-1 1:3200 1:204800 73 40

G6-2 1:3200 1:204800 89 52

G6-3 1:3200 1:409600 98 79

G6-4 1:3200 1:409600 86 57

G6-5 1:1600 1:409600 97 72

G6-6 1:3200 1:409600 83 53 median l:3200^c 1:409600 88 55

^a All the MUCl conjugates in this table were made by the MBS method. Mice immunized with 15 g MUCl-KLH /mouse or MUCl peptide 15 g/mouse plus QS-21 10 g/mouse or BCG 3 x 10^/mouse were bled 7 days after the 3rd vaccines.

Test value obtained from pooled sera from all 6 mice in this group.

^C Compared with MUCl (30A) -KLH and MUC1(30)-KLH groups, p < 0.01 and p < 0.05 by Mann-Whitney/Wilcoxon non-parametric statistics . Absorbance values of the sera from unprimed mice were used as background values for substraction.

Comparison of conjugation methods (Table 3) . The median IgG titer of sera from mice immunized with MUC1(20)-KLH conjugated using glutaraldehyde and MUCl (20) -KLH and MUCl (30A) -KLH conjugated using SPDP were 1:1350, 1:50 and 1:2700, respectively. The median IgG titer after immunization with MUCl (20) -KLH and MUCl (30A) -KLH conjugated using MBS were 1:12150 and 1:12150 respectively, significantly higher than those prepared using glutaraldehyde or SPDP (p < 0.01) . Likewise, the median % positive cells by flow cytometry with sera from mice immunized with MUCl (20) -KLH (glutaraldehyde) , MUCl (20) - KLH (SPDP) and MUCl (30A) -KLH (SPDP) were 59%, 32% and 61% respectively. Both the MUCl (20) -KLH (MBS) and MUCl (30A) -KLH (MBS) groups had 97% positive cells, significantly higher than those conjugates prepared by glutaraldehyde or SPDP (p < 0.05) . For MUC1(20A) conjugates, on the other hand, no obvious difference in titer or % positive cell were observed between the MBS and SPDP methods. Reactivity against the MUCl-negative cell line 410.4 was minimal in all groups.

Comparison of different length MUCl peptides (Table 3) . Median IgG titers against the immunizing peptides were similar for sera from mice immunized with MUCl (20A) -KLH (1:4500 (SPDP) and 1:4500 (MBS)) and MUCl (30A) -KLH (1:2700 (SPDP) and 1:12150 (MBS)) . Although the median % positive cells for MUCl (30A) -KLH (SPDP) (61%) was slightly but not significantly higher than for MUCl (20A) -KLH (SPDP) (32%) , the median % positive cells for MUCl (30A) -KLH (MBS) (97%) was significantly higher than for MUCl (20A) -KLH (MBS) (54%) . When MUCl (50) -KLH was compared with MUCl (30) -KLH and MUCl (20) -KLH (Tables 2 and 3) , no significant difference in % positive cells was found. Table 3. Serological Response with Antisera from Mice Immunized with MUCl conjugated to KLH by Different Methods³

ELISA Flow Cytometry (IgG) peptide 410.4 cell E4 cell

(IgG)

Mixed normal blank 4 2 mAb HMFG II 1:320 1 99

Glutaraldehyde

MUCl (20) -: KLH

1 4050 4 77

2 1350 14 53

3 1350 9 59 median 1350 9_ 59

SPDP method

MUCl (20A) - ^■KLH

1 4050 4 4

2 4050 14 32

3 4050 10 91 median 4050 10. 32

MUCl (20) -: KLH

1 1350 5 96

2 50 27 43

3 50 7 16

4 50 2 55

5 150 3 2

6 50 11 21 median 5j0 6. 12

MUCl (30A) - ^•KLH

1 4050 11 85

2 4050 10 96

3 4050 4 56

4 150 11 44

5 450 2 30

6 1350 3 65 median 2700 7 §1

MBS method

MUC(20A) - KLH

1 4050 11 61

2 4050 6 54

3 12150 8 54 median 4050 8 54.

MUCl (20) - KLH

1 12150 6 89

2 12150 14 99

3 12150 8 95

4 36450 6 99

5 109350 5 99

6 12150 10 92 median 12150^b 2 97^b

MUCl (30A) - -KLH

1 12150 8 95

2 12150 6 99

3 12150 12 86

4 12150 3 98

5 12150 9 60

6 36450 3 ⁹ median 12150^b 7 97 ^a Ten days after the 3rd immunization with 10 g MUC1- KLH/mouse plus QS-21 10 g/mouse. Number 1-3 of mice in each group are strain CB6F1 while number 4-6 are BALB/c. ^b Compared with the corresponding MUCl-KLH conjugated by SPDP or Glutaradehyde method, using Mann-Whitney/Wilcoxon non-parametric statistics, p < 0.01 for ELISA titer, p < 0.05 for % positive cells. Serological response to conjugated MUCl with different sequences (Table 3) . The median IgG titer against the immunizing peptides of sera from mice immunized with MUCl (20) -KLH (SPDP) (1:50) was obviously lower than that from mice immunized with MUCl (20A) -KLH (SPDP) (1:4050) . However, the median % positive cells by flow cytometry with sera from these two groups was the same (32%) . Both median IgG titer (1:12150) and % positive cells (97%) from mice immunized with MUCl (20) -KLH (MBS) were significantly higher than that for mice immunized with MUCl (20A) -KLH (MBS) , 1:4050 and 54% respectively (p < 0.05) . When MUC1(30)-KLH was compared with MUCl (30A) -KLH (Table 2) , no obvious difference in serological response was found.

Effect of MUCl immunization on T cell responses. All the mice in Tables 3 and 4 were tested for DTH. Forty eight hours after footpad injection with 5 μg MUCl peptide, the average footpad thickness for mice immunized with MUCl peptides and MUCl-KLH constructs were 1.72 +/- 0.05 mm

(n=12) and 1.73 +/- 0.06 mm (n=28) respectively and not different from 1.73 +/- 0.06 mm (n=20) of mice injected with PBS alone. After DTH testing with 5 g KLH (positive control) , however, the average footpad thickness for mice immunized with MUCl-KLH was 2.1 +/- 0.17 mm (n=8) , significantly increased above the KLH DTH response for unprimed mice and mice immunized with MUCl peptides, 1.71

+/- 0.02 mm (n=6) and 1.71 +/- 0.03 mm (n=6) respectively.

The results of proliferative assays were similar to that of

DTH assays. Spleen cells from 5 mice for each group immunized with MUCl (20) -KLH, MUCl (30) -KLH and MUCl (50) -KLH or the corresponding peptide did not show increased lymphocyte proliferation above unprimed mice (4530cpm) when these cells were incubated with the corresponding peptide. Increased proliferation (14500-18600 cpm) was observed only when the spleen cells from mice immunized with MUCl-KLH conjugates were incubated with KLH.

An average of 30% specific release in 3 CTL assays on E4 cells (E:T, 60:1) was observed when the effector cells were obtained from BALB/c x C3H Fl mice immunized with mitomycin-treated E4 cells, but no significant specific release (0-2%) were seen from the mice immunized with MUCl peptides or MUCl-KLH conjugates.

Effect of Active Immunization with MUCl-KLH on E4 cell Lung Metastasis in BALB/c x C3H Mice (Table 4) . Ten mice immunized with MUCl (30) -KLH produced IgG antibody at a mean titer of 1:5940 with good E4 cell surface reactivity (mean % positive cells, 52%) . Four weeks after i.v. challenge with E4 cells, the mean number of lung colonies from 10 mice was 19 and 20 for PBS and KLH control groups respectively, while only 1 for the MUCl (30A) -KLH group (p < 0.01) .

Table 4. The Effect of Active Immunization with MUCl (30)-

KLH Plus QS-21 on E4 Cell Lung Metastasis³

Sera or mAb ELISA Flow Cytometry No. of reciprocal % positive cells colonies titer (IgG) in lungs

(IgG) 410.4 cell E4 cell

mAb HMFG-2 320 1 81

PBS Control

1-1 - - - 44 1-2 - 51

1-3 - - - 6

1-4 - - - 1

1-5 5 - 21 -

1-6 - - - 39

1-7 - - - 2

1-8 - - - 30

1-9 - - - 3

1-10 - - - 4 mean^b blank 5 5 19

KLH Control

2-1 - - - 51

2-2 - - - 8

2-3 - - - 14

2-4 - - - 5

2-5 - - - 48

2-6 - - - 17

2-7 - - - 8

2-8 - - - 11

2-9 - - - 23

2-10 - - - 14 mean^b 0 5 5 20

MUCl (30A) -

KLH

3-1 8100 5 50 4

3-2 8100 5 62 5

3-3 2700 6 21 3

3-4 2700 4 84 1

3-5 2700 5 26 0

3-6 2700 5 49 0

3-7 810C 6 35 0

3-8 8100 10 63 1

3-9 8100 5 84 0

3-10 8100 6 41 0 mean 5940 6_ 52 l^c

^a BALB/c x C3H Fl were immunized twice at one week interval with 8 g MUCl (30A) -KLH plus 8 g QS-21 /mouse and boosted one week after the 2nd vaccine with 8 g MUC1(30A) peptide plus 8 g QS-21. KLH group were given the same amount of KLH as the MUCl (30A) -KLH group. Three days after 2nd vaccine, 2 x 10^s E4 cells were injected i.v. to all the groups. Twenty eight days after E4 cell injection, mice were sacrificed to check the matastastic colonies in lungs . Mice were bled 10 days after 2nd vaccine for ELISA and flow cytometry assay. ^b The test values of ELISA and flow cytometry were obtained from a pool of sera from 10 mice in this group. ^c Compared with PBS and KLH control groups, p < 0.01, by Mann- Whitney/Wilcoxon non-parametric statistics. EXPERIMENTAL DISCUSSION

MUCl specific antibodies have been detected in sera from occasional breast, pancreatic and colon cancer patients (2, 23) . This suggests that MUCl can be recognized by the human immune system, and raised the possibility that immunity against tumor cells expressing MUCl might be induced by properly constructed MUCl vaccines. Applicants' explored here vaccines containing synthetic MUCl peptides of different lengths and sequences mixed with various adjuvants or covalently attached using different linker methods to KLH. Vaccines containing unconjugated MUCl peptides plus QS-21 or BCG failed to induce antibody. However, when the MUCl peptides were conjugated to KLH, plus QS-21, high titer IgM and, especially, IgG antibodies against MUCl antigen were successfully induced. The reactivity of these antisera with MUCl-expressing mouse tumor E4 and human breast tumor MCF7 cells was strong, similar to mAb HMFG-2. When conjugation methods utilizing MBS, SPDP and glutaraldehyde were compared, MBS was found to be the best linker for preparing MUCl-KLH conjugates, inducing the most favorable antibody in both titer and specificity. Although conclusions on the effect of MUCl peptide length on immune response can not be drawn at this time, 30 or 50 amino acid MUCl conjugates seemed to induce antisera with higher titer against MUCl positive tumor cells than 20 amino acid MUCl conjugates. For MUCl peptide with a single tandem repeat (20 amino acids) , APDTR within the peptide, MUCl (20) , induced antibody with stronger reactivity against MUCl-expressing E4 cells than MUC1(20A) which contains APDTR at the N-terminal of the peptide. This effect was lost, however, when longer peptides (30 amino acids) were tested.

T lymphocyte immunity against MUCl peptide in unimmunized cancer patients has been documented (24, 25) . In this study, applicants' tested T cell responses in mice immunized with synthetic MUCl peptides conjugated to KLH plus QS-21. QS-21 is an immunological adjuvant known to be capable of inducing CTL against other soluble protein antigens (29) . However, applicants' did not observe T cell responses (DTH, lymphocyte proliferation or CTL) to MUCl antigen or MUCl positive cells after immunization. Others have attempted to induce T cell responses with synthetic MUCl peptide, MUCl conjugates, vaccinia expressing MUCl or MUCl expressed on whole tumor cells (5, 26-28) . By immunizing mice with MUCl (20 amino acids) coupled to diphtheria-toxoid or fused with glutathione-S-transferase, or a natural mucin (human milk fat globule) plus complete Freund's adjuvant, Apostolopoulos et al (26) also failed to induce T cell responses in mice. However, T cell responses were observed after immunizing mice with whole tumor cells expressing MUCl antigen, as applicants' were also able to demonstrate. DTH but not in vitro immune responses in mice immunized with MUCl (16 amino acids) -KLH were reported by Ding et al (27) . These different observations may be due to the use of different strains of mice. While failing to induce T cell responses in mice with MUCl peptide corresponding to one tandem repeat (20aa) , Finn et al found MUCl peptide corresponding to five tandem repeats has the capacity for CTL induction (30) . Whether longer MUCl peptides, conjugated to KLH or not, also have this capacity is the focus of further study. Since amino acid sequences of natural mucins and motifs of peptides associated with Class I MHC molecules are different in mice than humans (6, 31) , T cell responses to MUCl antigens in humans may be significantly different than those seen in mice. The CTL against MUCl already identified in unimmunized patients with breast, ovary and pancreatic cancers suggest that this is so (24, 25) .

Despite the lack of detected T cell immunity to MUCl after immunization, protection from challenge with MUCl positive E4 cells was seen. The number of lung metastases in mice immunized with MUC(30)-KLH plus QS-21 was significantly lower than that for the PBS or KLH plus QS-21 control groups. This suggests that humoral immunity may be responsible for this resistance to challenge with MUC1- expressing E4 tumor cells. Others have also described protection from challenge with MUCl positive tumor cells in rodents after immunization. Significant rejection of mucin- expressing tumor occurred in Fisher rats immunized with a vaccinia recombinant expressing MUCl (5) , and prolonged survival of CAFl mice immunized with MUCl conjugate BP-1-7- KLH (GVTSAPDTRPAPGSTA) (SQ ID. 1) was described after challenge with MUCl-expressing E3 cells (27) . While DTH responses were seen in the BP-1-7-KLH immunized mice, CTL responses were not described. Consequently it is difficult to know from the results in rodent models which arm of the immune system is primarily responsible for protection from tumor challenge, and so it is difficult to predict which assays of immunity will be most important to follow in the clinic in Phase I/II trials designed for optimizing the immunogenicity of MUCl vaccines.

CTL and antibody responses against MUCl have been described in unimmunized patients with breast, ovary or pancreatic • cancer (2, 23-25) . It remains to be determined whether immunity against MUCl in patients can be augmented by treatment with tumor vaccines. To date only one trial has been conducted, immunization of advanced disease breast cancer patients with a 105 amino acid MUCl peptide (5 tandem repeats) mixed with BCG (30) . While the final results are not available, it appears that neither anti MUCl antibodies nor DTH were consistently augmented. There was, however, a 2-3 fold augmentation of HLA unrestricted anti- MUCl CTL precursors in post-immunization blood compared to pre-immunization blood (O. J. Finn, personal communication) . However, these results in patients with advanced disease may not be applicable to an adjuvant setting. Our results demonstrate that MUCl-KLH conjugates are far more potent than MUCl plus BCG for inducing IgM and IgG antibodies against MUCl. Although T cell responses to MUCl antigen were not observed in our study, antibodies with high titer against synthetic MUCl and MUCl-expressing murine and human tumor cells were successfully induced in all mice and this was associated with significant protection from challenge with MUCl-expressing E4 tumor cells. Based on this, applicants' have initiated a clinical trial with MUCl-KLH (30 amino acid) prepared using an MBS linker plus QS-21 and have plans to test a 90 amino acid MUCl peptide in this setting as well. Assays for humoral, DTH, proliferative and CTL responses will be followed.

Human mucin MUCl is abundantly expressed in many cancers of epithelial origin and is largely restricted to the apical surface of secretary cells in normal tissues. It is therefore a potential target for cancer immunotherapy. In preparation for clinical trials, vaccines containing synthetic MUCl peptides of different lengths and sequences, and mixed with various adjuvants or covalently attached

(using different linker methods) to protein carrier keyhole limpet hemocyanin (KLH) were studied in mice. MUCl peptides (containing 20 or 30 amino acids) plus adjuvants

QS-21 or BCG did not induce antibody. However, MUCl peptides conjugated to KLH (MUCl-KLH) , especially by linker m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) , plus QS-21, induced high titer antibody against the immunizing peptides, median titer 1:800 for IgM and 1:307,200 for IgG. In addition, these antisera showed strong reactivity with MUCl-expressing mouse tumor E4 cells and human breast MCF-7 cells, similar to the MUCl- reactive mAb HMFG-2. Based on these studies, a vaccine containing MUCl-KLH conjugate prepared with linker MBS, plus QS-21, has been constructed for testing in clinical trials. References

1. Arklie, J., Taylor-Paradimitriou, J., Bodmer, W. , Egan, M. , and Millis, R. Differentiation antigens expressed by epithelial cells in the lactating breast are also detectable in breast cancers. Int. J. Cancer, 28:23-29, 1981.

2. Kotera Y., Fontenot, J. D. , Pecher, G., Metzgar, R. S., and Finn, 0. J. Human immunity against a tandem repeat epitope of human mucin MUC-1 in sera from breast, pancreatic and colon cancer patients. Cancer Res., 54:2856- 2860, 1994.

3. Devine, P. L., Layton, G. T., Clark, B. A., Birrell, G.

W. , Ward, B. G., Xing, P. X., andMcKenzie, F.C. Production of MUCl and MUC2 mucins by human tumor cell lines. Biochem. Biophys. Res. Commun., 178:593-599, 1991.

4. Hollingsworth, M. A., Strawhecker, J. M., Caffrey T. C, and Mack, D. R. Expression ofMUCl, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines. Int. J. Cancer, 57:198-203, 1994.

5. Hareuveni, M. , Gautier, C, Kieny, M.-P., Wreschner, D., Chambon, P., and Lathe, R. Vaccination against tumor cells expressing breast cancer epithelial tumor antigen. Proc. Natl. Acad. Sci. USA., 87:9498-9502, 1990.

6. Gendler, S. J., Spicer, A. P., Lalani, E.-N., Duhig, T. , Peat, N. , Burchell, J., Pemberton, L., Boshell, M. , and Taylor-Papadimitriou, J. Structure and biology of a carcinoma-assaciated mucin, MUCl. Am. Rev. Respir. Dis., 144:S42-S47, 1991.

7. Burchell, J., Taylor-Papadimitriou, J., Boshell, M., Gendler, S., and Duhig, T. A short sequence, within the amino acid tandem repeat of a cancer-associated mucin, contains immunodominant epitopes. Int. J. Cancer, 44:691- 696, 1989.

8. Fontenot, J. D. , Tjandra, N. , Bu, D., Ho, C, Montelaro, R. C, and Finn, O.J. Biophysical characterization of one-, two-, and three-tandem repeats of human mucin (muc-1) protein core. Cancer Res., 53:5386-5394, 1993.

9. Perez, L., Hayes, D. F., Maimonis, P., Abe, M. , O'Hara, C , and Kufe, D. W. Tumor selective reactivity of a monoclonal antibody prepared against a recombinant peptide derived from the DF3 human breast carcinoma-assaciated antigen. Cancer Res., 52:2563-2568, 1992.

10. Taylor-Papadimitriou, J., Peterson, J. A., Arklie, J., Burchell, J. , Ceriani, R. L., and Bodmer, W. F. Monoclonal antibodies to epithelium specific components of the milk fat globule membrane: production and reactions with cells in culture. Int. J. Cancer, 28:17-21, 1981.

11. Miller, F. R. , Miller, B. E., and Heppner, G. H. Characterization of metastatic heterogeneity among . subpopulations of a single mouse mammary tumor: heterogeneity in phenotypic stability. Invasion Metastasis, 3:22-31, 1983.

12. Lalani, E.-N., Berdichevsky, F., Boshell, M., Shearer, M., Wilson D., Stauss, H., Gendler, S. J. , and Taylor- Papadimitriou, J. Expression of the gene coding for a human mucin in mouse mammary tumor cells can affect their tumorigenicity. J. Biol. Chem., 266:15420-15426, 1991.

13. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan, M. A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 51:1409-1416, 1973. 14. Maloy, W. L., Coligan, J.E., and Paterson, Y. Production of antipeptide antisera. In Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds.) pp. 9.4.1- 9.4.11. John Wiley & Sons, Inc. New York, 1994.

15. Carlesson, J., Drevin, H., and Axen, R. Protein thiolation and reversible protein-protein conjugation N- succinimidyl 3- (2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. , 173:727-737, 1978.

16. Kensil, C.R., Patel, U. , Lennick, M., and Marciani, D. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria molina cortex. J. Immunol., 146:431-437, 1991.

17. Livingston, P.O., Ritter, G. , and Calves, M.J. Antibody response after immunization with the gangliosides GM1,GM2, GM3 , GD2, and GD3 in the mouse . Cancer Immunol . Immunother., 29:179-184, 1989.

18. Zhang, S., Helling, F., Lloyd, K. 0., and Livingston, P. 0. Increased tumor cell reactivity and complement- dependent cytotoxicity with mixtures of monoclonal antibodies against different gangliosides. Cancer Immunol. Immunother., 40:88-94, 1995.

19. James S. P. Measurement of proliferative responses of cultured lymphocytes. In Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds.) pp. 7.10.1-7.10.10. John Wiley & Sons, Inc. New York, 1994.

20. Luo, Y, and Dorf, M. E. Delayed-type hypersensitivity. In Current Protocols in Immunology (J. E. Coligan, A. M.

Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds.) pp. 4.5.1-4.5.5. John Wiley & Sons, Inc. New York, 1994 .

21. Wunderlich, J. and Shearer, G. Induction and measurement of cytotoxic T lymphocyte activity. In Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds.) pp. 3.11.1-3.11.15. John Wiley & Sons, Inc. New York, 1994.

22. Dohi, T., Nores, G. A., Oguchi, H., Inufusa, H. , and Hako ori, S. GD3 lactone as an immunogen associated with melanoma: effect of immunization with GM3 lactone on melanoma growth in vivo. In Gangliosides and Cancer ( H. F. Oettgen, eds.) pp 275-281, VCH Verlagsgesellschaft , Weinhei (Germany) and VCH Publishers ( New York, U.S.A.) , 1988.

23. Rughetti A., Turchi V., Ghetti C. A., Scambia, G. , Panici, P. B., Roncucci, G., Mancuso, S., Frati, L., and Nuti, M. Human B-cell immune response to the polymorphic epithelial mucin. Cancer Res., 53:2457-2459, 1993.

24. Jerome, K. R. , Barnd, D. L. , Bendt , K. M. , Boyer, C. M. , Taylor-Papadimitriou, J., McKenzie, I. F. C, Bast, R. ' C. Jr., and Finn, O. J. Cytotoxic T-lymphocytes derived from patients with breast adenocarcinoma recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells. Cancer Res., 51:2908-2916, 1991.

25. Ioannides, C. G. , Fisk, B., Jerome, K. R. , Irimura, T., Wharton, J. T., and Finn, 0. J. Cytotoxic T cells from ovarian malignant tumors can recognize polymorphic epithelial mucin core peptides. J. Immunol., 151:3693-3703, 1993.

26. Apostolopoulos, V., Xing, P-X., and McKenzie, F. C. Murine immune response to cells transfected with human MUCl: immunization with cellular and synthetic antigens. Cancer Res., 54:5186-5193, 1994.

27. Ding, L., Lalani, E-N. , Reddish, M., Koganty, R., Wong, T. , Samuel, J. , Yacyshyn, M. B., Meikle, A., Fung, P. Y.

S., Taylor-Papadimitriou, J., and Longenecker, B. M. Immunogenicity of synthetic peptides related to the core peptide sequence encoded by the human MUCl mucin gene: effect of immunization on the growth of murine mammary adenocarcinoma cells transfected with the human MUCl gene. Cancer Immunol. Immunother., 36:9-17, 1993.

28. Bu, D. , Domenech, N. , Lewis, J. , Taylor-Paradimitriou, J. , and Finn, O. J. Recombinant vaccine mucin vector: in vitro analysis of expression of tumor-associated epitopes for antibody and human cytotoxic T-cell recognition. J. Immunol., 14:127-135, 1993.

29. Newmann, M. J., Wu, J-Y., Gardner B. H., Munroe, K. J., Leombruno, D., Recchia, J. , Kensil, C.R., and Coughlin, R.

T. A sponin adjuvant induction of ovalbumen-specific CD8+ cytotoxic T-lymphocyte responses. J. Immunol., 148:2357- 2360, 1992.

30. Finn, 0. J. Immunity to epithelial tumors and mucin- based vaccine design. Proc. Am. Assoc. Cancer Res., 36:675, 1995.

31. Engelhard, V. H. Structure of peptides associated with class I and Class II MHC molecules. Annu. Rev. Immunol., 12:181-207, 1994. SECOND SERIES OF EXPERIMENTS

The mucin MUC-1 is expressed on breast carcinomas in an under glycosylated configuration and is therefore a target for immune recognition. A 30 amino acid (aa) sequence of MUC-1 has been synthesized and in order to agument its immunogenicity, has been covalently linked to KLH and mixed with the immune adjuvant QS21. Eligiblity criteria include: patients with stage 4 NED (no evidence of disease) , elevated CEA or CA15-3 levels and NED, or initially unresectable stage 3 post adjuvant therapy (tx) . Five vaccines, each containing 100 meg of MUC-1 peptide, were given on weeks 1, 2, 3, 7, 19. Thus far five pts are on study, although only 5 patients have received >_ 3 vaccinations. Local erythema and induration at the injection site and mild flu-like symptoms most prominent after vaccination # 2 were observed in all pts. Patient sera were analyzed by ELISA for IgG and IgM antibodies against purified MUC-1 and by an immune adherenece rosetting assay against MUC-1 positive or negative cell lines. IgM/IgG titers by ELISA for the first five patients were :

Week# 0 3 8 20

Pt#l 0/0 10240/40 2560/160 320/1280

Pt#2 0/0 180/640 2560/2560

Pt#3 0/0 2560/320 2560/2560

Pt#4 0/0 1280/80 2560/2560

Pt#5 0/0 320/320 640/320

For Pt# 1 to Pt# 4 immune adherence assays measuring IgM reactivity against MCF-7 cells gave a titer of 0 pre therapy which have risen to a titer of 160 by week 9. The fifth patient (Pt# 5) started with a titer of 80 pre therapy and increased to 160 by week 9. The MUC-1 mucin is strongly immunogenic in breast cancer patients when presented in a vaccine containing KLH and QS 21. THI RD SER I ES OF EXPERIMENTS

Stage IV no evidence of disease (NED) breast cancer patients (BCPts) or earlier stage BCPts except for rising CEA or BR2729 levels are at high risk for overt recurrence and might benefit from immunotherapy. Mucin MUC-1, found on most breast carcinomas, is a potential target. A synthetic 30 amino acid (aa) sequence of MUC-1 has been conjugated with KLH and mixed with the immune adjuvant QS- 21 to increase immune recognition. Nine patients (ages 43- 61) have been vaccinated: eight stage IV NED, one stage II with increased CEA level and NED, all but one stage IV NED patient on hormonal treatment. All patients received five doses of 100 meg MUC-1 s.c. given on weeks 1, 2, 3, 7, and 19. All patients had transient grade 2 local toxicity at the vaccine site and most had grade 1-2 flu-like symptoms. All patients remain NED (median follow up 55 weeks) although one patient had a chest wall recurrence which was excised. For all patients, the range of IgM and IgG reciprocal titers against purified MUC-1 by ELISA are:

Week # IgM Titers IgG Titers

0 0-160 0-10

3 10-20,480 0-320

5 1280-20,480 40-20,480

13 10-20,480 160-2560

21 320-30,480 640-10,240

Five patients maintain IgG titers (range 320-1280) between six-twelve months following the last vaccine. Analysis of IgG subclass in eight patients reveal predominantly IgGl and IgG3. Immune adherence rosetting against MCF-7 cell lines revealed an increase in IgM titers in 6/7 patients. Inhibition assays demonstrate that all sera react exclusively with the APDTRPA determinant of MUC-l. No evidence for augmentation of T cell immunity was found. This MUC-1 vaccine is immunogenic in breast cancer patients who are NED.

FOURTH SERIES OF EXPERIMENTS

The mucin MUC-1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer immunotherapy. MUC-1 contains multiple tandem repeats of the 20 amino acid peptide (VTSAPDTRPAPGSTAPPAHG) . The APDTR epitope is particularly immunogenic since it is recognized by a variety of murine monoclonal antibodies and immune sera, and by some sera and cytotoxic T cells from unimmunized patients with epithelial cancers. A 30 amino acid peptide VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA was prepared with cysteine at the N-terminal end for chemical conjugated to keyhole limpet hemocyanin (KLH) . Six breast cancer patients immunized in the adjuvant setting with this conjugate plus the immunological adjuvant QS-21 have all produced high titer (by ELISA) IgG and IgM antibodies against the 30 amino acid MUC-1 peptide. A series of smaller peptides were prepared to determine the epitopes recognized by these immune sera in inhibition assays. Only peptides containing APDTRPA were able to inhibit ELISA reactivity for the full 30 amino acid peptide. No sera were inhibited by APDTR, APDTRP, PDTRPA or any other peptides that did not contain the full APDTRPA epitope. Remarkably, sera from all six patients recognized this same epitope and only this epitope. Reactivity was greatest, however, when the APDTRPA was at the C-terminal end of the peptide, raising the possibility that it is recognized preferentially because it was terminal in the immunogen as well. An additional group of patients are planned to be immunized with a conjugate vaccine containing MUC-1 peptide with other amino acids at the C-terminal end. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT: Livingston, Philip O Zhang, Shengle

⁽ii) TITLE OF INVENTION: Conjugated Mucin Peptide Vaccines (ill) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Cooper & Dunham LLP

(B) STREET: 1185 Avenue of the Americas

(C) CITY: New York

(D) STATE: NY

(E) COUNTRY: U.S.A. (F) ZIP: 10036

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(vill) ATTORNEY/AGENT INFORMATION: (A) NAME: White, John P (B) REGISTRATION NUMBER: 28,678

(C) RE ERENCE/DOCKET NUMBER: 50397-A-PCT/JPW/AKC

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 212-278-0400 (B) TELEFAX: 212-391-0525

(2^X INFORMATION FOR SEQ ID NO:1: (1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 arr no acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 1 5 10 15

(2^V INFORMATION FOR SEQ ID NO:2:

(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(il) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 1 5 10 15

Gly Vai Thr Ser Cys 20

(2) INFORMATION FOR SEQ ID NO: 3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro l 5 10 15

Ala Pro Gly Ser Cys 20 (2) INFORMATION FOR SEQ ID NO: :

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 ammo acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 1 5 10 15

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Cys 20 25 30

(2) INFORMATION FOR SEQ ID NO:5:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 am o acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Cys Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

1 5 10 15

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala 20 25 30

(2) INFORMATION FOR SEQ ID NO: 6:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ll) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Cys Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 1 5 10 15

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 20 25 30

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 35 40 45

Arg Pro Ala 50

(2) INFORMATION FOR SEQ ID NO:7:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(il) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly 1 5 10 15 Val Thr Ser Ala 20

Claims

What is claimed is:

1. A vaccine capable of producing an immune response which recognzies a mucin, comprising an amount of mucin peptide conjugated to an immunogenic protein effective to stimulate or enhance immune response in the subject, an effective amount of an adjuvant and a pharmaceutically acceptable vehicle.

2. The vaccine of claim 1, wherein the subject is a human.

3. The vaccine of claim 1, wherein the immunogenic protein is Keyhole Limpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin.

4. The vaccine of claim 1, wherein the mucin is MUCl.

5. The vaccine of claim 1, wherein the mucin is selected from a group consisting of MUC2, MUC3 ,

MUC4 and MUC5.

6. The vaccine of claim 4, wherein the mucin peptide ranges from ten amino acids to three hundred amino acids in length.

7. The vaccine of claim 4, wherein the mucin peptide is selected from the group consisting of APDTRPAPGSTAPPAHGVTS, TAPPAHGVTSAPDTRPAPGS, APDTRPAPGSTAP PAHGVTSAPDTRPAPGS ,

VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA, and

(VTSAPDTRPAPGSTAPPAHG) ₂VTSAPDTRPA.

8. The vaccine of claim 7, wherein the mucin peptide is VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA.

9. The vaccine of claim 6, wherein the effective amount of conjugated mucin peptide is an amount between about 1 μg and about lmg.

10. The vaccine of claim 1, wherein the adjuvant is QS- 21.

11. The vaccine of claim 10, wherein the effective amount of QS-21 is an amount between about lOμg and about 200μg.

12. The vaccine of claim 11, wherein the effective amount of QS-21 is about 100 μg.

13. The vaccine of claim 1, wherein the subject is afflicted with cancer and the immune response produced in the subject upon administration of the vaccine effectively treats the cancer.

14. The vaccine of claim 1, wherein the subject is susceptible to cancer and the immune response produced in the subject upon administration of the vaccine effectively prevents the cancer.

15. The vaccine of claim 14, wherein cells of the cancer have the mucin on their surface.

16. The vaccine of claim 14, wherein the cancer is a breast cancer, prostate cancer, colon cancer, lung or pancreas cancer.

17. A method for stimulating or enhancing in a subject production of an immune response which recognizes the mucin comprising administering to the subject an effective dose of the vaccine of claim 1.

18. A method for treating cancer in a subject afflicted with cancer comprising administering to the subject an effective dose of the vaccine of claim 1.

19. A method for preventing cancer in a subject susceptible to cancer comprising administering to the subject an effective dose of the vaccine of claim 1.

20. A method for preventing the recurrence of a cancer in a subject susceptible to cancer comprising administering to the subject an effective dose of the vaccine of claim 1.

21. The method of claim 17, 18, 19 or 20 wherein the immunogenic protein is Keyhole Limpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin.

22. The method of claim 17, 18, 19 or 20, wherein the adjuvant is QS-21.

23. The method of claim 17, 18, 19 or 20, wherein the mucin is MUCl .

24. The method of claim 17, 18, 19 or 20, wherein the mucin is selected from a group consisting of MUC2 , MUC3, MUC4 and MUC5.

25. The method of claim 23, wherein the mucin peptide ranges from ten amino acids to three hundred amino acids in length.

26. The method of claim 25, wherein the mucin peptide is selected from the group consisting of APDTRPAPGSTAPPAHGVTS , TAPPAHGVTSAPDTRPAPGS , AP DTR PAP G S TAP PAH GVT SAPDT R PAP G S , VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA, and

(VTSAPDTRPAPGSTAPPAHG)₂ TSAPDTRPA.

27. The method of claim 26, wherein the mucin peptide is VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA.

28. The method of claim 23, wherein the effective amount of conjugated mucin peptide is an amount between about 1 μg and about lmg.

29. The method of claim 25, wherein the adjuvant is QS- 21.

30. The method of claim 29, wherein the effective amount of QS-21 is an amount between about lOμg and about 200μg.

31. The method of claim 30, wherein the effective amount of QS-21 is about 100 μg.

32. The method of claim 18, 19 or 20, wherein cells of the cancer have the mucin on their surface.

33. The method of claim 18, 19 or 20, wherein the cancer is a breast cancer, prostate cancer, colon cancer, lung or pancreas cancer.