WO2001075179A2

WO2001075179A2 - Preprocalcitonin as tumor rejection antigen precursor and uses thereof

Info

Publication number: WO2001075179A2
Application number: PCT/US2001/040288
Authority: WO
Inventors: Hamid Echchakir; Fathia Mami-Chouaib; Isabelle Vergnon; Salem Chouaib; Jean François BAURAIN; Pierre G. Coulie; Thierry Boon-Falleur
Original assignee: Ludwig Institute For Cancer Research; Institut National De La Sante Et De Recherche Medicale; Institut Gustave Roussy
Priority date: 2000-03-31
Filing date: 2001-03-14
Publication date: 2001-10-11
Also published as: AU2001253852A1; WO2001075179A3; EP1301624A2

Abstract

The invention relates to the identification of a tumor rejection antigen precursor. It has been established that preprocalcitonin protein functions as a tumor rejection antigen precursor, and is processed to at least one tumor rejection antigen. Complexes of the tumor rejection antigen and an MHC molecule are recognized by cytolytic T lymphocytes, and lysed.

Description

PREPOCALCITONIN AS TUMOR REJECTION ANTIGEN PRECURSOR

AND USES THEREOF

FIELD OF THE INVENTION

This invention relates to various therapeutic methodologies derived from the recognition that certain abnormal cells present complexes of human leukocyte antigens and peptides derived from preprocalcitonin protein on their surfaces. In addition, it relates to the ability to identify those individuals diagnosed with conditions characterized by cellular abnormalities whose abnormal cells present this complex, the presented peptides, and the ramifications thereof.

BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one. An important facet of the system is the T cell response. This response requires that T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens ("HLA"), or major histocompatibility complexes ("MHCs"), and peptides. The peptides are derived from large molecules which are processed by the cells which also present the HLA/MHC molecule. See in this regard Male et al., Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters 6-10. The interaction of T cell and complexes of HLA/peptide is restricted, requiring a T cell specific for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. This mechanism is involved in the immune system's response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities. Recently, much work has focused on the mechanisms by which proteins are processed into the HLA binding peptides. See, in this regard, Barinaga, Science 257:880 (1992); Fremont et al., Science 257:919 (1992); Matsumura et al., Science 257:927 (1992); Latron et al., Science 257:964 (1992). The mechanism by which T cells recognize cellular abnormalities has also been implicated in cancer. For example, in PCT application PCT US92/04354, filed May 22, 1992, published on Nov. 26, 1992, and incorporated by reference, a family of genes is disclosed, which are processed into peptides which, in turn, are expressed on cell surfaces, which can lead to lysis of the tumor cells by specific CTLs. The genes are said to code for "tumor rejection antigen precursors" or "TRAP" molecules, and the peptides derived therefrom are referred to as "tumor rejection antigens" or "TRAs". See Traversari et al., Immunogenetics 35:145 (1992); van der Bruggen et al., Science 254:1643 (1991), for further information on this family of genes.

In U.S. Pat. No. 5,405,940 the disclosure of which is incorporated by reference, nonapeptides are taught which bind to the HLA-A1 molecule. The reference teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. As a result, the fundamental discoveries made by Boon, van der Eynde, van der Bruggen, et al, have been extended to other MHC systems. In this regard, see, e.g., U.S. Patent Nos. 6,034,214; 6,013,765; 6,013,481; 5,997,870; 5,952,228; 5,939,526; 5,928,938 and others. Also see, e.g., U.S. Patent No. 6,037,135.

The work by Boon et al. has focused, in part, on the identification and isolation of genes which were not recognized previously. Additional work has discovered that known molecules can function as tumor rejection antigen precursors, or "TRAPs", as described in many of the patents cited supra as well as in U.S. Patent No. 5,342,774, which is incorporated by reference, as are all of the patents listed supra.

One of the first molecules to be recognized as functioning as a tumor rejection antigen precursor was the enzyme tyrosinase. See, e.g., U.S. Patent No. 5,487,974. Others include the molecule referred to in the patent literature as "HOM-MEL 40," which was found to be identical to the known molecule SSX-2, and U.S. Patent No. 5,888,751, which teaches that the protein known as SCP-1 functions as a tumor rejection antigen precursor.

The lion's share of this work has used melanoma as the primary material for identifying tumor rejection antigen precursors. The inventors have identified an additional tumor rejection antigen precursor from lung cancer cells. This "TRAP" has been identified as the preprocalcitonin protein. This discovery, and the ramifications thereof, constitute the invention, as will be seen in the disclosure which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

EXAMPLE 1

A large cell lung carcinoma was taken from a patient who had been typed as HLA-A2, A68, B7, B35, C4, C7 positive. The cells were cultured under standard conditions, and a cell line, referred to hereafter as "IGR-Heu" was derived. This is a non-small cell lung carcinoma ("NSCLC") cell line. This line is used in examples presented infra.

EXAMPLE 2

This example details the isolation of cytolytic T cell line "Heu 161 CTL," also used in the examples which follow.

A fresh tumor sample was taken from the same patient referred to supra, and was dissociated in DMEM medium containing ImM Hepes, 0.3U/ml DNAse, 0.5 U/ml collagenase, and 0.28 U/ml hyaluronidase. The suspension was frozen, and then thawed.

Following thawing, viable tumor infiltrating lymphocytes and tumor cells were isolated from the suspension, using standard techniques (Ficoll-Hypaque density gradient centrifugation). Tumor infiltrating lymphocytes were seeded at 10⁴ cells/microwell, and then stimulated by adding irradiated, autologous tumor cells (3xl0³ cells/well, 10000 rads), as well as irradiated, autologous EBV transformed B cells (4xl0⁴cells/well), in RPMI supplemented with 10% human AB serum, and recombinant IL-2. Cells were fed every 3 days with new medium and recombinant IL-2, and were restimulated every other week with irradiated tumor and EBV transformed B cells, as described supra. After 6 weeks, the lymphocytes were cloned via limiting dilution, and were stimulated with cells as described supra, in DMEM medium containing rIL-2 (100 U/ml) and 3% (v/v) of conditioned medium from PHA activated blood lymphocytes. One of the resulting T cell cloness was clone "161". All clones were amplified with the restimulation protocol supra, every week. The ability of clone 161 to lyse tumor cells was tested, and was confirmed. In additional experiments, it was determined that CTL 161 recognized complexes of a peptide and an HLA-A2 molecule. This was determined by combining cells of clone 161 with autologous tumor cells, and monoclonal antibodies specific for HLA-A2. The mAbs abrogated the ability of CTL 161 to lyse target cells, indicating that the presenting molecule was HLA-A2.

EXAMPLE 3

This example describes the isolation of the DNA which encoded the protein processed to the antigen recognized by CTL 161.

About 3μg of poly (A)⁺RNA was isolated from the cell clone described in example 1, supra. This RNA was converted to cDNA, using oligo(dT) primer:

5^' - ATAAGAATGCGGCCGCTAAACTA(T)₁₈VN (SEQ ID NO: 1).

This primer contains a Notl site at its 5^' end. The degenerate 3^' end of this primer favors annealing at the 5^' end of the polyA tail of mRNA molecules, thus diminishing the proportion of cDNA clones containing long poly (A) stretches.

The resulting cDNA was ligated to Hindlll - EcoRI adaptors, phosophorylated, digested with Notl, and litgated to plasmid pCEP4. This plasmid contains EBV origin of replication oriP, as well as a sequence that encodes EBV nuclear antigen -1 (EBNA-1). This allows for episomal replication of plasmids which are transfected into mammalian clones.

The recombinant plasmids were electroporated into E. coli DH5α, and selected with 50μg/ml of ampicillin.

The resulting library was divided into 264 pools of about 100 cDNA clones, each of which was treated by amplifying and extracting the plasmid DNA.

Following this, human embryonic kidney cells which had been stably transfected with an EBNAl construct ("293-EBNA" cells) were cofransfected with an HLA-A2 construct, and the plasmid DNA. In brief, 5-7xl0⁴ 293-EBNA cells were seeded in flat bottomed microwells, and combined with lOOng of plasmid DNA from the library described supra. 50ng of expression vector pcDNAI/Amp containing an HLA-A*0201 cDNA clone, and 1.3 μl of lipofectamine. Transfections were carried out in duplicate. After 24 hours, 3000 cells of CTL 161 were added to each well, and 24 hours later, half of the medium was collected, and TNF content was measured by testing the cytotoxic effect of the medium on WEHI- 164c 13 cells in an MTT colorimetric assay, which is described by Hansen, et al, J. Immunol Meth 119:203-210 (1989), incorporated by reference.

Eighty five pools were positive. One pool was subcloned, and one clone, referred to as "cDNA clone 150," was found to transfer expression of the relevant antigen into 293- EBNA cells. This clone was analyzed further.

EXAMPLE 4

The sequence of the cDNA clone referred to supra was secured following standard methods. Comparison to GENBANK revealed that the 956 base pair sequence is transcribed from the calcitonin /α-CGRP gene, identified in GENBANK as XI 5943. The 956 base sequence, SEQ ID NO: 2 herein, contains a polyadenylation signal and a polyA sequence at its 3' end.

The calcitonin/α CGRP gene encodes calcitonin, a calcium lowering hormone, as well as the neuropeptide α-CGRP. See Broad, et al, Nucl. Acids Res. 17:6999-7011 (1989). Both of these molecules are processed from precursor proteins encoded by the calcitonin / α CGRP mRNA. The transcripts are both generated from a common primary transcript, via tissue specific, alternative RNA processing events. See, Amara, et al, Nature 298: 240-244 (1982); Jonas, et al, Proc. Natl. Acad. Sci USA 82:1994-1998 (1985). The α CGRP molecule is encoded by a transcript that contains exons 1, 2, 3, 5 and 6, while calcitonin is encoded by a transcript containing exons 1-4.

Analysis of SEQ ID NO:2 shows that nucleotidesl and 2 are of unknown origin. Nucleotides 3-216 are a portion of intron 1, i.e. nucleotides 2085-2298 of X15943, with the exception of one change at position 13 of SEQ ID NO:2. This is T, whereas position 2095, the corresponding base in XI 5943, is A.

Nucleotides 217-311 correspond to exon 2 of X15943, nucleotides 312-452 to exon 3 of X15943, and nucleotides 453-947 to exon 4 of X15943. EXAMPLE 5

These experiments detail identification of the region of SEQ ID NO:2 which encodes the antigenic peptide, i.e. the HLA-A2 binding partner that is recognized by the CTLs. To do this, a forward primer, i.e.:

ggtgtcatgg gcttccaaaa gt (SEQ ID NO:3) located at the 5' end of SEQ ID NO:2, and reverse primers, ctgaatggtg ctgcatgg ag (SEQ ID NO:4) gcactagtc ctgcaccag (SEQ ID NO: 5) gcaagtactc agattaccgc ac (SEQ ID NO: 6) tcgctggaca atatccctttt c (SEQ ID NO:7) and gatcagcaca ttcagaagca gg (SEQ ID NO: 8) which are located at the 3' end, were used to generate PCR fragments, or "mini-genes," consisting of a portion of the nucleotide sequence of SEQ ID NO: 2, i.e., the portion including the primers and located in between them.. SEQ ID NO: 4 and one of SEQ ID NOS:5-8 were paired in a PCR reaction, which involved 3 minutes of incubation at 94°C, followed by 30 cycles of amplification, with a cycle being defined as 1 minute at 94°C, 2 minutes at 65°C, and 3 minutes at 72°C, followed by final elongation for 10 minutes at 72°C.

Template plasmids were diluted by diluting PCR products 100,000 fold, after which aliquots were used as templates for additional PCR amplification (again, 30 cycles). These PCR products were cloned into expression plasmid pcDNA3.1, using standard methods. The plasmids were then cofransfected, together with HLA-A2 cDNA clones as described supra, into 293-EBNA cells, using lypofectamine.

CTL 161, described supra, was added to the transfectants in the same manner as is described above, and TNF release was measured.

The results indicated that the presented peptide was encoded by a region which corresponds to nucleotides 292-403 of SEQ ID NO: 2. This is a region that is common to both calcitonin and α-CGRP mRNA. EXAMPLE 6

These experiments detail work on expression of the construct of SEQ ID NO:2 in tumor samples. Calcitonin is normally expressed by the C cells of the thyroid, and at high levels in medullary thyroid carcinoma. (Edbrooke, et al, EMBO J 4:715-724 (1985)). It is also expressed ectopically in lung carcinoma (Edbrooke, et al, supra; Kelley, et al Cancer Res 81:19-25 (1994)). CGRP is expressed in neural cells (Rosenfeld, et al Science 225: 1315- 1320 (1984)). Expression was determined by using primers: ggtgtcatgg gcttccaaaa gt (SEQ ID NO: 9) and tcctgcttct gaatgtgctg at (SEQ ID NO: 10) in a mixture which also contained 15.75 μl H₂O, 2.5 μl of DNA polymerase buffer, 2 μl of dNTPs (10M each), 1 μl of each primer, 0.25 μl of Taq polymerase, and 2.5μl of cDNA, which have been derived from 50ng of total RNA. PCR was carried out for 5 minutes at 94°C, followed by 30 cycles, where a cycle is defined as 1 minute at 94°C, 2 minutes at 63°C, and 2 minutes at 72°C, followed by an elongation step of 10 minutes at 72°C. Amplified DNA was assessed visually via agarose gels and ethidium bromide staining. The results are summarized as follows:

Tumor Tumor

Samples Cell Lines

Non-small cell lung carcinoma

squamous carcinoma 7/22 adenocarcinoma 10/61 undifferentiated carcinoma 2/8 large cell carcinoma 1/3

Small cell lung cancer 3/5 4/23

Neuroendocrine tumors 3/6

Bronchioalveolar carcinoma 1/4

EXAMPLE 7 The peptide encoded by nucleic acids 292-403 of SEQ ID NO: 2 has amino acid sequence:

LHAAPFRSALESSPADPATLSEDEARLLLAALVQDYV (SEQ ID NO: 11). This sequence was screened using NTH algorithm "HLA binding predictions" found at http://bimas.drct.nih.gov/molbio/hla_bind/ which is based upon Parker, et al., J. Immunol 152: 163 (1994), both of winch are incorporated by reference. See Rammensee, "A Database of MHC Ligands And Peptide Motifs" at http://l 1342.296.221/ also incorporated by reference as well, to identify potential HLA binding peptides. These are set forth infra, with reference to the amino acids of SEQ ID NO: 11.:

PREDICTED HLA-A2 BINDERS FROM AMINO ACID SEQUENCE ENCODED BY NUCLEOTIDES 292-403 OF SEQ ID NO:2:

Position Peptide

19-27 TLSEDEARL

29-37 LAALVQDYV

21-29 SEDEARLLL

28-36 LLAALVQDY

27-35 LLLAALVQD

12-20 SSPADPATL

10-18 LESSPADPA

2-10 HAAPFRSAL

25-33 ARLLLAALV

7-15 RSALESSPA

26-34 RLLLAALVQ

23-31 DEARLLLAA

20-28 LSEDEARLL

24-32 EARLLLAAL

11-19 ESSPADPAT Position Peptide

8-16 SALESSPAD

1-9 LHAAPFRSA

13-21 SPADPATLS

4-12 APFRSALES

9-17 ALESSPADP

28-36 LLAALNQDYV

19-28 TLSEDEARLL

18-3.6 ATLSEDEARL

27-35 LLLAALVQDY

21-29 SEDEARLLLA

10-19 LESSPADPAT

9-17 ALESSPADPA

26-35 RLLLAALVQD

1-10 LHAAPFRSAL

23-32 DEARLLLAAL

20-29 LSEDEARLLL

11-20 ESSPADPATL

24-33 EARLLLAALV

6-14 FRSALESSPA

8-17 SALESSPADP

4-12 APFRSALESS

16-24 DPATLSEDEA

3-11 AAPFRSALES

These peptides can be tested for binding to HLA molecules and stimulation of CTLs, in accordance with, e.g., Parker, et al, J., Immunol 152:163, and Ruppert, et al., Cell 74: 929- 937 (1993), incorporated by reference. Also see Van derBruggen, et al, Euro. J. Immunol 24: 3038-3043 (1994, incorporated by reference for an alternate way to do this. Also, it is known that alternate reading frames exist for tumor rejection antigen precursors, leading to the generation of entirely new famielies of relevant tumor rejection antigens. Se, e.g., Wang, et al., J. Immunol 161(7): 3598-606 91998)? Robbins, et al, j. Immunol 159(1): 303-308 (1997); Couli, et al, Proc. Natl. Acad Sci USA 92(17): 7976-80 (1995), all incorporated by reference.

EXAMPLE 7

Similarly, the amino acid sequences for calcitonin (SEQ ID NO: 12) and CGRP (SEQ ID NO: 13), as described by, e.g., Broad, et al, Nucl. Acids Res. 17(17); 6999-7011 (1989); Amara, et al., Nature 298: 240-44 (1982); Jonas, et al., Proc. Natl. Acad Sci USA 82(7): 1994-8 (1985), all incorporated by reference.

SEQ ID NO: 12

MGFQKFSPFLALSILVLLQAGSLHAAPFRSALESSPADPATLSEDEARLLLAALVQDY

VQMK

ASELEQEQEREGSSLDSPRSKRCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPGKK

RDMSS

DLERDHRPHVSMPQNAN

SEQ ID NO: 13:

MGFQKFSPFLALSILVLLQAGSLHAAPFRSALESSPADPATLSEDEARLLLALVQDYV

QMK

ASELEQEQEREGSRIIAQKRACDTATCVTHRLAGLLSRSGGVVK-NNFVPTNVGSKAF

GRRRRDLQA were screened, leading to the following:

Predicted HLA binding Peptides encoded by both calcitonin and CGRP

HLA Position Peptide

HLA-A1 42-50 LSEDEARLL HLA Position Peptide

63-72 ASELEQEQER

42-51 LSEDEARLLL

31-39 ALESSPADPA

HLA-A2 9-17 FLALSILVL

58-66 YVQMKASEL

17-25 LLQAGSLHA

53-61 ALVQDYVQM

10-18 LALSILVLL

18-26 LQAGSLHAA

15-23 LVLLQAGSL

7-15 SPFLALSIL

3-11 FQKFSPFLA

16-24 VLLQAGSLH

22-30 SLHAAPFRS

55-63 VQDYVQMKA

9-18 FLALSILVLL

16-25 VLLQAGSLMA

17-26 LLQAGSLHAA

1-10 MGFQKFSPFL

11-20 ALSILVLLQA

14-23 ILNLLQAGSL

54-63 LVQDYVQMKA

22-31 SLHAAPFRSA HLA Position Peptide

7-16 SPFLALSILV

3-12 FQKFSPFLAL

6-14 FSPFLALSIL

HLA-A3 54-62 LVQDYVQMK

50-58 LLAALVQDY

9-17 FLALSILVL

41-49 TLSEDEARL

17-25 LLQAGSLHA

53-62 ALVQDYVQMK

49-58 LLLAALVQDY

9-18 FLALSILVLL

11-20 ALSILVLLQA

16-25 VLLQAGSLHA

HLA-A24 2-10 GFQKFSPFL

10-18 LALSILVLL

42-50 LSEDEARLL

58-66 YVQMKASEL

34-42 SSPADPATL

15-23 LVLLQAGSL

46-54 EARLLLAAL

57-65 DYVQMKASEL

5-13 KFSPFLALSI

42-51 LSEDEARLLL Position Peptide

14-22 ILVLLQAGSL

6-14 FSPFLALSIL

40-48 ATLSEDEARL

9-17 FLALSILVLL

3-11 FQKFSPFLAL

46-54 EARLLLAAL

7-15 SPFLALSIL

15-23 LVLLQAGSL

58-66 YVQMKASEL

24-32 HAAPFRSAL

10-18 LALSILVLL

40-48 ATLSEDEARL

52-60 AALVQDYVQM

46-54 EARLLLAALV

3-11 FQKFSPFLAL

46-54 EARLLLAAL

58-66 YVQMKASEL

46-55 EARLLLAALV

3-11 FQKFSPFLAL

7-15 SPFLALSIL

46-54 EARLLLAAL HLA Position Peptide

34-42 SSPADPATL

52-60 AALVQDYVQM

33-41 ESSPADPATL

6-14 FSPFLALSIL

Predicted HLA binding peptides unique to calcitonin

HLA Position . Peptide

HLA-A1 123-131 SSDLERDHR

76-84 SLDSPRSKR

94-102 GTYTQDFNK

88-96 LSTCMLGTY

104-113 HTFPQTAIGV

119-128 KRDMSSDLER

HLA-A2 87-95 NLSTCMLGT

97-105 TQDFNKFHT

126-134 LERDHRPHV

89-97 STCMLGTYT

96-104 YTQDFNKFH

96-105 YTQDFNKFHT

76-84 SLDSPRSKRC

68-77 QEQEREGSSL

88-97 LSTCMLGTYT

86-95 GNLSTCMLGT HLA Position Peptide

HLA-A3 94-102 GTYTQDFNK

92-100 MLGTYTQDF

76-84 SLDSPRSKR

110-118 AIGVGAPGK

112-120 GVGAPGKKR

91-100 CMLGTYTQDF

94-103 GTYTQDFNKF

87-96 NLSTCMLGTY

110-119 AIGVGAPGKK

109-118 TAIGVGAPGK

HLA-A24 95-103 TYTQDFNKF

85-93 CGNLSTCML

69-77 EQEREGSSL

102-111 KFHTFPQTAI

84-93 RCGNLSTCML

HLA-B7 79-88 SPRSKRCGNL

HLA-B8 79-87 SPRSKRCGN

114-122 GAPGKKRDM

79-88 SPRSKRCGNL HLA Position Peptide

HLA-B35 88-96 LSTCMLGTY

79-87 SPRSKRCGN

79-88 SPRSKRCGNL

131-140 RPHVSMPQNA

Predicted HLA binding peptide unique to CGRP

HLA Position Peptide

HLA-A1 83-91 ACDTATCVT

113-21 NVGSKAFGR

67-76 EQEQEREGSR

HLA-A2 96-104 GLLSRSGGV

97-105 LLSRSGGVV

89-97 CVTHRLAGL

96-105 GLLSRSGGVV

HLA-A3 109-117 FVPTNVGSK

113-121 NVGSKAFGR

97-106 LLSRSGGVVK

HLA-A24 118-126 AFGRRRRDL

117-126 KAFGRRRRDL HLA Position Peptide

100-109 RSGGWKNNF

HLA-B7 89-97 CVTHRLAGL

86-94 TATCVTHRL

89-98 CVTHRLAGLL

117-126 KAFGRRRRDL

HLA-B8 89-97 CVTHRLAGL

96-104 GLLSRSGGV

86-94 TATCVTHRL

88-97 TCVTHRLAGL

117-126 KAFGRRRRDL

119-128 FGRRRRDLQA

HLA-B35 110-119 VPTNVGSKAF

100-109 RSGGVVKNNF

117-126 KAFGRRRRDL

The protocols referred to in example 5, supra, may be used for these peptides as well. All peptides described herein can be used to generate CTLs in vitro, following, e.g., Celis, et al., Proc. Natll Acad Sci 91: 2105-2109 (1994), and Salgaller, et al., Cancer. Immmunol Immunother 39: 105-116 (1994), both of which are incorporated by reference. Collectively, the references cited herein provide information on how to identify by relevant peptides for any MHC molecule. The foregoing examples show that the gene which encodes calcitonin/ α CGRP also functions as a tumor rejection antigen precursor, and is processed to a tumor rejection antigen .It shares properties with molecules like HOM-MEL- 40 and tyrosinase, described supra. Such molecules have known functions, but also function as tumor rejection antigen precursors. Hence, one aspect of the invention pertains to a method for determining cancer in a subject, by assaying a sample taken from the subject for expression of all or apart of the preprocalcitonin protein. As indicated supra, calcitonin is normally expressed by the C-cells of the thyroid, and is also found at high levels in medullary thyroid carcinoma, as well as being expressed ectopically in lung carcinoma. α-CGRP is expressed in neural cells. One of ordinary skill in the art can exclude these types of cells, if necessary, in order to determine if preprocalcitonin, calcitonin per se, or α- CGRP is expressed in the sample. For example, preprocalcitonin is not found in these cells, so expression of the molecules is indicative of cancer.

As indicated supra, both calcitonin and α-CGRP are generated from a common transcript. A transcript comprising at least nucleotides 292-403 of SEQ ID NO: 2 appears to be present in cancer cells, non small cell lung cancer cells in particular. Hence, a further feature of this invention is the identification of nucleic acid molecules which comprise at least nucleotides 292-403 of SEQ ID NO: 2 and as many as all of the nucleotides of SEQ LD NO: 2, or nucleic acid molecules which hybridize thereto, under stringent conditions. One can determine such expression via nucleotide hybridization and/or amplification assays, using oligonucleotide primers such as, but not being limited to, those set forth supra. Similarly, one can determine expression products, such as a polypeptide expressed by the nucleic acid molecules described supra using, e.g., antibodies specific for said proteins and polypeptides. One can also carry out the assay by using cytolytic T lymphocytes which are specific for complexes of peptides derived from proteins and polypeptides as described supra, and the MHC molecules with which they complex. As described supra, there are many such peptides which satisfy these criteria.

Any one of these methodologies can also be used in progression/regression studies, simply by monitoring levels of the protein, its expression, and so forth using any or all of the methods set forth supra. It should be clear that these methodologies may also be used to track the efficacy of a therapeutic regime. Essentially, one can take a baseline value for the protein, proteins or peptides being tested, using any of the assays discussed supra, administer a given therapeutic agent, and then monitor levels of the "marker" thereafter, observing changes in levels as indicia of the efficacy of the regime.

Regarding the progression/regression studies. One can monitor the course of an abnormality involving expression of the protein simply by monitoring levels of protein, its expression, antibodies against it and so forth using any or all of the methods set forth supra.

It should be clear that these methodologies may also be used to track the efficacy of a therapeutic regime. Essentially, one can take a baseline value for the protein or peptide, using any of the assays discussed supra, administer a given therapeutic agent, and then monitor levels of the protein, using any of the assays discussed supra, administer a given therapeutic agent, and then monitor levels of the protein thereafter, observing changes in the protein or peptide levels as indicia of the efficacy of the regime.

One can monitor these levels using, e.g., tetrameric peptide structures, such as structures based on the disclosures of Braud, et al., Nature 391 795-799 (1998); Airman, et al., Science 274: 94-96 (1996), and U.S. Patent application Serial No. 09/049,850, filed March 27, 1998, all of which are incorporated by reference.

The identification of preprocalcitonin protein as being implicated in pathological conditions such as cancer also suggests a number of therapeutic approaches to such conditions. Antibodies can be produced which bind to the protein, suggesting their use as a vaccine. Hence, a further embodiment of the invention is the treatment of conditions which are characterized by aberrant or abnormal levels of one or more preprocalcitonin proteins or peptides, via immunotherapeutic approaches. One of these approaches is the administration of an amount of protein, or an immunogenic peptide derived from the protein in an amount sufficient to provoke or augment an immune response. The protein or peptide may be combined with one or more of the known immune adjuvants, costimulatory molecules, or MHC helper binding peptides, such as saponins, GM-CSF, interleukins, LIF-3, emulsifying oils such as vitamin E, heat shock proteins and so forth. If the peptides are too small to generate a sufficient antibody response, they can be coupled to well-known conjugates used to stimulate responses.

Similarly, the immunotherapeutic approaches include administering an amount of inhibiting antibodies sufficient to inhibit the protein. These antibodies may be, e.g., antibodies produced via any of the standard approaches elaborated upon supra.

T cell responses may also be elicited by using peptides derived from the proteins which then complex, non-covalently, with MHC molecules, thereby stimulating proliferation of cytolytic T cells against any such complexes in the subject. It is to be noted that the T cells may also be elicited in vitro using immune responsive cells such as dendritic cells, lymphocytes, or any other immune responsive cells, and then reperfused into the subject being treated.

Note that the generation of T cells and/or antibodies can also be accomplished by administering cells, preferably treated to be rendered non-proliferative, which present relevant T cell or B cell epitopes for response.

The therapeutic approaches may also include gene therapies, wherein an antisense molecule, preferably from 10 to 100 nucleotides in length, is administered to the subject either "neat" or in a carrier, such as a liposome, to facilitate incorporation into a cell, followed by inhibition of expression of the protein. Such antisense sequences may also be incorporated into appropriate vaccines, such as in viral vectors (e.g., Vaccinia), bacterial constructs, such as variants of the well-known BCG vaccine, and so forth.

An additional DNA based therapeutic approach is the use of a vector which comprises one or more nucleotides sequences, preferably a plurality of these, each of which encodes an immunoreactive peptide derived from the expressed proteins. One can combine these peptide expressing sequences in all possible variations, such as one from each protein, several from one or more protein and one from each of the additional proteins, a plurality from some and none from others, and so forth.

Other therapeutic approaches include the administration of the preprocalcitonin- 1 proteins per se, one or more antigenic peptides derived therefrom, such as those presented supra, as well as so-called polytopic vaccines. These include a plurality of antigenic peptides, such as those supra, united together, preferably by linker sequences. The resulting peptides may bind to either MHC-Class I or Class II molecules. These proteins, peptides, or polytopic vaccines may be administered in combination with an appropriate adjuvant, costimulatory molecule, or binding helper peptide. They may also be administered in the form of genetic constructs which are designed to permit expression of the protein, the peptide, the polytopic structures, etc. Peptides and polytopic structures can be expressed by so-called "minigenes" i.e., DNA molecules designed to express portions of the entire preprocalcitonin molecule, or the various portions of the molecules, linked together as described supra.

The amount of agent administered and the manner in which it is administered will vary, based on the condition being treated and the individual. Standard forms of administration, such as intravenous, intradermal, subcutaneous, oral, rectal and transdermal administration can be used. With respect to formulations, the proteins and/or peptides may be combined with adjuvant and/or carriers such as a saponin, GM-CSF, one or more interleukin, vitamin E, LIF-3, one or more heat shock protein, etc.

When the nucleic acid approach is utilized, various vectors, such as Vaccinia or adenovirus based vectors can be used. Any vector useful in eukaryotic transfection, such as in transfection of human cells, can be used. These vectors can be used to produce, e.g., cells such as dendritic cells which present relevant peptide/MH complexes on their surface. The cells can then be rendered non-proliferative prior to their administration, using standard methodologies.

Polytopes, as used herein, are groups of two or more potentially immunogenic or immune stimulating peptides, which can be joined together in various ways, to determine if this type of molecule will stimulate and/or provoke an immune response.

These peptides can be joined together directly, or via the use of flanking sequences. See Thompson e tal. Proc. Natl. Acad. Sci USA 92(13): 5845-5849 (1995), teaching the direct linkage of relevant epitopic sequences. The use of polytopes as vaccines is well known. See, e.g., Gilbert et al., Nat Biotechnol. 15(12): 1280-1284 (1997); Thomson et al, supra; Thomson et al., J. Immunol. 157(2): 822-826 (1996); Tam et al., J. Exp. Med. 171(1): 299-306 (1990), all of which are incorporated by reference. The Tam reference in particular shows that polytopes, when used in a mouse model, are useful in generating both antibody and protective immunity. Further, the reference shows that the polytopes, when digested, yield peptides which can be and are presented by MHCs. Tam shows this by showing recognition of individual epitopes processed from polytope 'strings' via CTLs. This approach can be used, e.g., in determining how many epitopes can be joined in a polytope and still provoke recognition and also to determine the efficacy of different combinations of epitopes. Different combinations may be 'tailor-made' for the patients expressing particular subsets of tumor rejection antigens. These polytopes can be introduced as polypeptide structures, or via the use of nucleic acid delivery systems. To elaborate, the art has many different ways available to introduce DNA encoding an individual epitope, or a polytope such as is discussed supra. See, e.g., Allsopp et al., Eur J. Immunol. 26(8); 1951-1959 (1996), incorporated by reference. Adenovirus, pox- virus, Ty- virus like particulars, plasmids, bacteria, etc., can be used. One can test these systems in mouse models to determine which system seems most appropriate for a given, parallel situation in humans. They can also be tested in human clinical trials.

Other features of the invention will be clear to the skilled artisan, and need not be repeated here.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

Claims

WE CLAIM:

1. A method for determining presence of a transformed cell, comprising assaying a sample of cells for expression of a nucleic acid molecule comprising nucleotides 292-403 of SEQ ID NO:2, or a nucleotide sequence homologous thereto, wherein expression thereof is indicative of transformed cells in said sample.

2. The method of claim 1, wherein said nucleic acid molecule encodes preprocalcitonin.

3. The method of claim 1, wherein said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:2.

4. The method fo claim 1, wherein said nucleic acid molecule is mRNA.

5. The method of claim 3, comprising determining presence of mRNA via polymerase chain reaction.

6. The method of claim 1, comprising assaying said sample for a polypeptide which is encoded by said nucleic acid molecule.

7. The method of claim 6, wherein said polypeptide is preprocalcitonin.

8. The method of claim 6, comprising assaying said sample via an immunoassay.

9. The method of claim 1, wherein said transformed cell is a cancer cell.

10. The method of claim 9, wherein said cancer cell is a lung cancer cell.

11. The method of claim 10, wherein said lung cancer cell is a non small cell lung cancer cell.

12 A method for treating a subject afflicted with a disorder characterized by inappropriate amounts of a polypeptide which comprises an amino acid sequence encoded by nucleotides

292-403 of SEQ ID N 0:2, comprising:

(i) removing an immune responsive cell containing sample from said subject,

(ii) contacting the immune responsive cell containing subject to a cell line transfected with a nucleic acid molecule coding for said protein expressed by abnormal cells associated with said condition, under conditions favoring production of cytolytic T cells against a peptide derived from said protein, and

(iii) introducing said cytolytic T cells to said subject in an amount sufficient to lyse said cells.

13. The method of claim 12, wherein said polypeptide is preprocalcitonin.

14. A method for treating a subject with a condition characterized by abnormal amounts of a polypeptide comprising an amino acid sequence encoded by nucleotides 292-403 of SEQ ID NO:2, comprising administering to said subject an amount of a cell transfected with (i) a nucleic acid molecule which codes for said polypeptide and (ii) a nucleic acid molecule which codes for an MHC molecule which presents a peptide derived from said polypeptide, where in said peptide is presented by cells associated with said condition, sufficient to alleviate said condition.

15. The method of claim 14, further comprising treating said cell to render it non- proliferative.

16. A method for determining presence of a transformed cell, comprising assaying a sample of cells for presence of cytolytic T lymphocytes specific for a complex of an MHC molecule and a peptide consisting of an amino acid sequence found in α-CGRP, calcitonin, or preprocalcitonin, presence of said cytolytic T lymphocytes being indicative of transformed cells in said sample.

17. The method of claim 16, wherein said peptide consists of an amino acid sequence encoded by nucleotides 292-403 of SEQ ID NO:2