KR20030022293A - Compositions and methods for the therapy and diagnosis of ovarian cancer - Google Patents

Abstract

The present invention discloses compositions and methods for the treatment and diagnosis of cancer such as ovarian cancer. The composition may comprise one or more ovarian carcinoma proteins, immunogenic portions thereof, polynucleotides encoding such portions or antibodies or immune system cells specific for such proteins. The composition can be used, for example, for the prevention and treatment of diseases such as ovarian cancer. The present invention also provides a method for identifying tumor antigens secreted from ovarian carcinoma and / or other tumors. Polypeptides and polynucleotides as provided herein can also be used for the diagnosis and monitoring of ovarian cancer.

Description

Compositions and methods for the treatment and diagnosis of ovarian cancer {Compositions and methods for the therapy and diagnosis of ovarian cancer}

Ovarian cancer is a major health problem for women worldwide, including in the United States. Although progress has been made in the detection and treatment of ovarian cancer, no vaccines or other universally successful methods for the prevention or treatment of ovarian cancer are currently used. Treatment of the disease currently relies on a combination of aggressive treatments that may include one or more of a variety of therapies such as initial diagnosis and surgery, radiotherapy, chemotherapy and hormonal therapy. The course of treatment for a particular cancer is often selected based on a variety of prognostic variables, including analysis of specific tumor markers. However, the use of established markers often leads to inexplicable consequences, and high mortality rates continue to be observed in many cancer patients.

Immunotherapy has the potential to substantially improve cancer treatment and survival. Such therapies may include the generation or augmentation of an immune response against ovarian carcinoma antigens. However, relatively few ovarian carcinoma antigens are known so far, and the generation of an immune response to such antigens has not been shown to be therapeutically beneficial.

Accordingly, there is a need in the art for improved methods for identifying ovarian tumor antigens and for using these antigens in the treatment of ovarian cancer. The present invention fulfills this need and further provides other related advantages.

Summary of the Invention

Briefly, the present invention provides compositions and methods for the treatment of cancer, such as ovarian cancer. In one aspect, the invention provides immunogenicity of ovarian carcinoma proteins or ovarian carcinoma protein variants that differ by one or more substitutions, deletions, additions and / or insertions but do not substantially reduce the ability to react with ovarian carcinoma protein-specific antiserum. Provided is a polypeptide comprising a moiety. In certain embodiments, the ovarian carcinoma protein comprises a sequence encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 456-457, 460-477, 512-570 and the complements of these polynucleotides.

The present invention also provides polynucleotides encoding the above-described polypeptides or portions thereof, expression vectors comprising such polynucleotides, and host cells transformed or transfected with such expression vectors.

The present invention also provides a polypeptide composition comprising an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 394-455, 458-459, 478-511 and 571-596.

In another aspect, the present invention provides pharmaceutical compositions and vaccines. The pharmaceutical composition may comprise a physiologically acceptable carrier or excipient comprising (i) an amino acid sequence encoded by a polynucleotide comprising the sequence set forth in any one of SEQ ID NOs: 456-457, 460-477, and 512-570. A polypeptide comprising an immunogenic portion of a protein or ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce the ability to react with ovarian carcinoma protein-specific antiserum, (ii) Polynucleotides encoding such polypeptides, (iii) antibodies that specifically bind to these polypeptides, (iv) antigen presenting cells expressing such polypeptides, and / or (v) react specifically with such polypeptides In combination with one or more of the T cells. The vaccine may contain a nonspecific immune response enhancer (i) the amino acid sequence set forth in SEQ ID NOs: 394-455, 458-459, 478-511 and 571-596 or the sequence set forth in any of SEQ ID NOs: 456-457, 460-477 and 512-570 Ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide comprising or an ovarian carcinoma that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce the ability to react with ovarian carcinoma protein-specific antiserum A polypeptide comprising an immunogenic portion of a protein variant, (ii) a polynucleotide encoding such a polypeptide, (iii) an antiidiotype antibody that specifically binds to an antibody that specifically binds to said polypeptide, (iv ) Antigen presenting cells expressing such polypeptides and / or (v) T cells that specifically react with such polypeptides. In can be included with more than one.

The present invention also provides, in another aspect, fusion proteins comprising one or more polypeptides as described above, as well as polynucleotides encoding such fusion proteins.

In a related aspect, there is provided a pharmaceutical composition comprising a fusion protein or polynucleotide encoding the fusion protein with a physiologically acceptable carrier.

In another aspect, a vaccine is provided comprising a fusion protein or a polynucleotide encoding the fusion protein in combination with a nonspecific immune response enhancer.

In a further aspect, the present invention provides a method of inhibiting the expression of cancer in a patient, comprising administering to the patient a pharmaceutical composition or vaccine as described above.

In another aspect, the present invention provides a T cell comprising (a) the amino acid sequence set forth in SEQ ID NOs: 394-455, 458-459, 478-511, and 571-596 or any one of SEQ ID NOs: 456-457, 460-477, and 512-570. Substantially reduced ability to react with ovarian carcinoma protein-specific antiserum, with ovarian carcinoma proteins comprising an amino acid sequence encoded by a polynucleotide comprising the sequence set forth below, or with one or more substitutions, deletions, additions and / or insertions A polypeptide comprising an immunogenic portion of an ovarian carcinoma protein variant, (b) a polynucleotide encoding such a polypeptide, and / or (c) stimulating and / or antigen-presenting cells and T cells expressing such polypeptides Provided are methods for stimulating and / or proliferating T cells, including contacting under conditions and for sufficient time to allow proliferation. The polypeptide, polynucleotide and / or antigen presenting cell (s) may be present in a pharmaceutical composition or vaccine for use in stimulating and / or proliferating T cells in a mammal.

In another aspect, the present invention provides a method for inhibiting the expression of ovarian cancer from a patient, comprising administering to the patient a T cell prepared as described above.

In a further aspect, the present invention provides a method for the preparation of (a) a CD4 ⁺ and / or CD8 ⁺ T cell isolated from a patient, comprising (i) a polynucleotide comprising the sequence set forth in any one of SEQ ID NOs: Of an ovarian carcinoma protein comprising an amino acid sequence encoded by a nucleotide or an ovarian carcinoma protein variant that differs by one or more substitutions, deletions, additions and / or insertions but does not substantially reduce its ability to react with ovarian carcinoma protein-specific antiserum Incubating with a polypeptide comprising an immunogenic moiety, (ii) a polynucleotide encoding such polypeptide, or (iii) an antigen presenting cell expressing such polypeptide, and (b) an effective amount of proliferation in a patient Inhibiting the expression of ovarian cancer in the patient, the method comprising administering T cells, thereby inhibiting the expression of ovarian cancer in the patient. There is provided a method. Proliferated cells can be cloned prior to administration to the patient.

The present invention also provides a method for identifying tumor antigens secreted in another aspect. The method comprises (a) transplanting tumor cells into an immunodeficient mammal, (b) obtaining serum from the immunodeficient mammal after a time sufficient to allow tumor antigens to be secreted into the serum, and (c) immunocompetent mammals. Immunizing the animal with serum, (d) obtaining antisera from an immunocompetent mammal, and (e) selecting a tumor expression library with antisera to identify tumor antigens secreted therefrom. Preferred methods of identifying secreted ovarian carcinoma antigens include (a) transplanting ovarian carcinoma cells into SCID mice, (b) obtaining serum from SCID mice after a time sufficient to allow ovarian carcinoma antigens to be secreted into the serum, (c) immunizing immunocompetent mice with serum, (d) obtaining antiserum from an immunocompetent mammal, and (e) selecting an ovarian carcinoma expression library with antiserum to identify ovarian carcinoma antigens secreted therefrom. Steps.

The present invention also discloses antibody epitopes recognized by O8E polyclonal antiserum, set forth herein as SEQ ID NOs: 394-415.

Further disclosed by the present invention are the dimeric and hexameric peptides believed to bind HLA-0201, disclosed herein as SEQ ID NOs: 416-435 and 436-455, respectively.

These and other aspects of the invention will become apparent with reference to the following detailed description and the accompanying drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each were individually cited.

In another aspect of the present invention, we unexpectedly identified a series of novel repeat sequence elements at the 5'-end of a gene encoding O772P. Accordingly, the present invention includes sequences having at least 50% identity, preferably at least 70% identity, and more preferably at least 90% identity with X _n -Y, wherein X is an O772P repeat sequence set forth in SEQ ID NO: 596. An O772P polypeptide having the structure shown in Y typically comprises a sequence having at least 80% identity, preferably at least 90% identity and more preferably at least 95% identity with the O772P constant region sequence set forth in SEQ ID NO: 594. According to this embodiment, n can generally be an integer from 1 to 35, preferably an integer from 15 to 25, and X can be the same or different.

In one preferred embodiment, X comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 574-593, and Y comprises the sequence set forth in SEQ ID NO: 594.

In another preferred embodiment, an exemplary O772P polypeptide comprises the sequence set forth in SEQ ID NO: 595, which contains ₂₀ repeat sequence elements (ie, X ₂₀ ), wherein the X elements are arranged in the following order: From the N-terminus to the C-terminus in the O772P repeat region): SEQ ID NO: 574-SEQ ID NO: 575-SEQ ID NO: 576-SEQ ID NO: 577-SEQ ID NO: 578-SEQ ID NO: 579-SEQ ID NO: 580-SEQ ID NO: 581-SEQ ID NO: 583-SEQ ID NO: 584-SEQ ID NO: 585 -SEQ ID NO: 586-SEQ ID NO: 587-SEQ ID NO: 588-SEQ ID NO: 589-SEQ ID NO: 590-SEQ ID NO: 591-SEQ ID NO: 592-SEQ ID NO: 593.

According to another aspect of the invention, an O772P polynucleotide having the structure X _n -Y, wherein X comprises an O772P repeat sequence selected from the group consisting of any one of SEQ ID NOs: 512-540, 542-546 and 548-567 To provide. Y generally comprises a sequence having at least 80% identity, preferably at least 90% identity and more preferably at least 95% identity with the O772P constant region sequence set forth in SEQ ID NO: 568. In this embodiment, n is typically an integer from 1 to 35, preferably an integer from 15 to 25, and X may be the same or different.

In another embodiment, an exemplary O772P polynucleotide comprises the sequence set forth in SEQ ID NO: 596 containing ₂₀ repeat sequence elements (ie, X ₂₀ ).

According to another aspect of the invention, there is provided an O772P polypeptide comprising at least the antibody epitope sequence set forth in any one of SEQ ID NOs: 490-511.

According to another aspect of the invention, there is provided an O8E polypeptide comprising at least the antibody epitope sequence set forth in any one of SEQ ID NOs: 394-415.

Brief description of the sequence listing and drawings

SEQ ID NOs: 1-71 are ovarian carcinoma antigen polynucleotides shown in FIGS. 1A-1S.

SEQ ID NOs: 72-74 are ovarian carcinoma antigen polynucleotides set forth in FIGS. 2A-2C.

SEQ ID NO: 75 is 3 g of ovarian carcinoma polynucleotide (FIG. 4).

SEQ ID NO: 76 is Ovarian carcinoma polynucleotide 3f (FIG. 5).

SEQ ID NO: 77 is Ovarian carcinoma polynucleotide 6b (Figure 6).

SEQ ID NO: 78 is Ovarian carcinoma polynucleotide 8e (FIG. 7A).

SEQ ID NO: 79 is Ovarian carcinoma polynucleotide 8h (FIG. 7B).

SEQ ID NO: 80 is Ovarian carcinoma polynucleotide 12e (FIG. 8).

SEQ ID NO: 81 is Ovarian carcinoma polynucleotide 12h (FIG. 9).

SEQ ID NOs: 82-310 are ovarian carcinoma antigen polynucleotides set forth in FIGS. 15A-15EEE.

SEQ ID NO: 311 is the full length sequence of ovarian carcinoma polynucleotide O772P.

SEQ ID NO: 312 is an O772P amino acid sequence.

SEQ ID NOs: 313-384 are ovarian carcinoma antigen polynucleotides.

SEQ ID NO: 385 shows the cDNA sequence in form of clone O772P designated 21013.

SEQ ID NO: 386 shows the cDNA sequence in the form of clone O772P named 21003.

SEQ ID NO: 387 shows the cDNA sequence in form of clone O772P named 21008.

SEQ ID NO: 388 is the amino acid sequence corresponding to SEQ ID NO: 385.

SEQ ID NO: 389 is the amino acid sequence corresponding to SEQ ID NO: 386. SEQ ID NO: 390 is the amino acid sequence corresponding to SEQ ID NO: 387.

SEQ ID NO: 391 is the full length sequence of ovarian carcinoma polynucleotide O8E.

SEQ ID NOs: 392-393 are protein sequences encoded by O8E.

SEQ ID NOs: 394-415 are peptide sequences corresponding to OE8 antibody epitopes.

SEQ ID NOs: 416-435 are potential HLA-A2 depolymer binding peptides estimated using the full length open-reading frame from OE8.

SEQ ID NOs: 436-455 are potential HLA-A2 trimer binding peptides estimated using the full length open-reading frame from OE8.

SEQ ID NO: 456 is a truncated nucleotide sequence of the full length Genbank sequence showing homology to O772P.

SEQ ID NO: 457 is a full length Genbank sequence showing significant homology to O772P.

SEQ ID NO: 458 is a protein encoding a truncated modification of the full length Genbank sequence that shows homology to O772P.

SEQ ID NO: 459 is the full length protein sequence from Genbank, showing significant homology to the protein sequence for O772P.

SEQ ID NO: 460 encodes a unique N-terminal portion of O772P contained in residues 1-70.

SEQ ID NO: 461 contains a unique sequence and encodes residues 1-313 of SEQ ID NO: 456.

SEQ ID NO: 462 is the putative sequence for clone O772P.

SEQ ID NO: 463 is cDNA sequence for clone FLJ14303.

SEQ ID NO: 464 is partial cDNA sequence for clone O772P.

SEQ ID NO: 465 is the partial cDNA sequence for clone O772P.

SEQ ID NO: 466 is the partial cDNA sequence for clone O772P.

SEQ ID NO: 467 is partial cDNA sequence for clone O772P.

SEQ ID NO: 468 is the partial cDNA sequence for clone O772P.

SEQ ID NO: 469 is the partial cDNA sequence for clone O772P.

SEQ ID NO: 470 is the partial cDNA sequence for clone O772P.

SEQ ID NO: 471 is the partial cDNA sequence for clone O772P.

SEQ ID NO: 472 is partial cDNA sequence for clone O772P.

SEQ ID NO: 473 is a partial cDNA sequence for clone O772P.

SEQ ID NO: 474 is the partial cDNA sequence for clone O772P.

SEQ ID NO: 475 is a partial cDNA sequence for clone O772P.

SEQ ID NO: 476 is partial cDNA sequence for clone O772P.

SEQ ID NO: 477 shows the novel 5'-terminus of ovarian tumor antigen O772P.

SEQ ID NO: 478 is the amino acid sequence encoded by SEQ ID NO: 462.

SEQ ID NO: 479 is the amino acid sequence encoded by SEQ ID NO: 463.

SEQ ID NO: 480 is the amino acid sequence encoded by SEQ ID NO: 472.

SEQ ID NO: 481 is the partial amino acid sequence encoded by the possible open reading frame of SEQ ID NO: 471.

SEQ ID NO: 482 is the partial amino acid sequence encoded by the second possible open reading frame of SEQ ID NO: 471.

SEQ ID NO: 483 is a partial amino acid sequence encoded by SEQ ID NO: 467.

SEQ ID NO: 484 is a partial amino acid sequence encoded by the possible open reading frame of SEQ ID NO: 466.

SEQ ID NO: 485 is the partial amino acid sequence encoded by the second possible open reading frame of SEQ ID NO: 466.

SEQ ID NO: 486 is the partial amino acid sequence encoded by SEQ ID NO: 465.

SEQ ID NO: 487 is the partial amino acid sequence encoded by SEQ ID NO: 464.

SEQ ID NO: 488 shows extracellular, dural and cytoplasmic regions of O772P.

SEQ ID NO: 489 shows putative extracellular domain of O772P.

SEQ ID NO: 490 shows the amino acid sequence of Peptide # 2 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 491 shows amino acid sequence of Peptide # 6 corresponding to O772P specific antibody epitope.

SEQ ID NO: 492 shows the amino acid sequence of Peptide # 7 corresponding to the O772P specific antibody epitope.

SEQ ID NO: 493 shows amino acid sequence of peptide # 8 corresponding to O772P specific antibody epitope.

SEQ ID NO: 494 shows the amino acid sequence of Peptide # 9 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 495 shows amino acid sequence of peptide # 11 corresponding to O772P specific antibody epitope.

SEQ ID NO: 496 shows the amino acid sequence of Peptide # 13 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 497 shows the amino acid sequence of Peptide # 22 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 498 shows amino acid sequence of Peptide # 24 corresponding to O772P specific antibody epitope.

SEQ ID NO: 499 shows amino acid sequence of Peptide # 27 corresponding to O772P specific antibody epitope.

SEQ ID NO: 500 shows amino acid sequence of peptide # 40 corresponding to O772P specific antibody epitope.

SEQ ID NO: 501 shows amino acid sequence of Peptide # 41 corresponding to O772P specific antibody epitope.

SEQ ID NO: 502 shows the amino acid sequence of Peptide # 47 corresponding to the O772P specific antibody epitope.

SEQ ID NO: 503 shows the amino acid sequence of Peptide # 50 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 504 shows the amino acid sequence of Peptide # 51 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 505 shows the amino acid sequence of Peptide # 52 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 506 shows the amino acid sequence of Peptide # 53 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 507 shows the amino acid sequence of Peptide # 58 corresponding to the O772P specific antibody epitope.

SEQ ID NO: 508 shows the amino acid sequence of Peptide # 59 corresponding to an O772P specific antibody epitope.

SEQ ID NO: 509 shows amino acid sequence of peptide # 60 corresponding to O772P specific antibody epitope.

SEQ ID NO: 510 shows amino acid sequence of peptide # 61 corresponding to O772P specific antibody epitope.

SEQ ID NO: 511 shows the amino acid sequence of peptide # 71 corresponding to O772P specific antibody epitope.

SEQ ID NO: 512 (O772P repeat 1) shows an example of a cDNA sequence corresponding to repeat number 21 from the 5 ′ variable region of O772P.

SEQ ID NO: 513 (O772P repeat 2) provides an example of a cDNA sequence corresponding to repeat number 20 from the 5 ′ variable region of O772P.

SEQ ID NO: 514 (O772P repeat 3) provides an example of a cDNA sequence corresponding to repeat number 19 from the 5 ′ variable region of O772P.

SEQ ID NO: 515 (O772P repeat 4) provides an example of a cDNA sequence corresponding to repeat number 18 from the 5 ′ variable region of O772P.

SEQ ID NO: 516 (O772P repeat 5) shows an example of a cDNA sequence corresponding to repeat number 17 from the 5 ′ variable region of O772P.

SEQ ID NO: 517 (HP repeat 1) shows an example of a cDNA sequence corresponding to repeat number 21 from the 5 ′ variable region of O772P.

SEQ ID NO: 518 (HP repeat 2) shows an example of a cDNA sequence corresponding to repeat number 20 from the 5 ′ variable region of O772P.

SEQ ID NO: 519 (HP repeat 3) shows an example of a cDNA sequence corresponding to repeat number 19 from the 5 ′ variable region of O772P.

SEQ ID NO: 520 (HP repeat 4) shows an example of a cDNA sequence corresponding to repeat number 18 from the 5 ′ variable region of O772P.

SEQ ID NO: 521 (HP repeat 5) shows an example of a cDNA sequence corresponding to repeat number 17 from the 5 ′ variable region of O772P.

SEQ ID NO: 522 (HP repeat 6 5′-terminus) shows an example of a cDNA sequence corresponding to repeat number 16 from the 5 ′ variable region of O772P.

SEQ ID NO: 523 (1043400.1 repeat 1) provides an example of a cDNA sequence corresponding to repeat number 9 from the 5 ′ variable region of O772P.

SEQ ID NO: 524 (1043400.1 repeat 2) provides an example of a cDNA sequence corresponding to repeat number 10 from the 5 ′ variable region of O772P.

SEQ ID NO: 525 (1043400.1 repeat 3) provides an example of a cDNA sequence corresponding to repeat number 10/11 from the 5 'variable region of O772P.

SEQ ID NO: 526 (1043400.1 repeat 4) provides an example of a cDNA sequence corresponding to repeat number 11 from the 5 'variable region of O772P.

SEQ ID NO: 527 (1043400.1 repeat 5) shows an example of a cDNA sequence corresponding to repeat number 14 from the 5 ′ variable region of O772P.

SEQ ID NO: 528 (1043400.1 repeat 6) provides an example of a cDNA sequence corresponding to repeat number 17 from the 5 'variable region of O772P.

SEQ ID NO: 529 (1043400.3 repeat 1) shows an example of a cDNA sequence corresponding to repeat number 20 from the 5 ′ variable region of O772P.

SEQ ID NO: 530 (1043400.3 repeat 2) provides an example of a cDNA sequence corresponding to repeat number 21 from the 5 ′ variable region of O772P.

SEQ ID NO: 531 (1043400.5 repeat 1) shows an example of a cDNA sequence corresponding to repeat number 8 from the 5 ′ variable region of O772P.

SEQ ID NO: 532 (1043400.5 repeat 2) corresponds to repeat number 9 from the 5 ′ variable region of O772P and also provides an example of a cDNA sequence containing an intron sequence.

SEQ ID NO: 533 (1043400.5 repeat 2) shows an example of a cDNA sequence corresponding to repeat number 9 from the 5 ′ variable region of O772P.

SEQ ID NO: 534 (1043400.8 repeat 1) provides an example of a cDNA sequence corresponding to repeat number 17 from the 5 ′ variable region of O772P.

SEQ ID NO: 535 (1043400.8 repeat 2) provides an example of a cDNA sequence corresponding to repeat number 18 from the 5 'variable region of O772P.

SEQ ID NO: 536 (1043400.8 repeat 3) provides an example of a cDNA sequence corresponding to repeat number 19 from the 5 'variable region of O772P.

SEQ ID NO: 537 (1043400.9 repeat 1) shows an example of a cDNA sequence corresponding to repeat number 4 from the 5 ′ variable region of O772P.

SEQ ID NO: 538 (1043400.9 repeat 2) shows an example of a cDNA sequence corresponding to repeat number 5 from the 5 ′ variable region of O772P.

SEQ ID NO: 539 (1043400.9 repeat 3) provides an example of a cDNA sequence corresponding to repeat number 7 from the 5 'variable region of O772P.

SEQ ID NO: 540 (1043400.9 repeat 4) shows an example of a cDNA sequence corresponding to repeat number 8 from the 5 ′ variable region of O772P.

SEQ ID NO: 541 (1043400.11 repeat 1) provides an example of a cDNA sequence corresponding to repeat number 1 from the 5 ′ variable region of O772P.

SEQ ID NO: 542 (1043400.11 repeat 2) provides an example of a cDNA sequence corresponding to repeat number 2 from the 5 ′ variable region of O772P.

SEQ ID NO: 543 (1043400.11 repeat 3) provides an example of a cDNA sequence corresponding to repeat number 3 from the 5 ′ variable region of O772P.

SEQ ID NO: 544 (1043400.11 repeat 4) shows an example of a cDNA sequence corresponding to repeat number 11 from the 5 ′ variable region of O772P.

SEQ ID NO: 545 (1043400.11 repeat 5) shows an example of a cDNA sequence corresponding to repeat number 12 from the 5 ′ variable region of O772P.

SEQ ID NO: 546 (1043400.12 repeat 1) provides an example of a cDNA sequence corresponding to Repetition Number 20 from the 5 'variable region of O772P.

SEQ ID NO: 547 (PB repeat A) provides an example of a cDNA sequence corresponding to repeat number 1 from the 5 ′ variable region of O772P.

SEQ ID NO: 548 (PB repeat B) provides an example of a cDNA sequence corresponding to repeat number 2 from the 5 ′ variable region of O772P.

SEQ ID NO: 549 (PB repeat E) provides an example of a cDNA sequence corresponding to repeat number 3 from the 5 ′ variable region of O772P.

SEQ ID NO: 550 (PB repeat G) shows an example of a cDNA sequence corresponding to repeat number 4 from the 5 ′ variable region of O772P.

SEQ ID NO: 551 (PB repeat C) shows an example of a cDNA sequence corresponding to repeat number 4 from the 5 ′ variable region of O772P.

SEQ ID NO: 552 (PB repeat H) provides an example of a cDNA sequence corresponding to repeat number 6 from the 5 ′ variable region of O772P.

SEQ ID NO: 553 (PB repeat J) shows an example of a cDNA sequence corresponding to repeat number 7 from the 5 ′ variable region of O772P.

SEQ ID NO: 554 (PB repeat K) provides an example of a cDNA sequence corresponding to repeat number 8 from the 5 ′ variable region of O772P.

SEQ ID NO: 555 (PB repeat D) provides an example of a cDNA sequence corresponding to repeat number 9 from the 5 ′ variable region of O772P.

SEQ ID NO: 556 (PB repeat I) shows an example of a cDNA sequence corresponding to repeat number 10 from the 5 ′ variable region of O772P.

SEQ ID NO: 557 (PB repeat M) shows an example of a cDNA sequence corresponding to repeat number 11 from the 5 ′ variable region of O772P.

SEQ ID NO: 558 (PB repeat 9) shows an example of a cDNA sequence corresponding to repeat number 12 from the 5 ′ variable region of O772P.

SEQ ID NO: 559 (PB repeat 8.5) provides an example of a cDNA sequence corresponding to repeat number 13 from the 5 ′ variable region of O772P.

SEQ ID NO: 560 (PB repeat 8) shows an example of a cDNA sequence corresponding to repeat number 14 from the 5 ′ variable region of O772P.

SEQ ID NO: 561 (PB repeat 7) shows an example of a cDNA sequence corresponding to repeat number 15 from the 5 ′ variable region of O772P.

SEQ ID NO: 562 (PB repeat 6) shows an example of a cDNA sequence corresponding to repeat number 16 from the 5 ′ variable region of O772P.

SEQ ID NO: 563 (PB repeat 5) shows an example of a cDNA sequence corresponding to repeat number 17 from the 5 ′ variable region of O772P.

SEQ ID NO: 564 (PB repeat 4) shows an example of a cDNA sequence corresponding to repeat number 18 from the 5 ′ variable region of O772P.

SEQ ID NO: 565 (PB repeat 3) shows an example of a cDNA sequence corresponding to repeat number 19 from the 5 ′ variable region of O772P.

SEQ ID NO: 566 (PB repeat 2) shows an example of a cDNA sequence corresponding to repeat number 20 from the 5 ′ variable region of O772P.

SEQ ID NO: 567 (PB repeat 1) shows an example of a cDNA sequence corresponding to repeat number 21 from the 5 ′ variable region of O772P.

SEQ ID NO: 568 shows cDNA sequence from the 3 'constant region.

SEQ ID NO: 569 shows the cDNA sequence containing the consensus sequence of 21 repeats, a 3 'constant region and a 3' untranslated region.

SEQ ID NO: 570 shows the cDNA sequence of the consensus repeat sequence.

SEQ ID NO: 571 shows the consensus amino acid sequence of the first potential open reading frame of Repetition Number 1 from the 5 'variable region of O772P.

SEQ ID NO: 572 shows the consensus amino acid sequence of the second potential open reading frame of repeat number 1 from the 5 'variable region of O772P.

SEQ ID NO: 573 shows the consensus amino acid sequence of the third potential open reading frame of Repetition Number 1 from the 5 'variable region of O772P.

SEQ ID NO: 574 shows the consensus amino acid sequence of repeat number 2 from the 5 'variable region of O772P.

SEQ ID NO: 575 shows the consensus amino acid sequence of repeat number 3 from the 5 'variable region of O772P.

SEQ ID NO: 576 shows the consensus amino acid sequence of repeat number 4 from the 5 'variable region of O772P.

SEQ ID NO: 577 shows the consensus amino acid sequence of Repetition Number 5 from the 5 'variable region of O772P.

SEQ ID NO: 578 shows the consensus amino acid sequence of repeat number 6 from the 5 'variable region of O772P.

SEQ ID NO: 579 shows the consensus amino acid sequence of Repetition Number 7 from the 5 'variable region of O772P.

SEQ ID NO: 580 shows the consensus amino acid sequence of repeat number 8 from the 5 'variable region of O772P.

SEQ ID NO: 581 shows the consensus amino acid sequence of Repetition Number 9 from the 5 'variable region of O772P.

SEQ ID NO: 582 shows the consensus amino acid sequence of repeat number 10 from the 5 'variable region of O772P.

SEQ ID NO: 583 shows the consensus amino acid sequence of repeat No. 11 from the 5 'variable region of O772P.

SEQ ID NO: 584 shows the consensus amino acid sequence of repeat number 12 from the 5 'variable region of O772P.

SEQ ID NO: 585 shows the consensus amino acid sequence of repeat number 13 from the 5 'variable region of O772P.

SEQ ID NO: 586 shows the consensus amino acid sequence of repeat number 14 from the 5 'variable region of O772P.

SEQ ID NO: 587 shows the consensus amino acid sequence of repeat number 15 from the 5 'variable region of O772P.

SEQ ID NO: 588 shows the consensus amino acid sequence of Repetition Number 16 from the 5 'variable region of O772P.

SEQ ID NO: 589 shows the consensus amino acid sequence of repeat number 17 from the 5 'variable region of O772P.

SEQ ID NO: 590 shows the consensus amino acid sequence of repeat number 18 from the 5 'variable region of O772P.

SEQ ID NO: 591 shows the consensus amino acid sequence of repeat number 19 from the 5 'variable region of O772P.

SEQ ID NO: 592 shows the consensus amino acid sequence of repeat number 20 from the 5 'variable region of O772P.

SEQ ID NO: 593 shows the consensus amino acid sequence of repeat number 21 from the 5 'variable region of O772P.

SEQ ID NO: 594 shows the amino acid sequence of the 3 'constant region.

SEQ ID NO: 595 shows an amino acid sequence containing a consensus sequence of 21 repeats and a 3 'constant region.

SEQ ID NO: 596 shows the amino acid sequence of the consensus repeat sequence.

1A-1S (SEQ ID NOS: 1-71) show partial sequences of polynucleotides encoding exemplary secreted ovarian carcinoma antigens.

Figures 2A-2C show the total insert sequence for three of the clones of Figure 1. Figure 2A shows sequence 11731 (SEQ ID NO: 72) designated O7E. 2B shows sequence (11785; SEQ ID NO: 73) named O9E, and FIG. 2C shows sequence (13695; SEQ ID NO: 74) named O8E.

3 shows the results of microarray expression analysis of ovarian carcinoma sequences named O8E.

4 shows a partial sequence of a polynucleotide (named 3g; SEQ ID NO: 75) encoding the ovarian carcinoma sequence, a splice fusion between human T-cell leukemia virus type I carcinogenic protein TAX and osteonectin.

Figure 5 shows an ovarian carcinoma polynucleotide (SEQ ID NO: 76) named 3f.

Figure 6 shows an ovarian carcinoma polynucleotide (SEQ ID NO: 77) named 6b.

7A and 7B show ovarian carcinoma polynucleotides (SEQ ID NOS: 78 and 79, respectively) designated 8e and 8h.

8 shows an ovarian carcinoma polynucleotide (SEQ ID NO: 80) named 12c.

9 shows an ovarian carcinoma polynucleotide (SEQ ID NO: 81) named 12h.

10 shows the results of microarray expression analysis of the ovarian carcinoma sequence named 3f.

11 shows the results of microarray expression analysis of ovarian carcinoma sequence named 6b.

12 shows the results of microarray expression analysis of ovarian carcinoma sequence named 8e.

FIG. 13 shows the results of microarray expression analysis of the ovarian carcinoma sequence named 12c.

14 shows the results of microarray expression analysis of the ovarian carcinoma sequence named 12h.

15A-15EEE show partial sequences of additional polynucleotides (SEQ ID NOs: 82-310) encoding exemplary secreted ovarian carcinoma antigens.

16 is a diagram illustrating the location of several partial O8E sequences within the full length sequence.

17 is a graph depicting the results of epitope mapping studies on O8E protein.

18 is a graph of fluorescence activated cell sorting (FACS) analysis of OE8 cell surface expression.

19 is a graph of FACS analysis of OE8 cell surface expression.

20 shows the results of FACS analysis on O8E transfected HEK293 cells demonstrating cell surface expression of OE8.

21 shows the results of FACS analysis on SKBR3 breast tumor cells demonstrating cell surface expression of OE8.

22 shows OE8 expression in HEK 293 cells. The cells are probed with anti-OE8 rabbit polyclonal antiserum # 2333L.

23 shows the results of ELISA analysis of anti-OE8 rabbit serum.

24 shows the results of ELISA analysis of affinity purified rabbit anti-OE8 polyclonal antibodies.

FIG. 25 is a graph measuring antibody internalization of anti-OE8 mAb in which mAbs for amino acids 61-80 show ligand internalization.

The present invention generally relates to the treatment of ovarian cancer. More specifically, the present invention relates to polypeptides comprising at least a portion of ovarian carcinoma proteins and to polynucleotides encoding such polypeptides, as well as antibodies and immune system cells that specifically recognize said polypeptides. Such polypeptides, polynucleotides, antibodies and cells can be used in vaccines and pharmaceutical compositions for the treatment of ovarian cancer.

As noted above, the present invention generally relates to compositions and methods for the treatment of cancer, such as ovarian cancer. The compositions described herein may comprise immunogenic polypeptides, polynucleotides encoding such polypeptides, binding agents such as antibodies that bind the polypeptides, antigen presenting cells (APCs) and / or immune system cells (eg T cells). Can be.

Polypeptides of the invention generally comprise at least an immunogenic portion of an ovarian carcinoma protein or variant thereof. Certain ovarian carcinoma proteins have been identified using immunoassay techniques and are referred to herein as ovarian carcinoma antigens. An "ovarian carcinoma antigen" is a protein expressed by ovarian cancer cells (preferably human cells) at levels two or more times higher than in normal ovarian cells. Certain ovarian carcinoma antigens react to detectable levels (in immunoassays such as ELISA or Western blot) with antisera generated against serum from immunodeficient animals transplanted with human ovarian cancer. These ovarian carcinoma antigens are released or secreted from ovarian cancer into the serum of immunodeficient animals. Thus, certain ovarian carcinoma antigens provided herein are secreted antigens. Specific nucleic acid sequences of the invention generally include DNA or RNA sequences that encode all or part of such polypeptides, or sequences complementary to such sequences.

The invention also provides ovarian carcinoma sequences identified using techniques for assessing modified expression in ovarian cancer. The sequence can be a polynucleotide or a protein sequence. Ovarian carcinoma sequences are generally expressed at doubling levels, preferably at least five times higher than expression levels in normal ovarian tissue in ovarian cancer, as measured using the representative assays provided herein. Certain partial ovarian carcinoma polynucleotide sequences are provided herein. Proteins encoded by genes comprising such polynucleotide sequences (or complements thereof) are also contemplated as ovarian carcinoma proteins.

Antibodies are generally immune system proteins or antigen-binding fragments thereof capable of binding at least a portion of an ovarian carcinoma polypeptide as described herein. T cells that can be used in the compositions provided herein are T cells specific to such polypeptides (eg, CD4 ⁺ and / or CD8 ⁺ ). Certain methods described herein further utilize antigen presenting cells (eg, dendritic cells or macrophages) that express an ovarian carcinoma polypeptide as provided herein.

Ovarian Carcinoma Polynucleotide

Any polynucleotide encoding an ovarian carcinoma protein or portion or other variant thereof as described herein is included in the present invention. Preferred polynucleotides comprise at least 15 contiguous nucleotides, preferably at least 30 contiguous nucleotides, and more preferably at least 45 contiguous nucleotides encoding a portion of an ovarian carcinoma protein. More preferably, the polynucleotides encode an immunogenic portion of an ovarian carcinoma protein, such as an ovarian carcinoma antigen. Any polynucleotide complementary to such sequences is also included in the present invention. The polynucleotides may be single stranded (coding or antisense) or double stranded, and may be DNA (genome, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may be present in the polynucleotides of the present invention, although not necessary, and the polynucleotides may be linked to other molecules and / or supports, although not necessarily.

Polynucleotides may comprise a native sequence (ie, an endogenous sequence encoding an ovarian carcinoma protein or portion thereof) or may comprise a variant of such sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and / or insertions such that the immunogenicity of the encoded polypeptide is not reduced compared to native ovarian carcinoma proteins. The impact on the immunogenicity of the encoded polypeptide can generally be assessed as described herein. The variant preferably exhibits at least about 70% identity with a polynucleotide sequence encoding a native ovarian carcinoma protein or portion thereof, more preferably at least about 80%, and most preferably at least about 90%.

The percent identity for two polynucleotide or polypeptide sequences can be readily determined by comparing the sequences using computer algorithms well known to those skilled in the art, such as Megalign, using default variables. Comparisons between two sequences are typically performed by comparing sequences in a comparison window to identify and compare local regions of sequence similarity. As used herein, a “comparison window” is about 20 or more, usually 30 to about 75, or 40 that can optimally align two sequences and then compare one sequence with an equal number of contiguous reference sequences. To fragments of from about 50 consecutive positions. Optimal alignment of sequences for comparison can be performed using, for example, the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) Using default variables. Preferably, the proportion of sequence identity is determined by comparing two sequences that are optimally aligned through a comparison window of at least about 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window is (additional or deleted) Up to 20%, usually 5-15%, or 10-12%, as compared to the reference sequence (not containing). The ratio of sequence identity determines the number of positions where identical nucleic acid bases or amino acid residues are present in two sequences, yielding the number of positions matched, and the number of positions matched is the total number of positions in the reference sequence (ie, window size). Can be calculated by multiplying the result by 100 and calculating the proportion of sequence identity.

The variant may also or alternatively be substantially homologous to the native gene or portion or complement thereof. Such polynucleotide variants can hybridize under moderately stringent conditions with native DNA sequences (or complementary sequences) encoding native ovarian carcinoma proteins. Suitable moderately stringent conditions were pre-washed in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA, pH 8.0, hybridized overnight at 50 ° C.-65 ° C., 5 × SSC, then 20 at 65 ° C. each time. Washing twice with 2X, 0.5X and 0.2X SSC containing 0.1% SDS for minutes.

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, there are a large number of nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides possess minimal homology with the nucleotide sequences of all native genes. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are modified as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The resulting mRNAs and proteins are not necessary but may have a modified structure or function. Alleles can be identified using standard techniques (eg, hybridization, amplification, and / or database sequence comparison).

Polynucleotides can be prepared using any of a number of techniques. For example, ovarian carcinoma polynucleotides are generated for the serum of immunocompetent mice following injection of a terminally advanced ovarian cancer expression library into mice with serum from SCID mice transplanted with terminally advanced ovarian cancer as described in more detail below. Identification can be made by screening with antiserum. Ovarian carcinoma polynucleotides can also be identified using any of a number of techniques designed to assess differential gene expression. Alternatively, polynucleotides can be amplified from cDNA prepared from ovarian cancer cells. Such polynucleotides can be amplified via polymerase chain reaction (PCR). For this method, sequence-specific primers can be designed, purchased or synthesized based on the sequences provided herein.

The amplified portion can be used to isolate full length genes from suitable libraries (eg, ovarian carcinoma cDNA libraries) using well known techniques. Within this technique, libraries (cDNAs or genomes) are selected using one or more polynucleotide probes or primers suitable for amplification. Preferably, libraries are screened by size to include larger molecules. Random primed libraries may also be desirable for identifying the 5 'and upstream regions of genes. Genomic libraries are preferred for obtaining introns and stretching 5 'sequences.

In the case of hybridization techniques, well-known techniques can be used to label partial sequences (eg, by nick-detox or end-labeling using ³² P). Next, the bacterial or bacteriophage libraries are screened by hybridizing a filter containing denatured bacterial colonies (or ron containing phage plaques) with labeled probes. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989]. Colonies or plaques that hybridize are selected and propagated, and then DNA is isolated for further analysis. The cDNA clone can be analyzed by determining the amount of additional sequence, for example, by PCR using primers from a vector and primers from partial sequences. Restriction maps and partial sequences can be generated to identify one or more duplicate clones. The complete sequence can then be determined using standard techniques, which may include generating a series of deletion clones. The resulting duplicate sequences are then assembled into one continuous sequence. Full length cDNA molecules can be generated by linking suitable fragments using well known techniques.

Alternatively, there are a number of amplification techniques for obtaining full length coding sequences from partial cDNA sequences. Within this technique, amplification is generally performed via PCR. Any of a variety of kit commercials can be used to perform the amplification step. Primers can be designed using, for example, software well known in the art. The primer is preferably 22 to 30 nucleotides in length, has a GC content of at least 50%, and anneals to the target sequence at a temperature of about 68 ° C to 72 ° C. The amplified region can be sequenced as described above and the overlapping sequences can be assembled into contiguous sequences.

One such amplification technique uses reverse PCR to generate fragments in known regions of genes using Triglia et al., Nucl. Acids Res. 16: 8186, 1988. The fragment is then closed by intramolecular ligation and used as a template for PCR with different primers derived from known regions. Alternatively, the sequence adjacent to the partial sequence can be repaired by amplification with a primer for the linker sequence and a primer specific for a known region. The amplified sequence is typically subjected to secondary amplification with the same linker primer and a second primer specific for a known region. Modifications to this method using two primers that initiate stretching in opposite directions from known sequences are described in WO 96/38591. Further techniques can be found in capture PCR [Lagerstrom et al., PCR Methods Applic. 1: 111-19, 1991] and working PCR (Parker et al., Nucl. Acids. Res. 19: 3055-60, 1991. Other methods using amplification can also be used to obtain full length cDNA sequences.

In certain instances, full length cDNA sequences may be obtained by analysis of sequences provided in an expressed sequence tag (EST) database such as available from GenBank. Investigations of duplicate ESTs can generally be carried out using well-known programs (eg NCBI BLAST surveys), which can be used to generate continuous full length sequences.

Specific nucleic acid sequences of cDNA molecules encoding some of the ovarian carcinoma antigens are shown in FIGS. 1A-1S (SEQ ID NOS: 1-71) and FIGS. 15A-15EEE (SEQ ID NOs: 82-310). The sequences shown in FIGS. 1A-1S appear novel. For the sequences shown in Figures 15A-15EEE, database searches reveal a match with substantial identity. These polynucleotides are isolated by serological selection of an ovarian cancer cDNA expression library using techniques designed to identify secreted tumor antigens. Briefly, terminally advanced ovarian cancer expression libraries are prepared from SCID-induced human ovarian cancer (OV9334) on a vector λ-screen from Novagen. Serum used for selection is obtained by injecting serum from SCID mice transplanted with terminally advanced ovarian cancer into immunocompetent mice. This technique makes it possible to identify cDNA molecules that encode immunogenic portions of secreted tumor antigens.

In addition to the polynucleotides set forth herein, full length polynucleotides comprising such sequences, other portions of such full length polynucleotides, and sequences complementary to all or part of such full length molecules are specifically included in the present invention. It will be apparent to those skilled in the art that the technique can also be applied to identify antigens secreted from other types of tumors.

Other nucleic acid sequences of cDNA molecules encoding portions of ovarian carcinoma proteins are provided in FIGS. 4-9 (SEQ ID NOS: 75-81) and SEQ ID NOs: 313-384. These sequences are identified by selecting microarrays of cDNA for tumor-associated expression (ie, expression at least five times higher in ovarian cancer compared to normal ovarian tissue as measured using the representative assays provided herein). Such selection is in accordance with the manufacturer's instructions and essentially as described in Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94: 2150-2155, 1997, using Synteni microarrays (Palo Alto, Calif.). SEQ ID NOs: 311 and 391 are full length sequences into which some of these nucleic acid sequences are incorporated.

Any of a variety of well known techniques can be used to assess tumor-associated expression of cDNA. For example, hybridization techniques using labeled polynucleotide probes can be used. Alternatively or also, amplification techniques such as real-time PCR can be used. See Gibson et al., Genome Research 6: 995-1001, 1996; Heid et al., Genome Research 6: 986-994, 1996. Real-time PCR is a technique for evaluating the accumulation level of PCR products during amplification. This technique allows for quantitative assessment of mRNA levels in multiple samples. Briefly, mRNA is extracted from tumors and normal tissues, and cDNA is prepared using standard techniques. Real-time PCR can be performed using, for example, 7700 prism equipment (Perkin Elmer / Applied Biosystems; Foster City, CA). For example, matching primers and fluorescent probes can be designed for that gene using a primer expression program (Perkin Elmer / Applied Biosystems; Foster City, CA). Optimal concentrations of primers and probes can be initially determined by one skilled in the art and control (eg, β-actin) primers and probes can be purchased, for example, from Perkin Elmer / Applied Biosystems; Foster City, CA. . To quantify the amount of specific RNA in the sample, a standard curve is generated side by side using the plasmid containing the gene. Standard curves can be generated using Ct values generated in real-time PCR that correlate with the initial cDNA concentration used in the assay. Standard dilution ranges from 10 to 10 ⁶ copies of the gene of interest are generally sufficient. In addition, standard curves are generated for the control sequences. This allows for standardization of the initial RNA content of the tissue sample against the control amount for comparison purposes.

Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis by solid phase phosphoramidite chemical synthesis. Modifications in the polynucleotide sequence can also be accomplished using standard mutagenesis techniques such as oligonucleotide-directed site-specific mutagenesis (Adelman et al., DNA 2: 183, 1983). Alternatively, RNA molecules can be generated by in vitro or in vivo transcription of a DNA sequence encoding or a portion of an ovarian carcinoma antigen as long as the DNA is inserted into a vector with a suitable RNA polymerase promoter (T7 or SP6). As described herein, certain portions can be used to prepare encoded polypeptides. In addition, or alternatively, a portion may be administered to the patient to produce the encoded polypeptide in vivo.

Portions of sequences complementary to the coding sequence (ie, antisense polynucleotides) can also be used as probes or to control gene expression. CDNA constructs that can be transcribed into antisense RNA can also be introduced into cells or tissues to promote the production of antisense RNA. Antisense polynucleotides can be used as described herein to inhibit the expression of ovarian carcinoma proteins. Antisense technology can be used to regulate gene expression through triple-helix formation, which inhibits the ability of the double helix to sufficiently cleave for binding of polymerases, transcription factors or regulatory molecules. Gee et al., In Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY; 1994]. Alternatively, antisense molecules hybridize with regulatory regions (eg, promoters, enhancers or transcription initiation sites) of the gene and block transcription of the gene; It can be designed to block translation by inhibiting the binding of transcripts to ribosomes.

Any polynucleotide can be further modified to increase stability in vivo. Possible modifications include the addition of flanking sequences at the 5 'and / or 3'-ends, the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase bonds in the backbone, and / or inosine, cue Insertions of acetyl-, methyl-, thio- and other modified forms of adenine, cytidine, guanine and uridine as well as non-traditional bases such as eosin and waibutocin.

The nucleotide sequences described herein can be linked to many other nucleotide sequences using established recombinant DNA techniques. For example, polynucleotides can be cloned into various cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Particular corresponding vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In general, vectors contain origins of replication, convenient restriction endonuclease sites, and one or more selection markers that function in one or more organisms. Other elements may be used depending on the intended use, which will be apparent to those skilled in the art.

In certain embodiments, the polynucleotides may be introduced into cells of a mammal and formulated for expression. Such formulations are particularly useful for therapeutic purposes as described below. It will be appreciated by those skilled in the art that there are a number of methods for achieving expression of polynucleotides in a target cell, and that any suitable method may be used. For example, polynucleotides can be inserted into viral vectors such as, but not limited to, adenoviruses, adeno-associated viruses, retroviruses or vaccinia or other varicella virus (eg, avian pox virus). Techniques for inserting DNA into such vectors are well known to those skilled in the art. Retroviral vectors may additionally transfer or insert targeting moieties such as genes for selection markers (to aid in the identification or selection of transduced cells) and / or genes encoding ligands for receptors on specific target cells. Can be made specific to the target. Targeting can also be accomplished using antibodies by methods known to those of skill in the art.

Other formulations for therapeutic purposes include gelatinous dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads and lipid based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. Preferred colloidal systems for use as delivery vehicles in vitro or in vivo are liposomes (ie artificial membrane vesicles). The manufacture and use of such systems are well known in the art.

Ovarian Carcinoma Polypeptide

Within the context of the present invention, a polypeptide may comprise at least an immunogenic portion of an ovarian carcinoma protein or variant thereof as described herein. As noted above, certain ovarian carcinoma proteins are ovarian carcinoma antigens that are expressed by ovarian cancer cells and react to a detectable degree with antisera produced against serum from immunodeficient animals transplanted with ovarian carcinoma in an immunoassay (eg, ELISA). to be. Other ovarian carcinoma proteins are encoded by ovarian carcinoma polynucleotides provided herein. Polypeptides as described herein can be any length. There may be additional sequences and / or heterologous sequences derived from natural proteins, which sequences may retain (but not necessarily retain) additional immunogenic or antigenic properties.

As used herein, an “immunogenic portion” is that portion of an antigen that is recognized (ie, specifically bound) by B-cell and / or T-cell surface antigen receptors. Such immunogenic moieties generally comprise at least 5 amino acid residues of an ovarian carcinoma protein or variant thereof, more preferably at least 10, and even more preferably at least 20 amino acid residues. Preferred immunogenic portions are encoded by isolated cDNA molecules as described herein. In addition, immunogenic moieties are generally employed using well known techniques as summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. I can sympathize. Such techniques include screening polypeptides for their ability to react with ovarian carcinoma protein-specific antibodies, antisera and / or T-cell lines or clones. As used herein, antisera and antibodies are those in which they specifically bind to ovarian carcinoma proteins (ie, they react with ovarian carcinoma proteins in an ELISA or other immunoassay and do not react to detectably with unrelated proteins). "Ovarian carcinoma protein-specific". Such antiserum, antibodies and T cells can be prepared as described herein and using well known techniques. The immunogenic portion of the native ovarian carcinoma protein reacts at a level substantially less than the reactivity of such antiserum, antibodies and / or T-cells with full length polypeptides (eg in ELISA and / or T-cell reactivity assays). Part. Such immunogenic portions may respond at levels similar to or greater than the reactivity of the full-length protein in this assay. Such screening can generally be carried out using methods well known to those skilled in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the polypeptide can be immobilized on a solid support and contacted with the serum of the patient to allow antibodies in the serum to bind the immobilized polypeptide. Unbound serum can then be removed and the bound antibody can be detected using, for example, ¹²⁵ I-labeled Protein A.

As noted above, the composition may comprise variants of natural ovarian carcinoma proteins. As used herein, a polypeptide “variant” is a polypeptide that differs from a native ovarian carcinoma protein by one or more substitutions, deletions, additions and / or insertions, but without substantially reduced immunogenicity of the polypeptide. That is, the ability of the variant to react with ovarian carcinoma protein-specific antiserum may be increased or invariant compared to native ovarian carcinoma protein, or may be reduced to less than 50%, preferably less than 20%, relative to native ovarian carcinoma protein. . Such variants can generally be identified by modifying one of the polypeptide sequences to assess the reaction of the modified polypeptide with an ovarian carcinoma protein-specific antibody or antiserum as described herein. Preferred variants include variants in which one or more portions have been removed, such as an N-terminal leader sequence or transmembrane domain. Other preferred variants include variants in which the small portion (eg, 1-30 amino acids, preferably 5-15 amino acids) is removed from the N- and / or C-terminus of the mature protein.

Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90%, and most preferably at least about 95% identity with the native polypeptide. Preferably, variants contain conservative substitutions. A "conservative substitution" is a substitution in which an amino acid is substituted with another amino acid having similar properties, and consequently one skilled in the art of peptide chemistry can expect the secondary structure and degree of hydration of the polypeptide to be substantially unchanged. Amino acid substitutions can generally be achieved based on the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphotericity of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups exhibiting similar hydrophilicity values are leucine, Isoleucine and valine; Glycine and alanine; Asparagine and glutamine; And serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may exhibit conservative modifications include (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; And (5) phe, tyr, trp, his. Variants may also or alternatively contain non-conservative modifications. Variants may also be modified (or otherwise), for example, by deletion or addition of amino acids that minimally affect the immunogenicity, secondary structure and degree of hydration of the polypeptide.

As noted above, the polypeptide may comprise a signal (or leader) sequence at the N-terminus of the protein which directs the translation of the protein simultaneously with or after translation. The polypeptide may also be linked to a linker or other sequence for its convenient synthesis, purification or identification (eg poly-His), or to increase binding of the polypeptide to a solid support. For example, the polypeptide can be bound to an immunoglobulin Fc region.

Polypeptides can be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above can be readily prepared from DNA sequences using any of a variety of expression vectors known to those skilled in the art. Expression can be achieved in any suitable host cell transformed or transfected with an expression vector containing a DNA molecule encoding a recombinant polypeptide. Suitable host cells include prokaryotic, yeast and higher eukaryotic cells. Preferably, the host cell used is E. coli. E. coli, yeast or mammalian cell lines such as COS or CHO. Supernatants from suitable host / vector systems that secrete recombinant proteins or polypeptides into the culture medium may first be concentrated using commercially available filters. After concentration, the concentrate can be applied to a suitable purification matrix, such as an affinity matrix or an ion exchange resin. Finally, one or more reversed phase HPLC steps can be used to further purify the recombinant polypeptide.

Portions and other variants having up to about 100 amino acids, typically up to about 50 amino acids, can also be produced by synthetic means using techniques well known to those skilled in the art. For example, such polypeptides can be synthesized using any commercially available solid phase technique, such as the Merrifield solid phase synthesis method in which amino acids are added to sequentially growing amino acid chains. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Automated synthesis of polypeptides is available from suppliers such as Applied BioSystems (Foster City, Calif.) And can be operated according to the manufacturer's instructions.

In certain particular embodiments, the polypeptide comprises a plurality of polypeptides as described herein, or one polypeptide as described herein and an ovarian carcinoma protein or a known tumor antigen, such as a variant of such protein. May be a fusion protein. The fusion partner may, for example, assist in providing a T helper epitope, preferably a T helper epitope recognized by humans (immunological fusion partner), or assist in expressing the protein at a higher level than the native recombinant protein. (Expression enhancer). Certain preferred fusion partners are immunological and expression enhancing fusion partners. Other fusion partners can choose to increase the solubility of the protein or to allow the protein to target the desired intracellular compartments. Another additional fusion partner includes an affinity tag that facilitates purification of the protein.

Fusion proteins can generally be prepared using standard techniques, including chemical bonding. Preferably, the fusion protein is expressed as a recombinant protein, thereby allowing it to be produced at increased levels compared to unfused proteins in the expression system. Briefly, DNA sequences encoding polypeptide components can be assembled separately and linked into suitable expression vectors. The 3'-end of the DNA sequence encoding one polypeptide component is linked to the 5'-end of the DNA sequence encoding a second polypeptide component with or without a peptide linker so that the reading frame of the sequence is present in the image. do. This allows translation into one fusion protein that maintains the biological activity of both component polypeptides.

Peptide linker sequences can be used to separate the first and second polypeptide components at sufficient intervals so that each polypeptide reliably folds into its secondary and tertiary structures. Such peptide linker sequences are inserted into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) the ability to accept flexible extended structures; (2) the inability to accept secondary structures capable of interacting with functional epitopes on the first and second polypeptides; And (3) lack of hydrophobic or charged residues that can react with the polypeptide functional epitope. Preferred peptide linker sequences contain Gly, Asn and Ser residues. In addition, other neutral amino acids, such as Thr and Ala, may be used in the linker sequence. Amino acid sequences that may be usefully used as linkers are described in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8562, 1986; And US Pat. No. 4,935,233 and US Pat. No. 4,751,180. The linker sequence may be 1 to about 50 amino acids in length. The linker sequence is not necessary if the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and interfere with steric hindrance.

The linked DNA sequence is operably linked to a suitable transcriptional or translational regulatory element. The regulatory element responsible for the expression of the DNA is located only 5 'of the DNA sequence encoding the first polypeptide. Similarly, the stop codons required to terminate the translational and transcriptional termination signals are present only at 3 'of the DNA sequence encoding the second polypeptide.

Also provided are fusion proteins comprising the polypeptides of the invention together with unrelated immunogenic proteins. Preferably, the immunogenic protein may exhibit an immunorecall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins. See, eg, Stoute et al., New Engl. J. Med., 336: 86-91, 1997].

In a preferred embodiment, the immunological fusion partner is derived from protein D, which is the surface protein of Gram-negative bacterial Haemophilus influenza B (see WO 91/18926). Preferably, the protein D derivative comprises the first about one third of the protein (eg, the first N-terminal 100-110 amino acids) and the protein D derivative may be lipidated. In certain preferred embodiments, the first 109 residues of the lipoprotein D fusion partner are included at the N-terminus to provide a polypeptide having additional exogenous T-cell epitopes. Increase the expression level in E. coli, thus acting as an expression enhancer. Lipid tails ensure optimal presentation of antigen to antigen presenting cells. Other fusion partners include NS1 (hemagglutinin), a nonstructural protein from influenza viruses. Typically, 81 N-terminal amino acids are used, although different fragments comprising T-helper epitopes can be used.

In another embodiment, the immunological fusion partner is a protein known as LYTA or a portion thereof (preferably a C-terminal portion). LYTA is encoded by the amidase LYTA [LytA gene; See Gene 43: 265-292, 1986, which is derived from Streptococcus pneumoniae, which synthesizes N-acetyl-L-alanine amidase. LYTA is an automelting agent that specifically cleaves specific bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is involved in affinity for choline or for some choline analogs such as DEAE. This property allows E. coli to express plasmids useful for expression of fusion proteins. It has been studied for the development of E. coli C-LYTA. Purification of hybrid proteins containing C-LYTA fragments at the amino terminus has been described (Biotechnology 10: 795-798, 1992). In a preferred embodiment, the repeating portion of LYTA can be inserted into the fusion protein. The repeat portion is found in the C-terminal region starting at residue 178. Particularly preferred repeat moieties include residues 188-305.

Generally, polypeptides (including fusion proteins) and polynucleotides as described herein are isolated. A "isolated" polypeptide or polynucleotide has been removed from its natural environment. For example, natural proteins are isolated if they are separated from some or all of the materials present in nature together. Such polypeptides preferably have a purity of at least about 90%, more preferably at least about 95%, and most preferably at least about 99%. Polynucleotides are considered isolated, for example, when they are cloned into a vector that is not part of the natural environment.

Binder

The invention also provides agents such as antibodies and antigen-binding fragments thereof that specifically bind to ovarian carcinoma proteins. As used herein, an antibody or antigen-binding fragment thereof may cause an ovary if it reacts with ovarian carcinoma protein at a detectable level (eg, in an ELISA), and does not react to detectably with unrelated proteins under similar conditions. It is said to "specifically bind" with carcinoma proteins. As used herein, "bond" refers to a non-covalent bond between two separate molecules that cause a "complex" to form. Binding capacity can be assessed, for example, by determining the binding constant for the formation of the complex. The binding constant is the concentration of the complex divided by the value of the component concentration. In general, the two components are referred to herein as "binding" when the binding constant of the complex formation exceeds about 10 ³ L / mol. Binding constants can be determined using methods well known in the art.

Binding agents can also be used to distinguish patients suffering from or without cancer, such as ovarian cancer. That is, an antibody or other binding agent that binds to an ovarian carcinoma antigen produces a signal indicative of the presence of cancer in at least about 20% of patients suffering from the disease, and in the absence of the disease in at least about 90% of individuals who do not have cancer. Generates a voice signal pointing to. Biological samples (eg, blood, serum, leukocytes, urine and / or from patients without cancer and individuals with cancer (as determined using standard clinical trials) to determine whether the binder meets these requirements. Tumor biopsies) can be assayed as described herein for the presence of a polypeptide that binds the binder. It will be apparent that patients suffering from disease and individuals not suffering from disease should be tested in a statistically significant number. Each binder must meet the above criteria. However, one skilled in the art will recognize that a combination of binders may be used to enhance sensitivity.

Any formulation that meets the above requirements can be a binder. For example, the binding agent may be a ribosome with or without a peptide component, RNA molecule or polypeptide. In a preferred embodiment, the binding agent is an antibody or antigen-binding fragment thereof. Antibodies can be prepared by any of a variety of techniques known to those skilled in the art (see, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be prepared via cell culture techniques, including the production of monoclonal antibodies as described herein, or by transfection of the antibody gene into a bacterial or mammalian cell host suitable for the production of recombinant antibodies. have. As one technique, immunogens, including polypeptides, are initially injected into a variety of mammals (eg, mice, rats, rabbits, sheep or goats). In this step, the polypeptide of the present invention can act as an immunogen without modification. As an alternative, particularly for relatively short polypeptides, the polypeptides may exhibit good immune responses when linked to carrier proteins such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is preferably injected into the animal host according to a predetermined schedule, including one or more additional immunizations, and blood is taken periodically from the animal. Polyclonal antibodies specific for the polypeptide can then be purified from the serum, eg, by affinity chromatography using the polypeptide linked to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest are described, for example, in Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976, and improved techniques thereof. Briefly, these methods include the preparation of immortal cell lines capable of producing antibodies with the desired specificity (ie, reactivity with the polypeptide in question). Such cell lines can be generated, for example, from spleen cells obtained from an immunized animal as described above. The spleen cells are then immortalized, for example, by fusion with a myeloma cell fusion partner, preferably one that is genetically associated with the immunized animal. Various fusion techniques can be used. For example, splenocytes and myeloma cells can be combined with a nonionic detergent for several minutes and then plated at low density in a selection medium that supports the growth of hybrid cells but not myeloma cells. Preferred selection techniques use HAT (hypoxanthine, aminopterin, thymidine) selection. After sufficient time, usually about 1 to 2 weeks, colonies of the hybrid are observed. Single colonies are selected and their culture supernatants tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity are preferred.

Monoclonal antibodies can be isolated from the supernatants of growing hybridoma colonies. In addition, several techniques can be used to increase yield, such as injecting hybridoma cell lines into the abdominal cavity of a suitable vertebrate host such as a mouse. Monoclonal antibodies can then be harvested from ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation and extraction. Polypeptides of the invention can be used, for example, in the purification of affinity chromatography steps.

In certain embodiments, it may be desirable to use antigen-binding fragments of antibodies. Such fragments include Fab fragments that can be prepared using standard techniques. Briefly, immunoglobulins were purified from rabbit serum by affinity chromatography on a Protein A bead column (Harlow and Lane, Antiodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988), digested with papain and Fab and Fc fragments. Can be obtained. Fab and Fc fragments can be separated by affinity chromatography on a Protein A bead column.

Monoclonal antibodies of the invention may bind to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducing agents, drugs, toxins and derivatives thereof. Preferred radionuclides include ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At and ²¹² Bi. Preferred drugs include methotrexate and pyrimidine and purine analogs. Preferred differentiation inducing agents include phorbol esters and butyric acid. Preferred toxins include lysine, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and the Capricorn antiviral protein.

The therapeutic agent may be linked (eg covalently) directly or indirectly (eg via a linker group) to a suitable monoclonal antibody. Direct reaction between the agent and the antibody is possible when each has a substituent that can react with the other. For example, nucleophilic groups such as amino or sulfhydryl groups on one side may react with alkyl groups containing a good leaving group (eg halide) on the other, or carbonyl-containing groups such as anhydrides or acid halides. Can be.

Alternatively, it may be desirable to bind the therapeutic agent and the antibody through a linker group. The linker group can act as a spacer to isolate the antibody from the agent to avoid interference with binding. In addition, the linker group may act to increase the chemical reactivity of the substituent on the agent or antibody, thereby increasing the binding capacity. Increasing chemical reactivity can also promote the use of agents or functional groups on agents that are otherwise impossible.

It will be apparent to those skilled in the art that various di- or multi-functional reagents, such as those described in the catalog of Pierce Chemical Co. (Rockford, Ill.), Can be used as linker groups, regardless of homo and hetero functionality. The linkage can be made, for example, via an amino group, carboxyl group, sulfhydryl group or oxidized carbohydrate moiety. There are many references to this method, for example U.S. Patent 4,671,958 to Rodwell et al.

If the therapeutic agent is more effective than when liberated from the antibody portion of the immunobinding agent of the invention, it may be desirable to use a cleavable linker group during internalization or when internalizing. Many different cleavable linker groups have been published. Mechanisms for intracellular release of agents from these linker groups include the reduction of disulfide bonds (eg, US Pat. No. 4,489,710 to Spitler), the emission of photodegradable bonds (eg, Center, etc.). US Pat. No. 4,625,014], hydrolysis of derivatized amino acid side chains (eg US Pat. No. 4,638,045 to Kohn et al.), Serum complement-mediated hydrolysis [eg, to Rodwell et al. US Pat. No. 4,671,958] and acid-catalyzed hydrolysis (eg US Pat. No. 4,569,789 to Blattler et al.).

It may be desirable to bind one or more agents to the antibody. In one embodiment, multiple molecules of the agent are bound to one antibody molecule. In another aspect, one or more types of agents can be bound to one antibody. Regardless of certain embodiments, immunocombinants with one or more agents can be prepared by a variety of methods. For example, one or more agents can be directly linked to the antibody molecule, or linkers can be provided that provide multiple binding sites. Alternatively, carriers can be used.

The carrier can retain the agent in several ways, including covalent bonds, either directly or through a linker group. Suitable carriers are proteins, peptides such as albumin (eg, US Pat. No. 4,507,234 to Kato et al.) And polysaccharides such as aminodextran (eg, US Pat. No. 4,699,784 to Shih et al.). It includes. The carrier may also contain the agent by encapsulation or non-covalent linkage, such as in liposome vesicles (see, eg, US Pat. Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide preparations include radiohalogenated small molecules and chelated compounds. For example, US Pat. No. 4,735,792 describes exemplary radioactive halogenated small molecules and their synthesis. Radionuclide chelating agents can be formed from chelating compounds, including those containing nitrogen and sulfur atoms as donor atoms for binding metals or metal oxides, radionuclides. For example, US Pat. No. 4,673,562 to Davidson et al. Describes exemplary chelating compounds and their synthesis.

Several routes for administering antibodies and immunoconjugates can be used. Typically, the administration can be intravenous, intramuscular, subcutaneous or incised tumors. It is clear that the exact dose of antibody / immunoconjugate may vary depending on the antibody used, antigen density on the tumor and the removal rate of the antibody.

Also provided herein are anti-idiotype antibodies that mimic the immunogenic portion of ovarian carcinoma protein. Such antibodies can be derived for antibodies or antigen-binding fragments thereof that specifically bind to the antigenic portion of the ovarian carcinoma protein using well known techniques. An anti-idiotype antibody that mimics an immunogenic portion of an ovarian carcinoma protein is an antibody that specifically binds to an immunogenic portion of an ovarian carcinoma protein or an antigen-binding fragment thereof.

T cell

The immunotherapeutic composition also or alternatively comprises T cells specific for ovarian carcinoma protein. Such cells can generally be prepared in vitro or in vivo using standard procedures. For example, T cells are present in or isolated from bone marrow, peripheral blood, or fractions thereof from a mammal, such as a patient, using a cell isolation system commercially available, such as the CEPRATE ™ system (CellPro Inc., Botell WA). (US Pat. No. 5,240,856, US Pat. No. 5,215,926, WO 89/06280, WO 91/16116 and WO 92/07243). Alternatively, T cells can be derived from related or unrelated humans, nonhuman animals, cell lines or cultures.

T cells can be stimulated with ovarian carcinoma polypeptides, polynucleotides encoding them and / or antigen presenting cells (APCs) expressing such polypeptides. This stimulation is carried out under conditions and for a time sufficient to generate T cells specific for the polypeptide. Preferably, the ovarian carcinoma polypeptide or polynucleotide is present in a delivery vehicle such as microspheres to promote the production of specific T cells.

If a T cell kills a target cell coated with an ovarian carcinoma polypeptide or a target cell expressing a gene encoding such polypeptide, it is considered specific for the ovarian carcinoma polypeptide. T cell specificity can be assessed using any of a variety of standard techniques. For example, in chromium release assays or proliferation assays, the stimulation index of at least two-fold increase relative to negative control in lysis and / or proliferation indicates T cell specificity. Such assays are described, for example, in Chen et al., Cancer Res. 54: 1065-1070, 1994. Alternatively, detection of T cell proliferation can be accomplished by various known techniques. For example, T cell proliferation is detected by measuring the rate of increase in DNA synthesis (eg, by pulse-labeling the culture of T cells with tritium thymidine and measuring the amount of tritium thymidine inserted into the DNA). can do. Contact with the ovarian carcinoma polypeptide (200 ng / ml to 100 μg / ml, preferably 100 ng / ml to 25 μg / ml) for 3 to 7 days should increase the proliferation of T cells by more than twofold and / or as described above. Similarly, contacting for 2-3 days results in activation of T cells, as measured using standard cytokine assays, where a two-fold increase in the level of cytokine (eg, TNF or IFN-γ) release is Direct cell activation. Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998). T cells activated in response to ovarian carcinoma polypeptide, polynucleotide or ovarian carcinoma polypeptide-expressing APC may be CD4 ⁺ and / or CD8 ⁺ . Ovarian carcinoma polypeptide-specific T cells can be propagated using standard techniques. In a preferred embodiment, T cells are derived from the patient or related or unrelated donors and administered to the patient after stimulation and proliferation.

For therapeutic purposes, CD4 ⁺ or CD8 ⁺ T cells that proliferate in response to ovarian carcinoma polypeptide, polynucleotide or APC can be propagated numerically in vitro or in vivo. In vitro proliferation of such T cells can be achieved in several ways. For example, T cells can be reexposed to ovarian carcinoma polypeptides with or without the addition of stimulatory cells that synthesize ovarian carcinoma polypeptides and / or T cell growth factors such as interleukin-2. Alternatively, one or more T cells that proliferate in the presence of an ovarian carcinoma polypeptide can be propagated numerically by cloning. Methods for cloning cells are well known in the art and include limiting dilution. After proliferation, cells are described, for example, in Chang et al., Crit Rev. Oncol. Hemato. 22: 213, 1996, may be re-administered to the patient.

Pharmaceutical Compositions and Vaccines

In certain embodiments, polypeptides, polynucleotides, binders and / or immune system cells as described herein can be incorporated into pharmaceutical compositions or vaccines. Pharmaceutical compositions comprise one or more such compounds or cells and a physiologically acceptable carrier. The vaccine may comprise one or more such compounds or cells and nonspecific immune response enhancers. Nonspecific immune response enhancers can be any substance that enhances an immune response against foreign antigens. Examples of nonspecific immune response enhancers include adjuvants, biodegradable microspheres (eg polylactic acid galactide), and liposomes [compounds are incorporated into them; Reference Example: Fullerton, US Pat. No. 4,235,877]. Vaccine formulations are generally described in M.F. Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)", Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds that may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be incorporated into the fusion polypeptide in a composition or vaccine or exist as separate compounds.

The pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is produced in situ. As noted above, DNA may be present in a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Suitable nucleic acid expression systems contain the DNA sequences necessary for expression in a patient (eg, a suitable promoter and termination signal). Bacterial delivery systems include the administration of bacteria (eg, Bacillus-Calmette-Guerrin) that express the immunogenic portion of the polypeptide on the cell surface. In a preferred embodiment, the DNA can be introduced using a viral expression system (eg, vaccinia or other varicella virus, retrovirus or adenovirus), which can include the use of a nonpathogenic (defective) replication competent virus. Suitable systems are described, for example, in Fisher-Hoch et al., PNAS 86: 317-321, 1989; Flexner et al., Ann. N. Y. Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; U.S. Patents 4,603,112, 4,769,330 and 5,017,487; WO 89/01973; US Patent No. 4,777,127; British Patent 2,200,651; European Patent No. 0,345,242; WO 91/02805; Berkner, Biotechniques 6: 616-627, 1988; Rosenfeld et al., Science 252: 431-434, 1991; Kolls et al., PNAS 91: 215-219, 1994; Kass-Eisler et al., PNAS 90: 11498-11502, 1993; Guzman et al., Circulation 88: 2838-2848, 1993; And Guzman et al., Cir. Res. 73: 1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA is also described, for example, in Ulmer et al., Science 259: 1745-1749, 1993 and reviewed in Cohen, Science 259: 1691-1692, 1993 And may be "extracted" as such. Uptake of naked DNA can be increased by coating the DNA on biodegradable beads that are efficiently transported into the cell.

Any suitable carrier known to those skilled in the art may be used in the pharmaceutical compositions of the present invention, while the type of carrier may vary depending on the mode of administration. Compositions of the present invention may be formulated for suitable modes of administration, including, for example, topical, oral, intranasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, the above carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate can be used. Biodegradable microspheres (eg polylactate polyglycolate) can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are described in US Pat. Nos. 4,897,268 and 5,075,109.

Such compositions may also contain buffers (eg neutral buffered saline or phosphate buffered saline), carbohydrates (eg glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants , Chelating agents such as EDTA or glutathione, adjuvants such as aluminum hydroxide and / or preservatives. Alternatively, the compositions of the present invention can be formulated as lyophilisates. Compounds can also be encapsulated into liposomes using well known techniques.

Any of a variety of nonspecific immune response enhancers can be used in the vaccines of the invention. For example, an adjuvant may be included. Most adjuvants are substances designed to protect antigens from rapid catabolism, such as aluminum hydroxide or mineral oil, and stimulants of immune responses (eg, lipid A, Bortadella pertussis or Mycobacterium tubercules). Rothsis (Mycobacterium tuberculosis induced protein). Suitable adjuvants include, for example, Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories; Detroit, MI), Merck adjuvant 65 (Merck and Company, Inc .; Rahway, NJ), Alum, Commercially available as biodegradable microspheres, monophosphoryl lipid A and quill A. Cytokines such as GM-CSF or interleukin-2, -7 or -12 can also be used as an adjuvant.

In the vaccines provided herein, the adjuvant compositions can be designed to predominantly induce an Th1 type immune response. High levels of Th1-type cytokines (eg, IFN-γ, IL-2 and IL-12) tend to assist in the induction of cell mediated immune responses to administered antigens. In contrast, high levels of Th2-type cytokines (eg, IL-4, IL-5, IL-6, IL-10 and TNF-β) tend to assist in the induction of humoral immune responses. After application of the vaccine as provided herein, the patient will maintain an immune response including Th1- and Th2-type responses. In a preferred embodiment where the response is predominantly Th1-type, the level of Th1-type cytokines will increase to a level greater than the level of Th2-type cytokines. The levels of these cytokines can be readily assessed using standard assays. For an overview of the classes of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

Preferred adjuvants for use to induce a predominant Th1-type reaction are, for example, monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-) together with aluminum salts. MPL) combinations. MPL adjuvant is available from Ribi ImmunoChem Research Inc .; Hamilton, MT. US Pat. No. 4,436,727; 4,877,611; 4,877,611; 4,866,034 and 4,912,094]. AS-2 (SmithKline Beecham) is also preferred. CpG-containing oligonucleotides (CpG dinucleotides are unmethylated) also induce a predominant Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555. Another preferred adjuvant is saponin, preferably QS21, which can be used in combination with other adjuvant or alone. For example, an enhanced system is a combination of monophosphoryl lipid A with a saponin derivative, for example a combination of QS21 and 3D-MPL as described in WO 94/00153 or described in WO 96/33739. As can be seen QS21 includes lower reactive compositions that are inhibited by cholesterol. Other preferred formulations include oil-in-water emulsions and tocopherols. Particularly potent adjuvant formulations including QS21, 3D-MPL and tocopherols in oil-in-water emulsions are described in WO 95/17210. Any of the vaccines provided herein can also be prepared using well known methods to generate combinations of antigens, immune response enhancers and suitable carriers or adjuvants.

The compositions described herein can be administered as part of a sustained release formulation (such as a capsule or sponge that slowly releases the compound after administration). Such formulations may generally be prepared using well known techniques and may be administered, for example, by oral, rectal or subcutaneous transplantation, or by implantation into a target site of interest. Sustained release formulations may contain polypeptides, polynucleotides, or antibodies dispersed in a carrier matrix and / or contained in a reservoir surrounded by a rate controlling membrane. Carriers for use in such formulations are biocompatible and can also be biodegradable; Preferably, the formulation provides a relatively constant level of active ingredient release. The amount of active compound contained in a sustained release preparation depends on the site of implantation, the rate of release and the expected sustained release and the nature of the condition to be treated or prevented.

Any of several delivery vehicles can be used in pharmaceutical compositions and vaccines to facilitate the generation of antigen-specific immune responses that target tumor cells. Delivery vehicles include antigen presenting cells (APCs) such as dendritic cells, macrophages, B cells, monocytes and other cells that can be engineered to be efficient APCs. Such cells are not necessary but may increase the ability to present antigens, enhance the activation and / or maintenance of T cell responses, and have anti-tumor effects and / or be immunologically suitable for inoculators (ie , Matched HLA haplotype). APCs can generally be isolated from a variety of biological fluids and organs, and can be autologous, allogeneic, allogeneic, or heterologous.

Certain preferred embodiments of the invention utilize dendritic cells or progenitor cells thereof as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and have been shown to be effective as physiological adjuvants for exhibiting prophylactic or therapeutic anti-tumor immunity. Timmerman and Levy, Ann. Rev. Med. 50: 507-529, 1999]. In general, dendritic cells are B cells (CD19 and CD20), T cells (as measured using their typical shapes [radial in situ, distinct cytoplasmic dendritic projections visible in vitro] and standard assays). CD3), monocytes (CD14) and natural killer cells (CD56) can be identified based on the lack of differentiation markers. Dendritic cells can of course be engineered to express specific cell-surface receptors or ligands not commonly found in dendritic cells in vivo or ex vivo, and such modified dendritic cells are included in the present invention. As an alternative to dendritic cells, secreted vesicle antigen-loaded dendritic cells (so-called exosomes) can be used in vaccines. See Zitvogel et al., Nature Med. 4: 594-600, 1998].

Dendritic cells and progenitor cells can be harvested from peripheral blood, bone marrow, tumor-infiltrating cells, peri-tumoral tissue-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or other suitable tissues or body fluids. For example, dendritic cells can be differentiated in vitro by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and / or TNFα to a culture of monocytes harvested from peripheral blood. Alternatively, culturing CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow with a combination of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and / or other compounds that induce maturation and proliferation of dendritic cells By addition to the medium can be differentiated into dendritic cells.

Dendritic cells are typically classified as "mature" and "mature" cells, which allows a simple way to distinguish two well-characterized phenotypes. However, this nomenclature should not be construed as excluding all possible intermediate steps of differentiation. Immature dendritic cells are characterized as APCs with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptors, mannose receptors and DEC-205 markers. Mature phenotypes are typically cells responsible for lower expression of these markers and T cell activation, such as type I and II MHCs, adsorption molecules (eg CD54 and CD11) and costimulatory molecules (eg CD40, CD80 and CD86). Characterized by high expression of surface molecules.

APCs can generally be transfected with polynucleotides encoding ovarian carcinoma antigens (or portions thereof or other variants), such that the antigen or immunogenic portion thereof is expressed on the cell surface. Such transfection can occur ex vivo, and compositions or vaccines containing such transfected cells can be used for treatment as described herein. Alternatively, a gene delivery vehicle that targets dendritic or other antigen presenting cells can be administered to the patient, resulting in transfection in vivo. In vivo or ex vivo transfection of dendritic cells is described, for example, in the methods or literature generally described in WO 97/24447 (Mahviet al., Immunology and cell Biology 75: 456-460, 1997). It can be carried out using methods known in the art, such as the gene gun method described. Antigen loading of dendritic cells can be used to culture dendritic cells or progenitor cells with polypeptides, DNA (in naked or plasmid vectors) or RNA, or to express antigen-expressing recombinant bacteria or viruses (eg vaccinia, chicken pox, adenovirus or lentivirus). Vector). Prior to loading, the polypeptide may be covalently linked to an immunological partner (eg, a carrier molecule) that provides T cell help. Alternatively, dendritic cells can be pulsed separately or in the presence of a polypeptide with an unbound immunological partner.

Cancer therapy

In a further aspect of the invention, the compositions described herein can be used for immunotherapy of cancer such as ovarian cancer. In such methods, pharmaceutical compositions and vaccines are typically administered to the patient. As used herein, "patient" refers to all warm-blooded animals, preferably humans. The patient may or may not have cancer. Thus, the pharmaceutical compositions and vaccines can be used to prevent the onset of cancer or to treat cancer patients. In certain preferred embodiments, the patient is an ovarian cancer patient. Such cancers can be diagnosed using criteria generally accepted in the art, including the presence of malignant tumors. The pharmaceutical compositions and vaccines may be administered before or after surgery to remove the initial tumor and / or before or after treatment, such as the administration of radiotherapy or conventional chemotherapeutic drugs.

In certain embodiments, immunotherapy is active immunotherapy in which the treatment relies on in vivo stimulation of the endogenous host immune system in response to tumors by administration of immune response-modifying agents (eg, tumor vaccines, bacterial adjuvants and / or cytokines). Can be.

In another embodiment, the immunotherapy may be passive immunotherapy, wherein the treatment comprises delivery of a reagent (eg, effector cell or antibody) with an established tumor immune responsiveness that can directly or indirectly mediate antitumor effects. However, it is not necessarily dependent on the complete host immune system. Examples of effector cells include T lymphocytes (eg, CD8 ⁺ cytotoxic T lymphocytes, CD4 ⁺ T-helper tumor-infiltrating lymphocytes) expressing the polypeptides provided herein, killer cells (eg, natural killer cells, lymphokine-activation). Killer cells), B cells or antigen presenting cells (eg dendritic cells and macrophages). T cell receptors and antibody receptors specific for the polypeptides provided herein can be cloned, expressed and transferred into other vectors or effector cells for quantum immunotherapy. In addition, polypeptides provided herein for passive immunotherapy (as described above and in US Pat. No. 4,918,164) can be used to generate antibodies or antiidiotype antibodies.

Effector cells can generally be obtained in sufficient amounts for quantum immunotherapy by in vitro growth as described herein. Culture conditions for proliferating billions of single antigen-specific effector cells while maintaining antigen recognition in vivo are well known in the art. These in vitro culture conditions typically utilize intermittent stimulation with antigen, often in the presence of cytokines (eg, IL-2) and non-dividing feeder cells. As noted above, the immunoreactive polypeptides provided herein can be used to rapidly propagate antigen-specific T cell cultures to produce a sufficient number of cells for immunotherapy. In particular, standard techniques known in the art can be used to pulse antigen presenting cells such as dendritic cells, macrophages or B cells with an immunoreactive polypeptide or transfect with one or more polynucleotide sequences. For example, antigen presenting cells can be transfected with polynucleotides having promoters suitable for increasing expression in recombinant viruses or other expression systems. Cultured effector cells for use in therapy should be able to grow and distribute widely and be able to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and survive long-terms while maintaining a substantial number upon repeated stimulation with an antigen supplemented with IL-2. See, eg, Cheever, et al. , Immunological Reviews, 157: 177, 1997].

Alternatively, a vector expressing a polypeptide provided herein can be introduced into stem cells taken from a patient and cloned in vitro for autologous transplantation to the same patient.

The dosage as well as the route and frequency of administration vary from person to person and can be easily established using standard techniques. In general, pharmaceutical compositions and vaccines can be administered by injection (eg intradermal, intramuscular, intravenous or subcutaneous), intranasal (eg inhalation) or orally or incised tumors. One to ten doses may be administered for a 52 week period. Preferably six doses are administered at 1 month intervals, followed by periodic boosting. Another protocol may be appropriate for individual patients. Suitable dosages are amounts of compounds which, when administered as described above, may promote an anti-tumor immune response and are at least 10-50% above the base (ie untreated) level. This response can be monitored by measuring anti-tumor antibodies in the patient or by vaccine-dependent production of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of eliciting an immune response that results in improved clinical outcomes (eg, more frequent alleviation, complete, partial or longer disease-free survival) in vaccinated patients compared to non-vaccinated patients. Generally, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in the dose is about 100 μg to about 5 mg per kg of host. Suitable dosage sizes vary depending on the size of the patient, but typical ranges are from about 0.1 ml to about 5 ml.

In general, suitable doses and therapeutic regimens provide the active compound (s) in an amount sufficient to provide therapeutic and / or prophylactic benefits. Such responses can be monitored by establishing improved clinical outcomes (eg, more frequent alleviation, complete, partial or longer term disease free survival) in treated patients compared to untreated patients. Increases in the already present immune response to ovarian carcinoma antigens generally correlate with improved clinical outcomes. Such immune responses can generally be assessed using standard proliferation, cytotoxicity or cytokine assays that can be performed using samples taken from patients before and after treatment.

Screening to Identify Secreted Ovarian Carcinoma Antigens

The present invention provides a method for identifying secreted tumor antigens. Within this method, tumors are transplanted into immunodeficient animals, such as SCID mice, and maintained for a time sufficient for tumor antigens to be secreted into serum. In general, tumors can be maintained for 1 to 9 months, preferably 1 to 4 months, implanted subcutaneously or into germline fat pads in immunodeficient animals. Implantation can generally be performed as described in WO 97/18300. Antisera are then prepared in immunocompetent mice using standard techniques and using serum containing secreted antigens as described herein. Briefly, 50-100 μl of serum (each set was collected from three sets of immunodeficiency mice containing different SCID-induced human ovarian cancer) was prepared using RIBI-MPL or MPL + TDM (Sigma Chemical Co. 1: 1 (volume: volume) with a suitable adjuvant such as St. Louis, MO), and can be injected intraperitoneally into a winter-based immunocompetent animal for 5 months. Antisera from animals immunized in this manner can be obtained by taking blood after third, fourth and fifth immunizations. The antisera produced is usually not. Safety is verified against E. coli and phage antigens and used for serological expression screens (generally diluted after 1: 200).

The library is typically an expression library containing cDNA from one or more tumors of the type implanted in SCID mice. Such expression libraries can be prepared with any suitable vector, such as λ-screen from Novagen. CDNAs encoding polypeptides that react with antisera can be identified and sequenced using standard techniques. Such cDNA molecules can be further characterized to assess expression in tumors and normal tissues, and to assess antigen secretion in patients.

The methods provided herein have advantages over other methods for tumor antigen development. In particular, all antigens identified by this method must be secreted or released through necrosis of tumor cells. Such antigens may be present on the surface of tumor cells for a time sufficient to be targeted and killed by the immune system after vaccination.

Detection method of cancer

Generally, cancer is detected from a patient based on the presence of one or more ovarian carcinoma proteins and / or polynucleotides encoding such proteins in biological samples taken from the patient (eg, blood, serum, urine and / or tumor biopsies). can do. That is, such a protein can be used as a marker indicating the presence or absence of cancer such as ovarian cancer. Such proteins may also be useful for the detection of other cancers. The binding agents provided herein generally allow for the detection of the level of protein that binds the agent in a biological sample. Polynucleotide primers and probes can be used to detect levels of mRNA encoding tumor proteins, which also indicates the presence or absence of cancer. In general, ovarian carcinoma-associated sequences should be present at levels three or more times higher in tumor tissue than in normal tissue.

Various assays are known to those skilled in the art for detecting polypeptide markers in a sample using a binder (see, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, the presence or absence of cancer in a patient includes (a) contacting a biological sample taken from the patient with a binder, (b) detecting the level of polypeptide binding to the binder in the sample, and (c) previewing the level of the polypeptide in advance. It can be measured by comparing with the measured cutoff value.

In a preferred embodiment, the assay comprises binding to and removing polypeptide from the residue of the sample using a binder immobilized on a solid support. The bound polypeptide can then be detected using a detection agent containing a reporter group and specifically binding to the binder / polypeptide complex. Such detection reagents may include, for example, a binding agent that specifically binds to a polypeptide or an antibody or other agent that specifically binds to a binding agent such as anti-immunoglobulin, protein G, protein A or lectin. Alternatively, competitive assays can be used, wherein the polypeptide is labeled with a reporter group and binding to the immobilized binder after incubation of the sample with the binder. The extent to which the components of the sample inhibit the binding of the labeled polypeptide to the binder indicates the reactivity of the sample with the fixed binder. Suitable polypeptides for use in such assays include full length ovarian carcinoma proteins and portions thereof to which the binder binds as described above.

Solid supports are known to those skilled in the art and may be any material to which a tumor protein may be attached. For example, the solid support can be a test well in a microtiter plate or nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disk, for example glass, fiber glass, latex or plastic material, for example polystyrene or polyvinylchloride. The support may also be a magnetic particle or fiber optical sensor, for example as described in US Pat. No. 5,359,681. Binders can be immobilized to a solid support using a variety of techniques known to those skilled in the art, which are extensively described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to non-covalent bonds and covalent attachments, such as adsorption, which may be direct linkages between the agent and the functional groups on the support or through crosslinking agents. Immobilization by adsorption to wells or membranes in microtiter plates is preferred. In this case, adsorption can be achieved by contacting the binder in a suitable buffer with a solid support for a suitable time. Contact time varies with temperature, but is typically about 1 hour to 1 day. In general, contacting the amount of binder from about 10 ng to about 10 μg, preferably from about 100 ng to about 1 μg, with the wells of the plastic microtiter plate (eg, polystyrene or polyvinylchloride) to secure a suitable amount of binder. Suffice.

Covalent bonding of the binder to the solid support can generally be achieved by first reacting the support with a bifunctional reagent that reacts with both the support and a functional group such as a hydroxyl or amino group on the binder. For example, the binder can be covalently bound to a support having a suitable polymer coating by using benzoquinone or by condensing aldehyde groups on the support with active hydrogens and amines on the binding partner. See, eg, Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13].

In certain embodiments, the assay is a two-antibody sandwich assay. This assay can be performed by first contacting an antibody immobilized on a well of a solid support, often a microtiter plate, with the polypeptide to bind the immobilized antibody. The unbound sample is then removed from the immobilized polypeptide-antibody complex and a detection reagent containing a reporter group (preferably a second antibody capable of binding other sites on the polypeptide) is added. The amount of detection reagent that remains bound on the solid support is then measured using a method suitable for the particular reporter group.

More specifically, once the antibody is immobilized on the support as described above, the protein binding site remaining on the support is typically blocked. Any suitable blocking agent known to those skilled in the art can be used, such as bovine serum albumin (BSA) or Tween 20 ™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample to allow the polypeptide to bind with the antibody. The sample may be diluted with a suitable diluent such as phosphate-buffered saline (PBS) prior to incubation. In general, a suitable contact time (ie, incubation time) is a time sufficient to detect the presence of the polypeptide in a sample taken from an ovarian cancer patient. Preferably, the contact time is sufficient to achieve a binding level that is at least 95% of the binding level reached in equilibrium between the bound polypeptide and the unbound polypeptide. Those skilled in the art will appreciate that the time required to achieve equilibrium can be readily determined by assaying the level of binding that occurs at any given time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound samples can then be removed by washing the solid support with a suitable buffer such as PBS containing 0.1% Tween 20 ™. Subsequently, a second antibody containing a reporter group can be added to the solid support. Preferred reporter groups include those described above.

The detection reagent is then incubated for a time sufficient to detect the polypeptide bound to the immobilized antibody-polypeptide complex. Suitable amounts of time can generally be determined by assaying the level of binding that occurs over time. The unbound detection reagent is then removed and the bound detection reagent is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation coefficients or autoradiography are generally suitable. Spectroscopic methods can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin linked to different reporter groups (often radioactive or fluorescent groups or enzymes). Enzyme reporter groups can generally add a substrate (usually for a certain period of time) and then detect the reaction product by spectroscopic analysis or other analysis.

To determine the presence of cancer, such as ovarian cancer, the signal detected from the reporter group bound to the solid support is generally compared with a signal corresponding to a pre-measured cutoff value. In one preferred embodiment, the cutoff value for detection of cancer is the average signal obtained when the immobilized antibody is incubated with a sample from a patient without cancer. In general, a sample that generates a signal that exhibits three standard deviations above a pre-measured cutoff value is considered to be positive for cancer. In another preferred embodiment, the cutoff value is described in Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107], using the receiver operator curve. Briefly, in this embodiment, the cutoff value can be determined from the plot of true positive rate (ie, sensitivity) and false positive rate (100% -specificity) corresponding to each possible cutoff for diagnostic test results. The cutoff value on the plotting closest to the upper left corner (ie, the value that occupies the largest area) is the most precise cutoff value, and a signal that produces a signal larger than the cutoff value determined by this method can be considered positive. Alternatively, the cutoff value can be shifted to the left along the plot if the false positive rate is minimized or to the right if the false negative rate is minimized. In general, samples that generate signals greater than the cutoff value determined by this method are judged positive for cancer.

In a related embodiment, the assay is carried out by a flow-through or strip test method in which the binder is fixed to a membrane such as nitrocellulose. In the flow-through test, the polypeptide in the sample binds to the fixed binder as the sample passes through the membrane. The second labeled binder is then bound to the binder-polypeptide complex while the solution containing the second labeled binder is passed through the membrane. Detection of the bound second binder may then be performed as described above. In the strip test method, one end of the membrane to which the binder is bound is immersed in the solution containing the sample. The sample moves along the membrane through the region containing the second binder and into the region of the fixed binder. The concentration of the second binder in the region of the immobilized antibody indicates the presence of cancer. Typically, the concentration of the second binder at this site forms a line-like pattern that is visually readable. The absence of such a pattern indicates a negative result. In general, the amount of binder immobilized on the membrane is such that a biological sample contains a two-antibody sandwich assay, i.e., a level of polypeptide sufficient to generate a positive signal in the method discussed above, to form a visually discernible pattern. Choose. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane is from about 25 ng to about 1 μg, more preferably from about 50 ng to about 500 ng. Such testing can typically be performed with very small amounts of biological samples.

Of course, there are many other assay protocols suitable for use with the tumor proteins or binders of the invention. The above description is merely illustrative. For example, it will be apparent to one skilled in the art that the protocol can be readily modified to detect antibodies that bind the polypeptide in a biological sample using an ovarian carcinoma polypeptide. Detection of such ovarian carcinoma protein specific antibodies may be correlated with the presence of cancer.

Cancer may also or alternatively be detected based on the presence of T cells that specifically react with ovarian carcinoma proteins in biological samples. In certain methods, a biological sample comprising CD4 ⁺ and / or CD8 ⁺ T cells isolated from a patient is used to express an ovarian carcinoma protein, a polynucleotide encoding such a polypeptide and / or an APC expressing at least an immunogenic portion of such polypeptide. Incubated with and detect the presence or absence of specific activation of T cells. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from patients by conventional techniques (eg, Ficoll / Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells can be cultured in vitro with ovarian carcinoma proteins (eg, 5-25 μg / ml) at 37 ° C. for 2-9 days (typically 4 days). It may be desirable to culture other aliquots of T cell samples in the absence of ovarian carcinoma proteins serving as controls. In the case of CD4 ⁺ T cells, activation is preferably detected by assessing the proliferation of T cells. In the case of CD8 ⁺ T cells, activation is preferably detected by assessing cytolytic activity. The level of proliferation at least 2 times higher and / or at least 20% greater cytolytic activity than disease free patients indicates the presence of cancer in the patient.

As noted above, cancer may also be detected based on the level of mRNA encoding ovarian carcinoma protein in a biological sample. For example, two or more oligonucleotide primers can be used in the polymerase chain reaction (PCR) basic assay to amplify a portion of the ovarian carcinoma protein cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is an ovary Specific for polynucleotides encoding carcinoma proteins (ie hybridization). Next, amplified cDNA is isolated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to polynucleotides encoding ovarian carcinoma proteins can be used in hybridization assays to detect the presence of polynucleotides encoding tumor proteins in biological samples.

In order for hybridization to occur under assay conditions, the oligonucleotide primers and probes must be at least about 60%, preferably at least about 75%, with a portion of an ovarian carcinoma protein encoding polynucleotide that is at least 10 nucleotides in length, preferably at least 20 nucleotides in length. Or, more preferably, at least about 90% identity with an oligonucleotide sequence. Preferably, the oligonucleotide primers and / or probes hybridize to polynucleotides encoding polypeptides described herein under moderately stringent conditions as defined herein. Oligonucleotide primers and / or probes that can be usefully used in the diagnostic methods described herein are preferably at least 10 to 40 nucleotides in length. In a preferred embodiment, the oligonucleotide primer comprises at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides of a DNA molecule having a sequence provided herein. Techniques for PCR based assays and hybridization assays are well known in the art. See, eg, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989].

One preferred assay uses RT-PCR where PCR is applied in combination with reverse transcription. Typically, RNA is extracted from biological samples, such as biopsy tissue, and reverse transcribed to produce cDNA molecules. PCR amplification with one or more specific primers results in cDNA molecules, which can be isolated and visualized using, for example, gel electrophoresis. Amplification can be performed on biological samples taken from the subject and the subject not suffering from cancer. The amplification reaction can be performed on several cDNA dilutions within a 2-fold range. It is typically considered positive if expression increases more than twofold in several dilutions of the test patient sample relative to the same dilution of the non-cancer sample.

In other embodiments, ovarian carcinoma proteins and polynucleotides encoding such proteins can be used as markers to monitor cancer progression. In such embodiments, the above assays for diagnosing cancer can be performed over time and can assess changes in the level of reactive polypeptide (s). For example, the assay may be performed every 24 to 72 hours for a period of six months to one year, and thereafter as needed. In general, cancer is progressing in patients in which the level of polypeptide detected by the binder increases over time. In contrast, cancer does not progress when the level of reactive polypeptide is constant or decreases over time.

Certain in vivo diagnostic assays can be performed directly on tumors. One such assay involves contacting tumor cells with a binder. The bound binder can then be detected either directly or indirectly through the reporter group. Such binders can also be applied to histology. Alternatively, polynucleotide probes can be used for this application.

As noted above, to improve sensitivity, multiple ovarian carcinoma protein markers can be assayed in provided samples. It will be apparent that binders specific for the different proteins provided herein may be combined in a single assay. In addition, multiple primers or probes may be used simultaneously. The selection of tumor protein markers can be made based on routine experimentation to determine the combination that leads to optimal sensitivity. In addition or alternatively, assays for tumor proteins provided herein can be combined with assays for other known tumor antigens.

Diagnostic kit

The invention also provides kits for use in any of the above diagnostic methods. Such kits typically contain two or more components necessary to perform a diagnostic assay. The component may be a compound, a reagent, a container and / or equipment. For example, one container in the kit may contain a monoclonal antibody or fragment thereof that specifically binds to an ovarian carcinoma protein. Such antibodies or fragments may be provided attached to the support material described above. One or more additional containers may contain elements such as reagents or buffers to be used in the assay. Such kits may also or alternatively contain the above-described detection reagents containing reporter groups suitable for direct or indirect detection of antibody binding.

Alternatively, the kit can be designed to detect the level of mRNA encoding ovarian carcinoma protein in a biological sample. Such kits generally comprise one or more oligonucleotide probes or primers as described above that hybridize to a polynucleotide encoding an ovarian carcinoma protein. Such oligonucleotides can be used, for example, in PCR or hybridization assays. Additional components that may be present in such kits include a second oligonucleotide and / or a diagnostic reagent or container for facilitating detection of polynucleotides encoding ovarian carcinoma proteins.

The following examples are provided for the purpose of illustration of the invention, but not to limit it.

Example 1

Identification of Representative Ovarian Carcinoma Protein cDNA

This example illustrates the identification of cDNA molecules encoding ovarian carcinoma proteins.

Anti-SCID mouse serum (generated for serum from SCID mice with terminal ovarian carcinoma); Safety is validated in advance for E. coli and phage antigens and used at a 1: 200 dilution in serological expression screens. A library selected from SCID-induced human ovarian tumor (OV9334) is prepared using a directional RH oligo (dT) priming cDNA library construction kit and a λScreen vector from Novagen. Use a bacteriophage lambda screen. Amplified OV9334 library of about 400,000 pfu is selected.

Isolate 196 positive clones. Certain sequences that appear novel are provided in FIGS. 1A-1S and SEQ ID NOs: 1-71. Three complete insert sequences are shown in FIGS. 2A-2C (SEQ ID NOS: 72-74). Other clones with known sequences are shown in FIGS. 15A-15EEE (SEQ ID NOs: 82-310). Database searches identify the following sequences that are substantially identical to the sequences set forth in FIGS. 15A-15EEE.

These clones are further characterized using microarray technology to measure mRNA expression levels in various tumors and normal tissues. This analysis is performed using Synteni (Palo alto, CA) microarrays according to the manufacturer's instructions. PCR amplification products are aligned on slides, each product occupying a specific position in the alignment. MRNA is extracted from the tissue sample and reverse transcribed to generate a fluorescently-labeled cDNA probe. Microarrays are probed with labeled cDNA probes and the slides are scanned to measure fluorescence intensity. Analyze the data using the GEMtools software provided by Synteni. The results for a particular clone (13695, also named O8E) are shown in FIG. 3.

Example 2

Identification of Ovarian Carcinoma cDNA Using Microarray Technology

This example illustrates the identification of ovarian carcinoma polynucleotides by PCR subtraction and microarray analysis. Microarrays of cDNA were synthesized using Synteni (Palo alto, Calif.) microarrays according to the manufacturer's instructions and essentially for ovarian cancer-specific expression. Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94: 2150-2155, 1997].

PCR subtraction is performed using a tester comprising cDNA of four ovarian tumors (three of which are metastatic tumors) and a driver of cDNA from five normal tissues (adrenal, lung, pancreatic, spleen and brain). The cDNA fragments recovered from this subtraction are subjected to DNA microarray analysis, where the fragments are PCR amplified, attached to the chip, and hybridized with fluorescently labeled probes derived from mRNA of human ovarian tumors and various normal human tissues. Let's do it. In this analysis, slides are scanned, fluorescence intensity is measured, and the data is analyzed using Synteni GEMtools software. In general, sequences that exhibit at least five-fold increase in expression (relative to normal cells) in tumor cells are considered ovarian tumor antigens. Fluorescence results are analyzed and clones showing increased expression in ovarian cancer are further characterized by DNA sequencing and database investigation to determine the novelty of the sequence.

Using this assay, the ovary is a splice fusion between human T cell leukemia virus type I carcinogenic protein TAX (Jin et al., Cell 93: 81-91, 1998) and an extracellular matrix protein called osteonectin. Identify cancer antigens. The splice conjugation sequence is at the fusion point. The sequence of this clone is shown in Figure 4 and SEQ ID NO: 75. Osteonectin that is not spliced and unmodified independently from this assay is also identified.

Additional clones identified by this method are referred to herein as 3f, 6b, 8e, 8h, 12c and 12h. The sequences of these clones are shown in FIGS. 5-9 and SEQ ID NOs: 76-81. Microarray analysis is performed as described above and shown in FIGS. 10-14. By selecting an ovarian tumor (SCID-derived) cDNA library, full length sequences including clones 3f, 6b, 8e and 12h are obtained. This 2996 base pair sequence (named O772P) is shown in SEQ ID NO: 311 and the encoded 914 amino acid protein sequence is shown in SEQ ID NO: 312. PSORT analysis indicates type 1a dura protein located on the plasma membrane.

In addition to the above specific sequences, these selections identify the following sequences detailed in Table 1.

order Remark OV4vG11 (SEQ ID NO: 313) Human clone 1119D9 on chromosome 20p12 OV4vB11 (SEQ ID NO: 314) Human UWGC from Chromosome 6p21: y14c094 OV4vD9 (SEQ ID NO: 315) Human clone 1049G16 chromosome 20q12-13.2 OV4vD5 (SEQ ID NO: 316) Human KIAA0014 Gene OV4vC2 (SEQ ID NO: 317) Human KIAA0084 Gene OV4vF3 (SEQ ID NO: 318) Human Chromosome 19 Cosmid R31167 OV4VC1 (SEQ ID NO: 319) new OV4vH3 (SEQ ID NO: 320) new OV4vD2 (SEQ ID NO: 321) new O815P (SEQ ID NO: 322) new OV4vC12 (SEQ ID NO: 323) new OV4vA4 (SEQ ID NO: 324) new OV4vA3 (SEQ ID NO: 325) new OV4v2A5 (SEQ ID NO: 326) new O819P (SEQ ID NO: 327) new O818P (SEQ ID NO: 328) new O817P (SEQ ID NO: 329) new O816P (SEQ ID NO: 330) new OV4vC5 (SEQ ID NO: 331) new 21721 (SEQ ID NO: 332) People lumican 21719 (SEQ ID NO: 333) Human Retinoic Acid-binding Protein II 21717 (SEQ ID NO: 334) Human 26S Proteasome ATPase Subunit 21654 (SEQ ID NO: 335) Person Copine I 21627 (SEQ ID NO: 336) Human Neuron Specific Gamma-2 Enolase 21623 (SEQ ID NO: 337) Human Geranylgeranyl Transferase II 21621 (SEQ ID NO: 338) Human cyclin-dependent protein kinases 21616 (SEQ ID NO: 339) Human prepro-megakaryotic enhancer 21612 (SEQ ID NO: 340) Person UPH1 21558 (SEQ ID NO: 341) Human RalGDS-Type 2 (RGL2) 21555 (SEQ ID NO: 342) Human autoantigen P542

order Explanation 21548 (SEQ ID NO: 343) Human Actin-Related Protein (ARP2) 21462 (SEQ ID NO: 344) Human huntingtin interacting protein 21441 (SEQ ID NO: 345) Human 90K product (tumor related antigen) 21439 (SEQ ID NO: 346) Human guanine nucleotide regulatory protein (tim1) 21438 (SEQ ID NO: 347) Human Ku autoimmune (p70 / p80) antigens 21237 (SEQ ID NO: 348) Person S-Laminin 21436 (SEQ ID NO: 349) Person Ribophorin I 21435 (SEQ ID NO: 350) Human cytoplasmic chaperonin hTRiC5 21425 (SEQ ID NO: 351) Person EMX2 21423 (SEQ ID NO: 352) Human p87 / p89 gene 21419 (SEQ ID NO: 353) Person HPBRII-7 21252 (SEQ ID NO: 354) Person T1-227H 21251 (SEQ ID NO: 355) Person Coolin I 21247 (SEQ ID NO: 356) Kunitz-type protease inhibitors (KOP) 21244-1 (SEQ ID NO: 357) Human protein tyrosine phosphatase receptor F (PTPRE) 21718 (SEQ ID NO: 358) Human LTR Repeat OV2-90 (SEQ ID NO: 359) new Man Zinc Finger (SEQ ID NO: 360) Human PolyA Binding Proteins (SEQ ID NO: 361) Person Panropin (SEQ ID NO: 362) Human PAC Clone 278C19 (SEQ ID NO: 363) Human LLRep3 (SEQ ID NO: 364) Human Kunitz-type protease inhibitors (SEQ ID NO: 365) Human KIAA0106 gene (SEQ ID NO: 366) Human keratin (SEQ ID NO: 367) Human HIV-1TAR (SEQ ID NO: 368) Human glial induced nexin (SEQ ID NO: 369) Human Fibronectin (SEQ ID NO: 370) Human ECM Pro BM40 (SEQ ID NO: 371) Human collagen (SEQ ID NO: 372) Human Alpha Enolase (SEQ ID NO: 373) Human Aldolase (SEQ ID NO: 374) Human Transition Growth Factor BIG H3 (SEQ ID NO: 375) Human SPARC Osteonectin (SEQ ID NO: 376) Human SLP1 Leukocyte Protease (SEQ ID NO: 377) Human Mitochondrial ATP Synthetase (SEQ ID NO: 378) Human DNA Sequence Clone 461P17 (SEQ ID NO: 379) Man dpbB Pro Y Box (SEQ ID NO: 380) Person 40kDa keratin (SEQ ID NO: 381) Human arginosuccinate synthetase (SEQ ID NO: 382) Human Acid Ribosome Phosphoprotein (SEQ ID NO: 383) Human Colon Carcinoma Laminin Binding Protein (SEQ ID NO: 384)

This screening further identifies multiple forms of clone O772P (herein named 21013, 21003 and 21008). PSORT analysis indicates that 21003 (SEQ ID NO: 386; translated into SEQ ID NO: 389) and 21008 (SEQ ID NO: 387; translated into SEQ ID NO: 390) represent the type 1a transmembrane protein form of O772P. 21013 (SEQ ID NO: 385; translated into SEQ ID NO: 388) appears to be a truncated form of the protein, presumed to be a secreted protein by PSORT analysis.

Further sequencing yields a full length clone for O8E (2627 bp consistent with the message size observed by Northern analysis; SEQ ID NO: 391). This nucleotide sequence is obtained as follows: The original O8E sequence (OrigO8Econs) was found to overlap the sequence from the EST clone (IMAGE # 1987589) with as many as 33 nucleotides. This clone provides 1042 additional nucleotides upstream of the original O8E sequence. Linkage between EST and O8E is verified by sequencing multiple PCR fragments generated from ovarian early tumor libraries using primers for specific EST and O8E sequences (ESTxO8EPCR). If the immobilized PCR from the ovarian cancer library provides several clones terminated upstream of putative starting methionine (AnchoredPCR cons), the full-length status appears further, but no further sequence information is obtained. 16 shows a diagram depicting the location of each subsequence within the full length O8E sequence.

Two protein sequences can be translated from full length O8E. “a” (SEQ ID NO: 393) starts with putative starting methionine. The second form “b” (SEQ ID NO: 392) comprises 27 additional upstream residues at the 5′-end of the nucleotide sequence.

Example 3

This example discloses the identification and characterization of antibody epitopes recognized by O8E polyclonal antiserum.

this. Rabbit antiserum against O8E recombinant protein from E. coli is generated to test the epitope recognition response of the antibody against a 20- or 21-mer peptide corresponding to the O8E amino acid sequence. Peptides spanning amino acid regions 31-65, 76-110, 136-200 and 226-245 in the full length O8E protein are recognized by acid eluted peaks and / or salt eluted peaks from affinity purified anti-O8E serum do. As such, the amino acid sequence corresponding to the peptide constitutes an antibody epitope recognized by an affinity purified anti-O8E antibody.

The result of ELISA analysis of anti-O8E rabbit serum is shown in FIG. 23, and the result of ELISA analysis of affinity purified rabbit anti-O8E polyclonal antibody is shown in FIG.

For epitope mapping, 20- or 21-mer peptides corresponding to O8E proteins are synthesized. For antibody affinity purification, rabbit anti-O8E serum was run out on an O8E Sepharose column, and then the antibody was eluted with salt buffer containing 0.5M NaCl and 20mM PO ₄ , followed by 0.2M glycine, pH 2.3. Is applied to the acid elution step. Purified antibody is neutralized by the addition of 1M Tris (pH 8) and the buffer is exchanged with phosphate buffered saline (PBS). For enzyme-linked immunosorbent assay (ELISA) analysis, O8E peptide and O8E recombinant protein are coated in 96-well flat plates at 2 μg / ml for 2 hours at room temperature (RT). Plates are then washed 5 times with PBS + 0.1% Tween 20 and blocked with PBS + 1% bovine serum albumin (BSA) for 1 hour. Acid or salt eluted fractions of affinity purified anti-O8E antibodies are added to the wells at 1 μg / ml and then incubated for 1 hour at RT. After washing the plate again, donkey anti-rabbit-Ig-horseradish peroxidase (HRP) antibody is added and reacted for 1 hour at RT. The plates are washed and developed by addition of the chromogenic substrate 3,3 ', 5,5'-tetramethylbenzidine (TMB). Bos et al., J. of Immunoassay 2 : 187-204 (1981). ; From Sigma (St. Louis, MO). The reaction is incubated for 15 minutes at RT and then stopped by addition of 1N H ₂ SO ₄ . The plate is read at optical density 450 (OD450) using an automatic plate reader. The sequence of peptides corresponding to O8E antibody epitopes is disclosed herein as SEQ ID NOs: 394-415. Antibody epitopes recognized by the O8E polyclonal antiserum are shown in FIG. 17.

Example 4

This example discloses IHC analysis of O8E expression in ovarian cancer tissue samples.

For immunohistochemistry studies, paraffin-sealed formalin fixed ovarian cancer tissues are divided into 8 micron sections. Steam heat induced epitope recovery (SHIER) in 0.1 M sodium citrate buffer (pH 6.0) is used for optimal staining conditions. Sections are incubated with 10% serum / PBS for 5 minutes. A primary antibody (anti-O8E rabbit affinity purified polyclonal antibody) is added to each section for 25 minutes and then incubated with the anti-rabbit biotinylated antibody for 25 minutes. Endogenous peroxidase activity is blocked by incubating with hydrogen peroxidase three times for 1.5 minutes. An avidin biotin complex / horseradish peroxidase system is used in conjunction with a DAB chromophore to visualize expression of antigen. The slides are then counterstained with hematoxylin. One of the six ovarian cancer tissue sections (serous papilla carcinoma) shows O8E immunoreactivity. In optimizing staining conditions, 4/5 ovarian cancer samples are positively stained using O8E polyclonal antibodies. O8E expression is localized in the plasma membrane.

Six ovarian cancer tissues are analyzed with anti-O8E rabbit polyclonal antibodies. One of six ovarian cancer tissue samples (serous papilla carcinoma) is stained positive for O8E expression. O8E expression is localized to the surface membrane.

Example 5

This example discloses O8E peptides that bind to HLA-A2 and are presumed immunogenic to CD8 T cell responses in humans.

Potential HLA-A2 binding peptides of O8E are estimated by using the full-length open-reading frame (ORF) from O8E and working through "Episeek", a program used to estimate MHC binding peptides. The program used is described in Parker, KC et al., J. Immunol. 152 (1) : 163-175 (1994), the entire contents of which are incorporated herein by reference. Demonomers and hexameric peptides suspected of binding to HLA-0201 are disclosed herein as SEQ ID NOs: 416-435 and SEQ ID NOs: 436-455, respectively.

Example 6

This example discloses O8E cell surface expression measured by fluorescence activated cell sorting.

For FACS analysis, cells are washed with ice cold staining buffer (PBS / 1% BSA / azide). Cells are then incubated for 30 minutes on ice with 10 μg / ml of affinity purified rabbit anti-B305D polyclonal antibody. The cells are washed three times with staining buffer and incubated for 30 minutes on ice with goat anti-rabbit Ig (H + L) -FITC reagent (Southern Biotechnology) 1: 100 dilution. After washing three times, the cells are resuspended in staining buffer containing prodium iodide, a biostaining agent that allows the identification of permeable cells and analyzed by FACS. O8E surface expression is confirmed on SKBR3 breast cancer cells and HEK293 cells stably overexpressing cDNA for O8E. None of the MB415 cells or HEK293 cells stably transfected with unrelated plasmid DNA for control showed surface expression of O8E (FIGS. 18 and 19).

Example 7

This example further evaluates expression and surface localization of O8E.

For expression and purification of antigens used for immunization, E. O8E expressed in E. coli recombinant expression system is added to LB medium with the appropriate antibiotics and allowed to grow overnight at 37 ° C. in shake incubator. The next morning, 10 ml of overnight cultures are added to 500 ml of 2 × YT + suitable antibiotics in a 2 L-baffle equipped Erlenmeyer flask. When the optical density (at 560 nm) of the culture reaches 0.4 to 0.6, the cells are induced with IPTG (1 mM). After induction for 4 hours with IPTG, cells are harvested by centrifugation. Cells are then washed with phosphate buffered saline and centrifuged again. The supernatant is discarded and the cells frozen or immediately processed for later use. 20 ml of lysis buffer is added to the cell pellet and vortex. Next, this. To cleave E. coli cells, the mixture is passed through a French Press under a pressure of 16,000 psi. Next, the cells are centrifuged again, and the supernatants and pellets are examined for fractionation of the recombinant protein by SDS-PAGE. For proteins localized to the cell pellet, the pellet is resuspended in 10 mM Tris pH 8.0, 1% CHAPS, the inclusion body pellets are washed and then centrifuged again. Repeat this process two or more times. Washed inclusion pellets are solubilized with 8M urea or 6M guanidine HCL + 10 mM Tris pH 8.0 + 10 mM imidazole. Solubilized protein is added to 5 ml of nickel-chelate resin (Qiagen) and incubated for 45 minutes to 1 hour at room temperature under continuous shaking. After incubation, the resin and protein mixtures are injected through a disposable column and the effluent is collected. The column is then washed with 10-20 column volumes of solubilization buffer. The antigen is eluted from the column using 8M urea, 10 mM Tris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. Next, run through an SDS-PAGE gel to determine which fraction to pool from the column for further purification. As a final purification step, a strong anion exchange resin such as Hi-Prep Q (Biorad) is equilibrated with suitable buffer and the pooled fractions from above are packed onto a column. Each antigen is eluted from the column with increasing salt gradient. Fractions are collected as the column is run, and separate SDS-PAGE gels are run to determine the fractions to be pooled from the column. The pooled fractions are dialyzed against 10 mM Tris pH 8.0. The material was then subjected to acceptable purity as measured by SDS-PAGE or HPLC, concentration as measured by Lowry assay or amino acid analysis, identity as measured by amino terminal protein sequencing and Limulus (LAL). Evaluate endotoxin levels as measured by the assay. The protein is then filtered through a 0.22 micron filter, placed in a vial and frozen until needed for immunization.

For the production of polyclonal antiserum, 400 μg of each prostate antigen is mixed with 100 μg of muramyldipeptide (MDP). Equal amounts of incomplete Freund's adjuvant (IFA) are added and then mixed. 100 μg of the antigen mixed with the same amount of IFA is additionally injected into the animals every four weeks. Animal blood is taken 7 days after each additional injection. Blood is incubated at 4 ° C. for 12-24 hours and then centrifuged to obtain serum.

For characterization of polyclonal antiserum, 96 well plates are coated with 50 μl of antigen (typically 1 μg) incubated at 4 ° C. for 20 hours. 250 μl of BSA blocking buffer is then added to the wells and incubated for 2 hours at RT. Plates are washed six times with PBS / 0.01% Tween. Anti-O8E rabbit serum or affinity purified anti-O8E antibody is diluted with PBS. 50 μl of diluted antibody is added to each well and incubated for 30 minutes at RT. The plates are washed as described above and then 50 μl of goat anti-rabbit horseradish peroxidase (HRP) at 1: 10000 dilution is added and incubated for 30 minutes at room temperature. Plates are washed as described above and 100 μl of TMB microwell peroxidase substrate is added to each well. After 15 minutes of incubation in the dark at room temperature, the color reaction is stopped with 100 μl of 1N H ₂ SO ₄ and immediately read at 450 nm. All polyclonal antibodies show immunoreactivity against the O8E antigen.

For recombinant expression in mammalian HEK293 cells, full length O8E cDNA is subcloned into mammalian expression vectors pcDNA3.1 + and pCEP4 (Invitrogen) modified to include His and FLAG epitope tags, respectively. These constructs are transfected into HEK293 cells (ATCC) using Fugene 6 reagent (Roche). Briefly, HEK293 cells are plated at a density of 100,000 cells / ml in DMEM (Gibco) containing 10% FBS (Hyclone) and propagated overnight. The next day, 2 μl of Fugene 6 is added to 100 μl of DMEM without FBS and incubated for 15 minutes at room temperature. Fugene 6 / DMEM mixture is then added to 1 μg O8E / pCEP4 or O8E / pcDNAA3.1 plasmid DNA and incubated for 15 minutes at room temperature. Fugene / DNA mixture is then added to HEK293 cells and incubated for 48-72 hours at 37 ° C. under 7% CO ₂ . Cells are washed with PBS, then harvested and pelleted by centrifugation. For Western blot analysis, cells are incubated for 30 minutes in lysis buffer containing Triton-X100 on ice to prepare whole cell lysates. The lysate is then clarified by centrifugation at 10,000 rpm for 5 minutes at 4 ° C. Samples are diluted with SDS-PAGE fill buffer containing beta-mercaptoethanol and then heated for 10 minutes before filling into SDS-PAGE gels. Proteins are transferred to nitrocellulose and probed using anti-O8E rabbit polyclonal serum # 2333L at a 1: 750 dilution. The blot is expressed as goat anti-rabbit Ig bound to HRP and cultured in ECL substrate.

For FACS analysis, cells are further washed with ice-cold staining buffer (PBS + 1% BSA + Azide). Cells are then incubated for 30 minutes on ice with 10 μg / ml of Protein A purified anti-O8E polyclonal serum. The cells are washed three times with staining buffer and then incubated for 30 minutes on ice with goat anti-rabbit Ig (H + L) -FITC reagent (Southern Biotechnology) at a 1: 100 dilution. After washing three times, the cells are resuspended in staining buffer containing propidium iodide (PI), a biostaining agent that allows the identification of permeable cells and analyzed by FACS.

From these experiments, O8E expression is detected on the surface of transfected HEK293 cells and SKBR3 cells by FACS analysis using rabbit anti-O8E serum, as shown in FIGS. 20 and 21. In addition, expression is detected by Western blot analysis in transfected HEK293 cell lysates (FIG. 22).

Example 8

Generation and Characterization of Anti-O8E mAbs

this. Mouse monoclonal antibodies are generated for the O8E protein from E. coli. Intraperitoneal (IP) injection of complete Freund's adjuvant (CFA) containing 50 μg recombinant O8E followed by further intraperitoneal injection of incomplete Freund's adjuvant (IFA) containing 10 μg recombinant O8E protein A / J immunize mice. Three days prior to removal of the spleen, mice were immunized by intravenous injection of about 50 μg of soluble O8E recombinant protein. Spleens of mice with positive titers for O8E are isolated, single cell suspensions are prepared, and then fused with SP2 / 0 myeloma cells to generate B cell hybridomas. Supernatants from hybrid clones are tested for specificity for recombinant O8E by ELISA and epitopes are mapped using peptides that span the entire O8E sequence. MAbs are also tested for their activity of detecting O8E present on the surface of cells stably transfected with O8E and the surface of breast tumor cell lines by flow cytometry.

For ELISA analysis, 96 well plates are coated at a concentration of 1 to 2 μg / ml or 10 μg / ml, respectively, of overlapping 20-mer peptides spanning the recombinant O8E protein or the entire O8E molecule. After coating, the plates are washed five times with wash buffer (PBS + 0.1% Tween-20) and blocked with PBS containing 0.5% BSA, 0.4% Tween-20. Next, hybrid supernatant or purified mAb is added and the plate is incubated at room temperature for 60 minutes. The plates are washed five times with wash buffer and reacted for 60 minutes by adding donkey antimouse Ig (Jackson ImmunoResearch) bound with horseradish peroxidase (HRP), a secondary antibody. The plate is washed again five times with wash buffer and then the peroxidase substrate is added. Of the hybridoma clones produced, 15 clones secrete mAbs that recognize the entire O8E protein. Epitope mapping revealed that 14 of these 15 clones secreted mAbs that recognize O8E amino acid residues 61-80, and one clone secreted mAbs that recognize amino acid residues 151-170.

For flow cytometry, HEK293 cells stably transfected with O8E and SKBR3 cells expressing O8E mRNA are harvested and washed with flow staining buffer (PBS + 1% BSA + azide). Cells are incubated for 30 min in the supernatant from mAb hybrid and on ice, then washed three times with staining buffer. Cells are incubated with goat-anti mouse Ig-FITC for 30 minutes on ice, then washed three times with staining buffer and then resuspended in wash buffer containing propidium iodide. Flow cytometry revealed that 15/15 mAb was able to detect O8E protein expressed on O8E transfected HEK293 cell surface. The 6/6 mAb tested for SKBR3 cells can recognize surface expressed O8E.

Example 9

Extended DNA and Protein Sequence Analysis of Sequence O772P

Full length sequences comprising clones 3f, 6b, 8e and 12 are obtained by selecting the ovarian tumor (SCID derived) cDNA libraries described in detail in Example 2. The 2996 base pair sequence designated O772P is shown in SEQ ID NO: 311, and the encoded 914 amino acid protein sequence is shown in SEQ ID NO: 312. The DNA sequence O772P was searched in a public database including Genbank, showing a significant hit with Genbank Accession No. AK024365 (SEQ ID NO: 457). This Genbank sequence was found to be 3557 base pairs in length and encode a 1156 amino acid long protein (SEQ ID NO: 459). Truncated residues 25-3471 of this sequence (residue 25 correspond to the first ATG start codon of Genbank sequence (SEQ ID NO: 456)) encode a protein of SEQ ID NO: 1148 amino acids (SEQ ID NO: 458). The published DNA sequence (SEQ ID NO: 457) differs from O772P in that it has a five base pair insert corresponding to bases 958-962 of SEQ ID NO: 457. This insert generates a frame shift so that SEQ ID NO: 457 encodes the additional N-terminal protein sequence (SEQ ID NO: 312) for O772P. O772P also encodes a unique N-terminus contained in residues 1-79 (SEQ ID NO: 460). In addition, residues 1-313, the N-terminal end of SEQ ID NO: 456, contain a unique sequence and are described as SEQ ID NO: 461.

Example 10

Generation of Polyclonal Antibodies for Immunohistochemistry and Flow Cytometry of Cell-Related Expression Patterns of Molecule O772P

O772P molecules are identified in Examples 2 and 9 herein. In order to investigate the subcellular localization and specificity of antigen expression in various tissues, polyclonal antibodies against O772P are generated. For the preparation of antibodies, O772P-1 (amino acids 44-772 of SEQ ID NO: 312) and O772P-2 (477-914 of SEQ ID NO: 312) were used. Expression in E. coli recombinant expression system and propagation overnight at 37 ° C. in LB medium. The next day, 10 ml of overnight cultures are added to 500 ml of 2x YT containing a suitable antibiotic. When the optical density (560 nm) of the culture reaches 0.4 to 0.6, the cells are induced with IPTG. After induction, cells are harvested, washed, lysed and passed through a French press under a pressure of 16000 psi. Cells are then centrifuged and pellets examined for fractionation of recombinant protein by SDS-PAGE. For proteins local to the cell pellet, the pellet is resuspended in 10 mM Tris, pH 8.0, 1% CHAPS, the inclusion body pellets are washed and then centrifuged. Washed inclusion bodies are solubilized with 8M urea or 6M guanidine HCl containing 10 mM Tris, pH 8.0 + 10 mM imidazole. Solubilized protein is then added to 5 ml of nickel-chelate resin (Qiagen) and incubated for 45 minutes at room temperature.

After incubation, the resin and protein mixtures are passed through a column and the effluent is collected. The column is washed with 10-20 column volumes of buffer and the antigen is eluted with 8M urea, 10 mM Tris, pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. Next, SDS-PAGE is performed to determine which fractions to pool for further purification. As a final purification step, the strong anion exchange resin is equilibrated with a suitable buffer and the collected fractions are packed onto a column. Each antigen is eluted from the column with increasing salt gradient. Fractions are collected and analyzed by SDS-PAGE to determine which fractions to pool from the column. The pooled fractions are dialyzed with 10 mM Tris, pH 8.0 and the resulting protein is subjected to quality control for final release. Release criteria are as follows: (a) purity as measured by SDS-PAGE or HPLC, (b) concentration as measured by Lowry analysis or amino acid analysis, and (c) as measured by amino terminal protein. Identity and (d) endotoxin levels as measured by Limulus (LAL) assay. The protein is then filtered through a 0.22 μM filter and frozen until needed for immunization.

To generate polyclonal antiserum, 400 μg O772P-1 or O772P-2 is mixed with 100 μg muramyldipeptide (MDP). Rabbits are immunized every four weeks with 100 μg of antigen mixed with the same amount of incomplete Freund's adjuvant (IFA). Seven days after the further antigen injection, the animal's blood is collected, the blood is incubated at 4 ° C. for 12-24 hours and then centrifuged to obtain serum.

To characterize this antiserum, 96 well plates are coated with antigen and then blocked with BSA. Rabbit serum is diluted with PBS and added to each well. The plate is then washed and chlorine anti-rabbit horseradish peroxidase (HRP) is added. The plate is washed again and TMB microwell peroxidase substrate is added. After incubation, the color reaction is stopped and the plate is read immediately at 450 nm. All polyclonal antibodies are immunoreactive against suitable antigens.

Immunohistochemical analysis of O772P expression is performed on tissues immobilized with paraffin-sealed formalin. O772P is expressed in normal ovary and ovarian tumors, but not in normal heart, kidney, colon, lung or liver. In addition, immunohistochemistry and flow cytometry showed that O772P is a molecule bound to the plasma membrane. O772P contains one plasma transmembrane domain that is assumed to be encoded by amino acids 859-880. The N-terminus of O772P is extracellular and is encoded by amino acids 1-859, while the C-terminus is intracellular. Sequence analysis shows that there are 17 potential N-linked glycosylation sites.

Example 11

O772P is expressed on the surface of primary ovarian tumor cells

For recombinant expression in mammalian cells, O772P-21008 (SEQ ID NO: 387) and O772P full length cDNA (SEQ ID NO: 311 encoding the protein of SEQ ID NO: 312) are subcloned into mammalian expression vector pBIB or pCEP4, respectively. These constructs are transfected into HEK293 cells with Fugene 6 (Roche). HEK cells are then plated at a density of 100,000 cells / ml in DMEM containing fetal calf serum (FBS) and allowed to grow overnight. The next day, 2 μl of Fugene 6 is added to 100 μl of DMEM without FBS and incubated for 15 minutes at room temperature. Fugene 6 / DMEM mixture is then added to 1 μg O772P / pBIB or O772P / pCEP4 plasmid DNA and incubated for an additional 15 minutes at room temperature. Fugene 6 / DNA mixture is then added to HEK293 cells and incubated for 48-72 hours at 37 ° C. under 7% CO ₂ . The cells are washed and pelleted by centrifugation.

For Western blot analysis, whole cell lysates are produced by culturing cells in lysis buffer and then clarifying by centrifugation. The sample is diluted and analyzed on SDS-PAGE. Next, the gel is transferred to nitrocellulose and probed using purified anti-O772P-2 rabbit polyclonal antibody. The blot is reacted with goat anti-rabbit Ig bound HRP and then expressed in culture in ECL substrate. Western blot analysis revealed that O772P-21008 can be detected in HEK293 cells transfected with O772P.

To measure the cell expression profile of O772P in cells, primary ovarian tumor cells are grown in SCID mice. Cells are recovered from the mice and analyzed by flow cytometry. Briefly, cells are washed with cold staining buffer containing PBS, 1% BSA and Na azide. Cells are incubated with 10 μg / ml of purified anti-O772P-1 and O772P-2 polyclonal serum for 30 minutes. After incubation, the cells are washed three times with staining buffer and incubated with goat anti-rabbit Ig (H + L) bound to Southern Biotechnology (FITC). The cells are washed and resuspended in staining buffer containing propidium iodide (PI), a biostaining agent that identifies non-viable cells. Cells are then analyzed using a fluorescence activated cell sorter (FACS). FACS analysis revealed that O772P is present on the cell surface. Surface expression of O772P on tumor cells will aid in immune targeting of therapeutic antibodies.

Example 12

Functional Characterization of Anti-O8E Monoclonal Antibodies

As described in Example 8. Mouse monoclonal antibodies (mAbs) generated against O8E from E. coli are tested for their ability to promote O8E antigen localization. Localization of antibodies is determined by in vitro cytotoxicity assays. Briefly, HEK293 and O8E / HEK transfected cells are plated in 96 well plates containing DME + 10% heat-inactivated FBS in the presence of purified anti-O8E antibody or control antibody 50 ng / well. The isotypes of the anti-O8E mAbs are as follows: 11A6-IgG1 / kappa, 15C6-IgG2b / kappa, 18A8-IgG2b / kappa and 14F1-IgG2a / kappa. W6 / 32, which acts as a positive control, is a pan-human MHC I group mouse monoclonal antibody, and negative controls include two unrelated mAbs, Ir-Pharm and Ir-Crxa. After incubation with an O8E specific antibody or related control antibody, mAb-zap, a goat anti-mouse Ig-saporin bound secondary antibody (Advanced Targeting Systems), was added to a half of the well at a concentration of 100 ng / well, Plates are incubated for 48-72 hours at 37 ° C. in a 7% CO ₂ incubator. This assay takes advantage of the toxicity of saporin, a ribozyme inactivating protein that exhibits cytotoxic effects upon internalization. After incubation with mAb-zap, MTS reagent is added, and then the OD490 of the plate is read by a microplate ELISA reader to quantify the endogenousness. 25 shows the results from these assays. The top panel shows HEK cells not transfected with O8E, so the O8E antibody should not be bound but inherent. Whether they are cultured with or without mAb-zap, all samples show the same proliferation level except W6 / 32, a positive control antibody. The lower panel shows cells that are transfected with O8E and bind to O8E specific antibodies. Antibodies from hybridomas 11H6, 14F1 and 15C6 that recognize amino acids 61-80 of O8E, as measured by reduced proliferation levels due to the toxicity of mAb-zap, may promote internalization of O8E surface proteins (FIG. 25). Antibodies produced by hybridoma 18A8, recognizing amino acids 151-170 of O8E, cannot promote internalization as measured by normal levels of proliferation in the presence or absence of mAb-zap.

Example 13

Characterization of the Ovarian Tumor Antigen, O772P

Various forms of ovarian tumor antigen, cDNA and protein sequences of O772P are described above (eg, Examples 2 and 9). Genbank investigations show that O772P is highly similar to FLJ14303 (Accession No. AK024365; SEQ ID NOs: 457 and 463). Protein sequences corresponding to O772P and FLJ14303 are disclosed in SEQ ID NOs: 478 and 479, respectively. FLJ14303 is mostly identical to O772P and most of the 3'-terminus shows 100% homology. However, the 5'-end of FLJ14303 is observed to be stretched further to 5 'than O772P. In addition, FLJ14303 contains a 5bp insert (SEQ ID NO: 457), indicating a frame shift of the amino terminal protein sequence whereby FLJ14303 will encode a different protein using a different starting methionine than O772P. Such inserts are present in the genomic sequence and are observed in all EST clones that show identity with this region, suggesting that FLJ14303 (SEQ ID NO: 457) is elongated, suggesting that it is a splice variant of O772P with an ORF containing other amino termini. Additional 5'-nucleotide sequences include repeat sequences identified during genomic mapping of O772P, with the 5'-terminus of O772P and the corresponding region of FLJ14303 exhibiting homology between 90 and 100%. Taken together, O772P and FLJ14303 are different splice variants of the same gene, suggesting that different unique repeat sequences are spliced at the 5'-end of the gene.

Another 10 or more repeat sequences appearing in the same region of chromosome 19 suggest that various forms of O772P may be present, each having a different 5'-end due to different splices of different repeat sequences. Northern blot analysis of O772P identifies multiple O772P hybridized transcripts of different sizes, in some cases 10kb or more.

Upon further analysis, 13 additional O772P related sequences are identified and their cDNA and amino acid sequences are shown in Table 2.

order Technology Dural domain 464 LS # 1043400.1 (cDNA) nd 465 LS # 1043400.10 (cDNA) 0 466 LS # 1043400.11 (cDNA) 2 467 LS # 1043400.12 (cDNA) 2 468 LS # 1043400.2 (cDNA) nd 469 LS # 1043400.3 (cDNA) 470 LS # 1043400.5 (cDNA) nd 471 LS # 1043400.8 (cDNA) One 472 LS # 1043400.9 (cDNA) 0 473 LS # 1043400.6 (cDNA) nd 474 LS # 1043400.7 (cDNA) nd 475 LS # 1043400.4 (cDNA) nd 476 LS # 1043400.1 (cDNA) 0 477 1043400.10 New 5 '(cDNA) - 480 LS # 1043400.9 (Amino acid) - 481 LS # 1043400.8B (amino acid) contains a transmembrane domain - 482 LS # 1043400.8A (Amino acid) - 483 LS # 1043400.12 (amino acid) contains the transmembrane domain - 484 LS # 1043400.11B (amino acid) contains the transmembrane domain - 485 LS # 1043400.11A (Amino acid) - 486 LS # 1043400.10 (amino acid) - 487 LS # 1043400.1 (Amino acid) - nd = not measured

First, these sequences represent overlapping and / or distinct sequences of O772P splice forms that can encode O772P native polypeptides of a particular splice form. However, as a result of nucleotide alignment of these sequences, no identical regions were identified within the repeating factors. This indicates that as long as these sequences contain 16 or more repeat domains, they may represent different specific regions of the O772P gene, all of which form one linear transcript. The 5'-end of sequence LS # 1043400.10 (Table 2; SEQ ID NO: 465) is specific for both O772P and FLJ14303 and does not contain any repeating elements, suggesting that this sequence may represent the 5'-end of O772P. do.

Previously, dural presumptive analysis revealed that O772P contains one to three dural domains. This is demonstrated by demonstrating the presence of plasma membrane binding molecules that exhibit O772P by the use of immunohistochemistry and flow cytometry. However, immunohistochemistry also suggests the presence of the secreted form of O772P, which is presumably due to alternative splice forms of O772P or post-translational cleavage process. Analysis of some of the sequences set forth in Table 2 shows that sequences 1043400B.12, 1043400.8B and 1043400.11B all contain dural regions, while 1043400.8A, 104300.10, 1043400.1, 1043400.11A and 1043400.9 all lack dural sequences. It suggests that proteins are secreted.

Assays show that some of O772P is expressed and / or retained in the plasma membrane so that O772P can produce desired targets, such as therapeutic antibodies, to induce specific immunotherapy for these proteins. The putative extracellular domain of O772P is shown in SEQ ID NO: 489 and it is expected that secretion of O772P will occur as a result of the cleavage reaction in the following sequence:

SLVEQVFLD K TLNASFHWLGSTYQLVDIHVTEMESSVYQP.

Proteolytic cleavage is most likely to occur at lysine (K) at position 10 of SEQ ID NO: 489. The extracellular, dural and cytoplasmic regions of O772P are all shown in SEQ ID NO: 488.

Extracellular:

SLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTRNGRSFPNLVGG

Dural:

WAVILIGLAGLLGLITCLICGVLVTT

cytoplasm:

RRRKKEGEYNVQQQCPGYYQSHLDLEDLQ

Example 14

Immunohistochemical (IHC) Analysis of O8E Expression in Ovarian Cancer and Normal Tissues

To examine tissues expressing ovarian cancer antigen O8E, IHC analysis is performed on a wide range of tissue sections using polyclonal and monoclonal antibodies specific for O8E. The generation of O8E specific polyclonal antibodies is described in detail in Example 8. The monoclonal antibodies used for staining are 11A6 and 14F1 specific for O8E and amino acids 61-80 of 18A8 which recognize amino acids 151-170 of O8E (see Example 12 for details on production).

To perform staining, tissue samples are fixed in formalin solution for 12-24 hours, embedded in paraffin, and then divided into fractions of 8 microns. Steam heat induced epitope recovery (SHIER) in 0.1 M sodium citrate buffer (pH 6.0) is used for optimal staining conditions. Sections are incubated for 5 minutes with 10% serum / PBS. Primary antibodies are added to each fraction and allowed to react for 25 minutes, followed by incubation for 25 minutes with anti-rabbit or antibody or anti-mouse biotinylated antibody. Endogenous peroxidase activity is blocked by three treatments of 1.5 minutes with hydrogen peroxidase. The avidin biotin complex / horseradish peroxidase (ABC / HRP) system is used in conjunction with a DAB chromogen to visualize antigen expression. The slides are then counterstained with hematoxylin to visualize the cell nuclei.

The results using rabbit affinity purified polyclonal antibodies to O8E (amino acids 29-283; see Example 3 for details of the production of these antibodies) are shown in Table 3. The results using three monoclonal antibodies are shown in Table 4.

Immunohistochemical Analysis of O8E Using Polyclonal Antibodies group O8E expression Ovarian Cancer positivity Breast cancer positivity Normal ovary positivity Normal breast positivity blood vessel positivity kidney voice lungs voice colon voice liver voice Heart voice

Immunohistochemical Analysis of O8E Using Monoclonal Antibodies Normal tissue 11A6 18A8 14F1 Endothelial epithelium Endothelial epithelium Endothelial epithelium skin 2 2 0 0 One One skin One One 0 0 One One breast 0 One n / a n / a One One colon 0 0 0 0 0 0 factory 0 0 0 0 0 0 colon 0 0 0 0 0 0 colon 0 0 0 0 0 0 ovary 0 0 0 0 One 0 colon 0 0 0 0 0 One liver 0 0 0 0 One 2 skin 0 0 0 0 One 0 Duodenum and pancreas 0 0 0 0 0 0 appendix 0 0 0 0 0 0 Chairman 0 0 0 0 0 0 0 = not dyed, 1 = weak dyed, 2 = medium dyed, n / a = not valid.

Example 15

Epitope Mapping of O772P Polyclonal Antibodies

To perform the mapping of epitopes of O772P, peptides with sequences derived from the sequences of O772P are generated. These peptides are 15-mers produced by chemical synthesis on a membrane support with overlapping five amino acids. The peptide is covalently bound to the Whatman 50 cellulose support via its C terminus and the N terminus is left unbound. To examine epitope specificity, the membranes were soaked with 100% ethanol for 1 minute and then TBS / Tween / Triton buffer (50 mM Tris, 137 mM NaCl, 2.7 mM KCl, 0.5% BSA, 0.05% Tween 20, 0.05% Triton X-100 , pH 7.5) for 16 hours. The peptides were then used as well as two O772P specific antibodies, O772P-1 (amino acids 44-772 of SEQ ID NO: 312) and O772P-2 (477-914 of SEQ ID NO: 312; see Example 10 for details on antibody preparation). Probing with control unrelated rabbit antibody. The antibody is diluted to 1 μg / ml and incubated for 2 hours at room temperature with the membrane. The membrane is then washed with TBS / Tween / Triton buffer for 30 minutes and then incubated with HRP-bound anti-rabbit secondary antibody at 1: 10,000 dilution for 2 hours. The membrane is washed again with TBS / Tween / Triton for 30 minutes and the ECL is used to visualize anti-peptide reactivity. Specific epitope binding specificities for each O772P-polyclonal antibody are described in Table 5.

Sequence number Peptide # Anti-O772P1 Anti-O772P2 Peptide sequence 490 2 *** - TCGMRRTCSTLAPGS 491 6 * * /- CRLTLLRPEKDGTAT 492 7 * - DGTATGVDAICTHHP 493 8 - - CTHHPDPKSPRLDRE 494 9 *** *** RLDREQLYWELSQLT 495 11 * /- - LGPYALDNDSLFVNG 496 13 **** - SVSTTSTPGTPTYVL 497 22 - - LRPEKDGEATGVDAI 498 24 ** * /- DPTGPGLDREQLYLE 499 27 * /- - LDRDSLYVNGFTHRS 500 40 * /- - GPYSLDKDSLYLNGY 501 41 - - YLNGYNEPGPDEPPT 502 47 *** *** ATFNSTEGVLQHLLR 503 50 - *** QLISLRPEKDGAATG 504 51 - ** GAATGVDTTCTYHPD 505 52 - * /- TYHPDPVGPGLDIQQ 506 53 - * LDIQQLYWELSQLTH 507 58 - * HIVNWNLSNPDPTSS 508 59 - * DPTSSEYITLLRDIQ 509 60 - * LRDIQDKVTTLYKGS 510 61 - *** LYKGSQLHDTFRFCL 511 71 - ** DKAQPGTTNYQRNKR * = Relative reactivity level,-; Not combined, ****; Max coupling

Example 16

Identification of New N-terminal Repeat Structures Associated with O772P

Various forms of O772P cDNA and proteins are identified and characterized as described above (eg, Examples 1, 2, 9 and 14). Importantly, O772P RNA and protein have been overexpressed in ovarian cancer tissues compared to normal tissues, and have proven to provide interesting targets for ovarian cancer diagnostic and therapeutic uses.

An open reading frame (ORF) derived from genomic nucleotide sequences identified as described above as homologous to O772P was examined by bioinformation analysis and identified a plurality of nucleotide repeat sequences in the 5 'region of the gene encoding the O772P protein. do. Many such repeat sequences are identified via RT-PCR using primers specific for each repeat. Fragments containing multiple repeats are amplified from cDNA to confirm the presence of specific repeats and to order these repeats.

Unexpectedly, when analyzing various sets of O772P sequences obtained from different databases and experimental data, at least 20 different repeat sequences (see Table 6) with significant levels of identity to each other (see Table 6) corresponded to the 5 'region of the O772P gene. Is identified in the N-terminal region of the O772P protein. Each repeat includes a contiguous open reading frame that encodes a polypeptide unit that can be spliced with one or more other repeats such that the concatomer of the repeat forms in various numbers and orders. Interestingly, other molecules with repeating structural domains similar to those described with respect to O772P herein have been described in the scientific journal. For example, mucin-based proteins, which are the main glycoprotein components of the mucus covering the cell surface in the respiratory, digestive and urogenital tracts, have been shown to consist of serial repeating sequences with different numbers, lengths, and amino acid sequences for each mucin. See Perez-Vilar and Hill, J. Biol. Chem. 274 (45): 31751-31754, 1999].

The various repeat structures identified herein are most likely to be expected to produce multiple O772P forms by alternative splicing. The cDNA sequences of the identified repeats are shown in SEQ ID NOs: 513-540, 542-546, and 548-567. The encoded amino acid sequences of these repeats are shown in SEQ ID NOs: 574-593. In many cases, these amino acid sequences represent consensus sequences derived from alignment of sequences derived from one or more experiments.

These splice forms may encode a unique O772P protein with multiple repeat domains attached to the carboxy terminal protein constant of O772P, each comprising a transmembrane region. The cDNA sequence of this O772P constant region is shown in SEQ ID NO: 568 and the encoded amino acid sequence is shown in SEQ ID NO: 594.

The useful O772P sequences purchased are digested into identifiable repeats and these sequences are compared by Clustal method (MegAlign software in the DNASTAR sequencing package) using weighted residue weight tables to identify relationships between repeat sequences. Using these information and sequence data provided via RT-PCR and sequence alignment (automatic and manual) via SeqMan (DNASTAR), one exemplary consensus full-length O772P contig sequence containing 20 unique repeat units is identified. do. The cDNA for these O772P cDNA contigs is shown in SEQ ID NO: 569 and the encoded amino acid sequence is shown in SEQ ID NO: 595. This form of O772P protein comprises the following consensus repeat structure in the following order:

SEQ ID NO: 572-SEQ ID NO: 574-SEQ ID NO: 575-SEQ ID NO: 576-SEQ ID NO: 577-SEQ ID NO: 578-SEQ ID NO: 579-SEQ ID NO: 580-SEQ ID NO: 581-SEQ ID NO: 582-SEQ ID NO: 583-SEQ ID NO: 584-SEQ ID NO: 585-SEQ ID NO: 588-SEQ ID NO: 589 -SEQ ID NO: 590-SEQ ID NO: 591-SEQ ID NO: 592-SEQ ID NO: 593.

Thus, SEQ ID NO: 595 shows one exemplary full length consensus sequence for the O772P protein. However, many other forms of O772P are more or less repeats, based on the scientific paper's description of proteins that include repeat structures similar to the current knowledge of these proteins as is commonly known as described above. It is expected to exist as having. In addition, many forms of O772P are assumed to be represented by different arrangements of these N-terminal repeating structures, eg, different orders. The presence of various forms of O772P with different number of repeats is evidenced by Northern analysis of O772P. In this study, Northern hybridization with O772P-specific probes resulted in smearing of a number of O772P-hybrid transcripts, some exceeding 10 kb.

Thus, the variable repeat region of the O772P protein may be exemplarily represented by the structural formula Xn-Y, wherein X comprises a repeat structure having at least 50% identity with the consensus repeat sequence described in SEQ ID NO: 596; n is the number of repeats present in the protein, generally expected to be an integer from 1 to about 35; Y comprises an O772P constant region sequence set forth in SEQ ID NO: 594 or sequences having at least 80% identity with SEQ ID NO: 594. Each X in the Xn repeat region of the O772P molecule is different.

To examine the consensus sequence of each 20 repeat regions, the sequences determined experimentally for the separate repeat regions are aligned and the consensus sequences are determined. In addition to examining the consensus sequence of each repeat region, the consensus repeat sequence is also examined. These sequences are obtained by aligning 20 separate consensus sequences. The variability of the repeats is determined by aligning the consensus amino acid sequence obtained in each repeat region with the entire repeat consensus sequence. Identity data is presented in Table 6.

Percent identity of repeat sequence with reference to consensus repeat sequence Repeat number (amino acid) Sequence number Percent identity for consensus repeat sequences 2 574 88 3 575 84 4 576 88 5 577 89 6 578 93 7 579 90 8 580 91 9 581 88 10 582 85 11 583 86 12 584 87 13 585 87 14 586 89 15 587 89 16 588 89 17 589 83 18 590 84 19 591 83 20 592 57 21 593 68

From the foregoing, while certain aspects of the invention have been described herein for purposes of illustration, it will be apparent that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is limited only by the appended claims.

Claims (35)

Structural Formula X _n -Y wherein X comprises a sequence having at least 50% identity to the consensus O772P repeat sequence set forth in SEQ ID NO: 596, each X present in the polypeptide is different; Y is O772P set forth in SEQ ID NO: 594 O772P polypeptide comprising a sequence having at least 80% identity with a constant region sequence; n is an integer from 1 to 35). The polypeptide of claim 1, wherein X comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 574-593. The polypeptide of claim 1, wherein Y comprises the sequence set forth in SEQ ID NO: 594. The polypeptide of claim 1, wherein n is an integer from 15 to 25. 3. The polypeptide of claim 1, wherein n is 20. The polypeptide of claim 1, comprising SEQ ID NO: 595. The polypeptide of claim 1 overexpressed in ovarian cancer cells as compared to normal tissue. Structural Formula X _n -Y wherein X comprises an O772P repeat sequence selected from the group consisting of any one of SEQ ID NOs: 574-593, each X present in the polypeptide is different; Y is an O772P constant region sequence set forth in SEQ ID NO: 594 And a sequence having at least 90% identity with n being an integer from 15 to 25). The polypeptide of claim 8, wherein n is 20. 10. The polypeptide of claim 8, comprising SEQ ID NO: 595. The polypeptide of claim 8, which is overexpressed in ovarian cancer cells as compared to normal tissue. Structural Formula X _n -Y, where n is 20, X is SEQ ID NO: 574-SEQ ID NO: 575-SEQ ID NO: 576-SEQ ID NO: 577-SEQ ID NO: 578-SEQ ID NO: 579-SEQ ID NO: 580-SEQ ID NO: 581-SEQ ID NO: 58-SEQ ID NO: 584-SEQ ID NO: 585 An O772P polypeptide comprising the O772P repeat sequence of SEQ ID NO: 586-SEQ ID NO: 587-SEQ ID NO: 588-SEQ ID NO: 589-SEQ ID NO: 591-SEQ ID NO: 592-SEQ ID NO: 593; and Y comprises the sequence set forth in SEQ ID NO: 594. The polypeptide of claim 12, comprising SEQ ID NO: 595. The polypeptide of claim 12 overexpressed in ovarian cancer cells as compared to normal tissue. Formula X _n -Y (where, X is each X containing O772P repeat sequence selected from the group consisting of any one of SEQ ID NO: 512-540, 542-546 and 548-567, and present in the polynucleotide are different and; Y is An O772P polynucleotide having a sequence having at least 95% identity to an O772P constant region sequence set forth in SEQ ID NO: 568; n is an integer from 1 to 35. The polynucleotide of claim 15 comprising the sequence set forth in SEQ ID NO: 569. The polynucleotide of claim 15 wherein n is 15-25. 16. The polynucleotide of claim 15, wherein n is 20. The polynucleotide of claim 15, which is overexpressed in ovarian cancer cells as compared to normal tissue. (a) the sequences set forth in SEQ ID NOs: 464-477 and 512-569; (b) the complement of the sequences set forth in SEQ ID NOs: 464-477 and 512-569; (c) a sequence consisting of 20 or more contiguous residues of the sequences set forth in SEQ ID NOs: 464-477 and 512-569; (d) sequences that hybridize with the sequences set forth in SEQ ID NOs: 464-477 and 512-569 under high stringency conditions; (e) a sequence having at least 75% identity with the sequences of SEQ ID NOs: 464-477 and 512-569; (f) a sequence having at least 90% identity with the sequences of SEQ ID NOs: 464-477 and 512-569; And (g) An isolated polynucleotide comprising a sequence selected from the group consisting of degenerate variants of the sequences set forth in SEQ ID NOs: 464-477 and 512-569. (a) a sequence encoded by the polynucleotide of claim 20; (b) a sequence having at least 80% identity with the sequence encoded by the polynucleotide of claim 20; And (c) An isolated polypeptide comprising an amino acid sequence selected from the group consisting of a sequence having at least 90% identity with a sequence encoded by the polynucleotide of claim 20. An expression vector comprising the polynucleotide of claim 20 operably linked to an expression control sequence. A host cell transformed or transfected with the expression vector according to claim 22. An isolated antibody or antigen-binding fragment thereof that specifically binds to the polypeptide of claim 21. (a) obtaining a biological sample from the patient; (b) contacting the biological sample with a binder that binds to the polypeptide of claim 21; (c) determining the amount of polypeptide binding to the binder in the sample; (d) comparing the amount of polypeptide to a pre-measured cutoff value, thereby determining the presence of cancer in the patient, A method of detecting the presence of cancer in a patient. A fusion protein comprising at least one polypeptide according to claim 21. T cells (a) a polypeptide according to claim 21; (b) a polynucleotide according to claim 20; And (c) at least one component selected from the group consisting of antigen presenting cells expressing a polynucleotide according to claim 20; Contacting under conditions and for a time sufficient to allow stimulation and / or proliferation of T cells, A method of stimulating and / or proliferating T cells specific for tumor proteins. An isolated T cell population comprising T cells prepared according to the method of claim 27. A first component selected from the group consisting of a physiologically acceptable carrier and an immunostimulant; (a) a polypeptide according to claim 21; (b) a polynucleotide according to claim 20; (c) the antibody according to claim 24; (d) a fusion protein according to claim 26; (e) the T cell population according to claim 28; And (f) A composition comprising a second component selected from the group consisting of antigen presenting cells expressing a polypeptide according to claim 21. A method of stimulating an immune response in a patient, comprising administering the composition of claim 29 to the patient. A method of treating ovarian cancer in a patient, comprising administering the composition of claim 29 to the patient. (a) obtaining a biological sample from the patient; (b) contacting the biological sample with an oligonucleotide that hybridizes to the polynucleotide sequence according to claim 21; (c) measuring the amount of polynucleotide hybridizing to the oligonucleotide in the sample; (d) comparing the amount of polynucleotide hybridizing to the oligonucleotide with a pre-determined cutoff value, thereby determining the presence of cancer in the patient, A method of measuring the presence of cancer in a patient. An O772P polypeptide comprising one or more antibody epitope sequences set forth in any one of SEQ ID NOs: 490-511. An O8E polypeptide comprising one or more antibody epitope sequences set forth in any one of SEQ ID NOs: 394-415. An isolated antibody or antigen-binding fragment thereof that specifically binds to the polypeptide of claim 1.