AU685539B2 - Antigen carbohydrate compounds and their use in immunotherapy - Google Patents

Description

Oui f.: 532830 P100/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Tle~. Attstin Researeh lflstittt -Tey- 7b u froffifB EBuiling Austi Hopia 3 6 -st IIIEIDILBER VIC J08 DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Address for Service: Invention Title: Antigen carbohydrate compounds and their use in imniunotherapy The following statement is a full description of this invention, including the best method of per-forming it known to me:- 5020 ANTIGEN CARBOHYDRATE COMPOUNDS AND THEIR USE IN IMMUNOTHERAPY This invention relates to the immunotherapy of disease states, and in particular, but not exclusively to the immunotherapy of carcinomas.

Cancer is a major cause of death and severe trauma in modem society. Cancer is no respecter of persons as the young, old, males, females and peoples of all races may contract cancer, although cancer in children is relatively rare, perhaps with the exception of childhood leukemia. In western society, cancer of the colon and lung cancer are major diseases. In women, breast cancer is the most common form of cancer.

Many cancers are accompanied by overproduction of human mucin. Mucins are heavily glycosylated proteins (greater than about 100Kd) which are produced by many epithelial cells and tumours Mucins found on cancer cells are different in some respects to those on normal epithelial cells, in that some mucins have a deficiency in their carbohydrate coat which leaves the protein core exposed. There are seven forms of known human mucin designated MUC1, MUC2, MUC3, MUC4, MUC5 MUC6 and MUC7 4, 26, 27).

MUC1 is the most ubiquitous. The various mucins all have very similar properties, that is, they are transmembrane glycoproteins, all having a variable number of repeated amino acid sequences, which have a high content of serine, threonine and proline. Overproduction of aberrantly glycosylated mucins (either non-glycosylated or a deficiency in glycosylation) is characteristic of tumours of the breast, ovary, pancreas, colon, lungs, prostate and other tumours of secretory tissue. The cDNA sequences of the respective protein cores of the human mucins MUC1 to MUC7 have been cloned and characterized and have been found to contain highly repetitive central portions of varying numbers of repeats of particular amino acid motifs (known as VNTR's). By way of example, MUC1 consists of unique amino and carboxyl terminal sequences separated by a highly repetitive central portion containing forty B:\AUST1N:PM32828.mmb21 Dcember 1994

I

to eighty tandemly arranged copies or repeats of a twenty amino acid motif. The VNTR's of MUC1 through MUC7 are set forth below: MUC1 VNTR SAPDTRPAPGSTAPPAHGVT MUC2 VNTR PTTTPISTTTMVTPTPTPTGTQT MUC3 VNTR HSTPSFTSSITTTETTS MUC4 VNTR TSSASTGHATPLPVTD VNTR 87 base pair degenerate tandem repeats without peptide repeats VNTR PTTSTTSA (494 base pair insert eight amino acid tandem repeat) MUC6 VNTR 169aa (507 base pair) repeat unit MUC7 VNTR TTAAPPTPPATTPAPPSSSAPPE The repeated subunit of MUC6 comprises 169 amino acids, although at this time the amino acid sequence of this repeat unit has not been fully characterized. The MUC7 sequence has recently been published (27).

g* Finn and colleagues have demonstrated that in the lymph nodes of patients with breast cancer cancer of the pancreas, ovary and other tumours, cytotoxic lymphocytes are present which react with human mucin. Antibodies to the MUC1 peptide can block the activity of these cytotoxic T-lymphocytes on MUC1' target cells Recently, cytotoxic lymphocytes to a murine lung cancer have also been described (28).

The surgery associated with tumour removal is traumatic to the patient, often disfiguring, and costly. Established chemotherapeutic and radiation procedures for tumour treatment which S 25 may be carried out in place of or in conjunction with surgical procedures are often debilitating and associated with severe side-effects. There is accordingly an urgent need for therapeutic compounds and methods for the prevention/treatment of tumours.

There is an urgent need for new compounds and methods for the treatment of cancer.

Similarly, there is a pressing need for alternative compounds and therapies for the treatment BM,"USTIN:PCnh53282.mtnb2I Deamber 1994 Illllhh r of other disease states such as type I allergies, malaria, IIIV, dental caries, flu, cholera, foot and mouth disease, meningitis, Leishmania infection, whooping cough, rabies, Streptococcus infection, respiratory infection, measles, Lyme disease, tuberculosis, bacterial meningitis, shingles, rubella, hepatitis, herpes, hepatitis A, polio, venereal disease/trachoma, hepatitis B, common cold, cervical cancer, meningitis/pneumonitis, chicken pox, small pox, pneumonia/PUO.

In accordance with the first aspect of the present invention, there is provided a compound comprising a conjugate between an antigen, and a carbohydrate polymer comprising oxidized mannose.

In accordance with another aspect of the present invention, there is provided a compound comprising a conjugate between the human mucin polypeptide, one or more repeated subunits thereof, or a fragment of said repeated subunits, with a carbohydrate polymer comprising 15 oxidized mannose.

By "oxidized mannose" it is meant that one or more mannose units of the carbohydrate polymer are oxidized to aldehydes for conjugation with antigen.

Insofar as the present invention is concerned, the antigen can be a human autoantigen or a peptide antigen derived from a virus, microorganism or plant or an amino acid subunit of at least five amino acids in length of a human autoantigen or a peptide antigen derived from a virus, microorganism or plant. The antigen of the present invention can also consist of more than one, five or more amino acid subunits (as mentioned above) linked together. These 25 linked subunits may be from the same or different origins within the bounds described above.

Examples of the antigens envisaged by the present invention are as follows: pollens, hepatitis C virus (HIV) core, El, E2 and NS2 proteins, Plasmodium falciparum circumsporozoite protein, HIV-gpl20/160 envelope glycoprotein, streptococcus surface protein Ag, influenza nucleoprotein, haemagglutinin-neuraminidase surface infection, TcpA pilin subunit, VP1 protein, LMCV nucleoprotein, Leishmania major surface glycoprotein (gp63), Bordetella pertussis surface protein, rabies virus G protein, Streptococcus M protein, Syncyticial virus I -1 IC L P- (RSV) F or G proteins, Epstein Barr virus (EBV) gp340 or nucleoantigen 3A, haemagglutinin, Borrelia burgdorferi outer surface protein (Osp) A, Mycobacterium tuberculosis 38kDa lipoprotein or Ag85, Neisseria meningitidis class 1 outer protein, Varicella zoster virus IE62 and gpl, Rubella virus capsid protein, Hepatitis B virus pre S1 ag, Herpes simplex virus type I glycoprotein G or gp D or CP27, Murray valley encephalitis virus E glycoprotein, Hepatitis A virus VP1, polio virus capsid protein VP1, VP2 and VP3, chlamydia trachomatis surface protein, Hepatitis B virus envelope Ag pre S2, Human rhinovirus (HRV) capsid, papillomavirus peptides from oncogene E6 and E7, Listeria surface protein, Varicella virus envelope protein, Vaccinia virus envelope protein, Brucella surface protein, a combination of one or more of said antigens, an amino acid subunit of said antigens comprising five or more amino acids in length or combinations of one or more of said subunits.

'The antigens of the present invention can also consist of whole cells or sub-cellular fractions thereof. Such cells or sub-cellular fractions thereof may be derived from any tumour type or other source. Examples of cancer types from which the whole cells or sub-cellular fractions may be derived are breast, lung, pancreas and colon cancer and melanoma. Some further examples of specific antigens obtained from tumours are melanoma specific antigen (for example, the MAGE series antigen), carcino embryonic antigen (CEA) from colon and other cancers or indeed antigens extracted from any tumour.

This invention includes any one or more of the antigens listed and in particular includes any one or more of the human mucins MUC1 through MUC7 which, as mentioned above, all comprise highly repetitive central portions of repeated amino acid sequences which are high in serine, threonine and proline. In particular, the compounds of this invention may comprise a human mucin polypeptide (containing a variable number of repeats associated with normal allelic variation), or may comprise one or more of the repeated sequences of human mucin, preferably two to eighty, more preferably two to twenty and even more preferably two to ten repeated subunits of human mucin. The human mucin and subunits thereof are preferably non-glycosylated or aberrantly glycosylated so as to provoke an immune response to the B:AUSTIN:PCT1532828.nmb21 December 1994 I I I mucins found on cancer cells which have a deficiency in their carbohydrate coat which leaves the protein core exposed. The use of human mucin MUC1 is particularly preferred although it is to be clearly understood that the invention extends to the use of any antigen and especially to the use of the human mucins MUC1 through MUC7. For the purpose of convenience, the term MUC will hereafter be used to refer to any of the human mucins MUC1 through MUC7 and repeated subunits thereof. While only the human mucins will be dealt with hereafter, it must be kept in mind that his invention equally relates to any other antigen as mentioned previously.

10 Fragments of MUC may also be conjugated to a carbohydrate polymer. These fragments would generally comprise from five to twenty amino acids from the repeated amino acid sequences of any of mucins MUC1 through MUC7. For example, a fragment of the mucin MUC1 may comprise the amino acid sequence APDTR, APDTRPAPG, DTRPAPGSTAPP, and the like. For convenience of description these fragments are also included with the definition MUC. Similarly, other antigen fragments comprising at least five amino acids may be conjugated to a carbohydrate polymer.

A specified antigen (such as MUC1, MUC2, MUC3, MUC4, MUC5, MUC6 or MUC7) may form part of a fusion protein in order to facilitate expression and purification on production of the fusion protein in recombinant host cells. The non-antigen portion of the fusion protein would generally represent the N-terminal region of the fusion polypeptide with the carboxy terminal sequences comprising antigen sequences. Fusion proteins may be selected from glutathione-S-transferase, f-galactosidase, or any other protein or part thereof, particularly those which enable affinity purification utilizing the binding or other affinity characteristics of the protein to purify the resultant fusion protein. The protein may also be fused to the Cterminal or N-terminal of the carrier protein. The nature of the fusion protein will depend upon the vector system in which fusion proteins are produced. An example of a bacterial expression vector is pGEX which on subcloning on a gene of interest into this vector produces a fusion protein consisting of glutathione-S-transferase with the protein of interest.

Examples of other vector systems which give rise to fusion proteins with a protein of interest B:\AUSTIN:PCT\532828.mmb21 December 1994 I I I d are described in Sambrook et al which is incorporated herein in its entirety by reference.

These can be included or cleaved; if included they could have a "carrier" function.

The protein or fusion protein maybe expressed in a number of prokaryotic coli or 0-sutilis) or eukaryotic (baculovirus, CHO cells, cos cells or yeast) expression systems. In some of these systems, for example, baculovirus or yeast, by introducing glycosylation motifs into the protein or fusion protein, the mannose rich glycosylation may be adequate; negating the need for chemically linking with mannose rich carbohydrate polymers. These novel fusion proteins may be used with or without mild periodate oxidation.

The number of repeated monomer units in the polymer is not important but generally carbohydrate polymers would comprise at least twenty monomer units, preferably in excess of one hundred monomer units, more preferably in excess of one thousand monomer units, and still more preferably in excess of ten thousand monomer units or more. Carbohydrate 15 polymers may be a mixture of polysaccharide chains of varying molecular weights. Most preferably the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer containing mannose units. While most preferably the carbohydrate polymer is mannan, mannose carbohydrate units may be combined, for example, with carbohydrate units selected from glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-0-methyl-D-galactose, 2-0-acetyl--D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, c-D-galactopyranose 6-sulphate, fructose and a abequose, conformation and configuration isomers thereof, or a carbohydrate formed of two or more different monomer units.

25 Antigens may be conjugated to a carbohydrate polymer according to standard processes well known in the art of carbohydrate chemistry for the derivatization and reaction of polysaccharides and monosaccharides. Mannose units of carbohydrate polymers are to be oxidized with conventional oxidizing reagents such as sodium periodate to give a polyaldehyde which is then directly reacted with the antigen (such as repeated subunits of MUC1) where amino functional groups on the protein chain (such as the e group of lysine) react with the aldehyde groups

(-Z

P -T 0 I L Sla~ssr~-rs I- to form a Schiffbase. Polysaccharide chains may be first activated with cyanogen bromide and the activated polysaccharide then reacted with a diamine, followed by conjugation to the antigen to form a conjugate which may optionally then be oxidized. The carbohydrate and polypeptide may be derivatized with bifunctional agents in order to cross-link the carbohydrate and polypeptide. Commonly used cross-linking agents include 1,1 -bis-(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicyclic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1, 8-octane. Derivatizing agents such as methyl-3-[(p-azido-phenyl)dithio] propioimidate yield photactivitable intermediates which are capable of forming cross-links in the presence of light. Oxidized carbohydrates may be reacted with hydrazine derivatives of antigens to give a conjugate. Alternatively, carbohydrates may be reacted with reagents such as carbonyl diimidazole, which after oxidation gives the desired conjugate.

S" The coupling of antigens to a carbohydrate involves converting any or all of the functional S. groups on the carbohydrate to reactive groups and thereafter reacting the reactive groups on the carbohydrate with reactive groups on the polypeptide. Carbohydrate polymers are replete with hydroxide groups, and in some instances, carboxyl groups (such as in idruionate), ester 20 gioups (such as methylgalacturonate) and the like. These groups may be activated according to standard chemical procedures. For example, hydroxyl groups may be reacted with hydrogen halides, such as hydrogen iodide, hydrogen bromide and hydrogen chloride to give the reactive halogenated polysaccharide. Hydroxy groups may be activated with phosphorous trihalides, active metals (such as sodium ethoxide, aluminium isopropoxide and potassium tert-butoxide), or esterified (with groups such as tosyl chloride or acetic acid) to form reactive groups which can be then be reacted with reactive groups on the polypeptide to form one or more bonds. Other functional groups on carbohydrates apart from hydroxyl groups may be activated to give reactive groups according to well known procedures in the art.

B:\AUSTIN:PC 532828.mmb21 December 1994 I ~LI~CPq ~ls~ 8 Polypeptides comprising MUC or other antigens may be produced according to well known procedures such as peptide synthesis, protein purification, or expression of polypeptides in host cells. Peptide synthesis may be employed for polypeptides containing up to about a hundred amino acids (for example, five repeated subunits of MUC1).- Generally, for polypeptide containing about twenty or more amino acids, the preferred means of production is recombinant expression in a host cell, preferably a prokaryotic host cell, and more preferably a bacterial host cell. However, as discussed earlier, eukaryotic systems may also be used. Procedures for expression of recombinant proteins in host cells are well established, see, for example, Sambrook, et al Carbohydrates may be purified from natural sources or synthesized according to conventional procedures, Carbohydrates are available commercially from many suppliers.

In another aspect, the invention relates to an immunogenic vaccine against human disease states and in particular against tumour cells expressing human mucin or a subunit thereof, which comprises a compound comprising a conjugate between an antigen and a carbohydrate polymer comprising oxidized mannose, in association with a pharmaceutically acceptable carrier. Antigens which may be used in this aspect of the invention are as previously described. The vaccine is administered to human patients to protect against various disease o: 20 states including cancer cell growth, and in particular, the growth of tumours of secretory •tissues, such as tumours of the breast, colon, lung, pancreas, prostate, and the like, Patients may be immunized with the vaccine to protect against tumrnour formation of secretory tissues.

Alternatively, patients suffering from tumours may be immunized with the vaccine as part of :o e a therapeutic regimen for tumrnour treatment. By way of example, to protect women from breast cancer, women may be immunized with the vaccine pre- or post-puberty and may receive one or more injections, preferably an initial immunization, followed by one or more booster injections separated by several months to several years. In one immunization 0 4 schedule, women may be immunized with the compounds of the invention and then receive a booster immunization at appropriate intervals. Further booster immunizations are then provided at regular intervals. The route of immunization is 1 no different from conventional human vaccine administration. Accordingly, vaccines may be administered subcutaneously, intramuscularly, orally, intravenously, and the like.

Some other disease states which may be protected against in this manner include, type I allergies, malaria, HIV, dental caries, flu, cholera, foot and mouth disease, meningitis, Leishmania infection, whooping cough, rabies, Streptococcus infection, respiratory infection, measles, Lyme i:;ease, tuberculosis, bacterial meningitis, shingles, rubella, hepatitis, herpes, hepatitis A, polio, venereal disease/trachoma, hepatitis B, common cold, cervical cancer, meningitis/pneumonitis, chicken pox, small pox, pneumonia/PUO.

The amount of compounds of the invention or compositions thereof delivered to a patient is not critical or limiting. An effective amount of a compound of the invention is that which will stimulate an immune response against the antigen component. The amount of compounds or compositions delivered may vary according to the immune status of the patient (depending on whether the patient is immunosuppressed or immunostimulated), the judgement of attending physician or veterinarian whether the compound is used as a vaccine to prevent or treat a disease state or as a vaccine to prevent tumour formation, or whether the vaccine is used in the treatment of an existing tumour. By way of example, patients may receive from 1lg to 10,000/g of the compounds of the invention, more preferably 50g to 5,000/ug, still more preferably 100[g to 1,000tg, and even more preferably 100/g to 500tg of the compounds of the invention. Adjuvants are not generally required. However, adjuvants may be used for immunization. Suitable adjuvants include alum, as well as any other adjuvant or adjuvants well known in the vaccine art for administration to humans.

Compounds of the invention may be administered to patients in concert with a cytokine or other immune regulator. By way of example, immune regulators which may be administered in concert with the compounds of the invention include one or more of GM-CSF, G-CSF, M- CSF, TNFc or 3, interferon a or y, any of IL1 through IL13, or any other cytokine. The immune regulator may be administered at the same time as the compounds of the invention, B:AUSTIN:PCT532828.mmb21 December 1994 I_ optionally part of a multi-component idministration form. Alternatively, the compounds of th;s invention and immune regulators may be administered at different time intervals.

In a still further aspect of this invention, there is provided a method for inducing a cell mediated immune response against antigens which comprises administering to an animal (including a human) a compound comprising a conjugate between said antigen and a carbohydrate polymer comprising oxidized mannose, optionally in association with a pharmaceutically acceptable carrier, The immunization of humans and animals with the compounds of this invention may provoke a potentiated cellular response of activated T-lymphocytes which are cytotoxic to cells expressing the antigen component. By way of example, humans and animals may be immunized against tumours which express human mucins. A potential benefit of this invention arises from the fact that animals may be protected against cancer prior to tumour growth, as the compounds of the invention may provoke a cellular immune response of cytotoxic T-cells which kill tumour cells expressing mucin or other antigenic determinants.

This invention is applicable to the immunization against tumours of secretory tissue, such as adenocarcinomas, more particularly, tumours of the breast, ovary, pancreas, colon, lung, prostate and the like.

The compounds of the invention may also be used as therapeutic agents for the treatmer 'f patients suffering from cancer, as a part of the overall treatment for eradication of the cancer.

Thus, the compounds of the invention may be administered to patients suffering from cancer either before or after surgery to remove the tumour. Preferably the compounds are administered as part of a chemotherapeutic regime following tumour excision. In these circumstances, the compounds of the invention are administered in amounts consonant with standard chemotherapeutic regimes for the administration of cytotoxic compounds for use in tumour treatment.

The compounds of this invention can also be used in immunization for therapy or prophylaxis of other disease states as mentioned earlier.

'Y2 Il,-17- 11 In a still further aspect, the invention relates to the use of a coni J comprising a conjugate between the human mucin polypeptide, one or more repeated subunits thereof, or a fragment of said repeated subunits and a carbohydrate polymer comprising oxidized mannose in the treatment of adenocarcinoma, particularly breast cancer.

The compounds of this invention possess the advantage of being substantially non-toxic on administration to animals or humans, and as a consequence the compounds are well tolerated by administration to patients.

The invention described herein is not restricted to the human mucin MUC1. The invention clearly extends to the use of other mucins expressed by cancer cells, as well as to the use of other antigens which on coupling to polysaccharides, can be used to provoke cytotoxic T-cell responses against tumour cells, which compounds may be used in vaccines to prevent tumour formation, as well as for the treatment of cancer, and/or the treatment or prophylaxis of other disease states as mentioned earlier.

4 The invention will now be described with reference to the following non-limiting Examples.

The following abbreviations are used in the Examples:

ABBREVIATIONS

SELISA: enzyme linked immunosorbent assay DTH: delayed type hypersensitivity FP: fusion protein GST: glutathione-S-transferase HMFG: human milk fat globule Kd: kilodalton KLH: keyhole-limpet haemocyanin PAGE: polyacrylamide gel electrophoresis PBS: phosphate buffered saline

I

SDS:

Tc:

VNTR:

CTL:

M-FP:

MHC:

MSA:

CASA:

FIGURE

Figure 1: sodium dodecyl sulphate cytotoxic T-lymphocytes variable number of tandem repeats cytotoxic T-cells mannan fusion protein major histocompatability complex mucin serum antigen circulating MUC1 serum antigen

LEGEND

Growth of 5 x 10 6 3T3 and MUC1+3T3 cells is BALB/c mice.

Sr

S*

to 0.006.

0 a 00 to 0 00C

S*

Figure 2: Dose response of MUC1I3T3 cells in mannan-fusion protein and non immunized BALB/c mice. Doses ranging from 10 6 5 x 10 7 cells. Growth of 5 x 10 6 3T3 tumor cells is also shown.

Figure 3: Mice immunized with mannan, mixture of mannan fusion protein, M-FP and a control group (immunized with PBS); 16.1FP-mannan, oxidized mannan, M-FP and PBS; dextran-FP M-FP and PBS, and challenged with 10 6 MUC1 3T3 cells.

Figure 4: BALB/c mice immunized with M-FP and treated with anti-CD3, anti-CD4 and anti-CD8 on 0, two days. Challenge with 10 6 MUC1'3T3 cells.

Figure 5: DTH response measured at forty eight hours in mice immunized with mannanfusion protein and challenged with dead (freeze-thaw 5 times) 3T3 and MUC1 3T3 cells, Cpl3-32-KLH, fusion protein, HMFG, mannan-fusion protein, GST, T4N1 and PBS in their hind footpads. Control (black box), mice treated with anti-CD4 (grey box) and mice treated with anti-CD8 (cross lines).

A:VAUSTIN:PCIN532828.mmb2I Dmcember 1994 c~ llllll~ I t- 1 ~1

I

13 Figure 6: Cytotoxic T-lymphocyte assay with P815 and MUC1 P815 51 Cr labelled target cells.

Figure 7: A: (DBA/2" x BALB/c)F1 mice were challenged with 5 x 10 6 MUC1IP815 cells. After thirteen days of tumour challenge (established tumours) mice were immunized with 5/g M-FP (5/pg corresponding to the amount of FP) once or every other day. Control mice were injected with PBS; B: DBA/2 mice were challenged with 5 x 106 MUC1+P815 cells. After fifteen days of tumour challenge (established tumours) mice were immunized with 5[g M-FP (5f/g corresponding to the amount of FP) once or every other day. Control mice were injected with PBS.

0.

Figure 8: CTL assay using 5 Cr MUC1-P815 and control P815 target cells BALB/c Smice were immunized I.P. weekly x 3 and then spleen cells were collected and combined with 5 Cr labelled target cells at various effector-to-target (E:T) ratios in 96 well U bottom plates and 5 'Cr release was measured: percent S specific release spontaneous release)/ (maximum release spontaneous release)] x 100 is shown at various E:T ratios using spleens from mice immunized with MUC1 tumor MFP-oxidized MFP-reduced 20 or FP when tested on MUC1-P815 targets or on control P815 targets:- MUC1 tumor MFP-oxidized MFP-reduced or SFP Antibody test by ELISA: Sera from immunized mice Tumor; MFP-oxidised; MFP-reduced; FP; saline (PBS)) were titrated on plates coated with 20Ag/ml FP, washed and the bound antibody was detected with sheep anti-mouse globulin conjugated to horseradish peroxidase and developed as described the absorbance at 405nm is recorded for various dilutions of antibody.

DTH responses were induced in cyclophosphamide treated mice (200mg/kg) by IP immunization as above; six days later, footpads were A:A\USTIN:PCTN 532828.mmh2I Deembcr 1994 I ii I I I I injected with 10 s killed (freeze/thawed five times) MUC1-3T3 and 3T3; FP or (PBS) and the thickness of injected and control footpads was recorded at forty eight hours. The increase in footpad size is recorded (cm 2 standard deviation) in mice immunized with Tumor cells MFP oxidised or FP and challenged as shown. There were five mice per group.

Figure 9: Growth of MUC1-3T3 tumors in five BALB/c mice per group after immunization with Tumor cells; MFP-oxidised; MFP reduced; FP or PBS, showing the size of the tumors (the product of two diameters), error bars, and the days after test tumor injection. (A) challenge with 10 6 MUC1-3T3 cells, challenge with 5 x 106 MUC1-3T3 cells.

Figure 10: CTL assay using SCr MUCI-P815(Tm211) (H2d) and control P815 (H2d) target cells. Various mice strains were immunized with MFP, spleen cells were collected and combined with 5 Cr labelled target cells at various effector:target ratios and chromium release was measured and calculated as a percentage NZB, B10D2, DBA2 and BALB/c are H2d. SJL H2s, CBA H2k, BALBb H2b, BL6 H 2 b, 129J H2b, NZW H22z *r *r Figure 11: CTL assay using 5 Cr MUCI-P815 (Tm211) (H2d) and control P815 (H2 2 d) target cells and E3 (H 2 b) either peptide or not peptide pulsed BALB/c, BALBb, BL6 mice were immunized with M-FP and were used as effector cells. 51 Cr BALB/cand BALB/b blasts, E3 and P815 MUC1 peptide pulsed.

BALB/c mice were effectors. 5 'Cr BALB/c, and BALB/b blasts, E3 and P815 MUC1 peptide pulsed BALB/b mice were used as effectors. Spleen cells were collected and combined with 'Cr labelled target cells at various effector: target ratios and chromium release was measured and calculated as a percentage.

BAAUSTSIN:PCT532828mmb21 December 1994 I I I I r w Figure 12: CTL assay using 5 Cr MUC1-P815 (Tm211) (H2d) and control P815 (H2 4 2R (KkDb), B6 (KbDb), CBA 5R (KbDd) spleen cells were made as blast cells and peptide pulsed were also target cells B10A(2R) (KkDb) were immunized with MFP, spleen cells were collected and combined with 51 Cr labelled target cells at various effector:target ratios and chromium release was measured and calculated as a percentage.

Figure 13: CTL assay using "Cr MUC1-P815 (Tm211) (H2d) and control P815 (H2d).

B6(KbDb), CBA 5R (KbDd) spleen cells were made as blast cells and peptide pulsed were also target cells. B10A(5R)(KbDd were immunized with MFP, spleen cells were collected and combined with 5'Cr labelled target cells at various effector: target ratios and chromium release was measured and calculated as a percentage.

Figure 14: CTL assay using 5 CrP815(H2d) and E3 (H2b) target cells. bm-1 (KbDb), B6(KbDd), 5R (KkDb) and bm-12 (KbDd), spleen cells were made as blast cells and peptide pulsed were also target cells. B6(KbD b were immunized with MFP, spleen cells were collected and combined with 5'Cr labelled target cells Sat various effector: target ratios and chromium release was measured and calculated as a percentage.

Figure 15: CTL assay using 5 'Cr P815 (H2d) and E3 (H2b) target cells, bm-1 (KkD), B6(KbDb), 5R(KbDd), and bm-12 (KbDd) spleen cells were made as blast cells and peptide pulsed were also target cells, bm-1 (KbDb) were immunized with MFP, spleen cells were collected and combined with "Cr labelled target cells at various effector:target ratios and chromium release was measured and calculated as a percentage.

Figure 16: CTL assay using tCr P81 5 (H2d) and E3 (H2b) target cells. bm-1 (KbDb), B6(KbDd), 5R (KbDd) and bm-12 (KbDd), spleen cells were made as blast cells B:AAUSTIN:PCT\532828.mmb21 December 1994 ~-~1111 1 11 Cr _1 and peptide pulsed were also target cells. bm-12 (KbDb) were immunized with MFP, spleen cells were collected and combined with 5 1 Cr labelled target cells at various effector: target ratios and chromium release was measured and calculated as a percentage.

Figure 17: CTL assay using 51 Cr P815 (H2d) MUC1 peptide pulsed, and SjL H2s) and NZW (H2z) spleen cells were made as blast cells and peptide pulsed were also target cells. SjL were immunized with MFP, spleen cells were collected and combined with 5 Cr labelled target cells at various effector:target ratios and chromium release was measured and calculated as a percentage.

Figure 18: CTL assay using 5 1 Cr P815 (H2d) MUC1 peptide pulsed, and SjL H2) and I NZW (H2z) spleen cells were made as blast cells and peptide pulsed were also target cells. NZW (H2z) were immunized with MFP, spleen cells were collected and combined with 51 Cr labelled target cells at various effector:target ratios and chromium release was measured and calculated as a percentage.

Figure 19: CTL assay using 5Cr P815 (H2d) MUC1 peptide pulsed cells. (KbDb), B6 20 (KbDb), 5R (KbDd), 2R (KkDb), bm-1 (KbDb) and NZW (H2Z) spleen cells were made as blast cells and peptide pulsed were also target cells. mice were immunized with MFP and their spleens were used as effector cells.

Figure 20: Mice immunized with different immunogens. Mice were bled and sera tested by ELISA for anti-MUC1 antibodies to Fusion protein and T4N1 (irrelevant peptide) coated plate. Absorbance values are at 1:100 antibody titer. Serum from mice immunized with T4N1 and the anti-MUC1 monoclonal antibody VA2 (isotope IgG1) were used as positive and negative controls.

B:\AUSTIN:PCT532828.mmb2l Dccnbr 1994 I I AIPlsA~- EXAMPLE 1 Materials and Methods Synthetic peptides, fusion protein, and HMFG production and immunization: Peptides C-p13-32 (MUC1 VNTR), p31-55 and p51-70 (N-terminal to VNTR) and p344-364 and p408-423 (C-terminal to VNTR) were synthesized using an Applied Biosystems Model 430A automated peptide synthesizer (sequences shown in Table The mouse CD4 Nterminal region peptide (T4N1) was also synthesized and used as a negative control peptide (Table HMFG, was isolated from human milk A fusion protein containing VNTR repeats was produced by subcloning the cDNA into the bacterial expression vector pGEX-3X (10) (Table 1).

BALB/c mice (females aged eight weeks) were immunized intraperitoneally with 50,g of either fusion protein, HMFG, C-p13-32 (coupled to diphtheria-toxoid or KLH (keyhole limpet hemocyanin) with glutaraldehyde) or T4N1 (coupled to diphtheria toxoid or KLH emulsified in complete Freund's adjuvant and this was repeated four and six weeks later in phosphate buffered saline. Prior to tumour injection and after tumour rejection mice were bled and the serum was tested on an ELISA for antibody production against the relevant immunogens.

20 MUC1 3T3 tumour injections (see later description for production of these cells) were given subcutaneously in 0.2mls containing the appropriate tumour dose. Mice treated with anti- CD3, anti-CD4, anti-CD8 and anti-y-interferon antibodies were given three intraperitoneal injections of 0.2mls on days 0 and +2 (0 day of tumour injection). Mice to be treated with antibody were injected subcutaneously with the tumour on day zero and on day five (tumour size approximately 0.15cm 2 when they were treated with rabbit complement (fresh serum 0.2mls intravenously) and antibody (0.2mls intraperitoneally), on days five and seven.

BAAUSTIN:TCS3228.mmb2I December 1994 I III II ISISSj~BSAllrr I- I 18 TABLE 1: Sequences of synthetic peptides .9 9 *9 9 4.

9.

Peptide Amino Acid Sequence MUC1 VNTR: Cp-13-32 C-PAHGVTSAPDTRPAPGSTAP Fusion protein (PAHGVTSAPDTRPAPGSTAP) x N-terminal region to MUC1: p31-55 TGSGHASSTPGGEKETSATORSSVP p51-70 RSSVPSSTEKNAVSMTSSVL C.terminal to MUC1: p334-364 NSSLEDPSTDVVOELORDISE p 4 0 8 423

TGFNOYKTEAASRVNL

Mouse C04: T4N1 KTLVLGKEQESAELPCEY Treatment of mice with antibodies: To ensure that the antibodies to CD3, CD4 and CD8 were depleting or blocking CD3 CD4 and CD8 T-cells, a serological analysis of residual cells was performed using the antibodies to CD3, CD4 and CD8. Spleen and lymph node cells were removed from normal and treated BALB/c mice, the lymphocytes were teased, washed in DME and incubated at 37 0 C for five minutes in 0.83% ammonium chloride to lyse red blood cells. Serology tests were performed where 2 x 105 spleen/lymph node cells from mice were added to a 1:500 dilution of anti-CD3, anti-CD4 and anti-CD8 ascites. Following extensive washing, the cells were incubated with (mouse thymus cell absorbed) rat anti-mouse IgG and incubated for thirty minutes on ice. Mice which had been treated with anti-CD3, anti-CD4, anti-CD8 or anti- CD4+CD8 were each tested with these antibodies. It was found that the CD3 cells were depleted and CD4' and CD8 cells had been blocked.

Preparation of soluble GST-MUC1 fusion protein A 309 base pair insert (PDF9.3) encoding a little more than 5 repeats of a 60 base pair motif from the VNTR region of MUC1 cDNA (10) was subcloned into the bacterial expression vector pGEX-3X, in the correct reading frame and orientation Fusion protein (FP), consisting of glutathione-S-transferase (GST, 26Kd) and MUC1 VNTR (12Kd), was induced with 0.1mM IPTG Cells were collected by centrifugation, washed and lysed by 8:1AUSTIN:PCTf32828.mmb21 December 1994 LL-I II sonication in buffer containing 1% Triton X-100. Supernatant containing the soluble FP was mixed with glutathione-agarose beads (sulphur-linked) (Sigma, St. Louis) and collected by centrifugation. The FP ((C-PAHGVTSAPDTRPAPGSTAP) x 5-GST) was eluted with buffer containing 5mM reduced glutathione, dialyzed against phosphate buffer and analyzed by SDS-PAGE.

Polyacrylamide gel electrophoresis Samples to be tested were mixed with SDS sample buffer, boiled for five minutes and then loaded onto a 12.5% SDS-PAGE gel. Gels were stained in 0.2% Coomasie blue and then destained in 7% acetic acid or were silver stained Molecular weight markers used: 200,000 myosin; 116,000 -galactosidase; 92,500 phosphorylase b: 66,200 bovine serum albumin; 43,000 hen egg white ovalbumin; 31,000 bovine carbonic anhydrase; 21,500, soybean trypsin inhibitor, and 14,400 hen egg white lysozyme.

15 Conjugation of Mannan to MUC1 fusion protein Mannan was oxidized to a poly-aldehyde by treating 14mg of mannan (a mannose containing *0 polysaccharide) in 1ml of 0.1M phosphate buffer pH6.0 with 100l1 0.1M sodium periodate in phosphate buffer for 30-60 minutes at 4°C. Following a further thirty minute incubation at 4°C with 10l ethandiol, the mixture was passed through a PD-10 column equilibrated in buffer pH6.0-pH9.0 and the mannan fraction collected; 900pg of MUC1 FP was added to the oxidized mannan, reacted overnight at room temperature and used for subsequent studies.

The fusion protein was radiolabelled with 125, using chloramine-T. The unlabelled fusion protein was mixed with radiolabelled fusion protein such that the specific activity was 1 x 10cpm//g and reacted with oxidized mannan as above. The mannan-FP was stabilized by reducing the Schiffs bases and residual aldehyde groups. The complex was then analyzed by SDS-PAGE, Autoradiography, coomasie (protein) stain, silver (protein +CHO) stain, and silver PAS (CHO) stain and by gel permeation chromatography using sepharyl S 208 column cm x 100 cm).

B:\AUSTIN:PCT532828.mmb22 December 1994 lWs k I L 1 L -Ulllsb-~- -111 Immunization schedule BALB/c mice (females aged eight weeks) were immunized intraperitoneally with (corresponding to amount of FP) mannan-FP, FP and a mixture of non-conjugated mannan FP in phosphate buffered saline (PBS) once weekly for three weeks. Mice were previously immunized with FP alone and this was used for a control for antibody production (see below). Prior to tumour injection, mice were bled and the serum tested by ELISA (see below) for antibody production against FP, (anti-mannan antibodies).

Tumours and antibodies The BALB/c mouse fibroblast cell line 3T3 transfected with the MUC1 cDNA transmembrane form with the ras gene and a cell line MUC1'3T3 was developed (obtained from Dr D Wreschner, Tel Aviv University, Israel). Mice received a 0.2ml subcutaneous injection of appropriate tumour cell dose in PBS and subsequent tumour growth measured. All S: measurements were performed with dial gauge callipers (Schnelltaster, H C Kroplin, Hessen, 15 Germany) and the size of the tumours were expressed by the area of the tumour size (cm 2 (diameter x diameter). The murine DBA/2 mastocytoma cell lines P815, and MUC1'P815 (containing the cDNA of the membrane anchored form of MUC1) were obtained from Dr B Acres (Transgene, Strasbourg, France).

Rat Mabs to murine CD3 (KT3.2), CD4 (H129.19) and CD8 (53-6.72) were prepared from S ascites and tissue culture supernatants (12 to 14). Ascites fluid were prepared in SCID mice S. as described previously Mice treated with anti-CD3, anti-CD4 and anti-CD8 antibodies were given three intraperitoneal injections of 0.2mls on days 0 and +2 (0=day of tumour injection). MUC1 antibodies used were VA1 and VA2, produced against a GST-MUC1 bacterial FP which contains five VNTR repeats (16).

Preparation of peptides and HMFG Peptides C-p13-32 (C-PAHGVTSAPDTRPAPGSTAP) (MUC1 VNTR) and T4N1 (KTLVLGKEQESAELPCEY) (mouse CD4 N-terminal region peptide) were synthesized using B:\AUSTIN:PC3532828.mmb21 December 1994 II I I I LII I I I I 1 an Applied Biosystems Model 430A automated peptide synthesizer. HMFG was isolated from human milk and prepared as previously described (17).

Enzyme linked immunosorbent assay (ELISA) Measurement of anti-fusion protein antibody: The ELISA test was performed (17), where 20I/g/ml of FP was coated in the wells of a microtitre plate, non-specific binding blocked with 2% bovine serum albumin, and 50p1 of serum from FP and mannan-FP immunized mice added for two hours at room temperature. Normal mouse serum (NMS) was used as negative control. After washing, sheep anti-mouse immunoglobulin conjugated to horseradish peroxidase conjugate (Amersham, United Kingdom) was added, incubated at room temperature and the plate was developed using 50[1, 0.03% 2,2'-azino-di(3-ethylbenzthiazoline sulphonate (Amersham, United Kingdom), 0.02% H202 (100 Volume, Ajax Chemical) in 0.1M citrate buffer, and incubated for ten to fifteen minutes at room temperature until the desired intensity 15 was achieved. Absorbency was read at 405nm in a plate reader.

C

Determination of the activity of fusion protein after conjugation to mannan: The ELISA test was performed as described above with the following modifications; of FP, mannan-FP and mannan were coated on the plate and the primary antibodies used were VA1 and VA2 (anti-FP Mabs).

Induction of DTH To induce DTH in mice, cyclophosphamide (Endoxan-Asta, Mead Johnston) at a dosage of 200mg/kg body weight, was injected into the peritoneal cavity two days before an intraperitoneal injection of 5ttg mannan-FP. Six days later, the hind footpads were injected with either 10s3T3 or MUC13T3 (freeze/thawed five times), 50/g of HMFG, FP, C-p13-32 (coupled to keyhole-limpet haemocynin using glutaraldehyde), T4N1 (an irrelevant peptide), mannan-FP, GST and mannan and an equivalent volume of PBS. The DTH response was measured at forty eight hours later, by measuring the width and the thickness B:\AUSTIN:PCT\532828.mmb21 December 1%4 9 II 22 of the footpad and calculating their product. All measurements of footpads were performed with dial gauge callipers (Schnelltaster, H C Kroplin, Hessen, Germany).

Cytotoxic T-lymphocyte assay BALB/c mice immunized with mannan-FP were sacrificed and their spleen cells were collected and washed in 2% foetal calf serum/PBS. The target cells, P815 and MUClP815 cells were either not treated or treated with 5mM phenyl N-acetyl-a-D-galactosaminide (pagal) for two days (to inhibit O-linked glycosylation) (Sigma, St Louis, USA) prior to use in a standard 5 Cr release assay. Tumour cells (10 6 cells) (target cells) were radiolabelled with 100/Ci of Na 2 5 CrO4 (Amersham Corp, Arlington Heights) for sixty minutes at 37 0

C,

followed by extensive washing. Spleen cells and target cells, were resuspended in culture medium, and then combined at various effector-to-target ratios in 96-well, U-bottom plates (Costar Corporation). The plates were then centrifuged at 100xg for three minutes to initiat, cell contact and incubated for four hours at 37 0 C in 10% C02. After incubation the 15 supernatants were collected and radioactivity was quantitated in a gamma counter (Beckman Instruments).

Spontaneous release of 51 Cr was determined by incubation of the target cells alone, while maximum release of 5 'Cr was determined by treatment with 10% sodium-dodecyl sulphate and percentage of specific release was determined as [(experimental spontaneous)/(maximum spontaneous)] x 100%.

T Proliferation Assay Mice immunized with M-FP were sacrificed, their spleen cells were collected, washed in 2% foetal calf serum/PBS, red blood cells lysed with 0.14% NH 4 C1 and duplicate cultures of x 105 spleen cells in 100/l of culture media were seeded in a 96-microwell plate. Spleen cells were stimulated with 100/l of the following: 10/xg T4N1, GST, mannan, HMFG, Cpl3-32, FP, MFP; and 105 breast cancer cells (pagal treated and untreated) of 3T3, MUC1 3T3, P815, MUC1'P815, and 105 human breast cancer cell lines T47D, MCF7 and ZR75. All tumour cells were treated with 25/g/ml of mitomycin-C (Sigma, Victoria Australia) for two B:\AUSTIN:PCT'32828mmb2 December 1994 gl hours at 37 0 C to inhibit proliferation of the tumour cells. Cultures were incubated at 37°C in 5% CO 2 for thirty six hours. [H]TdR (Amersham, United Kingdom) (6.7 Ci/mmol) incorporation was determined during the last four hours of culture (1/zCi/well).

EXAMPLE 2 Serological analysis of MUC13T3 cells: In vitro MUC1 3T3 cells did not appear to be different to normal 3T3 cells as they had the same appearance and growth characteristics. By serological analysis, MUC1+3T3 cells expressed high concentrations of MUC1 and were H-2d+. Antibodies to MUC1 VNTR peptides reacted with MUC1 3T3 and MUC1lP815 similarly to the human reast cancer cell lines T47D and MCF7 (typing with anti-HMFG: BC2 antibody, anti-fusion protein: VA1 and VA2 antibodies, and anti-MUC1 peptide antibodies: BCP7, BCP8, BCP9 and However the murine tumour was differently glycosylated than the human tumour as MUC1+3T3 and MUC1'P815 cells were reactive with anti-carbohydrate (3E1.2) antibody S 15 (epitope: glycolylsialyl-Tn) but not with other antibodies to carbohydrate (CC5 -epitope: blood group Lea). This shows that the protein antigens are intact, but the glycosylation is altered. This is not surprising as mice and humans have different glycosyl transferases and therefore different patterns of glycosylation. However, after removal of sugars by pagal ::treatmenm, t ;e antibodies to MUC1 VNTR (non-APDTR reacting antibodies) which previously 20 had weak or no reaction with cell lines, became reactive as their epitope has now exposed.

There was no difference noted with the (AP)DTR(PA) reactive antibodies. There was a major difference in reactivity with the carbohydrate reactive antibody (3E1.2) where the staining became weak or negative after pagal treatment, indicating that the pagal was indeed removing O-linked sugars as the epitope of 3E1.2 is O-linked to the protein core of the mucin (18).

25 The typing was repeated at different times and the same results were obtained, which indicated that the phenotype was stable.

In vivo growth of MUC1 3T3 cells: BALB/c mice received a subcutaneous injection of 5 x 106 MUC1 3T3 or 3T3 cells and the subsequent growth measured; 3T3 cells grew progressively and were not rejected, as would B:\AUSTIN:PCT532828.mmb21 December 1994 24 be expected in BALB/c mice. By contrast the MUC1I3T3 cells grew progressively until day when they started to shrink and had gradually disappeared by day eighteen. Thus, the human MUC1' gene product appears to confer an immunogenicity on 3T3 cells, leading to their rejection. This was indeed the case as the subsequent challenge with 5 x 10 6 MUC1* 3T3 or 3T3 cells demonstrated the total resistance in immunized mice to the growth of MUC1I 3T3 cells, whereas 3'13 cells grew that is, the immunogenicity was found only in MUC1 bearing tumours and was specific for this antigen. Specificity and memory indicate an immune response to MUC1 1 and not some other effects such as MUC1 effecting the growth properties of 3T3. After several weeks of repeated experiments using tumours passaged in vivo, we noted that not all of the mice rejected their tumours and up to 30% of MUC1+ tumours continued to grow. When these tumours were excised and MUC1+ measured serologically, a proportion of cells in the tumours were MUC1', that is, some of the MUC1+ transfected cells had lost their capacity to express MUC1* in vivo (we did not 'determine whether the genes were still present). Such observations have been reported elsewhere with rat tumours presumably due to unstable expression of MUC1. In all our future studies we ensured that tumours were 100% MUC1 when used, by serologically testing the MUC1 expression with the anti-HMFG antibody BC2.

T-cell immune responses to MUC1*3T3 cells: S 20 Cellular immunity was most likely to be the mode of rejection as it is the cotmonest mode of rejecting tumour allografts in mice. This was confirmed by the ability of anti-CD3 antibodies to totally abrog'te immunity. To determine whether CD4 or CD8 cells were responsible for rejection, mice received multiple doses of anti-CD4 or anti-CD8 antibody as these were known to cause immunosuppression in other models (20, 21). Functional CD4 25 cell depletion by blocking had a transient effect on tumour growth, and tumours were rejected in a similar fashion to untreated mice. By contrast, anti-CD8 treatment led to prolonged tumour growth. We conclude that CD3+ cells are totally responsible for rejection, CD4 cells have a minimal effect and CD8+ cells are the major effectors of graft rejection. It was noted that in anti-CD8 treated mice, the tumours were smaller than those receiving anti-CD3 clearly the anti-CD8 antibody was not as effective as total T-cell removal with anti-CD3 BAAUSTIN:PCl32828.mmb2 Dcembcr 1994

I

antibody. CD4 cells having a minor effect was unlikely as the combined action of anti-CD4 and anti-CD8 was no better than anti-CD8 alone. However, we noted that anti-y-interferon (yIFN) treatment (of no effect used alone) combined with anti-CD8 gave a similar effect with anti-CD3; thus yIFN plays a role in tumour graft rejection, which is mediated by CD8 cells and yIFN.

Thus, MUC1+3T3 cells could immunize BALB/c mice against MUC1 carried on the 3T3 cells and gave rise to cellular immunity expressed by CD3*8* cells but not by CD3*4 cells; there was little humoral immunity as no anti-MUC1 antibodies were found. To measure the various parameters of the immune response, we examined delayed type hypersensitivity and cytotoxic T-lymphocytes.

Delayed type hypersensitivity: Clearly the immune response was cellular and due to CD8 1 cells. To determine whether this also involved a DTH response (usually considered to be mediated by CD4 1 cells) or a cytotoxic T-cell response (usually CD8 1 mice were immunized with MUC1 3T3 cells and a DTH was performed by injecting the hind footpads with various antigens. Preliminary studies demonstrated that in the absence of cyclophosphamide no measurable DTH responses occurred. A DTH response was detected in the footpads injected with killed (freeze/thawed five times) MUC13T3 cells and a weaker response when challenged with either HMFG, fusion protein-GST and Cpl3-32-KLH. These responses were clearly specific as 3T3 cells elicited no response. To determine whether the DTH response was mediated by CD4* or CD8* cells, mice were injected with anti-CD4 and anti-CD8 antibodies and the DTH response measured. Anti-CD4 totally blocked DTH reactions, anti-CD8 25 partially blocked DTH reactions, but to a lesser extent, when challenged with MUC1+3T3 cells, Cpl3-32, HMFG and fusion protein. Thus the cells which cause the effective immune response to human MUC1 (CD8) were not the same as those eliciting a DTH response, although CD8 cells certainly contributed to the DTH.

BIAAUSTIN:PCT532828.mmb21 December 1994

I

rrsEWPr~e~ I 'I r Cytotoxic T-lymphocytes: Cytotoxic assays were performed and after MUC1+3T3 cell immunization there was up to 60% lysis of MUC1+P815 targets treated with pagal. Untreated MUClP815 targets and non-transfected P815 targets were not lysed. Pagal treated and non-treated 3T3 and MUC13T3 targets also gave no lysis possibly as 3T3 cells are poor targets for Tc assays. To determine the phenotype of the Tc, anti-CD4 and anti-CD8 antibodies were used in blocking studies the anti- CD8 reagent (53-6.7) was known to be capable of blocking T-cell lysis by CD8 cells. This proved to be the case in these studies as anti-CD8 could block Tc, whereas anti-CD4 and a control antibody had little effect. Since only Tc were found to pagal treated MUC1IP815 targets, and since non-APDTR reactive anti-MUC1 antibodies (VA1, BCP7, BCP9 and BCP10) became reactive with pagal treated MUC1+3T3, MUCI P815, T47D and MCF7 cells, it is clear that both the antibody reactive and T-cell reactive epitopes are hidden, and both exposed after pagal *"'treatment.

S" Mice resistant to MUC1l3T3 cells have CD8 T-cell immunity, CD4 DTH, a detectable Tc response due to CD8 cells, and no antibody (see below). As the Tc response (at least at the level of the T-cell phenotype) correlated with the effector cell phenotype in rejecting tumours, it would appear to be the more appropriate response to measure.

Immune responses B cells: While it was shown above that cellular immunity was effective and little antibody was made, the role of antibody was not clear. Further, mice generally make poor antibodies and S 25 mobilize complement so poorly that they are not the species of choice on which to study antibody mediated destruction of grafts, unless certain conditions are met a) the provision of sufficient antibody (be it polyclonal or monoclonal); b) the provision of sufficient complement; c) high density of surface antigens. The MUC1 1 antigen density is high so additional antibody and complement were provided. In addition, the mice were immunosuppressed with CD3 to remove any component of cellular immunity In spite B:\AUSTIN:PCT'532828.mmb21 December 1994 1 IIII I of large amounts of antibody and complement (as described in materials and methods) (capable of rejecting skin allograft and xenografts), the tumours grew progressively indeed, at the same rate as in mice not receiving antibody. Thus, antibody and complement are unable to cause rejection of MUC1 3T3 cells.

Immunization with HMFG, peptides and fusion protein: The preceding defines a model of the murine immune response to human MUC1 transfected into 3T3 cells and forms the basis for using various immunogens to generate a similar or greater immune response with synthetic materials as that produced with cellular MUC1. The aim was clearly to substantially decrease tumour growth after immunization. As immunogens, natural mucin (HMFG), synthetic products MUC1 peptides made of VNTR dimers, and a x VNTR repeat fusion protein were used. It should be noted that without prior immunization, tumours are rejected after eighteen days such mice then being resistant to a subsequent challenge. Thus there is a "window" of approximately eighteen days when tumours will be rejected. So that, immunization could lead either to no tumours appearing or to reduced size during this time.

To examine the immunogenicity of HMFG, fusion protein and synthetic peptides groups of fifteen 15 BALB/c mice were immunized with 50p/g of these materials and challenged with 20 1 5 x 10 6 3T3 or MUC13T3 cells. The 3T3 cells had the same progressive growth in all immunized and non-immunized mice, so there were no non-specific effects of the immunization procedures. When mice were challenged with the lower dose of 1 x 10 6 cells, significant differences were noted as compared to the non-immunized control. Thus, on day six, mice immunized with either the peptide or fusion protein had tumours approximately 25 25% that of controls; immunizing with HMFG was less effective, tumours being approximately 60% the size of controls. However when challenged with 5 x 106 MUC1+3T3 cells, there was some difference in tumour size, compared to the controls, but not as obvious by challenging with a lower dose. As expected with subsequent tumour challenge, the peptide immunized mice which had rejected the tumour were now resistant to tumour challenge.

Thus, immunizing mice with peptides, fusion proteins or HMFG and challenging with a low B:\AUST1N:PCP3282.mmh 21 Decembcr 1994 II 'II 28 dose of MUC1+3T3 cells gave rise to some anti-tumour effect. Although the VNTR containing peptide, fusion protein and HMFG gave some degree of protection, mice immunized with the N- and C-terminal peptides of MUC1 had no significant protection indicating that these peptides do not induce immunity to MUC1, and also showing that the immunization procedure itself was without effect. To measure the various parameters of the immune response, we examined MUC1 antibody production, delayed type hypersensitivity and cytotoxic T-lymphocytes.

Antibody: Immunized mice with peptides, fusion protein or HMFG had high levels of anti-MUC1 antibody both before and after tumour injection. Thus, immunization gave rise to high levels of antibody, but apparently little cellular immunity as shown by a minor effect on the tumours. It was of interest that mice immunized with the control peptide (T4N1), and which had rejected the tumour did not produce antibodies against MUC1; nor did the mice immunized with peptide and other immunogens have S 15 an increase in antibody titre after rejecting the tumour.

DTH: Mice immunized with HMFG, Cpl3-32 and fusion protein-GST had DTH responses to the various MUC1 antigens and which could be inhibited by CD4 (totally) and CD8 (partially) antibodies. Thus, immunization with the three agents 20 gave rise to some degree of cellular immunity but not sufficient to greatly inhibit tumour growth.

Cytotoxic T-lymphocvte assay: Tc assays were performed from spleen and lymph node cells of immunized mice and no cytotoxic cells were detected. Thus the various 25 immunization procedures appeared to bias the immune response to antibody production, rather than cellular immunity.

Table 2 summarizes the differences in immunizing with cellular and synthetic antigens.

B:XAlSTIN:PCM32828.mmb21 Decmber 1994 TABLE 2: Differences in immunizing with cellular and synthetic antigens Immunogens Tumour rejection Antibody DTH Tc Tumour MUC1'3T3 Peptide Fusion protein HMFG (mucin) o r

D

high; low; absent EXAMPLE 3 Analysis of M-FP The MUC1 FP was bound to mannan using periodate as described in the materials and methods. The amino groups of the FP reacts with aldehyde residues of the oxidized mannan to form the labile Schiff base (Scheme Free mannan and FP was not separated from conjugated mannan. Elution profiles for 25 1-FP and 25 -M-FP obtained by gel filtration chromatography demonstrated that the mannan-fusion protein eluted as two peaks (201Kd and 73Kd). The FP eluted as two peaks: 38Kd and 20Kd (this lower peak may be GST due to cleavage of FP). Autoradiography analysis of 25 I-FP and 1 I-M-FP showed that most of the FP has been conjugated to mannan. Gel staining analysis of coommassie (protein), silver PAS) (carbohydrate) and silver (carbohydrate protein) showed that M-FP conjugate runs as a smear and that it is a heterogeneous molecule. It showed that the coupling efficiency of FP to mannan was almost 100% (not shown) The activity of FP after conjugation to mannan determined by an ELISA test showed that the FP had retained all its activity.

In vivo growth of MUC1+3T3 cells BALB/c mice which received a subcutaneous injection of 5 x 106 MUC1 3T3 cells grew progressively until day ten when they started to shrink and disappeared by day eighteen, whereas 3T3 cells were not rejected as expected by BALB/c mice as set out in Example 2 BIXAUSTN:PCnM32828.mmb2l December 1994 (Figure Thus, the human MUC1+ gene product confers an immunogenicity on 3T3 cells, leading to their rejection, and such mice were totally resistant to subsequent challenge.

Cellular immunity was the mode of rejection as anti-CD3 and anti-CD-8 antibodies totally abrogated immunity.

Immunization with Mannan-Fusion Protein To examine the immunogenicity of the mannan-FP, groups of ten BALB/c mice were immunized with 5/g mannan-FP (5jg corresponding to the amount of FP) and challenged with 10 6 5 x 10 7 MUC1+3T3 cells. There was no obvious tumour growth in immunized mice (Figure 2A) as compared to non immunized mice (Figure 2B). To show that the mannan-FP conjugate gave rise to specific anti-tumour immunity and that mannan or FP alone were without effect, mice were immunized with an equivalent dose of mannan (as in the conjugate 7mg/ml), a mixture of mannan and FP and a group of non immunized mice and were challenged with 106 MUC1+3T3 cells. Mice immunized with mannan-FP conjugate, no tumour growth was observed whereas mice immunized with mannan alone and a mixture of mannan and FP, tumours grew no different as compared to non immunized mice (Figures 3A, 3B and 3C). Thus, protection of tumour growth was specific for the conjugate and mannan and FP alone was without effect. Mannan-FP immunized mice were challenged with 10 6 3T3 cells and the 3T3 cells had the same progressive growth in immunized and nonimmunized mice (Figure indicating that there were no non-specific effects of the S: immunization procedures.

oo Immune response to M-FP Anti-CD3 antibody could totally abrogate immunity in mice immunized with M-FP (Figure 4) and mice which received anti-CD4 or anti-CD8 antibodies showed the following effect: CD4 immunosuppression had a minor effect on tumour growth (Figure by contrast anti- CD8 treatment led to prolonged tumour growth. Thus CD3+/CD8 cells are totally responsible for the immunity and tumour protection, CD4+ cells had a minimal effect (Figure Thus M-FP could immunize against MUC1 carried on the 3T3 cells giving rise to cellular immunity expressed by CD3+/CD8 cells but not by CD3+/CD4 cells. To measure B:\AUSTIN:PCT322828.mmb21 December 1994 the various parameters of the immune response, we examined delayed type hypersensitivity, cytotoxic T-lymphocytes T-cell proliferation and antibody production.

Delayed type hypersensitivity: DTH responses (usually considered to be mediated by CD4 cells) were performed by injecting the hind footpads with the various antigens (Figure A DTH response was detected in the footpads challenged with killed (freeze/thawed five times) MUC1+3T3 cells, HMFG, FP-GST, Cpl3-32-KLH, mannan-FP and a weaker response to GST (as GST is part of the FP) (Figure These responses were clearly specific as killed 3T3 cells, mannan alone, an irrelevant peptide (T4N1) and PBS could elicit no responses. To determine whether the DTH response was mediated by CD4* or CD8* cells, mice were injected with anti-CD4 and anti-CD8 antibodies and the DTH response measured. Anti-CD4 totally inhibited DTH reactions, anti-CD8 inhibited but to a lesser extent (Figure Thus the cells which caused the effective immune response to human MUC1 (CD8 cells as shown in Figure 4) were not the same as those eliciting a DTH response, although CD8S cells certainly contributed to the DTH.

Cytotoxic T-lymphocytes: Cytotoxic assays were performed and it was shown that after M-FP immunization there was 50% MUC1 specific lysis of MUC1+P815 targets (Figure 6) down to ratio of 12:1. Non-transfected P815 targets were not lysed (Figure It is likely that these cells were CD8+ (12).

T cell proliferation: Proliferation assays were performed and it was shown that after 25 M-FP immunization there were proliferative T-cells to M-FP, FP, Cpl3-32, HMFG, and to paga! treated and untreated MUC13T3, MUC1 P815 cells. Other stimulants had no effect. In another assay different peptides were added at a range of peptide doses (1lg-80[ig). A dose response was observed.

B;\AUSTIN:PCT532828.mmb21 December 1994 cpl3-32, Cp-24, p13-32, pl-24, Apl-15 and p5-20 were positive of the peptides p5-20, p14- 24 and p16-24, p5-20 was positve but p14-24 and p16-24 were negative. It seems that the epitope is not likely to be the antibody epitope APOTR, but is in p14-24 possibly GSTAP.

the epitope of the T cell is further being investigated.

The sequence numbering is as follows: 1 5 10 15 20 21 next repeat PDTRPAPGSTAPPAHGVTSA Antibodies to Mannan GST-MUC1 fusion protein conjugate: Mice were bled and their sera tested by ELISA for anti-FP antibodies. No anti-FP antibodies were detected compared to mice immunized with FP alone. Plates coated with mannan coupled to BSA were used to detect anti-mannan antibodies and no anti-mannan antibodies were 5 detected. Normal mouse serum was as a negative control.

Thus, mice made totally resistant to MUC1+3T3 cells by immunizing with FP coupled to mannan have CD8 T-cell immunity, CD4+/CD8 DTH, a detectable Tc response due to CD8+ cells, proliferative T-cells to the specific to the immunizing antigen, and little humoral immunity as no anti-MUC1 antibodies were found M-FP appears to be able to induce an 20 anti-tumour response, similar to that shown with tumour cell rejection (12).

M-FP in therapy To determine the effectiveness of M-FP as a therapeutic agent against established tumours, Sinjections of MFP were delayed until tumours were established. MUC1 P815 cells grow 25 progressively in (DBA/2 x BALB/c)F1 mice, when given subcutaneously, until day sixteen to twenty when they start to shrink and usually disappear by day twenty-eight (Figure 7A).

To examine the effectiveness of M-FP on established tumours, groups of five (DBA/2 x BALB/c)F1 mice were injected with 5 x 106 MUC1P815 cells; thirteen days later, the mice were injected with 5/xg M-FP (51/g corresponding to the amount of FP) once, or every other day. Tumours in control mice (injected with PBS) were rejected by day twenty-eight.

BAAUSTIN:PCT\532828,mmb21 December 1994 However, mice injected regularly began to reject their tumours immediately, the tumours rapidly disappearing by day twenty. A single injection also gave rise to more rapid rejection (Figure 7A).

Another model was examined using MUC1 P815 cells in DBA/2 mice, which grow until day twenty-two to thirty when they shrink and usually disappear by day forty two. MUC1+P815 cells were injected subcutaneously and on day fifteen DBA/2 mice were injected with MFP.

In the control group mice injected with PBS, tumours were rejected by day forty-two whereas mice immunized with MFP once on day fifteen tumours were rejected by day thirty-three (Figure 7B) compared with mice immunized every other day, tumours were rejected by day twenty-seven with a rapid reversal in their growth after a single injection. Thus, not only do pre-immunized mice have an anti-tumour effect, this can be obtained with established tumours.

EXAMPLE 4 A patient suffering from breast cancer was injected with 50g of the mannan fusion protein produced according to Example 1. No side-effects were seen following immunization. The therapeutic treatment of the patient's cancer is currently under evaluation and it has already been observed that several lesions in the bone have disappeared.

S EXAMPLE The VNTR's of mucins MUC1 through MUC7 can be coupled to mannan and other carbohydrates according to standard procedures such as described in Example 1. Table 3 sets S out a description of the various mucin core proteins.

B:AUSTIN:PCT1532828.mmb21 December 1994 34 TABLE 3: Description of human mucin core proteins, cDNA's and genes MUMi MUC2 MUC3 MUC4 MUCKC MIIC6 MUC7 Tissue Breast, ovary, Trachea pancreas 'Gl, 'GU GI and resp. GI tract Resp. tract Bronchus Stomach gall bladder Salivary gland and 'resp. tract ±Stomach Polymorphism Yes Yes Yes???? Chromosome 1q21 11 p5.5 7q 3 11piS 11015.415 4 Molecular weight of protein kI~a 120 -240 160 80-320 39 Base pairs 60 60 51 48 24 507 69 VNTRI'aalrepeat) 20 23 17 16 8 j169 23 Number of repeats 40-80 14 20 39 -20 >6 6 Gastrointestinal; 'GU Gastrourinary; cResp. Respiratory; MUCI VNTR SAPDTRPAPGSTAPPAHVT MUC2 VNTR PTTTPISTTTMVTPTPTPTGTQT MUC3 VNTR HSTPSFTSSITTTETTS MUC4 VNTR TSSASTGHATPLPVTD VNTR 87 base pair degenerate tandem repeats without peptide. repeats.

VNTR PTTSTTSA (494 base pair insert eight amino acid tandem repeat) MIJC6 VNTR 169aa repeat unit MUC7 VNTR TTAAPPTPPATTPAPPSSSAPPE d aa amino acids EXAMPLE 6 MHC restriction of CTL's after MFP immunization To determine whether the CTL's produced in mice were MHC or indeed, Class I MHC restricted, mice were immunized with MFP (5Ag weekly x 3) and their spleen cells removed and used as CTL's against various 5 'Cr labelled target cells. The results demonstrate that: a) Immunizing H-2d mice (DBA/2, NZB, BALB/c or B10.D2) gave CTL's against P815-Tm211 (MUC1 P815 cells but not against P815-(MUC1) cells.

b) When mice of other H-2 haplotypes were immunized, no CTL's were found on testing the H-2 d P815 MUC1+ cells (in particular H-2b; C57BL/6; 129 and BALB.B; H-2k: CBA; H-2 5 SJL and H-2: NZW. Of interest in these studies is the finding that: BALB/c (H-2 d was these are a congenic pair differing only in H-2 BALB.B (H-2b) was 0 *0 B10.D2 (H-2 d was C57BL/6 (H-2 b was these are a congenic pair differing only in H-2 This maps the reactivity to the H-2 MHC complex.

c) In other studies it was shown that mice of the H-2b haplotype had activity for H-2 b (E3 MUC1 tumour cells), but not for other H-2 haplotypes.

00 0 0 00 0 Thus, CTL responses in mice to MFP are H-2 (MHC) restricted.

BAAUSTIN:PCn532828.mmb2l Decumber 1994 EXAMPLE 7 T-cell proliferation to MFP in mice Mice were immunized with various MFP (5A/g/week x 3) and tested on a range of peptides at different doses in a proliferation assay. In this assay, different peptides are added in different amounts to splenic cells in tissue culture and after forty-eight hours 3 H-thymidine is added for twenty-four hours. The cells are harvested and the incorporated radioactivity measured. The studies show that: i) MFP stimulates the proliferation of T-cells from immunized mice in the presence of peptides.

ii) There is a dose response so that peptides C-p13-32, C-pl-24: 5mcM is the optimal dose p13-32, pl-24: 10mcM is the optimal dose 15 Apl-15 1.OmcM is the optimal dose p5-20 1.OmcM is the optimal dose The sequence numbering is such that: 1 5 10 15 20 21 next repeat 20 P D T R P A P G S T A P P AH G V T S A iii) Of the peptides used: p 5 2 0 is p14-24 are 25 p16-24 The epitope is not likely to be the antibody epitope APDTR, but is in p14-24 possibly

GSTAP.

BAAUSTIN:PCTL532828.mmb21 December 1994 EXAMPLE 8 MANNAN CONJUGATION WITH ANTIGEN PREFERENTIALLY INDUCES CTL RESPONSES Immunotherapy of cancer requires the definition of a suitable target and for solid tumors either the induction of a CD8 cytotoxic lymphocyte response (CTL) or possibly a DTH (delayed type h3 persensitivity) reaction. In breast cancer mucin, MUC1 peptides of the variable number of tandem repeat sequence (VNTR) are a suitable target. The major problem is to induce a cellular immune response. MUC1 peptides conjugated to a carrier (KLH) induce a humoral response and give poor protection in mice with little cellular immunity (that is, a TH 2 -type T-cell response). However when MUC1 peptides are conjugated to mannan under oxidizing conditions, a TH, response occurs with significant tumor protection, CD8 CTLs, a high CTLp frequency and little antibody. By contrast, mannan-peptide produced under reducing conditions gives a S TH 2 -type response. Thus the mode of conjugation (oxidation or reduction) can be used 15 to select the desired type of immune response (TH, or TH 2 A successful vaccine for cancer immunotherapy would require a suitable target antigen and the production of a cytotoxic T-cell response Cancer mucins, particularly MUC1, provide a suitable target in cancer as there is a 10-fold increase in mucin S 20 expression, a ubiquitous distribution to all of the cell surface, and altered glycosylation which reveals normally hidden peptide sequences (particularly an :ntibody epitope: the amino acids APDTR from the variable number of tandem repeat region (VNTR)).

These changes generate a new target for immunotherapy which is apparently absent in normal mucin The APDTR sequence is immunogenic in mice leading to antibody 25 formation whether the antigen is administered as MUC1 1 cancer cells, purified mucin (HMFG) or peptides Such studies of immunogenicity in mice would be of little relevance to humans, were it not for the findings that tumor specific CTLp exist in the lymph nodes of patients with cancers of either breast, ovary or pancreas These CTLp can be stimulated by antigen and IL-2 in vitro to become functional CD8 CTLs, BAAUSTIN:PM32828.mmb21 December 1994 the target antigen being either the APDTR sequence of MUC1 or an adjacent sequence in the VNTR The CTLs are unusual in that they have not been shown to be MHC Class I restricted, but can stimulate T-cells, apparently because the multiple repeated epitopes of the VNTR in tlhe MUC1 molecule can crosslink the T-cells receptor Thus, theoretically, pbi:?' could be immunized with MUC1 peptide sequences to convert their CTLp into functional CTLs which should have a therapeutic anti-cancer effect. In the present study we show in a murine MUC1 tumor model, that immunizations with synthetic MUC1 peptide conjugated to a carrier, keyhole limpet hemocyanin (KLH), while being highly immunogenic in terms of antibody production, have little tumor-protective effect. By contrast, a 20mer MUC1 VNTR peptide sequence (made as a GST fusion protein when coupled to oxidized mannan (MFPoxidized) gives protection against MUC1 mouse tumors and also leads to the rapid reversal of the growth of established MUC1 tumors; 'reduced' mannan coupled to FP (MFP-reduced) is the same as the peptide-KLH or peptide DT. Thess results translate 15 into stimulation of either TH, or TH 2 T-cells depending on the chemical state of the immunogen.

MUC1 (eithei MUC1-3T3 or MUC1-P815) mouse tumor cells were produced by transfection of the human MUC1 gene into 3T3 or P815 cell lines (34,35). MUC1-3T3 20 cells grow as tumors but are rejected by BALB/c mice in 20 days (due to the immune response to human MUC1); in contrast MUC1-P815 cells grow and are rejected more slowly in 40 days in DBA/2 mice. Thus there is a "window" between 0 to eleven days in which to observe accelerated rejection or absence of tumor growth in immunized BALB/c animals; this period is longer in DBA/2 mice. In syngeneic mice, MUC1 25 tumor rejection is accompanied by the generation of CD8 CTLs reactive with MUC1' target cells (36) (See footnote A) (Figure 8A), a high CTLp frequency (36) with a mean of 1/14,600 compared with 1/782,500 in non-immune BALB/c mice (Table little antibody formation (Figure 8B) and DTH mediated by CD4+ cells (Figure 8C) (36).

Furthermore such mice are resistant to re-challenge with large doses x 10 7 of B\AUSTIN:PC1532828.mmb21 December 1994 MUC1l cells and this is entirely due to conventional H-2 restricted CD8* cells (36); thus, the ideal type of anti-tumor immunity is to induce CD8' CTLs and not antibody, a DTH response mediated predominantly by CD4 cells is present but appears not to be relevant. Several different strategies were used to induce tumor immunity: mice were immunized with either MUC1 synthetic peptides (a 20 amino acid sequence of the VNTR which forms a dimer) coupled to KLH as a carrier (see footnote with a MUC1 containing fusion protein produced in the pGEX system consisting of five VNTRs (that is, 100 amino acids) or with natural mucin (HMFG). Three injections calculated to contain 50/g of the mucin component of the immunogen was injected, intraperitoneally at weekly intervals. Each of these procedures induced a similar immune response, and the data for fusion protein suffice to illustrate this. These procedures led to some degree of protection when the mice were challenged with 10 6 MUC1-3T3 cells when tumors had a delayed appearance and were smaller (Figure 9A), but not when the challenge dose was increased to 5 x 106 cells (Figure 9B). In each 15 case no CTLs could be detected (Figure 8A), but there was high antibody production (Figure 8B), that is, a typical TH2-type response. The tumor protection observed by challenging mice with 10 6 cells may have been mediated by the few CTL (note the Jow CTLp frequency of 1/87,400 (50xg) or 1/150,000 (Table which were too few to detect in the standard CTL assay which directly analyzed freshly isolated splenocytes.

To alter the immunogenicity of the MUC1 peptides aimed to induce a TH,-type response similar to that induced directly by tumor cells (that is, a predominant CD8' CTL response), various protocols were used, the most successful being the conjugation S 25 of the fusion protein to mannan, a carbohydrate carrier previously used to increase immune responses. In the past mannan was used to induce cellular immunity to murine candidiasis, to enhance the action of lipopolysaccharides on T-cells proliferation against protein antigens, and to induce anti-peptide antibody responses (37, 38, 39). The optimal immunogenic preparation (MFP) consisted of the MUC1 fusion protein (FP) B:\AUSTIN:PCI532828,mmb21 December 1994 with GST attached, coupled by Schiff bases to aldehyde groups on oxidized (periodate treated) mannan (see footnote It was found to be critical in the procedure that the Schiff bases and residual aldehyde groups were not reduced with borohydride. Mice were injected with MFP (5/g of the FP component three times intraperitoneally at weekly intervals) and analyzed for their immune status prior to tumor challenge: there were CD81 CTLs present (Figure 8A), a high CTLp frequency with a mean of 1/6,900 compared with 1/782,500 in non-immune BALB/c mice (Table no detectable antibody titre (Figure 8B), but significant DTH responses (Figure 8C). We believe this is the first description of tumor cell killing generated with synthetic MUC1 antigens.

When challenged with tumor cells, total protection occurred, even up to doses of 50 x 106 cells (Fig. 2A) (the largest dose which could be given) that is, at least fifty times the protection afforded by immunization with FP or free peptide. A number of controls for cell target and immunization specificity were used and no protection was observed with: a) mannan and FP were mixed (non-conjugated), b) mannan was conjugated to another irrelevant fusion protein or c) mannan was injected alone (Fig. 3) Clearly, the MUC1 FP must be conjugated to mannan to immunise against MUC1 1 tumors and the crucial part of the linkage procedure was to leave the MFP in the oxidized state to preserve the aldehyde groups and the Schiff bases. When non-oxidized MFP was used (that is, reduced with sodium borohydride) there neither was tumor protection nor were CTLs generated (Figure 8A and 8B, Table Indeed, reduced MFP stimulated immunity similar to FP or peptide not conjugated to mannan.

Immunizing mice prior to tumor cell challenge is not a realistic model for the treatment of patients who already have cancer (although, it could be feasible for future studies 25 with MFP as a "vaccine" to immunise patients in remission who do not have tumors).

Thus, mice with established MUC1-P815 tumors were treated once or three times with MFP (5A/g of FP antigen) given on days fifteen, seventeen and nineteen after tumor injection. After a single injection, rapid reversal of progressive tumor growth occurred within four days, and after several injections there was a rapid and early disappearance BtkUSTIN:PM32828mmb2l Dccmber 1994 41 of the tumors, indicating that MFP is highly immunogenic, even in the presence of a substantial tumor load.

Immunization with MUC1 peptides conjugated to mannan gives rise to CD8 1 tumor specific cytotoxic T-cells responses, similar to the response naturally produced to tumors and which is responsible for tumor rejection. Although not yet proven, we consider the mannan is likely to target antigens to mannose receptors on macrophages.

Indeed, it is the splenic sinusoidal cells in marginal sinuses which have the highest density of receptors for mannose Furthermore, the fusion protein when similarly linked under oxidizing conditions to dextran (a polysaccharide which lacks mannose residues) could not induce antitumor immunity (not shown). However, targeting to macrophages alone is insufficient: the MFP needs to be in oxidized form, probably due to the requirement for aldehyde groups which are considered to be important in the interaction of T-cell glycoproteins (for example, the T-cells receptor) and glycoproteins S 15 on the antigen presenting cells Thus, oxidized MFP is likely to do two things: a) the mannan specifically targets the antigen to macrophages, and b) in oxidized form the formation of Schiff bases augments interactions required for T-cells activation. We note that anti-peptide antibodies have been made by conjugating a peptide using cyanogen bromide (37, 38, 39); this would yield a product analogous to the reduced 20 MFP described above that is, findings in accord with our results with a TH 2 -type response.

In mice, oxidized MFP is clearly a superior immunization method for MUC1 synthetic peptides when CTLs are required. It is of interest that if MFP is reduced with sodium borohydride, then antibody responses preferentially occur, similar to using the peptide carrier immunization described herein and in Thus, at will, we can produce either CTL responses by oxidation or antibody responses by reduction of the same product. Although individual cytokines were not measured, it appears that oxidized mannan-antigen immunization preferentially stimulates a CD8 1

TH

1 -type response, BAtUSTN:PCM32828.mmbb2 December 1994 42 whereas the reduced form stimulates the production of antibodies, and alters to a TH z type response. Whether this can be a standard approach applied to other peptides is currently under examination and whether cancer patients can be induced to make an effective immune response to MUC1 conjugated to mannan and whether this is immunogenic enough to generate sufficient functional CTLs to cause cancer regression in cancer patients will require appropriate clinical trials.

Footnote A Cytotoxic T lymphocyte precursor cell (CTLp) frequency determinations For each cell suspension in which CTLp frequencies were determined, a minimum of thirty two replicates for each of at least six responder cell doses were cultured in U-bottomed microtiter trays, with 5 x 105 BALB/c spleen stimulator cells in modified Eagle's medium supplemented with 10% fetal calf serum, with 5iM synthetic MUC1 peptide and 10x/ml rIL-2. Seven days later, each microculture was assayed for cytotoxicity by 15 replacing 100pl of culture medium with 100ld target cell suspension containing 10 4 s Cr-labelled MUC1-P815 tumor target cells. Wells were regarded as containing cytotoxic activity if they yielded specific 51 Cr release three standard deviation above the mean isotope release from 10 4 cells cultured alone or with stimulators only, peptide only or rIL-2 only. A linear relationship existed between the dose of responder cells on 20 a linear scale and the frequency of negative wells on a logarithmic scale. CTLp frequencies were determined as the inverse of the inverse of responder cell dose required to generate 37% negative wells Footnote B Peptides were synthesized on an Applied Biosystems synthesizer. The sequence of the synthetic MUC1 VNTR was C-PDTRPAPGSTAPPAHGVTSA; the C added to N-terminal aided in conjugation and enabled homodimers to form. This peptide was conjugated to Keyhole limpet hemocyanin using glutaraldehyde The fusion protein B:\AUSTIN:PCTl532828.mmb2l Decenbr 1994 9

C

C..

43 consisted of five VNTRs (that is, 100 amino acids) produced in the pGEX expression system and cleaved with GST attached Footnote C Mannan (Sigma), 14mg/ml in 0.1M phosphate buffer pH6.0, was oxidized with sodium periodate (0.01M) for 30-60 minutes at 4 0 C. Ethandiol (10/l) was added and incubated for a further thirty minutes at 4 0 C and passed through a Sephadex-G25 column equilibrated in bicarbonate buffer pH6.0 to pH9.0. The oxidized mannan that eluted in the void volume (2mls) was mixed with 900zg of MUC1 fusion protein and incubated overnight at room temperature and used without further purification. To form reduced MFP the mixture was treated with 1mg/ml sodium borohydride for three hours. The purity of MFP was analyzed by SDS-PAGE and gel permeation chromatography TABLE 4: CTLp frequency in mice immunized with different immunogens: CTLp Frequency* Immunogens Mouse #1 Mouse #2 Mouse #3 Mean Mouse #1 Mouse #2 Mouse #3 Mean Non-immunized BALB/c mice 1/850,000 1,725,000 1/782,500 Mice immunized with: Tumour cells 1/12,500 117,500 1/14,600 MFP-oxidized 118,800"* 116,200 116,300 116,900 MFP-reduced 1/84,000 1/92,000 1187,800 FP (50pg) 1/85,000 1190,000 1/87,400 FP (5pg) 1,150,000 Cp13-32-KLH 1/150,000 1,150,000 1/150,000 HMFG 1/340,000 1/440,000 1/383,700

C

C.

Analysis of individual BALB/c female mice.

BAAUSTIN:PCT32828mmb2L Decmber 1994 CTLp frequency of two pooled BALB/c female mice.

EXAMPLE CTL IN MICE IMMUNIZED AGAINST HUMAN MUC1 ARE MHC RESTRICTED CD8 1 cytotoxic T-cells have been induced to endogenous peptides associated with MHC Class I molecules, the interacting complex also requiring reaction of T-cells receptor ac, 13 reacting with the antigenic peptide in the groove of the MHC. Such CTL's have been produced to endogenous peptides or exogenous infectious agents particularly viral peptides, all of which cycle through well described intracellular pathways and while it has been difficult to induce CTL's to exogenous peptides too, it is certainly well Cdescribed, in viral infections such as influenza, EBV and the like. It is the aim of many cancer immunotherapy studies to induce such CTL's reacting against tumour peptides.

In melanoma in humans such CTL's have also been MHC restricted associating 15 particularly with HLA-A1 and HLA-A2. However, recently a new type of CTL was described by O. Finn and colleagues, the description giving great impetus to immunizing humans against solid tumours of breast, pancreas and vwary. What was described was the presence, in the draining lymph nodes of women with breast cancer, precursors to CTL's which could in vitro be stimulated in the presence of IL-2 and 20 different breast cancer cell lines to develop CTL's to breast cancer cell lines. The cells were CD3+ CD8 but reacted in a non-MHC restricted manner to lyse target cells.

The findings were not unique to breast cancer and have subsequently been found in draining lymph nodes of patients with carcinomas of either the pancreas, ovary and more recently in the circulation of patients with multiple myeloma. The CTL's react with MUC1 molecules, indeed with the repeating peptide unit (found as a variable number of tandem repeats, but in the protein core) as antibodies to the highly immunogenic APDTR region of the peptide core block the reaction of the CTL's on MUC1* target cells. Furthermore, the specificity of the CTL's was confirmed by reactivity with MUC1+ target cells produced by transfection and non-reactivity on non- B:\AUSTIN:PCT532828.mmb21 December 1994 MUC1 cells. Although the presence of non-MHC restricted CTL's defied convention, their mode of action is likely to be by cross-linking of the T-cell receptor by the repeating units on MUC1 rather than requiring a CD8/Class I interaction, and compelling evidence for such cross-linking has recently been provided. With the observations that MUC1 is immunogenic in mice for the induction of antibodies and appears to be immunogenic in humans to induce CTL's, we have embarked upon a program with the preclinical immunization of mice with soluble recombinant MUC1 molecules and a clinical trial has commenced with such materials. MUC1 murine tumours (produced by transfection) are readily rejected by mice with the induction of memory T-cells (resistant to a challenge), little antibody and CTL response; Immunization with MUC1 peptides, however, produces a predominate antibody response without CTL's. By conjugation of the fusion protein with mannan a significant CTL response can be induced in mice and we now report the CTL response is of the conventional type in that it is mediated by CD3 CD8 cells which are MHC restricted indeed, MUC1 molecules appear to be able to associate with at least seven or eight different types of Class I molecules, but the association is necessary for CTL's to function.

Immunizing mice with human MUC1 (MFP) leads to MHC restricted CTL's. These were found in mice with haplotypes b, d, k, z and s and were clearly MHC restricted as shown in congenic strains. Further testing in recombinant strains demonstrated MUC1 peptides are able to associate with D or K Class I molecules of the b, d or k haplotype. Clearly, anti-MUC1 CTL's induced by immunization are different from those which arise spontaneously in patients with tumours and should be borne in mind in clinical studies with a view to immunizing patients against their tumours.

A summary of the H-2 restriction studies is shown in Table 5 and results are shown in Figures 10 to B:\AUSTIN:PCl532828.mmb22 December 1994 TABLE 5: Summary of the 11-2 restriction studies Immunised H-2 Target cell Target cell mouse f(MUCi1 cell line or lysis strain peptide pulsed blast) a) INBRED LINES bC57BLIS C5713116, 129J b (spleen or E3) b d BALB/c, Bi O.D2,NZB BALB/c, DBA/2, d d BALBIC, B10.02, 11.132 NZB NZB B C57131.16_______ CBA k k CBA b a above d as above SJL s s SJL z NZW d as above NZW z z NZW s SJL d B1)JD2 b) CONGENIC b b C57131.6 C57BL16 d B1O.D2 BALB/c d d BALB/c b B10.02 d d BALB/c d b C5713116 c) RE- K kDb K kD1310.02 COMBINANTS KkD' C57BL16 b1O.a 92r0 KkD' 131.132 K kD' B10.02 B1D.A (5R) K k Dd KkDb 5R II K CBA ftKkDh C57131_1 BALB/c 0 0* 0 *0 00.0 0* 0 00 0 0 0 00 0 0 0 B:\AUST1N:PCT'532828.mmb22 Dccrnbcr 1994 Immunised H--2 Target cell Target cell mouse IMUC I+ cell line or lysis strain peptide pulsed blast) MUTANTS Kbmflb KkD(bml) bin-1 (class I K kDb mutant) K k lb KkDd (bm. 12) K kob bm-12(lass 11 KbmDb K k D'bl) mutant) K k~b Kkld K kDbhM. 1 2) K kDb od ,62M-1- KkDb Kh b (82m.Il K kob K kod KkDh KbD(#m-i) fld

Z

o S

S

OS

EXAMPLE 11 10 THE IMMUNOGLOBULIN ISOTOPE OF ANTIBODIES The immunoglobulin isotope of Antibodies produced by the various immunogens was determined by an ELISA.

Mice immunized with: FP had IgG1, IgG2a, IgG2b, IgM antibodies 15 Predominantly IgGi Cp13-32 had IgGi and 1gM antibodies Predominantly IgGi HMFG had 1gM antibodies It should be noted that in the tumour protection studies the protection was: BA:AUS1N;PCT32828.nimb2l Dccember 1994 48 FP Cpl3-32 HMFG and this correlated with their CTLp frequencies. It seems that IgM gives the least protection and then IgG1. Where all isotopes (including IgG2a) were produced the protection and CTLp frequency was better (as seen be immunizing with FP).

Mice immunized with: Tumour cells no antibodies were produced MFP-oxidized had very little antibody produced and this was of the IgG2a isotope The above two immunogens gave the best protection, high CTLp frequencies and cellular immunity (presumably of the TH, type). Immunizing with FP, Cpl3-32 and HMFG gives humoral immunity (antibodies and no cytotoxic T-cells) presumably of the TH, type.

Mice immunized with: MFP-reduced IgG1 antibodies were produced.

This appears to be of TH2 type response (no CTL were found) (Example 9).

Thus depending on the mode of MFP (either reduced of oxidized) we are able to produce cellular or humoral immunity.

Figure showing Antibody production in mice immunized with the different immunogens 2F**2 is Figure t*o* B:\AUSTIN:PCT\532828.mmb21 Dccembcr 1994 49

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oo* BAAUSTIN;PCM3282S.mmb2I Dcvmber 1994

Claims (24)

1. A compound comprising a conjugate between an antigen, and a carbohydrate polymer comprising oxidized mannose.

2. A compound according to claim 1, wherein said antigen is selected from pollen, hepatitis C virus (HIV) core, El, E2 and NS2 proteins, Plasmodium falciparum circumsporozoite protein, HIV-gpl20/160 envelope glycoprotein, streptococcus surface protein Ag, influenza nucleoprotein, haemagglutinin-neuraminidase surface infection, TcpA pilin subunit, VP1 protein, LMCV nucleoprotein, Leishmania major surface glycoprotein (gp63), Bordetella pertussis surface protein, rabies virus G protein, Streptococcus M protein, Syncyticial virus (RSV) F or G proteins, Epstein Barr virus (EBV) gp340 or nucleoantigen 3A, haemagglutinin, Borrelia burgdorferi outer surface protein (Osp) A, Mycobacterium tuberculosis 38kDa lipoprotein or Ag85, Neisseria meningitidis class 1 outer protein, Varicella zoster virus IE62 and gpl, Rubella virus capsid protein, Hepatitis B virus pre S1 ag, Herpes simplex virus type I glycoprotein G or gp D or CP27, Murray valley encephalitis virus E glycoprotein, Hepatitis A virus VP1, polio virus capsid protein VP1, VP2 and VP3, chlamydia 20 trachomatis surface protein, Hepatitis B virus envelope Ag pre S2, Human rhinovirus (HRV) capsid, papillomavirus peptides from oncogene E6 and E7, Listeria surface protein, Varicella virus envelope protein, Vaccinia virus envelope protein, Brucella surface protein, a combination of one or more of said antigens, an amino acid subunit of said antigens comprising five or more amino acids in length or combinations of one or more of said subunits. S

3. A compound according to claim 1, wherein said antigen is the human mucin polypeptide, one or more repeated subunits thereof, or a fragment of said repeated subunits. 56

4. A compound according to claim 3 comprising two to eighty copies of the repeated subunits of said human mucin.

A compound according to either claim 2 or claim 3, wherein said one or more repeated subunits of said antigen comprise part of a fusion polypeptide.

6. An immunogenic vaccine against disease states which comprises a compound comprising a conjugate between an antigen and a carbohydrate polymer comprising oxidized mannose in association with a pharmaceutically acceptable carrier.

7. An irmnunogenic vaccine as claimed in claim 6, wherein said antigen is an antigen as defined in claim 2.

8. An immunogenic vaccine as claimed in claim 6, which is directed against tumour cells expressing human mucin or a subunit thereof, and wherein said anti'gen comprises the human mucin polypeptide, one or more repeated subunits thereof, or a fragment of said repeated subunits.

9. An immunogenic vaccine according to claim 8, wherein said conjugate comprises two to eighty copies of the repeated subunits of said human mucin.

10. An immunogenic vaccine according to either claim 8 or claim 9, wherein said one or more subunits repeated subunits of said antigen comprise part of a fusion polypeptide.

11. A method for inducing a cell-mediated immune response against an antigen, which comprises administering to an animal a compound comprising a conjugate between said antigen, and a carbohydrate polymer comprising oxidized mannose, optionally in association with a pharmaceutically acceptable carrier. L" 57

12. The method of claim 11, wherein said antigen is an antigen as defined in claim 2.

13. The method of claim 11, wherein said cell-mediated immune response is induced against cells expressing human mucin, or subunits thereof, and wherein said human antigen comprises the human mucin polypeptide.

14. The method according to claim 13, wherein said conjugate comprises from two to eighty copies of the repeated subunit of said human mucin.

A method according to either claims 12 or 13, wherein said one or more repeated subunits of antigen comprise part of a fusion polypeptide.

16. A compound according to any one of claims 1 to 5, wherein said carbohydrate polymer is mannan.

17. An immunogenic vaccine according to any one of claims 6 to 10, wherein said carbohydrate polymer is mannan. 20

18. A method according to any one of claims 11 to 15, wherein said carbohydrate polymer is mannan.

19. A compound according to either claim 3 or claim 4, wherein said human mucin is MUC1.

20. An immunogenic vaccine according to either claim 8 or claim 9, wherein said human mucin is MUC1.

21. A method according to either claim 13 or claim 14, wherein said human mucin is MUCI. I 58

22. A compound according to claim 1, wherein said antigen comprises whole cells or sub-cellular fractions thereof.

23. An immunogenic vaccine according to claim 6, wherein said antigen comprises whole cells or sub-cellular fractions thereof.

24. The method of claim 11, wherein said antigen comprises whole cells or sub- cellular fractions thereof. DATED this 26th day of September, 1997 ILEXUS PTY LTD by Their Patent Attorneys DAVIES COLLISON CAVE e* eS See tt M ABSTRACT Conjugates between one or more repeated subunits of an antigen and a carbohydrate polymer are desired. Also described are immunogenic vaccines against disease states which contain the conjugates and methods for inducing cell-mediated immune responses. The conjugates may especially contain polymers of the carbohydrate mannose and one or more repeated subunits of human mucin. 0* o C C BAAUSrN:PCT3928.mmb2I flember 1994