WO1997014795A1

WO1997014795A1 - Tumor associated polypeptides with carboxyl-terminal alanine-proline, proline-proline or glycine-proline and antibodies thereto

Info

Publication number: WO1997014795A1
Application number: PCT/US1995/013331
Authority: WO
Inventors: Reiko F. Irie; Yoshikazu Kikumoto
Original assignee: John Wayne Cancer Institute
Priority date: 1995-10-19
Filing date: 1995-10-19
Publication date: 1997-04-24
Also published as: AU4131396A

Abstract

An IgM human monoclonal antibody (HuMAb), JWCI L94, that reacts to a human melanoma cell line was developed using B cells obtained from a melanoma patient. Screening of expression libraries from 2 melanoma cell lines revealed a number of immunoreactive clones that all shared the carboxyl (C) terminal sequence alanine proline (Ala-Pro). HuMAb L94 reacted not only to C-terminal Ala-Pro-containing fusion proteins but also to the synthetic dipeptide Ala-Pro. Also disclosed is the cross-reactivity of HuMAb L94 with peptides containing a C-terminal Gly-Pro or Pro-Pro. The epitope of human monoclonal antibody L94, reacting with human tumors was defined as the peptides with the C-terminal sequence alanine-proline (Ala-Pro). A synthetic peptide with such a sequence, RVAALARDAP, SEQ ID NO:13, was evaluated for its immunogenicity and shown to significantly increase cytotoxicity of melanoma patients' PBMC after in vivo immunization with melanoma cell vaccines (MCV). In vitro restimulation of a vaccinated patient's PBMC with the peptide also enhanced cytotoxicity against autologous peptide-pulsed BCL. This cytotoxicity was specific to the C-terminal sequence Ala-Pro.

Description

DESCRIPTION

TUMOR ASSOCIATED POLYPEPTIDES WITH CARBOXYL-TERMINAL ALANINE-PROLINE, PROLINE-PROLINE OR GLYCINE-PROLINE

AND ANTIBODIES THERETO

BACKGROUND OF THE INVENTION

The government owns rights in the present invention pursuant to grant numbers CA 12582 and CA 30647 from the National Cancer Institute.

Field of the Invention

The present invention relates generally to the field of immunotherapy based on the production of human monoclonal antibodies and the identification and isolation of human tumor antigens recognized by the antibodies as well as cytotoxic-T-lymphocytes . The invention also relates to characteristics of tumor associated antigens and their uses for therapy, diagnosis and prognosis of human cancer.

Description of the Related Art

Elevated titers of autoantibodies are known to be present in hosts with various autoimmune diseases, neuropathological diseases and cancer. These autoantibodies have been used to screen cDNA clones from expression libraries to encode pathogenic antigens of such diseases (Tan, 1991; Amagai et al . , 1991; Dropcho et al . , 1987; Szabo et al . , 1991; Hayashibe et al . , 1991) . The difficulties of using this approach to identify human tumor associated antigens (TAA) include the unavailability of high titer human anti-TAA antibodies and the interference of serum antibodies unrelated to TAA (Hayashibe et al . , 1991) . These problems, which are inherent in polyclonal serum antibodies, could be avoided if HuMAbs to tumor antigens of known tumor specificity were available.

Many human tumor-associated antigens have been identified using murine monoclonal antibodies. The clinical utility of these antigens has been evaluated extensively in the diagnosis and therapy of human cancer. For example, these antigens, especially peptide antigens have been tested for the induction of cellular immune responses first in mouse models and then in man (Samonigg et al . , 1993; Maclean et al . , 1992; Estin et al . 1988) . Unfortunately, high immunogenicity of these antigens has not been observed in human clinical trials. In some cases, these antigens e.g., a carcinoembryonic antigen (Bhattacharya-Chatterhee et al . , 1991) , p97 melanoma antigen (Brown et al . , 1981) or high molecular weight melanoma-associated antigen (Mittelman et al . , 1992) , have been shown to be present at high levels in human tumors, and yet immunization with them has resulted in only weak or no immune response in human patients. One possible explanation is that these antigens may be weakly immunogenic and/or partially or completely tolerated in the human immune system.

Conversely, tumor antigens identified by human antibodies are capable of inducing immune responses in man, at least humoral responses (Tai et al . 1985; Livingston et al . 1987) . For example, gangliosides GM2 and GD2 were initially identified by human monoclonal antibodies (Irie et al . , 1982; Tai et al . , 1983; Cahan et al . , 1982) and later confirmed to be able to induce a strong immunity in man when administered in a purified form (Livingston et al . , 1987; 1989) or as a tumor cell vaccine (Tai et al . , 1985) . Attempts to identify CTL epitopes on human melanomas have also been made (Traversari et al . 1992; van der Bruggen et al . 1991) . Using human autologous melanoma-specific CTL clones, Traversari determined the target antigens of CTL and isolated the gene MAGE- 1 encoding the antigen and finally identified the CTL epitope as a MAGE- 1-encoded nonapeptide. Humoral immune responses against the antigen were formed after administration of a MAGE antigen positive melanoma cell vaccine ( CV) in patients with melanoma (Hoon et al . , submitted) .

The strategy used by the present inventors to identify immunogenic tumor antigens has been to use human monoclonal antibodies as detection probes. A number of human B-lymphoblastoid cell lines that produce HuMAbs to melanoma associated antigens (Irie et al . , 1982) have been established in the inventors' laboratory. Chemical analysis of antigen epitopes for three such HuMAbs has identified the antigens as glycolipid antigens: ganglioside GD2, GM2 , and GM3, respectively (Cahan et al . , 1982; Tai et al . , 1983; Yamamoto et al . , 1990) . These antibodies have been shown to have therapeutic potential (Irie et al . , 1986; Irie et al . , 1989) . Epitopes recognized by these HuMAbs consist of only 2-3 terminal sugar residues of these glycolipids. Unfortunately, carbohydrate epitopes cannot be "cloned" and produced by standard molecular biology techniques as peptide epitopes can, and therefore gangliosides are not purified or synthesized at a cost that is practical for clinical applications. There exists, therefore an immediate need for tumor associated peptide antigens that are capable of eliciting a strong immune response in humans, in order to define the mechanism of anti-tumor action, especially the nature of the targeted antigens or epitopes, and in order to develop new and more effective cancer immunotherapy agents. SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing an antibody and preferably a human monoclonal antibody that recognizes a tumor associated antigen and that is immunoreactive with a carboxy terminal amino acid sequence alanine-proline, glycine-proline or proline- proline. A part of the present discovery is also a purified polypeptide segment that is capable of eliciting a strong immune response in humans and has immunoreactivity with Hu.-ϊAb L94 as well as cytotoxic-T- lymphocytes .

The monoclonal antibody, HuMAb L94 was discovered to be immunoreactive to human melanoma cells. In the process of discovering the antigenic determinant for this antibody, the inventors found that a series of peptides were recognized by this protein and that all the recognized peptides ended in a C terminal alanine- proline. Subsequently, it was shown that glycine-proline and proline-proline were also recognized by the antibody. It was also discovered that the addition of alanine- proline at the C-terminus of a known protein sequence such as that of _/β-galactosidase was sufficient for antibody recognition, even though native _/S-galactosάdase was not recognized by the antibody. It was further discovered that removal of the C-terminal AP sequence destroyed antibody recognition of the protein. Therefore, it is contemplated that any macromolecule that is attached to the dipeptide would be immunoreactive with the antibody of the present invention. Such macromolecules would include, but would not be limited to proteins, peptides, glycoproteins, carbohydrates and the like. The "C terminus" or "carboxy terminus" are used herein interchangeably and are defined herein as they are normally used in the art. An amino acid structure contains a carbon atom known as the a carbon to which is bonded an amine group, a carboxylic acid group and a side chain. In the peptide bond polymerization, the α-amine group from one amino acid binds to the cx-carboxylic acid group of the adjacent amino acid leaving one α-amine group free at one end of the polymer and one α-carboxylic group free at the other end of the polymer. The C terminus or carboxy terminus is defined as the end of the polymer with the free α-carboxylic acid group.

One aspect of the invention is a monoclonal antibody immunoreactive with an antigen having the C-terminal amino acid sequence, alanine-proline, glycine-proline or proline-proline and wherein the antibody recognizes the C-terminal sequence. Preferably the antibody is a human monoclonal antibody and more preferably HuMAb L94. The antigen may be defined in certain embodiments as a tumor associated antigen or even a melanoma associated antigen. The invention may also be described as a purified antibody prepared against a polypeptide having an amino acid sequence comprising alanine-proline, glycine-proline or proline-proline in the C-terminal position wherein the C terminus is recognized by the antibody. In certain preferred embodiments, the purified antibody may be a monoclonal antibody. An antibody prepared against an antigen or a polypeptide indicates that antibody recognition is induced by the presence of the antigen or polypeptide in the serum of an animal by standard techniques well known in the art .

Another aspect of the invention is a purified peptide comprising an amino acid sequence having as its C terminal sequence the sequence consisting of SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15 or SEQ ID NO: 16. Although the dipeptide alanine-proline has been shown to immunoreact with HuMAb L94, the polypeptide segments of the present invention may be four amino acids in length, or up to six, up to eight, up to ten, up to twenty, up to thirty, or even forty five amino acids in length, or they may even be up to one hundred or up to one hundred fifty amino acids in length providing the two C-terminal amino acids are alanine-proline, glycine-proline or proline- proline.

As used herein a tumor associated antigen is a protein or even a glycoprotein that is produced by tumor cells and that may be present on the surface of certain melanoma cells to be available to immunoreact with an antibody or even with a cytotoxic lymphocyte and may in some cases be useful to distinguish between a tumor cell and a benign cell . By a fragment of a tumor associated antigen is meant any part of the antigen that retains the antigenically recognized C-terminal AP, GP or PP either as produced by normal mRNA translation or as a result of post translational processing such as proteolytic cleavage, chemical cleavage or by any other means including but not limited to genetic engineering techniques.

The term "purified polypeptide" as used herein, is intended to refer to a polypeptide composition, isolatable from other tumor cell associated proteins, wherein the polypeptide is purified to any degree relative to its naturally-obtainable state, i.e., in this case, relative to its purity within a tumor cell extract. A purified polypeptide therefore also refers to a polypeptide, free from the environment in which it may naturally occur in intact cells. In certain embodiments, "purified" will refer to a polypeptide composition which has been subjected to fractionation to remove various non- protein components such as other cell components. Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques .

In certain other embodiments, the purified polypeptide will be chemically synthesized by solid phase synthesis and purified away from the other products of the chemical reactions by HPLC for example. Alternatively, the polypeptide may be produced by the overexpression of a DNA sequence included in a vector in a recombinant cell . In this method of producing the polypeptide, purification would be accomplished by any appropriate technique mentioned in the preceding paragraph.

It is also understood that a peptide of the present invention may also be linked at its N-terminus either directly or by a peptide linker, generally by genetic techniques well known to those of skill in the art, to a carrier protein and that such a protein-protein or protein-peptide fusion which exhibits immunoreactivity with HuMAb L94 and which maintains a C-terminal sequence of alanine-proline (AP) , glycine-proline (GP) or proline- proline (PP) is also encompassed by the present invention. For example, a polypeptide that contains any of the amino acid sequences designated herein as SEQ ID NOS :6 , 8, 9, 11, 13, 14, 15 or 16 and further including the amino acid sequences of -galactosidase or glutathione-S- transferase, for example would also be an embodiment of the present invention. It is understood that these carrier protein sequences are mentioned by way of example only and that other known carrier protein sequences such as keyhole limpet hemocyanin (KLH) , bovine or human serum albumin (BSA) , other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin or any other suitable protein sequence may also be included as embodiments of the present invention. It is understood that the term "peptide" denotes an amino acid polymer segment that is less than a full protein sequence and can be from as small as several amino acids in length up to one hundred or even one hundred and fifty amino acids in length. The peptide may be synthesized chemically, or it may be produced from a genetic sequence in a cell or cell-free translation system, or it may be produced by proteolytic cleavage of a protein or by any other means known in the art .

One such technique would be the use of an expression vector encoding a peptide linker ending in AP, GP or PP followed by a stop codon such that a gene sequence or even a partial gene sequence could be inserted into the expression region upstream of said peptide linker in such a way that the expressed polypeptide would end in a C terminal AP. All such proteins or peptides ending in a C terminal AP, GP or PP would be encompassed by the present invention. For example, such a vector would typically encode a restriction enzyme site, or multiple restriction enzyme recognition sequences followed by a coding region that may encode two or more amino acid residues proving that the last two are AP, GP or PP followed by a stop codon. For example, the last two codons prior to a stop codon (UAA, UAG or UGA) would be any of GCU, GCC, GCA, GCG, GGU, GGC, GGA, GGG, CCU, CCC, CCA, or CCG, followed by any of CCU, CCC, CCA, or CCG. Such a vector may also comprise a polyadenylation site, a leader sequence or any other sequence known to those of skill in the art that would enable the expression of the peptide or protein in a cell or on a cell surface, and particularly on the surface of a human cell.

Certain aspects of the present invention are isolated nucleic acid segments encoding polypeptides having immunoreactivity with HuMAb L94 , or that are recognized by cytotoxic T lymphocytes. Isolated nucleic acid segments are defined herein to mean nucleic acid segments isolated away from the total nucleic acids of the organism or cell in which they naturally occur. Such segments may be genomic DNA or they may be cDNA, i.e. they would not contain introns, or they may even be RNA sequences that may be transcribed from DNA. In addition, complementary sequences or sequences that hybridize to the claimed nucleic acid sequences under high stringency conditions would also fall within the scope of the claimed invention. Certain of the nucleic acid sequences will encode polypeptides having amino acid sequences in accordance with amino acid sequences disclosed herein and designated as SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 9- or SEQ ID NO: 11 or SEQ ID NO:13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO:16 or their functional equivalents, or alternatively, the nucleic acid sequences may be defined as comprising a sequence wherein a contiguous sequence region consists of the sequence designated herein as SEQ ID NO:5 or its complement.

In certain embodiments, the present invention may be defined as a purified tumor associated antigen having a C-terminal dipeptide sequence of alanine-proline, glycine-proline or proline-proline and encoded by a nucleic acid sequence hybridizable to SEQ ID NO:5 or its complement under high stringency conditions. A tumor associated antigen is defined as an antigen expressed by tumor cells that is not expressed or is expressed at detectably lower levels in a normal cell .

High stringency conditions for forming hybrids would be relatively low salt and\or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C to 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand. It is generally appreciated that conditions can also be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature.

Functional equivalents of the disclosed peptides will be those that contain small changes in the amino acid sequence, such as one, two or even three amino acid changes such that the polypeptide retains its immunoreactive activity. In particular, in light of the present disclosure, any amino acids other than the two C- terminal residues may be changed without affecting the activity and all such polypeptides would fall within the scope of the present claimed invention. It is understood that these nucleic acid sequences may comprise the coding region of an entire protein including its transcription/translation control regions or even the fusion of two proteins, and further that the sequences may comprise a DNA vector capable of replication in a cell, or even an expression vector. Such a vector may be a viral vector or a plasmid vector and would comprise the sequences needed for replication and expression in a cell, such as the origins of replication, promoters/enhancers, ribosomal binding sites, translation initiation regions, polyadenylation sites, and any other necessary sequences.

A further embodiment of the present invention is a recombinant cell expressing a peptide having immunoreactivity with HuMAb L94 or a CTL that recognizes the same antigenic determinant, and preferably the recombinant cell is an E. coli cell. As used herein, the term "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a polypeptide immunoreactive with HuMAb L94 has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Recombinant cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene, a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene. The said cell line may preferably be a bacterial cell line, such as an E. coli cell line which contains within the cells a plasmid or viral vector comprising a DNA segment encoding the protein, peptide or fusion of the present invention and the control elements necessary for the replication of the vector and for expression of the polypeptide, protein or fusion. It is understood that the cell line may also be another bacterial cell, or a yeast, plant, animal or even a human cell line. The selection of the appropriate cell line and a compatible vector are well known to those of skill in the art and all such cell lines and vectors fall within the scope of the present invention. It is also understood that the expression of the gene encoding the protein may be under the control of an inducible promoter, for example the lac promoter and that expression may be controlled by exogenously applied inducers . Most preferably, the cell is a prokaryotic cell such as an E. coli cell and even E. coli strain Y1089 or Y1090.

Transformed cells are generally understood to be those cells that have an inserted plasmid vector capable of replication within the transformed cells. Transfected cells are generally understood to be those which have been infected with a viral vector or a virally derived vector. In both cases, the vector may carry a segment of DNA which encodes for the protein or peptide of interest and which is capable of being replicated and expressed along with the DNA of the plasmid or viral vector. It is sometimes possible through manipulation of the growth conditions of the cells to "overproduce" the desired protein or peptide such that it is the major protein expressed in the cell .

The present invention may, in certain embodiments, be an antigen composition comprising a peptide having immunoreactivity with HuMAb L94 as disclosed herein, in an amount effective to elicit an immune response and may preferably be a peptide comprising a contiguous region consisting essentially of the amino acid sequence of SEQ ID NO:13. An immune response may be an antibody (humoral) response or a cellular (CTL) response. An effective amount may be a polypeptide at a concentration of between about 1 mg/ml and about 10 mg/ml and is more preferably a polypeptide at a concentration of about 5 mg/ml. Although the specification describes preferred protein concentrations, it is understood that these concentrations are offered by way of example only and do not limit the invention in any way. The polypeptide composition discussed in the present paragraph may also be a component of a polyvalent tumor cell vaccine (TCV) .

A polyvalent tumor cell vaccine is for example, a vaccine which comprises several tumor cell lines which express multiple tumor associated antigens. The cells are rendered inviable, preferably by irradiation, and administered to a patient in order to elicit an immune response as discussed elsewhere (Morton et al . , 1992, incorporated herein by reference) .

The tumor cells of the TCV may be rendered more effective by pre-immunization with the antigenic peptide. Therefore, the cells may or may not be pretreated, and the polypeptide composition which includes the polypeptide sequences of the present invention may be administered in conjunction with the TCV. A preferred method of treating human cancer patients and preferably, human melanoma patients would comprise administering to said patients a polyvalent tumor cell vaccine about every two weeks for three times and then about once a month for about a year, followed by administration about every 3 months for about four times and then about every six months thereafter, and further comprising administering a composition comprising a polypeptide having a C-terminal AP, GP or PP or even a polypeptide sequence as designated herein as SEQ ID NO: 8 or SEQ ID NO: 9 or preferably SEQ ID NO: 13 about every four weeks for two to four times and then about every six months thereafter.

The present invention, in certain embodiments,- is a method of enhancing an immune response comprising contacting immune system cells with an effective amount of an antigenic composition comprising a peptide having the C-terminal sequence alanine-proline, glycine-proline or proline-proline, and the method may be practiced by administering the antigenic composition to a subject having an immune system. Alternatively, the method may comprise the steps of obtaining cytotoxic lymphocytes from a subject, contacting said cytotoxic lymphocytes with an effective amount of a polypeptide having an alanine-proline, glycine-proline or proline-proline C- terminal sequence and reintroducing said lymphocytes into said subject. The enhanced immune response may be an active or a passive immune response. Alternatively, the response may be part of an adoptive immunotherapy approach in which the lymphocytes are pulsed with the polypeptide and then reintroduced into the subject. In certain embodiments, the subject is a human cancer patient and more preferably a human melanoma patient . The lymphocytes may be obtained from the serum of the subject, or alternatively from tumor tissue to obtain tumor infiltrating lymphocytes as disclosed in Rosenberg et al . , Science, vol 233, page 1318, incorporated herein by reference, and in certain preferred embodiments, the lymphocytes are peripheral blood lymphocytes. The method of enhancing the immune response may also be practiced in conjunction with tumor whole cell therapy.

A certain embodiment of the invention is a method of purifying a polypeptide or a peptide from a mixture comprising the steps of causing the polypeptide or peptide to have the C-terminal amino acid sequence alanine-proline, glycine-proline or proline-proline, contacting the polypeptide with the HuMAb L94 under conditions effective for immunoreactive binding of the antibody and the polypeptide and separating the antibody from the mixture . The instant method may further comprise the step of separating the polypeptide from the antibody. In certain applications the antibody may be bound to a solid matrix. The matrix may be any suitable material such as a filter composed of nylon, nitrocellulose or the like, the well of a microtiter plate or even agarose beads or any other suitable column or slurry material .

A certain embodiment of the present invention is a method of diagnosing cancer in a subject comprising detecting the presence of a tumor associated antigen having a carboxy terminal alanine-proline, glycine- proline or proline-proline. The presence tumor associated antigens may be detected by the presence in a subject of cytotoxic T lymphocytes that respond to the antigen, by the use of a skin test for example. In such a test, one may inject an antigen composition comprising a peptide having the sequence of SEQ ID NO: 13, for example, intradermally into a subject suspected of having cancer and monitoring the subject for 48 to 96 hours to detect the presence of an induration at the site of the injection wherein an induration of 2-5 mm is indicative of a positive reaction.

An alternate embodiment of the present invention is a method of purifying a polypeptide having a carboxy terminal alanine-proline, glycine-proline or proline- proline from a mixture. The method comprises the steps of contacting a mixture containing a polypeptide with an antibody immunoreactive with the carboxy terminal sequence of the polypeptide to be purified, and preferably with human monoclonal antibody HuMAb L94 under conditions effective for immunoreactive binding of the antibody and the polypeptide and separating the antibody from the mixture. The method may also comprise the step of separating the bound polypeptide from the antibody. In certain applications, the antibody may be bound to a solid matrix such as agarose beads, for example.

A further embodiment of the invention is a polypeptide purified by this method. The polypeptide may be a short peptide fragment, either naturally occurring or produced synthetically, or a full length protein or even a peptide-protein or protein-protein fusion. Such a peptide may be produced by proteolytic cleavage of a larger protein or it may be produced by a vector designed to produce a polypeptide with an AP, GP or PP C-terminal sequence. It is also an aspect of the present invention that such a protein may be proteolytically cleaved after purification to remove the C terminal amino acids.

It is understood that although clinical applications for the polypeptides and nucleic acid sequences, antibodies and recombinant cell lines of the present invention are disclosed, that the various embodiments of the invention will have other important utilities, such as the screening of tissue and culture samples for the presence of cancer associated antigens, diagnosing various cancers, and in the important fields of protein purification and characterization. For example, there are as many as seventy known protein sequences that have a C terminal alanine-proline sequence. These include viral proteins and proteins involved in the immune system among others and the purification of such proteins using the antibodies and methods of the present invention will have a wide usefulness in the art. For example, any protein of known sequence that has the C terminus AP, GP or PP can be detected and isolated from a solution by those of skill in light of the present disclosure. In addition, other naturally occurring proteins may be proteolytically cleaved to produce the required C terminal sequence and such fragments can be easily purified by an antibody column for example.

It is also understood that any protein may be engineered by genetic fusion to contain the C terminal AP, GP or PP, and that such a protein will now be easily purified in light of the present disclosure. It is also understood that any of the antigenic peptides disclosed herein may be linked by chemical or other means to other types of materials and that any such materials that do not interfere with the antigenic recognition would be easily purified by the present methods. Further, the polypeptides and antibodies of the present invention are useful for advancing the general knowledge and use of antigenic sequences, antibodies and activated cellular immune systems in the search for immunogenic weapons against various diseases .

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. IA illustrates the binding of HuMAb L94 to synthetic dipeptide Ala-Pro in a solid phase ELISA. The ELISA plate (ReactiBind , PIERCE) was coated with a 2 fold serially diluted dipeptide Ala-Pro. HuMAb L94 was added to each well, and bound HuMAb was detected by a peroxidase labeled goat anti-human IgM.

FIG. IB illustrates binding of HuMAb L94 to a variety of synthetic peptides. The ELISA plate was coated with lμg of peptide/well . Peptides were 1) Ala- Pro; 2) Ala-Pro-Pro-Ala-Pro-Ala-Ala, SEQ ID NO: 11; 3) Ala-Pro-Gly; 4) Ala-Pro-Ala; 5) Ala-Pro-amide; 6) Ala- Thr; 7) Ala-Tyr; 8) Gly-hPro; 9) Gly-Pro; 10) Gly-Pro- Pro; 11) Gly-Pro-Pro; 12) Ser-Pro; 13) Val-Pro; 14) Glu- Pro; 15) Lys-Pro; 16) Met-Pro; 17) Leu-Pro; 18) Phe-Pro; and 19) S-Ala-Pro.

FIG. IC illustrates the inhibition of binding of HuMAb L94 to the dipeptide Ala-Pro by various peptides.

Before the ELISA, HuMAb L94 was mixed with lOmg/ml of the following peptides: 1) PBS (-) ; 2) Ala-Pro; 3) Gly-Pro; 4) Pro-Pro; 5) Pro; 6) _/S-Ala-Pro; 7) Ala-Pro-amide; and 8) Ala-Pro-Gly, and the residual antibody binding activity was tested using an ELISA plate coated with Ala-Pro peptide (lμg/well) .

FIG. 2A - FIG. 2D indicate the time course of cytotoxicity against autologous #707-pulsed BCL by PBMC from vaccinated melanoma patients. #707 is designated

SEQ ID NO: 13. Cytotoxicity assays were performed at E:T of 80:1 as described in Example 4. A broken line indicates the -threshold for a positive response (17% lysis) . Lysis of autologous BCL pulsed with #707 (-AP) (peptide #707 with the C-terminal Ala-Pro deleted, SEQ ID NO: 12) or without a peptide did not exceed 9% at this ratio by any effector.

FIG. 2A is the pattern exhibited by 8 of 19 patients .

FIG. 2B is the pattern exhibited by 6 of 19 patients .

FIG. 2C is the pattern exhibited by 2 of 19 patients .

FIG. 2D is the pattern exhibited by 3 of 19 patients .

FIG. 3A illustrates the cytotoxicity of a vaccinated melanoma patient's PBMC restimulated with the peptide of SEQ ID NO: 9 in vi tro . The graph depicts lyses of autologous BCL pulsed with #707, SEQ ID NO: 13 and with #707 (-AP) , SEQ ID NO:12. Cytotoxicity of #707- restimulated PMBC's is shown against autologous BCL pulsed with #707 (SEQ ID NO:13) filled circles) and with #707(-AP) (SEQ ID NO:12) (open circles) . Cytotoxicity of PBMC's cultured without #707 (SEQ ID NO: 13) is shown as a control against autologous BCL pulsed with #707 (SEQ ID NO:13) (filled triangles) and with #707 (-AP) (SEQ ID NO:12) (open triangles) .

FIG. 3B is an illustration of lyses of autologous BCL in the presence of peptides derived from the #707 sequence. Cytotoxicity assays were performed at E:T of 40:1 in the presence of peptides as follows: decamer (RVAALARDAP, SEQ ID NO:13) (filled circles) ; octamer (AALARDAP, SEQ ID NO:14) (open circles) ; hexamer (LARDAP, SEQ ID NO: 15) (filled triangles) ; tetramer (RDAP, SEQ ID NO:16) (open triangles) ; dimer (AP) (filled squares) ; and #707(-AP) (RVAALARD, SEQ ID NO:12) (open squares) . Data are representative of three separate experiments.

FIG. 4 Cytolyses of autologous melanomas and #707- pulsed BCL by #707 restimulated PBMC. PBMC from vaccinated melanoma patients A, B and C were restimulated with or without #707, SEQ ID NO:13 as described in FIG. 3 and assayed for cytolyses at E:T of 40:1. Autologous melanoma targets were labeled with ⁵¹Cr for 2 hours and used. All the melanoma cell lines expressed #707 mRNA in in si tu hybridization. Cytotoxicity of #707-restimulated PBMC's (filled squares) and that of control PBMC's (open squares) cultured without #707 are shown. Lysis of autologous BCL without a peptide or pulsed with #707 (-AP) did not exceed 7% by any effector. Data of each patient are representative of three separate experiments.

FIG. 5A illustrates cold target inhibition of cytolyses of autologous #707-pulsed BCL (SEQ ID NO:13) . PBMC's from vaccinated melanoma patient A were restimulated with #707 and assayed for cytolyses at E:T of 40:1. Unlabeled autologous BCL without a peptide (filled circles), pulsed with #707 (SEQ ID NO:13) (open circles) and melanomas (filled triangles) were used- as cold targets. Lysis of autologous BCL without a peptide or pulsed with #707 (-AP) did not exceed 6%. Data are representative of four experiments.

FIG. 5B illustrates cold target inhibition of cytolyses of melanomas as described in FIG. 5A.

FIG. 6A illustrates antibody inhibition of cytolyses by #707 (SEQ ID NO:13) -restimulated PBMC. #707- restimulated PBMC from vaccinated melanoma patient A were assayed for cytolyses of autologous #707-pulsed BCL at E:T of 40:1 in -the presence or absence of antibodies. The final concentration of each antibody was lOμg/ml. L612 antibody (anti-GM) was used as a negative control. Lysis of autologous BCL without a peptide or pulsed with #707 (-AP) did not exceed 6%. Data are representative of four experiments .

FIG. 6B illustrates antibody inhibition of cytolyses by #707 (SEQ ID NO: 13) -restimulated PBMC. #707- restimulated PBMC from vaccinated melanoma patient A were assayed for cytolyses of melanomas as described in FIG. 6A.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An important embodiment of the present invention is the development of a new human monoclonal antibody L94 that is immunoreactive to human cancer cells and other proteins or peptides with the C-terminal sequence, AP, GP or PP. The present invention also includes the isolation and characterization of epitopic sequences recognized by the human monoclonal antibody, HuMAb L9 . Using a cloned sequence isolated from a melanoma cell line cDNA library, the present inventors have demonstrated that the C- terminal alanine-proline sequence is necessary and sufficient for antibody recognition and that it is also important for the induction of a cell mediated immune response (CTL) specific to melanoma cells. Therefore, this discovery is an important advance in the current understanding of humoral and cellular immunological responses to tumors, and has wide clinical applications in the practice of cancer immunotherapy and immunodiagnosis as well as non-clinical applications.

A cDNA expression library obtained from cultured human melanoma cell line M14 was screened with a human monoclonal antibody (HuMAb L94) resulting in the isolation of 5 -unique clones. The sequence of each of these clones was compared with all reported sequences using the PC/GENE software (Intelligences, Menlo Park, CA) . No amino acid homology was found for three of the clones, designated herein as clones #703A, #707, and

#811. The sequence of one clone, #702A is homologous to the 3'- untranslated region of human cytoskeletal actin (Erba et al . , 1986) , and the sequence of another of the clones, #808 is homologous to a coding region of human melanocyte specific gene PMEL 17 (Kwon et al . , 1991) .

In addition to the five clones isolated from the M14 cDNA library, five immunoreactive clones were also isolated from a cDNA expression library constructed from another antigenic melanoma cell line, M12.

Interestingly, all of these 5 clones from the M12 library had alanine-proline as the two C-terminal amino acids. The inventors subsequently discovered that HuMAb L94 also immunoreacts with polypeptides having the C-terminal amino acids glycine-proline and proline-proline, but the isolated clones all have alanine-proline at their C terminals .

Although the invention is not limited by any particular theory, the propensity for AP may be explained by some artefact of the cDNA library, or the AP termini may be produced by some endoproteolytic cleavage by a protease that is activated in transformed cells. Some proteases are reported to be activated during malignant transformation, including those that cleave fibronectin (Hui et al . , 1983) and proteoglycan (Hughes et al . , 1992) . These proteins are proteolytically cleaved and present new antigenic determinants after transformation. Prolylendopeptidase (Bruggen et al . , 1991; Irie et al . , 1982; Micanovic et al . , 1988; Briggs et al . , 1992) is typical of this type of protease, because it specifically cleaves the C terminal side of the Pro-X peptide bond (Kalwant et al . , 1991) .

Seven human proteins have previously been reported with alanine-proline C-termini. These include the type I membrane protein, T-cell differentiation antigen CD6 (Aruffo et al . , 1991) , leukosialin CD43 (Pallant et al . , 1989) , extracellular signal regulated kinase 1 (Owaki et al . , 1992) , a DNA binding protein, interferon regulatory factor (Miyamoto et al . , 1988) , a plasma protein, Protein C (Foster et al . , 1985) , alkaline phosphatase precursor (Micanovic et al . , 1988) and endothelin 3 precursor (Bloch et al . 1989) . It is contemplated that any of these proteins would immunoreact with HuMAb L94. HuMAb L94 has been tested with two different synthetic peptides having C terminal alanine-proline, Bradykinin potentiator B and pEKWAP, (Sigma Chemical Company) and both exhibited strong immunoreactivity with the L94 antibody.

Therefore, a protein with a C-terminal sequence AP, GP or PP, whose presence is diagnostic for any condition, would be detected by HuMAb L94 and such detection would be diagnostic for the condition so indicated. Some examples would be tumor associated antigens or viral antigens and the like.

Antigenic determinants which include alanine-proline in an internal sequence have been reported. For example, monoclonal antibodies against carcinoma associated epithelial mucins bind to peptides containing the sequence RPAP, SEQ ID NO: 19 (Briggs et al . , 1992; Nuti et al . i 1992) and monoclonal antibodies against the human B cell differentiation marker CD24 recognize LAP (Weber et al . , 1993) . The antigenicity of these two sequences is not dependent on the C-terminal position of alanine- proline, whereas HuMab L94 recognizes the alanine-proline only at the C-terminal. There is at the present time, no consensus regarding the smallest sequence which can be recognized as a complete antigenic determinant. Most estimates for the minimum number of sequential amino acids is around six. Using a direct enzyme linked immunosorbent assay, a dipeptide has, however, been shown to be an epitope of certain antibodies (Appel et al . , 1990; Khachigian et al . , 1991) . These previous studies used murine monoclonal antibodies raised against peptides or whole cells. However, the antibody of the present invention, HuMAb L94, is believed to be the first human monoclonal antibody able to recognize a two amino acid segment . The antigen binding region of HuMAb L94 is thought to form a narrow binding pocket which is able to contain the C- terminal dipeptide alanine-proline. Even though proline is able to enter the pocket, this single amino acid does not exhibit immunoreactivity with HuMAb L94. The adjacent residue, which is preferably alanine, can be replaced by glycine or proline, which have side chains which are similar to or smaller than alanine. But residues which have a more bulky side chain, such as serine, valine and glutamate may not be able to enter the binding pocket because of steric hindrance.

CTL Response

As an embodiment of the present invention, it was determined that polypeptide #707, (SEQ ID NO: 13) which includes the C-terminal alanine-proline sequence acts as a CTL target on human melanoma cells and is contemplated to play a primary role in the recognition of tumor cells by CTL. The inventors have previously demonstrated that common melanoma associated antigens (MAAs) may be presented as CTL epitopes in association with distinct HLA-A molecules on individual melanoma cells, and that HLA-A2, All and A24 may function as such supposed MAA- presenting elements (Hayashi et al . 1992; 1992a) . This evidence is consistent with the present disclosure for polypeptide #707, (SEQ ID NO:13) and thus it is contemplated that this polypeptide is a candidate for an MAA.

The first indication that polypeptide #707 (SEQ ID NO: 13) is a melanoma associated antigen is that the mRNA was specifically expressed by the majority of human melanomas, and particularly by all three MCV cell lines, MlO, M24 and MlOl, but never by benign cells. The second indication is that MlO, M24 and MlOl express HLA-A24, All and A2, respectively, and all these HLA-A molecules appeared to present the polypeptide #707 (SEQ ID NO:13) antigen in the data reported herein. This suggests that the #707 (SEQ ID NO: 13) antigen may possibly be an endogenous degradation product from a putative membrane- located protein in each MCV cell and that it may be associated with a respective HLA-A molecule and presented on the cell surface of MCV.

This HLA-A molecule-associated peptide may effectively be recognized by the CTL of a vaccinated patient, if the patient bears any one of the A24, All or A2 HLA-A molecules. The frequency of HLA-A24, All and A2 expression in Caucasians is 15%, 10% and 49%, respectively (Marincola et al . 1992), suggesting that approximately three quarters of the whole patient population presents the #707 (SEQ ID NO: 13) polypeptide. In fact, a high frequency (84.2%) of patients showed increased CTL response to polypeptide #707 after MCV immunization, indicating that the #707 antigen is probably presented on a very high proportion of melanoma cells, though not all. It is also known that #707- specific CTL precursors may be primed with MCV immunization in vivo, and secondary CTL may be induced by restimulation with #707 in vi tro . An example of such secondary CTLs also exhibited cytotoxicity to autologous melanomas in one patient, patient A (HLA-A24+) , (See FIG. 4) while this cross-reactive cytotoxicity was rather weak in two other patients, B (A2+) and C (A11+) . It is likely that the efficacy of #707 for the induction of melanoma-killing CTL may be influenced by either the HLA typing of the melanoma hosts; i.e., HLA-A24 may contain higher affinity to #707 than A2 or All, or the density of antigen expression in the host's melanomas or both.

This CTL-induction by #707 was completely different from the non-specific T cell activation by some immunomodulators like interleukins. Although a pentapeptide fragment (VPLFP, SEQ ID NO:17) of a proline- rich polypeptide was shown to have immunoregulatory activity on murine thymocytes (Szewczuk et al . 1991) , cytotoxicity enhancement by this peptide was not reported and its immunological activity was abolished when the C- - terminal sequence was substituted by Ala-Pro (VPLAP, SEQ ID NO:18) (Szewczuk et al . 1991) . In addition, no other known immunomodulators contain the C-terminal alanine- proline sequence. Thus this sequence is very characteristic of the antigen #707, which may be associated with human melanomas.

Determination of the Minimal Recognition Sequence

The removal of the C-terminal alanine-proline sequence completely abolished the ability of the #707 polypeptide sequence to be recognized by #707-specific CTL. While the recognition remained when N-terminal residues other than the alanine-proline were removed, the CTL reactivity to the N-terminal truncated polypeptides decreased (see FIG. 3B) . This data presents strong evidence that the C-terminal alanine-proline sequence is necessary for the interaction of #707 with the TCR of CTL. It is contemplated that some conformational constraint imposed by the C-terminal alanine-proline sequence may be involved in the interaction with TCR, since proline residues are known to induce specific restricted secondary structure to the peptide backbone through their rigid pyrrolidine rings (Gairin and Oldstone, 1993; Maryanski et al . 1990) . Although this would not affect the interaction of the dipeptide alanine-proline with the human monoclonal antibody L94 , the reactivity of the antibody is somewhat different from the CTL. When a series of #707-related peptides with C- terminal alanine-proline sequences were compared, the binding activity of L94 in peptide-ELISA was never reduced with the N-terminal truncation indicating that this antibody may require only the C-terminal alanine- proline sequence for binding. Thus it appears that the epitope recognized by CTL is not completely identical to that recognized by the antibody, although the C-terminal alanine-proline is shared by both. This may reflect the difference in the mechanism of peptide recognition between the cellular and humoral immune systems, particularly related to the involvement of MHC molecules.

Regarding the recognition by CTL, the residues N- terminal to the alanine-proline sequence in #707 may act mainly as an agretope which binds to the MHC groove ^"and stabilizes the #707/MHC complex and may not contribute to the specificity of CTL recognition, although epitopic and agretopic residues in one peptide may not completely be segregated from each other. In the absence of any structural information, however, there is still some uncertainty about the minimal length of amino-acid residues required for effective presentation and recognition by CTL.

It is contemplated that a pentamer is the minimal antigenic peptide for MHC class I-restricted CTL (Whitton et al . 1989; Reddehase et al . 1989) , although an octamer or nonamer may be the optimal length for the stable binding to MHC molecules (Cerundolo et al . 1991; Fremont et al . 1992; Matumura et al . 1992) . Although #707- induced CTL appeared to recognize the dimer alanine- proline, a dimer would not be expected to be able to adopt an appropriate conformation to fit into the MHC groove and, even if bound to an MHC molecule, would not be accessible to TCR. However, very high concentrations of the dimer alanine-proline may allow weak but active interaction with the MHC groove. Predictably, on the target cell surface exposed to a large number of alanine- proline molecules, these small dimer molecules may easily invade and occupy the vacant peptide-binding pockets of MHC and may form Ala-Pro/MHC complexes mimicking, though not identical to, the original #707/MHC complexes. Such Ala-Pro/MHC complexes may interact with a small - proportion of bulk CTL, whose TCR may have extremely high affinity to the C-terminal alanine-proline sequence.

Antigenic Compositions

The polypeptide segments disclosed herein have potential therapeutic applications in vi tro and in vi vo as eliciting the mammalian immune responses against tumor cells. In addition to CTL induction, antibody responses leading to antibody-dependent cellular or complement- mediated lysis of melanomas are indicated by the data presented herein.

An example of an in vi tro method of use of the antigenic compositions of the present invention is the activation of autologous mononuclear cells with the antigenic compositions of the present invention and reintroduction of the activated cells into the patient as exemplified in US Patent No. 5,192,537 (incorporated herein by reference) . Immunizations with the antigenic compositions is also indicated because of the localization of the antigen presenting protein on the cell surface of tumor cells.

It is understood that administration of a peptide in vivo may also comprise some technical elaboration; e.g., chemical modification of a free peptide, like coupling to a lipid tail, or combination with an effective adjuvant and with biological response modifiers to inhibit suppressor or non-specific NK / lymphokine-activated killer cells. Nevertheless, the employment of peptide- based immunization is necessary to obtain sufficient amount of effective, safe and inexpensive vaccines with uniform-quality. Such peptide vaccines may generate specific immune responses in melanoma patients, and may prevent post-operative metastases and will result in the prolongation of survival.

Vectors, bacterial strains, cDNA library, and oligonucleotides

The bacteriophage λ-gtll, E. coli cell strains Y1089 and Y1090, and the plasmid BLUESCRIPT (Promega, Madison, WI) were purchased from Stratagene (San Diego, CA) . Expression cDNA libraries of human melanoma cell lines

M14 and M12 were constructed at the Eco RI site of λ-gtll using the oligo dT priming method (Invitrogen, San Diego, CA) . The cDNA cloned in λ-gtll were subcloned into plasmid BLUESCRIPT and analyzed for their sequences using a modified dideoxy chain termination method (USB, Cleveland Ohio) .

Human monoclonal antibody JWCI L94 (HuMAb L94)

An IgM human monoclonal antibody was established from a melanoma patient's peripheral blood lymphocyte (PBL) using the Epstein Barr virus (EBV) transformation technique as previously described (Irie et al . , 1982) . Melanoma cell lines UCLA M14 and M12 were used to monitor the antibody secreted in the spent tissue culture medium using immunohistochemical assays. Purification of HuMAb L94 from the spent medium of the EBV transformed cell culture was performed as previously described (Katano et al . , 1984) . To determine an approximate molecular size of the antigen, these cell lines were tested by SDS-PAGE Western blotting analysis with HuMAb L94 but produced no visible band. This indicates either that the antigenicity was altered, unexpressed, or became cryptic during the SDS-PAGE procedure, or that the antigenic epitope resides in a glycolipid. However, neither acidic nor neutral glycolipid fractions purified from these cell lines exhibited antigenicity.

The reactivity of purified HuMAb L94 against a series of human tumor cell lines was tested using immunoadhesion (IA) assays. Table 1 shows a variety of human tumor cell lines recognized by HuMAb L94, including melanoma (9/18) , lung carcinoma (2/3) , colon carcinoma (1/2) , renal carcinoma (1/4) , stomach carcinoma (l/l) , neuroblastoma (0/2) , and erythroleukemia (l/l) . Two of the antigenic melanoma cell lines, M14 and M12, were tested by SDS PAGE Western blotting analysis with HuMAb L94 but produced no visible band. This indicates either that the antigen molecule was too small to be detected on the SDS-PAGE Western blotting analysis or that the antigenicity was inactivated or cryptic during the SDS PAGE.

Table 1

Expression of mRNA for #707 Peptide by Human Cancer

Cell Lines and Normal Lymphocytes

In Si tu Cell Lines Histologic Types Hybridization*

MlO Melanoma + M12 Melanoma +

M14 Melanoma +

M15 Melanoma +

M24 Melanoma

M25 Melanoma +

MlOl Melanoma +

Mill Melanoma +

M12 Melanoma +

K562 Erythroleukemia

SHN Neuroblastoma

SW480 Colon Cancer

SW48 Colon Cancer

MKN45 Gastric Cancer +

MNK28 Gastric Cancer

B645 Breast Cancer

L130 Lung Cancer

L135 Lung Cancer

Peripheral Blood Lymphocyte Monocytes

B cells

T cells

*mRNA level for #707 peptide was analyzed using digoxigenin- labeled complementary oligonucleotide probes.

Screening of melanoma cell cDNA expression library with HuMAb L94

Screening of the λ-gtll cDNA library constructed from melanoma cell lines, M14 and M12 , was performed as previously described (Oka et al . ) . In this technique, nitrocellulose filters were exposed to a melanoma cDNA expression library. Nitrocellulose filter lifts were reacted sequentially with HuMAb L94 and a goat anti human IgM antibody conjugated with peroxidase (Boehringer Mannheim, Indianapolis, IN) . The antigen antibody complexes were visualized by chromogenic substrate 4- chloro-1-naphthol .

Immunologic Assays

SDS polyacrylamide gradient gel electrophoresis followed by Western blotting were carried out as previously described (Laemmli 1970; Towbin et al . , 1979) . Peptides, glycine-proline-proline (GPP) , alanine-proline (AP) , alanine-proline-amide (APamide) , alanine-proline (AP) , alanine-proline-glycine (APG) , alanine-proline- alanine (APA) , proline-proline (PP) , lysine-proline (KP) , serine-proline (SP) , methionine-proline (MP) , leucine- proline (LP) , glutamic acid-proline (EP) , glycine- hydroxyproline-proline (GhPP) , and valine-proline (VP) were purchased from Bachem Bioscience Inc. (Philadelphia, PA) , Sigma and Novabiochem USA (La Jolla, CA) . The solid phase ELISA was used to test the binding of HuMAb L94 to peptide antigens. Peptide (lμg/well) was coated on the Reacti-Bind™ plate (Pierce, Rockford, IL) and left overnight at 4°C. ELISA was performed using a goat anti- human IgM (μ chain specific) conjugated with peroxidase. Data was analyzed using a plate reader and Softmax (Molecular Devices, Menlo Park, CA) . Inhibition ELISA was performed to examine cross reactivity of HuMAb L94 with several synthetic antigens (lOmg of peptide/lμg of HuMAb) . Pre-incubated HuMAb L94 with peptides was tested using Ala-Pro coated plates.

Synthetic peptides

Peptides were synthesized in the Beckman Research Institute of the City of Hope (Duarte, CA) using the F- moc method. High-pressure liquid chromatography was used to purify these peptides (>95 % purity) . High resolution mass spectrum confirmed the identity of the peptides. The decamer derived from peptide #707 (RVAALARDAP) SEQ ID NO: 13 was mainly used in this study, since the cDNA clone encoding this amino-acid sequence was isolated from the melanoma M14 expression library, and the transcript of the gene fragment was detected by in si tu hybridization in most of human melanoma cell lines but not in benign cell types.

In si tu hybridization

A synthetic oligodeoxynucleotide sequence complementary to mRNA for #707 (RVAALARDAP, SEQ ID NO: 13) peptide was end-labeled with digoxigenin and used to detect mRNA in a panel of cells. Cells fixed on glass slides were prehybridized for 1 hour at 42°C in a solution containing deionized formamide, 20x standard saline citrate, Denhardt's solution, heat denatured sheared herring sperm DNA, yeast transfer RNA and dextran sulfate. The 707 antisense probe (Molecular Biology Institute, UCLA School of Medicine, Los Angeles, CA) was tail-labeled with digoxigenin-11-dUTP (Boehringer

Mannheim, Indianapolis, IN) using the DNA tailing kit (Boehringer Mannheim) . The #707 sense probe, which was complementary to the test (antisense) probe, was used as a negative control. A 27 base pair oligonucleotide probe specific to human fibroblast -actin (Clontech

Laboratories, Inc. Palo Alto, CA) was used as a positive control. A digoxigenin-labeled probe was placed on the cells and incubated at 42°C overnight. Cells containing #707 mRNA were detected using the Genius nonradioactive nucleic acid detection kit (Boehringer Mannheim) . Slides were incubated with 2% normal sheep serum and 0.3% Triton X-100 at room temperature for 30 minutes. Anti- digoxigenin antibody was applied to the cells for 3 hours at room temperature. A solution containing nitroblue tetrazolium, X-phosphate solution and Levamisole was placed on the cells at room temperature until they developed a satisfactory color (2-5 hours) . Tumor cell lines

Melanoma cell lines, erythroleukemia K562 and EBV- transformed BCL were cultured by RPMI 1640 medium (JRH Biosciences, Lenexa, KS) supplemented with 10% heat- inactivated fetal bovine serum (Gemini Bioproducts, Calabasas, CA) .

Cultured human melanoma cells, M12 and M14 were maintained in RPMI 1640 medium supplemented with 5% FCS, penicillin lOOu/ml and streptomycin lOOμg/ml.

Allogeneic whole MCV

Nineteen melanoma patients of Stage III (American

Joint Committee on Cancer) were tested. All patients in this study were immunized in vivo with MCV according to the protocol of Morton et al . (1992) incorporated herein by reference. MCV consisted of three melanoma cell lines (M10, M24 and MlOl) , which were irradiated and cryopreserved before use. All these melanoma cell lines have been shown to express mRNA for #707 antigen by in si tu hybridization. The antibody L94 also reacted with the surface of all these cell lines in immunoadherence assays. MCV was thawed, washed and mixed with Bacill us Calmette Guerin and then injected intradermally every 2 weeks x 3, then monthly for 1 year, and then every 3 months x 4, finally every 6 months.

PBMC from melanoma patients and normal donors

Patients were bled before (week 0) and at monthly intervals (weeks 4 and 8) after the initiation of vaccination. PBMC were separated by Ficoll-Hypaque gradient centrifugation and cryopreserved before testing. PBMC from normal donors were obtained from the American Red Cross (Los Angeles, CA) . BCL were prepared by transformation of those PBMC with EBV as described (Issekutz et al . 1982) . RPMI 1640 supplemented with 10% heat-inactivated human AB serum (Irvine Scientific, Santa Ana, CA) was used as the culture medium for cytotoxicity and proliferation assays.

Cytotoxicity assays

A standard ⁵¹Cr-release assay was performed as described (Hayashi et al . 1992) . Melanoma cell lines were harvested and labeled with lOOμCi ⁵¹Cr for 2h at 37°C and used as targets. The labeled target cells were washed, resuspended in culture medium and seeded in 96- well microtiter plates at 5xl0³ cells/well, to which effectors were added. All assays were done in triplicate. After 4h of incubation, supernatants were collected. The percentage of specific cytolysis was calculated as 100 x (experimental release- spontaneous release / 5% Triton X release- spontaneous release) . For peptide experiments, BCL were pulsed with a peptide at lOμM simultaneously labeled with lOOμCi ⁵¹Cr for 2h at 37°C and used as targets. In some experiments, peptides were added directly to the assays . Seventeen percent lysis at an E:T of 80:1 was defined as a positive response to a peptide, because that value was 3SD above the mean of negative control values.

Cold target inhibition assays

Unlabeled cold target cells (50μl) were seeded in 96- well plates. Effectors (lOOμl) were added to the wells and incubated for lh at 37°C before ⁵¹Cr-labeled hot targets were added at the desired cold:hot target ratios .

Monoclonal antibodies Human IgM monoclonal antibodies L94 (anti-peptide #707) and L612 (anti-ganglioside GM) (Yamamoto et al . 19990) were developed and purified in the inventors' laboratory. Murine IgG monoclonal antibodies, anti-HLA class I, anti-HLA-DR, anti-CD3, anti-CD4 and anti-CD8, were purchased from AMAC, Inc., Westbrook, ME. ⁵¹Cr- labeled targets were preincubated with anti-class I, anti-HLA-DR, L94 or L612 antibody for lh at 37°C before the addition of effector cells in the cytotoxicity inhibition assays. Effectors were preincubated with anti-CD3, CD4 or CD8 antibody for lh before the addition of targets .

In vi tro restimulation of PBMC with #707

PBMC (3xl0⁶/well) from melanoma patients 4 weeks after the initiation of MCV were incubated with #707 at lOμM in 2 ml culture medium in 24-well plates at 37°C. After 5 days of incubation, cells were harvested and assayed for cytotoxicity.

Proliferation assays

PBMC (1x10^s cells/well) were incubated with peptides in 96-well plates, pulsed with [³H] TdR (0.5μCi/well) during the last 18 hours of a 4 day period, then harvested and counted. SI was calculated as [mean counts of triplicate cultures with peptide] / [mean counts without peptide] . The maximal SI among various concentrations ≥ 2.0 was defined as a positive response to a peptide.

HLA typing

PBMC from patients were used for HLA typing by a complement-mediated microcytotoxicity assay in Dr. Paul Terasaki's laboratory (UCLA School of Medicine, Los Angeles, CA) .

Antibodies

In another aspect, the present invention contemplates an antibody that is immunoreactive with any of the polypeptides or peptides disclosed herein. An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (See, e.g.. Antibodies "A Labora tory Manual , E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988) .

One method of preparing a polyclonal antibody is by immunizing an animal with an immunogen comprising a - polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for the polypeptides of the present disclosure may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes having C- terminal alanine-proline, glycine-proline or proline- proline can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the polypeptide. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

To obtain monoclonal antibodies, one might also initially immunize an experimental animal, often preferably a mouse, with an antigenic composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal . The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired polypeptides.

Following immunization, spleen cells are removed and fused, using a standard fusion protocol (see, e . g. , The - Cold Spring Harbor Manual for Hybridoma Development, incorporated herein by reference) with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against the polypeptides. Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods and those are further screened to select those that recognize the C-terminal sequence.

It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures which may utilize antibody specific to melanoma associated epitopes. These melanoma-specific monoclonal antibodies are anticipated to be useful in various ways for the treatment of melanoma through, for example, their application in passive immunization procedures. Additionally, it is proposed that monoclonal antibodies specific to the particular melanoma associated antigen may be utilized in other useful applications. For example, their use in immunoabsorbent protocols may be useful in purifying native or recombinant antigens or variants thereof.

In general, both poly- and monoclonal antibodies against melanoma cells may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding melanoma associated antigens or related proteins. Anti-melanoma antibodies will also be useful in immunolocalization studies to analyze the distribution of the antigen during various cellular or transformation events, for example, to determine the appearance of the antigen in the nuclear, cytoplasmic or membrane regions - of the cell during the transformation event in which a cell becomes cancerous. A particularly useful application of such antibodies is in purifying native or recombinant tumor associated antigens, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.

Use of Peptides as Size Markers

The peptides disclosed herein, and in particular peptide #707 will also find use as size markers for gel electrophoresis and for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gradient gel electrophoresis in particular (Laemmli 1970; Towbin et al . , 1979) .

As is well known in the art, a purified peptide or protein migrates as a discrete band on SDS-PAGE in appropriate conditions. Since the molecular weight of the peptide is known, such a "standard" can be run in an adjacent lane to a peptide of unknown molecular weight. For example, by running a series of peptides or proteins of various molecular weights in a single lane, one can create a ladder of molecular weight markers that are then used to estimate the molecular weights of proteins or peptides in the adjacent lanes.

The gels may either be stained with a protein staining dye such as Coomassie Blue, or the peptides may be labeled with a fluorescent marker or even a radioactive marker, or alternatively they may be reacted with a labeled antibody that recognizes the markers in a Western blot analysis . The human monoclonal antibody L94 will be particularly useful in this application, because it will be a routine matter to create a series of peptides or proteins of various molecular weights, all having C-terminal AP, GP or PP sequences in light of the present disclosure. These markers could then be used in a Western blot analysis to determine the size of other proteins that are recognized by the antibody.

Statistical Analysis

The results were evaluated for statistical significance by the Student's t-test.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

Discovery and Sequence Analysis of cDNA Clones Encoding Peptides Immunoreactive with HuMAb L94

To isolate the cDNA encoding the epitope of HuMAb L94, a cDNA library constructed from M14 and M12 melanoma cell lines at the Eco RI site of λ-gtll was screened with HuMAb L94. Seven clones which stained specifically with HuMAb L94 were isolated. All seven positive clones were expressed in E. col i Y1090 as 3-galactosidase fusions. The expressed proteins were then reacted with HuMAb L94 and analyzed by SDS PAGE and Western blotting. All of the fused proteins showed strong, specific binding to - HuMAb L94 in both the SDS PAGE and Western blotting. β- galactosidase alone failed to react, indicating that HuMAb L94 recognized the cloned peptides and not β- galactosidase .

The cloned sequences were subcloned into the BLUESCRIPT plasmid at the Eco RI site and their DNA sequences were analyzed by dideoxy termination methods. The DNA sequences and their deduced amino acid sequences are as follows:

The genetic sequence GAA TTC GCG GCC GCT CCT TGA, SEQ ID NO:l, encodes peptide #702A, Glu Phe Ala Ala Ala Pro, SEQ ID NO:6. The genetic sequence GAA TTC GCG GCC GCG CCT TGA, SEQ ID NO:2, encodes peptide 703A, which has the same amino acid sequence as peptide #702A, SEQ ID NO: 6. The genetic sequence GAA TTC GCG GCC GCG CCT CCA GCT CCA TGA, SEQ ID NO:3, encodes peptide #808, Glu Phe Ala Ala Ala Pro Pro Ala Pro, SEQ ID NO: 7. The genetic sequence GAA TTC GCG GCC GCT GAA GGT GCT CCA TGA, SEQ ID NO:4, encodes the peptides #711 and #807, Glu Phe Ala Ala Ala Glu Gly Ala Pro, SEQ ID NO:8. The genetic sequence GAA TTC GCG GCC GCC TTT GCT TCC TCA GGA GTT CAG ATG TGT TCT AAG CCT GCT GGA GTG ACC ACA CTT CCA AGA CCT GAT GGA GGC CAG AGC TCA GAG TGG CAA CGG GTC GCA GCC CTT GCT CGA GAC GCC CCG TGA, SEQ ID NO:5 encodes the peptide #707 Glu Phe Ala Ala Ala Phe Ala Ser Ser Gly Val Gin Met Cys Ser Lys Pro Ala Gly Val Thr Thr Leu Pro Arg Pro Asp Gly Gly Gin Ser Ser Glu Trp Gin Arg Val Ala Ala Leu Ala Arg Asp Ala Pro, SEQ ID NO:9, and the same sequence encodes peptide #703B except that there is 42 base gap such that after the fifth amino acid. The first five bases of peptide #703B, Glu Phe Ala Ala Ala, are designated SEQ ID NO:10 and the remaining amino acids of peptide #703B, Val Thr Thr Leu Pro Arg Pro Asp Gly Gly Gin Ser Ser Glu Trp Gin Arg Val Ala Ala Leu Ala Arg Asp Ala Pro, are designated SEQ ID NO: 11. The underlined regions of the genetic sequences indicate the linker region.

Five clones out of the seven had unique sequences . Clones #711 and #807 had the same DNA sequence, SEQ ID NO:4. The amino acid sequence of clone #703B SEQ ID NOS: 10 and 11, is contained in clone #707, SEQ ID NO: 9. The actual protein encoding regions were a small proportion of the total cDNA inserts, which ranged from 0.6kb to 2.0kb. The size of the immunoreactive peptides was consistent with the molecular weights of the immunoreactive proteins which were similar to that of β- galactosidase . Surprisingly, the deduced amino acid sequence of each immunoreactive clone had alanine-proline at the C terminus. Of particular significance were the clones #702A and 703A SEQ ID NO: 6, whose amino acid sequences consisted only of the alanine-proline from the cDNA sequence linked to the -galactosidase and linker sequences. Five additional clones were isolated by the same procedures from a cDNA library constructed from melanoma cell line M12. All 5 of these clones also had the C terminal sequence alanine-proline. EXAMPLE 2

Immunoreactivity of HuMAb L94 to the Dipeptide Alanine-Proline

The surprising results discussed in Example 1 led the inventors to examine whether the dipeptide Ala-Pro alone was sufficient for immunoreactivity with HuMAb L94. A synthetic peptide was obtained consisting of alanine- proline and it was examined for immunoreactivity with HuMAb L94 by ELISA. As shown in FIG. IA, HuMAb L94 bound to the dipeptide Ala-Pro in a dose dependent manner.

Next, a series of synthetic peptides were obtained to determine whether the dipeptide Ala-Pro must be at the C-terminus of the peptide in order to react with the antibody. These peptides were contacted with the HuMAb L94 in an ELISA. As shown in FIG. IB (column 2-4) , the - addition of Gly, Ala, or Ala-Ala to the C-terminus of Ala-Pro abolished the immunoreactivity. Ala-Pro is the amino terminal (N-terminal) sequence of recombinant human IL 1-_/3. This protein was reacted with HuMAb L94 in a Western blot. The N-terminal Ala-Pro of the protein was not recognized by HuMAb L94. These data demonstrated that the C-terminal position of Ala-Pro is essential for the immunoreactivity with HuMAb L94.

A variety of dipeptides were also examined for the ability to immunoreact with HuMAb L94. Substitution of the C-terminal Pro to Thr, Tyr, or 4-hydroxyPro abolished the binding (FIG. IB column 6-8) , and substitution of Pro-amide significantly reduced the binding (FIG. IB column 5) . These results indicated that the C-terminal Pro residue was essential for recognition by the HuMAb L94 , although it was recognized less efficiently in the amide form. A series of dipeptides was also obtained with alternate amino acids substituted for the Ala in the N- terminal position of the dipeptide. When Ala was substituted by 3-Ala, Gly, and Gly-Pro (FIG. IB column 9, 10 and 19) , the antigenicity was found to be intact.

However, antibody binding was completely negative when Ala was substituted by hydroxy-Pro, Ser, Val, Glu, Lys, Met, Leu, and Phe (FIG. IB column 11-18) . Antibody binding appears to be dependent on the presence of a small amino acid side chain in the N-terminal position of the dipeptide. The peptide Pro-Pro exhibited very low binding affinity, which may be due to the lack of binding of the imino group of the proline residue with the maleic anhydride of the Reacti-Bind™ Plate (Pierce) .

A series of ELISA competitive assays were also performed to determine the ability of the immunopositive peptides from FIG. IB to inhibit antibody binding to Ala- Pro. As shown in FIG. IC, the binding of HuMAb L94 to Ala-Pro was completely inhibited by Ala-Pro, Gly-Pro, and Pro-Pro, and was significantly inhibited by 3-Ala-Pro and Ala-Pro-amide, but not by proline alone or by Ala-Pro- Gly. These data suggest that all the immunopositive peptides are recognized by the same binding pocket on the antibody, L94.

EXAMPLE 3

Expression of Antigen on Surface of Melanoma Cells and in si tu Hybridization

A series of immunoabsorbent assays demonstrated the expression of the Ala-Pro antigen or a cross-reactive antigen on the melanoma cell surface. Recombinant phage lysogens encoding peptide #711, SEQ ID NO: 8 were cultured at 30°C and protein expression was induced by IPTG treatment and a temperature shift to 42°C. The extract of the lysogen was used to detect the reactivity of HuMAb L94 in an SDS-PAGE Western blot. After HuMAb L94 was adsorbed to the antigenic melanoma cell line M14, the binding of HuMAb L94 to a ^-galactosidase fusion with peptide #711 was reduced significantly. HuMAb L94 absorbed with non-antigenic melanoma cell M15 reacted similarly to that of the negative control. Furthermore, pre-incubation of HuMAb L94 with the dipeptide Ala-Pro or treatment of target M14 cells with proteases such as trypsin or carboxypeptidases Y, and P (Boehringer Mannheim) significantly reduced the IA activity of HuMAb L94. These data indicate that a sequence antigenic for HuMAb L94 which has the C-terminal Ala-Pro or a cross reactive sequence is expressed on the cell surface of melanoma M14.

In si tu hybridization for mRNA encoding one of the cloned peptides, #707, SEQ ID NO: 13, was performed on 9 human melanoma cell lines, 9 other histologic types of human cancer cell lines, and normal lymphocytes. As shown in Table 1, 100% of the melanoma cell lines, including those negative for membrane antigen expression, such as M15, were positive for the mRNA. In addition, one third of the other types of cancer cells tested (1 colon cancer, 1 gastric cancer and 1 breast cancer cell line) were positive for the mRNA. Monocytes, B- lymphocytes and T lymphocytes obtained from peripheral blood lymphocytes were all negative.

EXAMPLE 4 Recognition of Peptide #707 by PBMC

From Vaccinated Melanoma Patients

PBMC from vaccinated melanoma patients were evaluated for recognition of the amino acid sequence #707 (SEQ ID NO: 13) and the results were compared among various phases (0, 4 and 8 weeks post vaccination) and with control normal donors (FIG. 2A - FIG. 2D) . In 16 of 19 melanoma patients (84.2%) , PBMC lysed autologous BCL pulsed with #707 (SEQ ID NO: 13) , either before or after vaccination, while similar cytotoxicity was observed in only one of 19 normal donors (5.3%) (Table 2) . C- terminal Ala-Pro seemed to be crucial for the recognition of the peptide, because autologous BCL pulsed with a C- terminal truncated peptide named #707 (-AP) (RVAALARD, SEQ ID NO:12) were hardly lysed. The cytotoxic response to the peptide was rather weak at week 0 (mean lysis at E:T of 80:1=17.47%) ; however, after in vivo immunization with MCV, 16 patients (84.2%) showed significantly increased responses (mean lysis = 35.32% at week 4; 28.22% at week 8) (FIG. 2A - FIG. 2C) . Overall, the time course of cytotoxic responses to the peptide can be classified into four patterns as shown in FIG. 2A to FIG. 2D as follows: (2A) the most typical pattern: positive but weak response at week 0, increased at week 4, reduced but still above - the prevaccination level at week 8; (2B) the second typical pattern: positive but weak at week 0, increased at week 4 and maintained even at week 8; (2C) a minor pattern: negative response at week 0, greatly enhanced at week 4 but immediately reduced to negative at week 8; (2D) a minor pattern: negative throughout the test period.

Table 2

Peptide #707-Specific Cytotoxicity of PBMC's From

Vaccinated Melanoma Patients

Autologous BCL targets pulsed with #707 #707(-AP)*

Mean ± SD Positive Mean ± SD Positive of lysist response (%) of % lysis response PBMC Donor (%)

Melanoma patients (n=19)

10 Week o§ 17.47 + 14 (73 .7) 6, .98 ± 1(5.3) Ch 5-02|| 3,.23 1

Week 4 35.32 + 16 (84 .2) 5, .40 + 0(0)

11.52 3.70

Week 8 28.22 14 (73 .7) 7, .21 ± 1(5.3)

10.39| 1 *1 5,.20

Total 16 (84.2) 1(5.3)

Enhanced 16 (84.2) 0(0)

15 response post vaccination **

Control 6.21 ± 1 (5.3) 5.68 ± 0(0)

(π=19) 3.38 4.50

5 *C-terminal AP deletion of #707, octapeptide [RVAALARD] . tE:T=80:l. tl7% lysis which was 3SD above the mean lysis of controls was defined as a positive response to a peptide. § eeks after the initiation of vaccination. |Significant difference vs values of control (p<0.001) . Hsignificant 10 difference vs values of week 0 (p<0.001) . **Significantly increased response either at week 4 or 8 compared to week 0 (p<0.05) . Statistical significance was analyzed by student's test .

I I

These results suggest that PBMC's from melanoma patients may be presensitized in vivo with the antigen, possibly derived from autologous melanomas, and that cytotoxic responses to the peptide may be recalled in vi tro . The antigen present in MCV cells may further enhance the memory of PBMC's in contrast to PBMC's from normal donors, which may not be sensitized with the antigen, indicating that melanomas may present the antigen.

EXAMPLE 5 In Vi tro Restimulation With Peptide #707

PMBC restimulation in vi tro with polypeptide #707, SEQ ID NO: 13 was investigated to determine the PBMC responses, especially the cytotoxic responses in vaccinated melanoma patients. The cytotoxicity against autologous peptide-pulsed BCL was greatly enhanced by in vi tro restimulation of PBMC with the peptide (FIG. 3A) . This cytotoxicity was specific to peptide #707 (SEQ ID

NO: 13) and different from that of non-specific NK cells, since cytolysis of K562 (NK target) or autologous BCL without a peptide was not enhanced. Lysis of autologous BCL without a peptide did not exceed 8% at E:T of 80:1 by either effector. Lyses of K562 by #707-restimulated PBMC and control at E:T of 80:1 were ~ 14% and 25%, respectively. Data are representative of four separate experiments .

The C-terminal Ala-Pro amino acid sequence of the peptide again seemed to be necessary for the recognition. In order to evaluate the requirement of other N-terminal residues, a series of C-terminal Ala-Pro-containing peptides, a decamer, octamer, hexamer, tetramer and dimer, were compared for their ability to induce lysis of autologous BCL by #707-restimulated effectors (FIG. 3B) . #707 (-AP) (SEQ ID NO: 12) was used as a negative control. The most efficient peptide was the decamer (SEQ ID NO:13) , which induced half maximal lysis (30% at E:T of 40:1) of BCL at O.OlμM. The octamer was a little less potent (30% lysis at 0.025μM) . Variation in the length of N-terminal residues was associated with negative shifts in the dose-response curve. The hexamer and tetramer were less efficient (30% lysis at 0.1 and 0.7μM, respectively) . The dimer Ala-Pro had a detectable effect at only at a very high concentrations (30% lysis at

>100μM) . Lysis of BCL without a peptide was 1% and lysis of BCL with dipeptides other than AP (AV, GP or PA) did not exceed 5% at concentrations up to 500μM. Thus the original decamer (SEQ ID NO: 13) proved to be the most effective among C-terminal Ala-Pro-containing peptides. This decapeptide was used in the subsequent assays described below.

EXAMPLE 6 Cytotoxicity Against Autologous Melanomas by #707-Restimulated PBMC

To determine whether in vi tro #707-restimulation also enhances cytotoxicity against autologous melanomas, PBMC from vaccinated patients A, B and C were examined. These patients were selected because their melanomas showed #707 mRNA expression in in si tu hybridization and because #707-restimulation of their PBMC increased cytotoxic responses to #707. In all three patients, in vi ro #707-restimulation simultaneously enhanced cytotoxicity against autologous melanomas (FIG. 4) . In a high responder, patient A, the cytotoxicity against autologous melanomas was almost at the same level as that against #707-pulsed BCL. In the less responsive patients, B and C, however, cytolysis of autologous melanomas was less impressive but significant. This cross-reactive cytotoxicity of #707- restimulated PBMC indicates that the epitope of peptide #707 may be presented on the surface of autologous melanomas in a manner similar to that on #707-pulsed BCL. This possibility was examined by cold-target inhibition tests (FIG. 5A- FIG. 5B) . Cold autologous melanomas inhibited lysis of autologous #707-pulsed BCL, although less efficiently than cold #707-pulsed BCL (FIG. 5A) . Conversely, cold autologous #707-pulsed BCL completely blocked lysis of autologous melanomas (FIG. 5B) . This suggests that the epitope identical to or cross-reactive with #707 may be presented on the surface of melanoma cells and may be recognized as a target of cytolysis .

EXAMPLE 7

Antibody Inhibition of Cytolysis

In order to identify the effectors and the mechanism of cytolysis, antibody inhibition assays were performed (FIG. 6A- FIG. 6B) . Lysis of both autologous #707-pulsed BCL and melanoma targets was completely blocked by anti- MHC class I, CD3 and CD8 monoclonal antibodies; the lysis was partially, but significantly inhibited by anti-#707 antibody L94 ; but it was not inhibited by anti-human histocompatibility leukocyte antigen (HLA-DR) and CΩ.4 antibodies (FIG. 6A- FIG. 6B) . This indicates that both targets may be lysed by CD8+ CTL in a class I-restricted manner and that #707-induced CTL may recognize #707 antigen, which is associated with a class I MHC molecule on a melanoma.

EXAMPLE 8 Use of the cDNA to Isolate the Full Length Gene

Full-length peptide analysis of the antigenic protein is important in order to evaluate its biological function and pathogenic significance for the disease. The sequences of the cDNA's disclosed herein, and particularly clone #707 are useful as primers or probes to isolate the full length gene encoding the melanoma associated antigen. The techniques to accomplish the isolation of the full length gene are well known in the art .

For example, a genomic library could be constructed by well known techniques and screened with the cDNA clones. The library would be separated, for example by polyacrylamide gel electrophoresis, or agarose gel electrophoresis and then transferred to a filter such as a nitrocellulose filter. The clones would then be labeled with ³ P by enzymatic labelling with polynucleotide kinase, for example. The clone could also be radioactively labeled by nick translation or in a polymerase chain reaction that included radiolabeled nucleotides. Alternatively, the probe could be labeled with a fluorescent marker such as biotin or any fluorophore. Such labelling techniques are well known in the art .

The labeled probe would then be hybridized to the denatured DNA on the filter and washed under increasingly stringent conditions, incrementally higher temperatures for example, until the positive clones can be identified by the difference in intensity of autoradiography or fluorescence compared to the negative clones. These positive clones would then be rescreened and sequenced to determine the full gene sequence encoding the antigen. This process would be repeated until the full length gene was isolated.

The full protein could then be expressed in an E. coli cell, for example, and used for further analysis. It is understood that the protein could also be truncated or altered by site directed mutagenesis, for example and that such altered proteins or partial sequences would also fall within the scope of the present invention.

EXAMPLE 9 A Peptide Vaccine

A peptide which comprises as part of its amino acid sequence, a sequence immunoreactive with HuMAb L94 may be clinically very important as an effective vaccine in inducing anti-tumor humoral and cell-mediated immune responses in cancer patients. The inventors' results have shown that such peptides have the ability not only to stimulate the proliferation of lymphocytes of melanoma patients who received melanoma cell vaccine, but also to induce cytotoxic T cells in vi tro against autologous melanoma cells.

As is well known in the art, a given polypeptide may vary in its immunogenicity. It is often necessary therefore to couple the immunogen (e.g., a polypeptide of the present invention) with a carrier. Exemplary and' preferred carriers are keyhole limpet hemocyanin (KLH) and human serum albumin. Other carriers may include a variety of lymphokines and adjuvants such as INF, IL2 , IL4 , IL8 and others.

Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine. It is also understood that the peptide may be conjugated to a protein by genetic engineering techniques that are well known in the art. As is also well known in the art, immunogenicity to a particular immunogen can be enhanced by the use of non^¬ specific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete BCG, Detox, (RIBI, Immunochem Research Inc.) ISCOMS and aluminum hydroxide adjuvant (Superphos, Biosector) .

The preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4.578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, .or adjuvants which enhance the effectiveness of the vaccines.

The peptide may be formulated into the vaccine in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccine will be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e . g. , the capacity of the individual's immune system to respond. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application in a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the determination of the practitioner for each particular patient.

EXAMPLE 10 Diagnostic Immunoassays and Skin Tests

Immunoassays that measure the degree of specific humoral and cellular immune responses in patients during active, specific immunotherapy can be developed in light of the present disclosure. The peptides and protein of this invention can be evaluated for their in vivo immunogenicity and therapeutic efficacy in immunotherapy. A variety of humoral and cell-mediated assays have been developed to define the system with the greatest ability to predict clinical responses. The humoral assays include the immune adherence, membrane immunofluorescence, FACS analysis, ELISA, and radioimmunoassays.

In light of the present disclosure, the synthetic peptide antigens may be used to establish more accurate serologic assay systems and skin tests that have the ability to predict immune and clinical responses induced by these newly discovered antigens.

Of particular interest to the present disclosure is a diagnostic skin test for the presence of cancer and preferably for melanoma using the peptide antigens disclosed herein. The techniques of diagnostic skin tests are well known in the art as explained in Remington ' s Pharmaceu tical Sciences , 18th Ed., Mack

Publishing Company, Easton, PA, (incorporated herein by reference) . In such a test, an antigenic preparation would be injected intradermally into a subject, and the subject would then be monitored for hypersensitivity after a period of 48 to 96 hours. A palpable induration of from 2-5 mm may be indicative of a positive reaction.

While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT:

(A) NAME: John Wayne Cancer Institute

(B) STREET: 2200 Santa Monica Blvd.

(C) CITY: Santa Monica

(D) STATE: CA (E) COUNTRY : USA

(F) POSTAL (ZIP) CODE: 90404

(ii) INVENTORS: IRIE, Reiko F.

KIKUMOTO, Yoshikazu

(iii) TITLE OF INVENTION : Tumor Associated Polypeptides with Carboxyl-Terminal Alanine-Proline, Proline-Proline or Glycine-Proline and Antibodies Thereto

(iv) NUMBER OF SEQUENCES: 18

(v) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Arnold, White & Durkee (B) STREET: P. 0. Box 4433

(C) CITY: Houston

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(B) TYPE: nucleic acid 20 (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

25. GAATTCGCGG CCGCGCCTTG A 21

(2) INFORMATION FOR SEQ ID NO:3 :

30 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

35

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : GAATTCGCGG CCGCGCCTCC AGCTCCATGA 30

40

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs 45 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

50 GAATTCGCGG CCGCTGAAGG TGCTCCATGA 30 (2) INFORMATION FOR SEQ ID NO: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 138 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

10

GAATTCGCGG CCGCCTTTGC TTCCTCAGGA GTTCAGATGT GTTCTAAGCC TGCTGGAGTG 60

ACCACACTTC CAAGACCTGA TGGAGGCCAG AGCTCAGAGT GGCAACGGGT CGCAGCCCTT 120 15 GCTCGAGACG CCCCGTGA 138

(2) INFORMATION FOR SEQ ID NO: 6 : '

20 (i) SEQUENCE CHARACTERISTICS: ι

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

25

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

(2) INFORMATION FOR SEQ ID NO:7 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

Glu Phe Ala Ala Ala Pro Pro Ala Pro 1 5

(2) INFORMATION FOR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

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

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 Glu Phe Ala Ala Ala Glu Gly Ala Pro 1 5

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 :

10

Glu Phe Ala Ala Ala Phe Ala Ser Ser Gly Val Gin Met Cys Ser Lys 1 5 10 15

Pro Ala Gly Val Thr Thr Leu Pro Arg Pro Asp Gly Gly Gin Ser Ser

15 20 25 30 σ so.

Glu Trp Gin Arg Val Ala Ala Leu Ala Arg Asp Ala Pro 35 40 45

20

[ 2 ) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids 25 (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

30 Glu Phe Ala Ala Ala

(2) INFORMATION FOR SEQ ID NO: 11

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

10

Val Thr Thr Leu Pro Arg Pro Asp Gly Gly Gin Ser Ser Glu Trp Gin 1 5 10 15

Gin Arg Val Ala Ala Leu Ala Arg Asp Ala Pro

15 20 25

I

(2) INFORMATION FOR SEQ ID NO: 12:

20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

25

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Arg Val Ala Ala Leu Ala Arg Asp

1 5

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Arg Val Ala Ala Leu Ala Arg Asp Ala Pro 1 5 10

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14

Ala Ala Leu Ala Arg Asp Ala Pro 1 5

\ 2 ) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15

Leu Ala Arg Asp Ala Pro 1 5

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Arg Asp Ala Pro

1

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17

Val Pro Leu Phe Pro 1 5

INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18

Val Pro Leu Ala Pro 1 5

Claims

CLAIMS :

1. A purified antibody prepared against a polypeptide having an amino acid sequence comprising alanine-proline, glycine-proline or proline-proline in the C-terminal position, wherein the C terminus of the polypeptide is recognized by said antibody.

2. The antibody of claim 1 wherein said antibody is further defined as a monoclonal antibody.

3. A purified peptide comprising an amino acid sequence having as its C-terminal sequence SEQ ID NO: 6 , SEQ ID - NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

4. A purified tumor associated antigen having a C- terminal dipeptide sequence of alanine-proline, glycine- proline or proline-proline and encoded by a nucleic acid sequence hybridizable to SEQ ID NO: 5 or its complement under high stringency conditions.

5. The peptide of claim 3, further defined as comprising a polypeptide linked to the N-terminal of said peptide .

6. The peptide of claim 5, wherein said polypeptide is -galactosidase or glutathione-S-transferase.

7. The peptide of claim 5, wherein said polypeptide is joined to said peptide by a peptide linker.

8. The peptide of claim 3, further defined as comprising a T cell receptor antigen.

9. An isolated segment of DNA encoding a peptide in accordance with claim 4.

10. The DNA segment of claim 9, further defined as comprising SEQ ID NO: 5 or its complement.

- 11. The DNA segment of claim 9, further defined as a DNA vector.

12. The vector of claim 11, further defined as an expression vector capable of expressing a peptide of claim 4 in a host cell .

13. A recombinant cell expressing a peptide in accordance with claim 4.

14. The recombinant cell of claim 13 further defined as an E. coli cell .

15. The recombinant cell of claim 13, further defined as containing the vector of claim 11.

16. The recombinant cell of claim 13, further defined as a human melanoma cell.

17. A method of purifying a polypeptide having a carboxy terminal alanine-proline, glycine-proline or proline- proline from a mixture comprising the steps of:

contacting said mixture with the antibody of claim 1 under conditions effective for immunoreactive binding of the antibody and the polypeptide; and

separating the antibody from the mixture

18. The method of claim 17, further comprising the step of separating the bound polypeptide from the antibody.

19. The method of claim 17 wherein said antibody is bound to a solid matrix.

20. The method of claim 19 wherein said matrix is agarose beads .

21. A polypeptide obtained by the method of claim 17

22. An antigen composition comprising a peptide in accordance with claim 3 or claim 4 , in an amount effective to elicit an immune response.

23. The antigen composition of claim 22 wherein the immune response is a CTL response.

24. The antigen composition of claim 22 wherein the peptide is present at a concentration of between 1 mg/ml and about 10 mg/ml.

25. The antigen composition of claim 22 wherein the peptide is present at a concentration of about 5 mg/ml

26. The antigen composition of claim 22 wherein said peptide is a component of a polyvalent tumor cell vaccine.

27. A method of treating a human cancer patient comprising administering the antigen composition of claim 22 in conjunction with a cancer whole cell vaccine therapy.

28. The method of claim 27 wherein said human cancer patient is a human melanoma patient.

29. A method of enhancing an immune response comprising contacting immune system cells with an effective amount of an antigenic composition comprising a peptide having the C-terminal sequence alanine-proline.

30. The method of claim 29, wherein an effective amount of the antigenic composition is administered to a subject .

31. The method of claim 29, wherein the method is performed ex vivo, and comprises the steps of:

obtaining cytotoxic lymphocytes from a subject;

contacting said cytotoxic lymphocytes with the antigenic composition; and

reintroducing said lymphocytes into said subject

32. The method of claim 31 wherein said subject is a human cancer patient.

33. The method of claim 32 wherein said patient is a human melanoma patient .

34. The method of claim 31 wherein said lymphocytes- are obtained from the serum of said patient .

35. The method of claim 31 wherein said lymphocytes are peripheral blood lymphocytes.

36. The method of claim 31, further comprising whole tumor cell vaccine therapy.

37. A method of purifying a polypeptide from a mixture comprising the steps of :

causing said polypeptide to have the C-terminal amino acid sequence alanine-proline, glycine- proline or proline-proline;

separating the antibody from the mixture.

38. The method of claim 37, further comprising the step of separating the bound polypeptide from the antibody.

39. The method of claim 37 wherein said antibody is bound to a solid matrix.

40. The method of claim 39 wherein said matrix is agarose beads.

41. A method of diagnosing cancer in a subject comprising detecting the presence of a tumor associated antigen having a carboxy terminal alanine-proline, glycine-proline or proline-proline in said subject.

42. The method of claim 41 further defined as comprising the steps of: injecting an antigenic composition of claim 22 intradermally into a subject suspected of having cancer; and

monitoring said subject for 48 to 96 hours to detect the presence of an induration at the site of injection;

wherein an induration of 2-5 mm is indicative of a positive reaction.