EP1135491A2 - Methodes et compositions permettant d'inhiber la croissance de cellules neoplasiques - Google Patents

Methodes et compositions permettant d'inhiber la croissance de cellules neoplasiques

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Publication number
EP1135491A2
EP1135491A2 EP99965077A EP99965077A EP1135491A2 EP 1135491 A2 EP1135491 A2 EP 1135491A2 EP 99965077 A EP99965077 A EP 99965077A EP 99965077 A EP99965077 A EP 99965077A EP 1135491 A2 EP1135491 A2 EP 1135491A2
Authority
EP
European Patent Office
Prior art keywords
seq
acid sequence
polypeptide
sequence identity
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99965077A
Other languages
German (de)
English (en)
Inventor
Jian Chen
Audrey Goddard
Austin L. Gurney
Kenneth Hillan
Mary Napier
William I. Wood
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1998/025108 external-priority patent/WO1999028462A2/fr
Application filed by Genentech Inc filed Critical Genentech Inc
Publication of EP1135491A2 publication Critical patent/EP1135491A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention concerns methods and compositions for inhibiting neoplastic cell growth.
  • the present invention concerns antitumor compositions and methods for the treatment of tumors.
  • the invention further concerns screening methods for identifying growth inhibitory, e.g., antitumor compounds.
  • Malignant tumors are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin.. 43:7 (1993)).
  • Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
  • neoplastic cells such as cancer cells.
  • the present invention relates to methods and compositions for inhibiting neoplastic cell growth. More particularly, the invention concerns methods and compositions for the treatment of tumors, including cancers, such as breast, prostate, colon, lung, ovarian, renal and CNS cancers, leukemia, melanoma, etc., in mammalian patients, preferably humans.
  • cancers such as breast, prostate, colon, lung, ovarian, renal and CNS cancers, leukemia, melanoma, etc.
  • the present invention concerns compositions of matter useful for the inhibition of neoplastic cell growth comprising an effective amount of a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof, in admixture with a pharmaceutically acceptable carrier.
  • the composition of matter comprises a growth inhibitory amount of a PR0655. PR0364 or PR0344 polypeptide, or an agonist thereof.
  • the composition comprises a cytotoxic amount of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof
  • the compositions of matter mav contain one or more additional growth inhibitory and/or cytotoxic and/or other chemotherapeutic agents
  • the present invention concerns compositions of matter useful for the treatment of a tumor in a mammal comprising a therapeutically effective amount of a PR0655 PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof
  • the tumor is preferabiv a cancer
  • the invention concerns a method for inhibiting the growth of a tumor cell comprising exposing the cell to an effective amount of a PR0655 PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof
  • the agonist is an ant ⁇ -PR0655, ant ⁇ -PR0364 or ant ⁇ -PR0344 agonist antibody
  • the agonist is a small molecule that mimics the biological activity of a PR0655, PR0364 or PR0344 polypeptide
  • the method may be performed vitro or in vivo
  • the invention concerns an article of manufacture comprising
  • composition comprising an active agent contained within the container wherein the composition is effective for inhibiting the neoplastic cell growth e growth of tumor cells, and the active agent in the composition is a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof, and
  • the agonist may be an antibody which binds to the PR0655, PR0364 or PR0344 polypeptide
  • the agonist is an ant ⁇ -PR0655 ant ⁇ -PR0364 or ant ⁇ -PR0344 agonist antibody
  • the agonist is a small molecule that mimics the biological activity of a PR0655, PR0364 or PR0344 polypeptide
  • Similar articles of manufacture comprising a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof in an amount that is therapeutically effective for the treatment of tumor are also within the scope of the present invention
  • articles of manufacture comprising a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof,
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PR0655, PR0364 or PR0344 polypeptide
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity ⁇ et more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity more preferably at least about 93% sequence identity yet more preferably at least about 94% sequence identity vet more preferablv at least about 95% sequence identity yet more preferablv at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferablv at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferablv at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferablv at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PR0655 PR0364 or PR0344 polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane doma ⁇ n(s) of such polypeptide are disclosed herein Therefore, soluble extracellular domains of the herein described PR0655 PR0364 or PR0344 polypeptides are contemplated Another embodiment is directed to fragments of a PR0655, PR0364 or PR0344 polypeptide coding sequence, or the complement thereof, that may find use as.
  • nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 1 10 nucleotides in length, yet more preferably at
  • the invention concerns an isolated PR0655, PR0364 or PR0344 polypeptide.
  • the invention concerns an isolated PR0655, PR0364 or PR0344 polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81% positives, more preferably at least about 82% positives, yet more preferably at least about 83% positives, yet more preferably at least about 84% positives, yet more preferably at least about 85% positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives, yet more preferably at least about 90% positives, yet more preferably at least about 91% positives yet more preferably at least about 92% positives v et more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives yet more preferably at least about 97% positives, yet more preferably at least about 98% positives and yet more
  • PR0364 or PR0344 polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described
  • Processes for producing the same are also herein described, wherein those processes comprise cultu ⁇ ng a host cell comprising a vector which comp ⁇ ses the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PR0655, PR0364 or PR0344 polypeptide and recovering the PR0655, PR0364 or PR0344 polypeptide from the cell culture
  • Another aspect of the invention provides an isolated PR0655 PR0364 or PR0344 polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated Processes for producing the same are also herein described, wherein those processes comprise cultu ⁇ ng a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PR0655, PR0364 or PR0344 polypeptide and recovering the PR0655, PR0364 or PR0344 polypeptide from the cell culture
  • the invention concerns agonists of a native PR0655, PR0364 or PR0344 polypeptide as defined herein
  • the agonist is an ant ⁇ -PR0655, ant ⁇ -PR0364 or anti- PR0344 agonist antibody or a small molecule
  • the invention concerns a method of identifying agonists to a PR0655, PR0364 or PR0344 polypeptide which comprise contacting the PR0655, PR0364 or PR0344 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PR0655, PR0364 or PR0344 polypeptide.
  • the PR0655, PR0364 or PR0344 polypeptide is a native PR0655, PR0364 or PR0344 polypeptide
  • the invention concerns a composition of matter comprising a PR0655, PR0364 or PR0344 polypeptide, or an agonist of a PR0655, PR0364 or PR0344 polypeptide as herein described, or an ant ⁇ -PR0655, ant ⁇ -PR0364 or ant ⁇ -PR0344 agonist antibodv in combination with a carrier
  • the carrier is a pharmaceutically acceptable carrier
  • PR0655 Another embodiment of the present invention is directed to the use of a PR0655.
  • the invention provides vectors comprising DNA encoding any of the herein described polypeptides
  • Host cell comprising any such vector are also provided
  • the host ceils may be CHO cells. E coli. yeast, or Baculovirus-infected insect cells
  • a process for producing any of the herein described polypeptides is further provided and comprises cultu ⁇ ng host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture
  • the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence
  • Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
  • the invention provides an antibody which specifically binds to any of the above or below described polypeptides
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
  • the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
  • Figure 1 shows a nucleotide sequence (SEQ ID NO 1 ) of a native sequence PR0655 cDNA, wherein SEQ ID NO 1 is a clone designated herein as "DNA50960- 1224"
  • Figure 2 shows the ammo acid sequence (SEQ ID NO 2) derived from the coding sequence of SEQ ID NO 2
  • Figure 3 shows a nucleotide sequence (SEQ ID NO 6) of a native sequence PR0364 cDNA, wherein SEQ ID NO 6 is a clone designated herein as "DNA47365-1206"
  • Figure 4 shows the amino acid sequence (SEQ ID NO 7) derived from the coding sequence of SEQ ID NO 6 shown in Figure 3
  • Figure 5 shows a nucleotide sequence (SEQ ID NO 16) of a native sequence PR0344 cDNA, wherein SEQ ID NO 16 is a clone designated herein as "DNA40592- 1242"
  • Figure 6 shows the amino acid sequence (SEQ ID NO 17) derived from the coding sequence of SEQ ID NO 16 shown in Figure 5
  • PR0655 refers to native sequence PR0655.
  • PR0364 or PR0344 and PR0655, PR0364 or PR0344 variants which are further defined herein
  • the PR0655, PR0364 or PR0344 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods
  • a "native sequence PR0655”, “native sequence PR0364” or “native sequence PR0344” comprises a polypeptide having the same ammo acid sequence as the PR0655 PR0364 or PR0344 polypeptide as derived from nature
  • Such native sequence PR0655, PR0364 or PR0344 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means The term "native sequence” PR0655.
  • PR0364 or PR0344 specifically encompasses naturally-occurring truncated or secreted forms (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring allelic variants of the PR0655 PR0364 and PR0344 polypeptides
  • the native sequence PR0655, PR0364 or PR0344 polypeptide is a mature or full-length native sequence PR0655, PR0364 or PR0344 polypeptide as shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7) or Figure 6 (SEQ ID NO 17), respectively
  • the PR0655 PR0364 and PR0344 polypeptides disclosed in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), or Figure 6 (SEQ ID NO 17) respectively are shown to begin with the methionine residue designated therein as amino acid position 1 , it is conceivable and possible that another methionine residue located either upstream
  • PR0655 variant polypeptide means an active PR0655 polypeptide (other than a native sequence
  • PR0655 polypeptide as defined below having at least about 80% ammo acid sequence identity with the amino acid sequence of (a) residues 1 or about 22 to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) X to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 17 to 26 ofF ⁇ gure 2 (SEQ ID NO 2) or (c) another specificallv derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2)
  • PR0364 variant polypeptide means an active PR0364 polypeptide (other than a native sequence PR0364 polypeptide) as defined below having at least about 80% ammo acid sequence identity with the amino acid sequence of (a) residues 1 or about 26 to 241 of the PR0364 polv peptide shown in Figure 4 (SEQ ID NO 7), (b) X to 241 of the PR0364 polypeptide shown in Figure 4 (SEQ ID NO 7) wherein X is any ammo acid residue from 21 to 30 of Figure 4 (SEQ ID NO 7), (c) 1 or about 26 to X of Figure 4 (SEQ ID NO 7), wherein X is any ammo acid from ammo acid 158 to amino acid 167 of F ⁇ gure 4 (SEQ ID NO 7) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 7)
  • PR0344 variant polypeptide means an active PR0344 polypeptide (other than a native sequence PR0344 polypeptide) as defined below, having at least about 80% ammo acid sequence identity with the ammo acid sequence of (a) residues 1 or about 16 to 243 of the PR0344 polv peptide shown in Figure 6 (SEQ ID NO 17), (b) X to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID NO 17), wherein X is any amino acid residue from 1 1 to 20 of Figure 6 (SEQ ID NO 17), or (c) another specificaliv derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO 17)
  • PR0655, PR0364 and PR0344 variants include, for instance, PR0655, PR0364 and PR0344 polypeptides wherein one or more amino acid residues are added or deleted, at the N- or C-termmus, as well as within one or more internal domains of the native sequence
  • a PR0655 variant will have at least about 80% amino acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% ammo acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% ammo acid sequence identity, more preferably at least about 90% ammo acid sequence identity, more preferably at least about 91% ammo acid sequence identity, more preferably at least about 92% am
  • a PR0364 variant will have at least about 80% amino acid sequence identity, more preferably at least about 81% ammo acid sequence identity, more preferably at least about 82% ammo acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% ammo acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% am o acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91%) amino acid sequence identity more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% ammo acid sequence identity more preferably at least about 95% amino acid sequence identity, more preferably at least about 96%> amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably
  • a PR0344 variant will have at least about 80% ammo acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% am o acid sequence identity, more preferably at least about 89% am o acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91%) amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% am o acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% am o acid sequence identity,
  • PR0655, PR0364 and PR0344 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 ammo acids in length, more often at least about 60 amino acids in length, more often at least about 70 am o acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 ammo acids in length, more often at least about 150 amino acids in length, more often at least about 200 am o acids in length, more often at least about 250 amino acids in length, more often at least about 300 amino acids in length, or more
  • Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program
  • Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO” represents the amino acid sequence of a hypothetical PR0655, PR0364 or PR0344 polypeptide of interest.
  • “Comparison Protein” represents the amino acid sequence of a polypeptide against which the "PRO” polypeptide of interest is being compared
  • PRO-DNA represents a hypothetical PR0655-, PR0364- or PR0344- encoding nucleic acid sequence of interest
  • “Comparison DNA” represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA” nucleic acid molecule of interest is being compared
  • "X”, “ Y”, and “Z” each represent different hypothetical amino acid residues and "N", “L” and “V” each represent different hypothetical nucleotides.
  • I* c *l -2,-4, 15,-5, -5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4.0,-2, 0,-2.-8, 0, 0,-5 ⁇ ,
  • filel and file2 are two dna or two protein sequences.
  • Max file length is 65535 (limited by unsigned short x in the jmp struct)
  • a sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
  • the program may create a tmp file in /tmp to hold info about traceback.
  • static nm matches in core — for checking */ static lmax; /* lengths of stripped file names */ static UP]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current elem number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char out[2][P LINE] /* output line */ static char star[P LINE]; /* set by stars() *//
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + + ; ps[ ⁇ ] + + ,
  • *py+ + *px; else if ( ⁇ slower(*px))
  • *py++ toupper(*px), if ( ⁇ ndex("ATGCU",*(py-l))) natgc + + , ⁇
  • Page2ofnwsubr.c ...readjmps if 0 ⁇ 0 && dx[dmax] ottset && fj) ⁇
  • Percent (° o) am o acid sequence identity is defined as the percentage of ammo acid residues in a candidate sequence that are identical with the amino acid residues in a PR0655, PR0364 or PR0344 sequence, after aligning the sequences and introducing gaps, if necessarv to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN ALIGN-2 or Megalign (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source
  • ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
  • % amino acid sequence identity of a given am o acid sequence A to, with, or against a given ammo acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzvmology, 266 460-480 (1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values.
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the ammo acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison am o acid sequence of interest (/ e , the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of ammo acid residues of the PRO polypeptide of interest
  • the amino acid sequence A is the comparison am o acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest
  • PR0655 variant polynucleotide or "PR0655 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PR0655 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 17 to 26 of Figure 2 (SEQ ID NO 2), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the am o acid sequence shown in Figure 2 (SEQ ID NO 2)
  • a PR0655 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about
  • nucleic acid sequence identity more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID NO
  • PR0364 variant polynucleotide or 'PR0364 variant nucleic acid sequence ' means a nucleic acid molecule which encodes an active PR0364 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 26 to 241 of the PR0364 polypeptide shown in Figure 4 (SEQ ID NO 7), (b) a nucleic acid sequence which encodes ammo acids X to 241 of the PR0364 polypeptide shown m Figure 4 (SEQ ID NO 7), wherein X is any ammo acid residue from 21 to 30 of Figure 4 (SEQ ID NO 7), (c) a nucleic acid sequence which encodes amino acids 1 or about 26 to X of Figure 4 (SEQ ID NO 7), wherein X is any amino acid from amino acid 158 to amino acid 167 of Figure 4 (SEQ ID NO 7) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the
  • PR0344 variant polynucleotide or "PR0344 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PR0344 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 16 to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID NO 17), (b) a nucleic acid sequence which encodes amino acids X to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID NO 17), wherein X is any amino acid residue from 1 1 to 20 of Figure 6 (SEQ ID NO 17), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the am o acid sequence shown in Figure 6 (SEQ ID NO 17)
  • a PR0344 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at
  • PR0655, PR0364 and PR0344 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length more often at least about 450 nucleotides in length more often at least about 600 nucleotides in length more often at least about 900 nucleotides in length or more
  • Percent (%) nucleic acid sequence identity with respect to the PR0655.
  • PR0364 and PR0344 polypeptide-encodmg nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PR0655, PR0364 or PR0344 polypeptide-encodmg nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled m the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALI
  • % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows
  • % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al . Nucleic Acids Res , 25 3389-3402 (1997))
  • NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov
  • % nucleic acid sequence identity of a given nucleic acid sequence C to. with, or against a given nucleic acid sequence D is calculated as follows
  • a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide- encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide- encodmg nucleic acid and the comparison nucleic acid molecule of interest (i e , the sequence against which the PRO polypeptide-encodmg nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide- encodmg nucleic acid molecule of interest
  • an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid
  • PR0655, PR0364 and PR0344 variant polynucleotides are nucleic acid molecules that encode an active PR0655, PR0364 or PR0344 polypeptide, respectively, and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PR0655 polypeptide shown in Figure 2 (SEQ ID NO 2), to nucleotide sequences encoding the full- length PR0364 polypeptide shown in Figure 4 (SEQ ID NO 7), to nucleotide sequences encoding the full-length PR0344 polypeptide shown in Figure 6 (SEQ ID NO 17), respectively PR0655, PR0364 and PR0344 variant polypeptides may be those that are encoded by a PR0655, PR0364 or PR0344 variant polynucleotide
  • the term "positives", in the context of the amino acid sequence identity comparisons performed as described above includes amino acid residues in the sequences compared that are not onlv identical but also those that that are
  • isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other protemaceous or non-proteinaceous solutes
  • the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PR0655, PR0364 or PR0344 natural environment will not be present Ordinarily, however isolated polypeptide will be prepared
  • An "isolated" nucleic acid molecule encoding a PR0655, PR0364 or PR0344 polypeptide or an "isolated" nucleic acid molecule encoding an ant ⁇ -PR0655, ant ⁇ -PR0364 or ant ⁇ -PR0344 antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PR0655- PR0364- or PR0344-encod ⁇ ng nucleic acid orthe ant ⁇ -PR0655-, ant ⁇ -PR0364- or ant ⁇ -PR0344-encod ⁇ ng nucleic acid
  • the isolated nucleic acid is free of association with all components with which it is naturally associated
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and. in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • antibody is used in the broadest sense and specifically covers, for example, single anti- PR0655, anti-PR0364 and anti-PR0344 monoclonal antibodies (including agonist antibodies),anti-PR0655,anti- PR0364 and anti-PR0344 antibody compositions with polyepitopic specificity, single chain anti-PR0655, anti- PR0364 and anti-PR0344 antibodies, and fragments of anti-PR0655, anti-PR0364 and anti-PR0344 antibodies (see below).
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 °C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyv ⁇ nylpyrrol ⁇ done/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride 75 mM sodium citrate at 42 °C, or (3) employ 50% formamide, 5 x SSC (0 75 M NaCl, 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0 1% SDS and 10%
  • Modely stringent conditions may be identified as described by Sambrook et al , Molecular Cloning A Laboratory Manual, New York Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength and % SDS) less stringent that those described above
  • An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising 20% formamide, 5 x SSC ( 150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like
  • the term "epitope tagged" when used herein refers to a chimeric polypeptid
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused
  • the tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes
  • Suitable tag polypeptides generally have at least six ammo acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)
  • the term “lmmunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesm”) with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (.
  • the adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand
  • the immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG- 1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM
  • “Active” or “activity” for the purposes herein refers to form(s) of PR0655, PR0364 or PR0344 which retain a biological and/or an immunological activity of native or naturally-occurring PR0655, PR0364 or PR0344, wherein "biological” activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring PR0655, PR0364 or PR0344 other
  • Bio activity in the context of an antibody or another agonist that can be identified by the screening assays disclosed herein (e.g., an organic or inorganic small molecule, peptide. etc.) is used to refer to the ability of such molecules to invoke one or more of the effects listed herein in connection with the definition of a “therapeuticallyeffecti ve amount.”
  • biological activity is the ability to inhibit neoplastic cell growth or proliferation.
  • a preferred biological activity is inhibition, including slowing or complete stopping, of the growth of a target tumor (e.g., cancer) cell.
  • Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor (e.g. , cancer) cell.
  • Yet another preferred biological activity is the induction of apoptosis of a target tumor (e.g., cancer) cell.
  • immunological cross-reactivity means immunological cross-reactivity with at least one epitope of a PR0655, PR0364 or PR0344 polypeptide.
  • immunological cross-reactivity means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PR0655, PR0364 or PR0344 polypeptide having this activity with polyclonal antisera raised against the known active PR0655, PR0364 or PR0344 polypeptide.
  • antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds.
  • the immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologically cross-reactive molecule (e.g., antibody) identified, to the corresponding PR0655, PR0364 or PR0344 polypeptide is significantly higher (preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 6-times, most preferably at least about 8-times higher) than the binding affinity of that molecule to any other known native polypeptide.
  • Tuor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma. liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
  • the "pathology” of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response etc
  • an “effective amount” of a polypeptide disclosed herein or an agonist thereof in reference to inhibition of neoplastic cell growth is an amount capable of inhibiting, to some extent, the growth of target cells
  • the term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells
  • An "effective amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • a “therapeutically effective amount ' in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects (1) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest, (2) reduction in the number of tumor cells, (3) reduction in tumor size, (4) inhibition (; e , reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs, (5) inhibition (/ e , reduction, slowing down or complete stopping) of metastasis, (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor, and/or (7) relief, to some extent, of one or more symptoms associated with the disorder
  • a "therapeutically effective amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes of treatment of tumor may be determined empirically and in a routine manner
  • a “growth inhibitory amount” of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof is an amount capable of inhibiting the growth of a cell, especially tumor, _? g , cancer cell, either in vitro or in vivo
  • a “growth inhibitory amount” of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and m a routine manner
  • a "cytotoxic amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof is an amount capable of causing the destruction of a cell, especially tumor, e g , cancer cell, either in vitro or in vivo
  • a "cytotoxic amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells
  • the term is intended to include radioactive isotopes (e , I 131 , I l2 ⁇ Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof
  • chemotherapeutic agent is a chemical compound useful in the treatment of tumor, e g , cancer
  • chemotherapeutic agents include ad ⁇ amycin.
  • taxoids e g , paclitaxel (Taxol, Bristol- Myers SquibbOncology, Princeton, NJ), and doxetaxel (Taxotere, Rh ⁇ ne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatm, melphalan, vinblastine, bleomycin, etoposide.
  • lfosfamide mitomycin C, mitoxantrone, vinc ⁇ stine, vinorelbme, carboplatin, teniposide, daunomycin, carminomycin, aminopte ⁇ n, dactinomycin, mitomycins, esperami ⁇ ns (see, U S Patent No 4,675, 187), melphalan and other related nitrogen mustards
  • hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onap ⁇ stone
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially tumor, e.g. , cancer cell, either in vitro or in vivo.
  • the growth inhibitory agent is one which significantly reduces the percentage of the target cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G 1 arrest and M- phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin. daunorubicin, etoposide, and bleomycin.
  • DNA alky lating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds., Chapter 1 , entitled “Cell cycle regulation, oncogens, and antineoplastic drugs” by Murakami et al., (WB Saunders: Philadelphia, 1995), especially p. 13.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor: prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin- associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor-I and
  • growth hormone
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions, .14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press ( 1985).
  • the prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, glycosylated prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be derivatized into a prodrug form for use in this invention include, but are not limited to. those chemotherapeutic agents described above.
  • agonist is used in the broadest sense and includes any molecule that mimics a biological activity of a native PR0655, PR0364 or PR0344 polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PR0655, PR0364 or PR0344 polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a PR0655, PR0364 or PR0344 polypeptide may comprise contacting a tumor cell with a candidate agonist molecule and measuring the inhibition of tumor cell growth.
  • Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
  • Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
  • Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin,
  • “Native antibodies” and “native immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR).
  • CDRs complementarity-determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et al., NIH Publ. No.91 -3242, Vol. I, pages 647-669 (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD. [1991]) and/or those residues from a "hypervariable loop" (i.e.
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1 ) of the heavy chain.
  • Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • immunoglobulins The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the am ino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. , IgG 1 , IgG2, IgG3, IgG4, IgA, and IgA2.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. , IgG 1 , IgG
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, /. e. , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparationswhich typically includedifferent antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al.. Nature.256:495 [1975], or may be made by recombinant DNA methods (see, e.g., U.S. PatentNo.4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature. 352:624-628 [1991] and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA. 81:6851- 6855 [1984]).
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequence
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PRIM ATIZEDTMantibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H - V L ).
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161 ; and HoIIinger et al., Proc. Natl. Acad. Sci. USA. 90:6444-6448 (1993).
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody.
  • the label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • the label may also be a non-detectable entity such as a toxin.
  • solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere.
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. , an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.
  • a “liposome” is a small vesicle composed of various types of lipids, phosphol ⁇ ids and/or surfactant which is useful for delivery of a drug (such as a PR0655, PR0364 or PR0344 polypeptide or antibody thereto) to a mammal
  • a drug such as a PR0655, PR0364 or PR0344 polypeptide or antibody thereto
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes
  • a "small molecule” is defined herein to have a molecular weight below about 500 Daltons
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0655, PR0364 and PR0344
  • cDNAs encoding PR0655, PR0364 and PR0344 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below
  • cDN A clones encoding PR0655, PR0364 and PR0344 polypeptides have been deposited with the ATCC
  • the actual nucleotide sequences of the clones can readily be determined by the skilled artisan by sequencing of the deposited clones using routine methods in the art
  • the predicted ammo acid sequences can be determined from the nucleotide sequences using routine skill For the PR0655, PR0364 and PR0344 polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time
  • PR0655, PR0364 and PR0344 variants can be prepared PR0655, PR0364 and PR0344 variants can be prepared by introducing appropriate nucleotide changes into the PR0655, PR0364 or PR0344 DNA, and/or by synthesis of the desired PR0655, PR0364 or PR0344 polypeptide Those skilled m the art will appreciate that ammo acid changes may alter post-translational processes of the PR0655, PR0364 or PR0344 polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
  • Variations in the native full-length sequence PR0655, PR0364 or PR0344 or in various domains of the PR0655, PR0364 or PR0344 described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U S Patent No 5,364,934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PR0655, PR0364 or PR0344 that results in a change in the am o acid sequence of the PR0655, PR0364 or PR0344 as compared with the native sequence PR0655, PR0364 or PR0344
  • the variation is by substitution of at least one amino acid with any other ammo acid in one or more of the domains of the PR0655, PR0364 or PR0344
  • Guidance in determining which ammo acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PR0655, PR0364 or PR0344 with
  • PR0655 PR0364 and PR0344 polypeptide fragments are provided herein Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein Certain fragments lack ammo acid residues that are not essential for a desired biological activity of the PR0655, PR0364 or PR0344 polypeptide
  • PR0655, PR0364 and PR0344 fragments may be prepared by any of a number of conventional techniques Desired peptide fragments may be chemically synthesized
  • An alternative approach involves generating PR0655, PR0364 and PR0344 fragments by enzymatic digestion, e , by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment
  • Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR) O gonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR
  • PR0655, PR0364 and PR0344 polypeptide fragments share at least one biological and/or immunological activity with the native PR0655, PR0364 or PR0344 polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), and Figure
  • conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened
  • Substantial modifications in function or immunological identity of the PR0655, PR0364 or PR0344 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • hydrophobic norleucine, met, ala, val. leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
  • the variations can be made using methods known in the art such as oligonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 ( 1986); ZoIIer et al.. Nucl. Acids Res.. .10:6487 ( 1987)]
  • cassette mutagenesis [Wells et al., Gene.34:315 ( 1985)]
  • restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA. 317:415 ( 1986)] or other known techniques can be performed on the cloned DNA to produce the PR0655, PR0364 or PR0344 variant DNA.
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • Such amino acids include alanine, glycine, serine, and cysteine.
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main- chain conformation of the variant [Cunningham and Wells. Science. 244: 1081 -1085 ( 1989)].
  • Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins. (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
  • One type of covalent modification includes reacting targeted amino acid residues of a PR0655, PR0364 or PR0344 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PR0655, PR0364 or PR0344.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking PR0655, PR0364 or PR0344 to a water-insoluble support matrix or surface for use in the method for purifying anti-PR0655, anti-PR0364 or anti-PR0344 antibodies, and vice-versa.
  • crosslinking agents include, e.g., l, l-bis(diazoacetyl)-2-phenylethaneglutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosaIicylicacid,homobifunctionalimidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
  • esters with 4-azidosaIicylicacid,homobifunctionalimidoesters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents
  • Another type of covalent modification of the PR0655, PR0364 or PR0344 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PR0655, PR0364 or PR0344 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PR0655, PR0364 or PR0344.
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present
  • Addition of glycosylation sites to the PR0655, PR0364 or PR0344 polypeptide may be accomplished by altering the amino acid sequence
  • the alteration may be made, for example, by the addition of, or substitution by, one or more se ⁇ ne or threonine residues to the native sequence PR0655, PR0364 or PR0344 (for O-linked glycosylation sites)
  • the PR0655, PR0364 or PR0344 ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PR0655, PR0364 or PR0344 polypeptide at preselected bases such that codons are generated that will translate into the desired ammo acids.
  • PR0364 or PR0344 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation
  • Chemicai deglycosylation techniques are known in the art and described. for instance, by Hakimuddm. et al . Arch Biochem Biophvs , 259 52 (1987) and by Edge et al . Anal Biochem .
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth Enzvmol . 138 350 (1987)
  • PR0655, PR0364 or PR0344 comprises linking the PR0655, PR0364 or PR0344 polypeptide to one of a variety of nonproteinaceous polymers, e , polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640,835, 4,496,689, 4,301, 144, 4,670,417, 4,791,192 or 4, 179,337
  • the PR0655, PR0364 or PR0344 poiypeptide of the present invention may also be modified in a way to form a chimeric molecule comprising PR0655, PR0364 or PR0344 fused to another, heterologous polypeptide or am o acid sequence
  • a chimeric molecule comprises a fusion of the PR0655, PR0364 or PR0344 polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind
  • the epitope tag is generally placed at the amino- or carboxyl- terminus of the PR0655, PR0364 or PR0344 polypeptide
  • the presence of such epitope-tagged forms of the PR0655, PR0364 or PR0344 polypeptide can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the PR0655, PR0364 or PR0344 polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epi
  • tag polypeptides include the Flag-peptide [Hopp et al , BioTechnology, 6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science, 255 192-194 (1992)], an ⁇ -tubulin epitope peptide [Skinner et al J Biol Chem , 266 15163- 15166 ( 1991 )], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA, 87 6393-6397 (1990)]
  • the chimeric molecule may comprise a fusion of the PR0655, PR0364 or PR0344 polypeptide with an immunoglobulin or a particular region of an immunoglobulin
  • an immunoglobulin or a particular region of an immunoglobulin
  • the immunoglobulin fusion preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PR0655, PR0364 or PR0344 polypeptide in place of at least one variable region within an Ig molecule
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule
  • PR0655, PR0364 or PR0344 by cultu ⁇ ng cells transformed or transfected with a vector containing PR0655, PR0364 or PR0344 nucleic acid It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PR0655.
  • PR0364 or PR0344 For instance, the PR0655, PR0364 or PR0344 polypeptide sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e g , Stewart et al , Solid-Phase Peptide Synthesis. W H Freeman Co , San Francisco, CA ( 1969), Merrifieid, J Am Chem Soc .
  • In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosvstems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions Various portions of the PR0655, PR0364 or PR0344 polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PR0655, PR0364 or PR0344 polypeptide
  • DNA encoding PR0655, PR0364 or PR0344 may be obtained from a cDNA library prepared from tissue believed to possess the PR0655, PR0364 or PR0344 mRNA and to express it at a detectable level Accordingly, human PR0655, human PR0364 or human PR0344 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples
  • the PR0655-, PR0364- or PR0344-encod ⁇ ng gene may also be obtained from a genomic library or by known synthetic procedures (e , automated nucleic acid synthesis) Libraries can be screened with probes (such as antibodies to the PR0655, PR0364 or PR0344 or oligonucieotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it.
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989)
  • An alternative means to isolate the gene encoding PR0655, PR0364 or PR0344 is to use PCR methodology [Sambrook et al , supra, Dieffenbach et al PCR Primer A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
  • the Examples videow describe techniques for screening a cDNA library.
  • the oligonucieotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
  • the oligonucieotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32 P-Iabeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PR0655, PR0364 or PR0344 production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperamre, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991 ) and Sambrook et al., supra.
  • Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl 2 , CaP0 4 , liposome-mediated and electroporation.
  • transformation is performed using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes.
  • Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al.. Gene. 23:315 (1983) and WO 89/05859 published 29 June 1989.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells
  • Suitable prokaryotes include but are not limited to eubacte ⁇ a such as Gram-negative or Gram-positive organisms, for example, Enterobacte ⁇ aceae such as E coli
  • E coli strains are publicly available, such as £ colt K 12 strain MM294 (ATCC 31 446) E cot/ X 1776 (ATCC 31.537), £ co// strain W31 10 (ATCC 27,325) and K5 772 (ATCC 53 635)
  • Other suitable prokaryotic host cells include Enterobacte ⁇ aceaesuch as Eschenchia, e g , E coli, Enterobacter, Erwinia klebstella, Proteus Salmonella, e g Salmonella tvp tmurium, Serratia, e g , Serratia marcescans.
  • Strain W31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations
  • the host cell secretes minimal amounts of proteolytic enzymes
  • strain W31 10 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including £ coli W31 10 strain 1 A2, which has the complete genotype tonA , £ to// W31 10 strain 9E4 which has the complete genotype tonA ptri.
  • E co// W31 10 strain 27C7 ATCC 55,244 which has the complete genotype tonA ptr 3 pho A E15 (argF-lac) 169 degP ompT kar , E coli W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA El 5 (argF-lac) 169 degP ompT rbs7 ilvG karf, E coli W31 10 strain 40B4, which is strain 37D6 with a non- kanamycin resistant degP deletion mutation, and an £ coli strain having mutant pe ⁇ plasmic protease disclosed in U S Patent No 4,946,783 issued 7 August 1990 Alternatively, in vitro methods of cloning, e g , PCR or other nucleic acid polymerase reactions, are suitable
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PR0655-, PR0364- or PR0344-encod ⁇ ng vectors Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290 140 [1981 ], EP 139 383 published 2 May 1985), Kluyxeromyces hosts (U S Patent No 4,943,529, Fleer et al , Bio/Technology, 9 968-975 ( 1991 )) such as, e g K lactis (MW98-8C, CBS683, CBS4574, Louvencourt et al , J Bacte ⁇ ol , 737 [1983]), K fragilis (ATCC 12,424) K bulgaricus (ATCC 16,045), K wtckeramii (ATCC 24,178), K waltn (
  • thermotolerans and K marxianm yarrow ⁇ a(E ⁇ 402.226), Pichiapastons (EP 183,070, Sreek ⁇ shna et al , J Basic Microbiol , 28 265-278 [ 1988])
  • Candida Tnchoderma reesia EP 244,234
  • Neurospora crassa Neurospora crassa
  • Schwanniomyces such as Schwanniomyces occidentals
  • filamentous fungi such as, e g , Neurospora, Penicillium, Tolypocladium (WO 91/00357 published l O January 1991 ) and Aspergillus hosts such as A nidulans (Ballance et al , Biochem Biophys Res Commun 1 12 284-289 [1983], Tilburn et al , Gene.
  • Methylotropic yeasts are suitable herein and include but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula Candida Kloeckera Ptchia. Saccharonnces, Torulopsis and Rhodotorula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony The Biochemistry ot Methv lotrophs 269 ( 1982)
  • Suitable host cells for the expression of glycosylated PR0655, PR0364 or PR0344 are derived from multicellular organisms
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CV 1 line transformed by SV40 (COS-7, ATCC CRL 1651 ).
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J Gen Virol . 36 59 (1977)), Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasm, Proc Natl.
  • mice sertoli cells TM4, Mather. Biol Reprod , 23 243-251 (1980)
  • human lung cells W138, ATCC CCL 75
  • human liver cells Hep G2, HB 8065
  • mouse mammary tumor MMT 060562, ATCC CCL51
  • the selection of the appropriate host cell is deemed to be within the skill in the art.
  • the nucleic acid (e g , cDN A or genomic DNA) encoding PR0655, PR0364 or PR0344 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression
  • a replicable vector for cloning (amplification of the DNA) or for expression
  • the vector may, for example, be in the form of a plasmid. cosmid. viral particle, or phage.
  • nucleic acid sequence may be inserted into the vector by a variety of procedures
  • DNA is inserted into an appropriate restriction endonuclease s ⁇ te(s) using techniques known in the art
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
  • the PR0655, PR0364 or PR0344 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide.
  • a heterologous polypeptide which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide
  • the signal sequence may be a component of the vector, or it may be a part of the PR0655- PR0364- or PR0344-encod ⁇ ng DNA that is inserted into the vector
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders
  • the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Saccharomvces and Kluyveromvces ⁇ -factor leaders, the latter described in U S Patent No 5.010,
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are well known for a variety of bacteria, yeast, and viruses
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, poiyoma. adenovirus VSV or BPV) are useful for cloning vectors in mammalian cells
  • Selection genes will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicillm, neomycin, methotrexate, or tetracyc ne. (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e . the gene encoding D-alanme racemase for Bacilli
  • selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PR0655- PR0364- or PR0344-encod ⁇ ng nucleic acid, such as DHFR or thymidine kinase
  • DHFR thymidine kinase
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al . Proc Natl Acad Sci USA. 77 4216 (1980).
  • a suitable selection gene for use in yeast is the trp ⁇ gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282 39 (1979), Kingsman et al , Gene, 7 141 ( 1979), Tschemper et al , Gene, JO 157 (1980)]
  • the trp] gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 [Jones, Genetics 85. 12 ( 1977)]
  • Expression and cloning vectors usually contain a promoter operably linked to the PR0655-, PR0364- or PR0344-encodmg nucleic acid sequence to direct mRNA synthesis
  • Promoters recognized by a variety of potential host cells are well known Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al , Nature, 275 615 ( 1978), Goeddel et al , Nature. 28 . 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res .
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding PR0655, PR0364 or PR0344
  • suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al , J Biol Chem , 255 2073 ( 1980)] or other glycolytic enzymes [Hess et al , J Adv Enzyme Reg .2 149 ( 1968), Holland, Biochemistry. 7 4900 (1978)], such as enolase, glyceraldehyde- 3-phosphate dehydrogenase. hexokinase, pyruvate decarboxylase. phosphofructokinase. glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, t ⁇ osephosphate isomerase, phosphoglucose isomerase, and glucokinase
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein. giyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73.657
  • PR0655 PR0364 or PR0344 transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e g , the actm promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
  • viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cyto
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription
  • Many enhancer sequences are now known from mammalian genes (globin, eiastase albumin, ⁇ -fetoprotem and insulin)
  • an enhancer from a eukaryotic cell virus examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers
  • the enhancer may be spliced into the vector at a position 5' or 3' to the PR0655, PR0364 or PR0344 coding sequence, but is preferably located at a site 5' from the promoter
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DN As or cDN As These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PR0655, PR0364 or PR0344
  • Gene amplification and/or expression may be measured in a sample directly, for example, by conventional
  • Southern blotting Northern blotting to quantitate the transcription of mRNA [Thomas, Proc Natl Acad Sci USA, 77 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal. and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PR0655, PR0364 or PR0344 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PR0655, PR0364 or PR0344 DNA and encoding a specific antibody epitope
  • PR0655, PR0364 or PR0344 may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e , T ⁇ ton-X 100) or by enzymatic cleavage Cells employed in expression of PR0655, PR0364 or PR0344 can be disrupted by various physical or chemical means, such as freeze-thavv cvcling. sonication, mechanical disruption, or cell lysing agents It may be desired to purify PR0655. PR0364 or PR0344 from recombinant cell proteins or polypeptides
  • Some drug candidates for use in the compositions and methods of the present invention are antibodies and antibody fragments which mimic the biological activity of a PR0655, PR0364 or PR0344 polypeptide
  • polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and. if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrape ⁇ toneal injections
  • the immunizing agent may include the PR0655, PR0364 or PR0344 polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanm, serum albumin, bovine thyroglobulm.
  • adjuvants examples include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate)
  • MPL-TDM adjuvant monophosphoryl Lipid A, synthetic trehalose dicorynomycolate
  • the immunization protocol may be selected by one skilled in the art without undue experimentation
  • the antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein. Nature, 256 495 ( 1975)
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in ⁇ itro
  • the immunizing agent will typically include the PR0655.
  • PR0364 or PR0344 polypeptide or a fusion protein thereof
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies Principles and Practice.
  • Immortalized cell lines are usually transformed mammalian cells, particularly myelomacells of rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells
  • the parental cells lack the enzyme hypoxanthine guanine phospho ⁇ bosyl transferase (HGPRT or HPRT).
  • HGPRT or HPRT hypoxanthine guanine phospho ⁇ bosyl transferase
  • the culture medium for the hyb ⁇ domas typically will include hypoxanthine, aminopte ⁇ n, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium
  • More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia Human myeloma and mouse-human heteromyeloma cell lines also have been described forthe production of human monoclonal antibodies [Kozbor, J Immunol . 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications, Marcel Dekker. Inc , New York, (1987) pp 51-63].
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PR0655.
  • PR0364 or PR0344 Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by lmmunoprecipitation or by an m vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding. supra] Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S. Patent No 4.816,567
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucieotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies)
  • the hybridoma cells of the invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells Chinese hamster ovarv (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also mav be modified for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U S Patent No 4 816,567 Morrison e
  • the antibodies may be monovalent antibodies
  • Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at an ⁇ point in the Fc region so as to prevent heavy chain crosslinking Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking
  • the antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins. immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') : or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity in some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues Humanized antibodies mav also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or
  • a humanized antibody has one or more ammo acid residues introduced into it from a source which is non-human These non- human amino acid residues are often referred to as import" residues which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers [Jones et al , Nature 321 522-525 ( 1986), Riechmann et al Nature, 332 323-327 (1988), Verhoeyen et al , Science, 239 1534- 1536 ( 1988)] by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized” antibodies are chimeric antibodies (U S Patent No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species In practice humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substitute
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 ( 1991 ) Marks et al , J Mol Biol . 222 581 (1991)] The techniques of Cole et al , and Boerner et al , are also available for the preparation of human monoclonal antibodies (Cole et Alan R Liss, p 77 ( 1985) and Boerner et al , J Immunol , 147( 1 ) 86-95 ( 1991 )] Similarly, human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects including gene rearrangement, assembly, and antibody repertoire This approach is described, for example in U S Patent Nos 5 545,807, 5,54
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens
  • one of the binding specificities is for the PR0655, PR0364 or PR0344
  • the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit
  • Methods for making bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-cham pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305 537-539 ( 1983)] Because of the random assortment of immunoglobulin heavy and light chains, these hyb ⁇ domas (quadromas) produce a potential mixture often different antibody molecules, of which onlv one has the correct bispecific structure The purification of the correct molecule is usually accomplished bv affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Tra
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain
  • one or more smail amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e , tyrosine or tryptophan)
  • Compensatory "cavities" of identical or similar size to the large side cha ⁇ n(s) are created on the interface of the second antibody molecule by replacing large am o acid side chains with smaller ones (e g , alanine or threonine)
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg , F(ab') 2 bispecific antibodies) Techniques for generating bispecific antibodies from antibody fragments have been described in the literature For example, bispecific antibodies can be prepared using chemical linkage Brennan et al , Science, 229 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') : fragments These fragments are reduced in the presence of the dithiol complexmg agent sodium arsenite to stabilize vicinal dithiols and prevent lntermolecular disulfide formation The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody
  • the bispecific antibodies produced can be used as agents
  • Fab' fragments may be directly recovered from £ coli and chemically coupled to form bispecific antibodies
  • Shalaby et al , J Exp Med . 175 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab'), molecule
  • Each Fab' fragment was separately secreted from £ coli and subjected to directed chemical coupling in vitro to form the bispecific antibody
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets
  • bispecific antibodies have been produced using leucine zippers Kostelny et al , J Immunol , 148(5) 1547- 1553 (1992)
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers
  • This method can also be utilized for the production of antibody homodimers
  • t ⁇ specific antibodies can be prepared Tutt et al , J Immunol 147 60 ( 1991 ) Exemplary bispecific antibodies may bind to two different epitopes on a given PR0655, PR0364 or
  • ant ⁇ -PR0364 or ant ⁇ -PR0344 polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (eg , CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular PR0655, PR0364 or PR0344 polypeptide
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PR0655, PR0364 or PR0344 polypeptide
  • These antibodies possess a PR0655-, PR0364- or PR0344-b ⁇ nd ⁇ ng arm and an arm which binds a cytotoxic agent or a radionuc de chelator, such as EOTUBE, DP
  • Heteroconjugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U S Patent No 4,676,980], and for treatment of HIV infection [WO 91/00360, WO 92/200373, EP 03089] It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include lmmothiolate and methyl-4-mercaptobuty ⁇ m ⁇ date and those disclosed, for example, in U S Patent No 4,676,980
  • Homodimeric antibodies with enhanced antitumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al , Cancer Research, 53 2560-2565 (1993)
  • an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See, Stevenson et al , Anti-Cancer Drug Design, 3 219-230 ( 1989) 7 Immunoconiugates
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (/ e a radioconiugate)
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof)
  • a radioactive isotope / e a radioconiugate
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbind g active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ⁇ cin A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleurites fordu proteins dianthin proteins, Phvtolaca amertcana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin crotin sapaona ⁇ a officinalis inhibitor, gelonin, mitogellin, restrictocm, phenomycin, enomycin, and the tricothecenes
  • a variety of radionuclides are available for the production of radioconjugated antibodies Examples include : ⁇ : B ⁇ lj l I, lj
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protem-coupiing agents such as N-succm ⁇ m ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol)prop ⁇ onate (SPDP) im othiolane (IT), bifunctional derivatives of lmidoesters (such as dimethy ladipimidate HCL), activ e esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-az ⁇ dobenzo ⁇ I ) hexanediam e), bis-diazonium derivatives (such as b ⁇ s-(p-d ⁇ azon ⁇ umbenzoyl)-ethylened ⁇ am ⁇ ne), d ⁇ socvanates (such as tolyene 2,6-dnsocyanate), and bis- active fluorine compounds (such as l ,5-d ⁇ flu
  • MX-DTPA Carbon- 14-labeled l- ⁇ soth ⁇ ocyanatobenzyl-3- methyldiethylene t ⁇ aminepentaacetic acid
  • the antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pretarget g wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e , avidin) that is conjugated to a cytotoxic agent (e g a radionucleotide)
  • a "receptor” such as streptavidin
  • a ligand e , avidin
  • cytotoxic agent e g a radionucleotide
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al Proc Natl Acad Sci USA, 82 3688 (1985), Hwang et al Proc Natl Acad Sci USA 77 4030 ( 1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholme, cholesterol, and PEG-de ⁇ vatized phosphatidylethanolamine (PEG- PE) Liposomes are extruded through filters of defined pore size to y leld liposomes with the desired diameter Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al , J Biol Chem .
  • chemotherapeutic agent such as Doxorubicin
  • a chemotherapeutic agent is optionally contained within the liposome.
  • the proteins disclosed in the present application have been assayed in a panei of 60 tumor cell lines currently used in the investigational, disease-oriented, in vitro drug-discovery screen of the National Cancer Institute (NCI)
  • NCI National Cancer Institute
  • the purpose of this screen is to identify molecules that have cytotoxic and or cytostatic activity against different types of tuniors NCI screens more than 10 000 new molecules per year (Monks et al . J. Natl Cancer Inst . 83 757-766 ( 1991 ) Boyd, Cancer Pnnc Pract Oncol Update, 3(10) 1- 12 (H989]
  • the tumorcell lines employed in this study have been described in Monks et al . supra
  • the cell lines the growth of which has been significantly inhibited by the proteins of the present application are specified in the Examples
  • cell-based assays and animal models for tumors can also be used to verify the findings of the NCI cancer screen, and to further understand the relationship between the protein identified herein and the development and pathogenesis of neoplastic cell growth
  • primary cultures derived from tumors m transgenic animals as described below can be used in the cell-based assays herein, although stable cell lines are preferred Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e g , Small et al , Mol Cell Biol , 5 642-648 [ 1985])
  • a variety of well known animal models can be used to further understand the role of the molecules identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other agonists of the native polypeptides, including small molecule agonists.
  • Animal models of tumors and cancers include both non- recombmant and recombinant (transgenic) animals
  • Non-recombinant animal models include, for example, rodent, e g , murine models
  • Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e g , subcutaneous injection, tail vein injection, spleen implantation, mtrapentoneal implantation, implantation under the renal capsule, or orthopin implantation, e g . colon cancer cells implanted in colonic tissue (See, e g , PCT publication No WO 97/33551 , published September 18, 1997)
  • nude mice Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice
  • the autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B 10 LP, C 17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS.
  • the cells introduced into such animals can be derived from known tumor/cancer cell lines, such as, any of the above-listed tumor cell lines, and, for example, the B 104- 1 - 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene), /- ⁇ s-transfected NIH-3T3 cells.
  • Caco-2 ATCC HTB-37
  • HT-29 ATCC HTB-38
  • Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al , Br J Cancer, 48 689-696 [1983]).
  • Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures
  • the subcutaneous (s c ) space in mice is very suitable for tumor implantation
  • Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions
  • tumor tissue fragments of suitable size are introduced into the s c space
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue Boven and Winograd ( 1991 ), supra
  • Animal models ofbreast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or neu- transformed NIH-3T3 cells into nude mice essentially as described by Drebin et al . Proc Natl Acad Sci USA
  • animal models of colon cancer can be generated by passaging colon cancer cells in animals, e , nude mice, leading to the appearance of tumors in these animals
  • An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al . Cancer Research. 54 4726-4728 ( 1994) and
  • Tumors that arise in animals can be removed and cultured m vitro Cells from the in vitro cultures can then be passaged to animals Such tumors can serve as targets for further testing or drug screening Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage ceils and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest Such passaging techniques can be performed with any known tumor or cancer cell lines
  • Meth A, CMS4, CMS5, CMS21 , and WEHI- 164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al , J Exp Med , 146 720 [1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Palladino et al , J Immunol .
  • tumor cells are propagated in vitro in cell culture Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about l O l O 6 to lOx lO 7 cells/ml The animals are then infected subcutaneously with 10 to 100 ⁇ of the cell suspension, allowing one to three weeks for a tumor to appear
  • the Lewis lung (3LL) carcinoma of mice which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL)
  • SCCL small cell carcinoma of the lung
  • This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al , Br J Cancer 4J_. suppi 4 309 [1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive For further information about this tumor model see Zacharski Haemostasis, 16 300-320 [1986])
  • One way of evaluating the efficacy ot a test compound in an animal model on an implanted tumor is to measure the size of the tumor before and after treatment Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important variable in the description of tumor growth is the tumor volume doubling time Computer programs for the calculation and description of tumor growth are also available such as the program reported by Rygaard and Spang-Thomsen, Proc 6th Int Workshop on Immune-Deficient Animals.
  • necrosis and inflammatory responses following treatment may actually resuit in an increase in tumor size, at least initially Therefore these changes need to be carefully monitored, by a combination of a morphomet ⁇ c method and flow cytomet ⁇ c analysis
  • Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals
  • Animals that can serve as a target for transgenic manipulation include without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g , baboons chimpanzees and monkeys
  • Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U S Patent No 4,873, 191 ), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Natl Acad Sci USA.
  • transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals")
  • the transgene can be integrated either as a single transgene, or in concatamers. e g , head-to-head or head-to-tail tandems
  • Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko etal , Proc Natl Acad Sci USA, 89 6232- 636 (1992)
  • transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level of mRNA expression can then be analyzed using techniques such as m situ hybridization, Northern blot analysis, PCR, or immunocytochemistry The animals are further examined for signs of tumor or cancer development
  • Screening assays for drug candidates are designed to identify compounds that competitively bind or complex with the receptor(s) of the polypeptides identified herein or otherwise signal through such receptor(s)
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates
  • Small molecules contemplated include synthetic organic or inorganic compounds, including peptides.
  • soluble peptides preferably soluble peptides, (poly)pept ⁇ de- lmmunoglobulin fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art
  • a receptor of a polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e g , on a microtiter plate, by covalent or non-covalent attachments
  • Non-covalent attachment generally is accomplished bv coating the solid surface with a solution of the polypeptide and drying
  • an immobilized antibody e a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface
  • the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e g , the coated surface containing the anchored component
  • the non-reacted components are removed, e , by washing, and complexes anchored on the solid surface are detected
  • the originally non- immobilized component carries a detectable label
  • the candidate compound interacts with but does not bind to a particular receptor, its interaction with that polypeptide can be assayed bv methods well known for detecting protein-protein interactions
  • Such assays include traditional approaches, such as. cross-linking, co-immunoprecipitation. and co-purification through gradients or chromatographic columns
  • protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song Nature (London).340 245-246 ( 1989). Chien et al , Proc Natl Acad Sci USA. 88 9578-9582 ( 1991 )] as disclosed by Chevray and Nathans [Proc Natl Acad. Sci USA.
  • transc ⁇ ptional activators such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain
  • yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-bindmg domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain
  • the expression of a GAL l-/ocZ reporter gene under control of a GAL4-act ⁇ vated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ -galactosidase
  • MATCHMAKERTM for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech This system can
  • polypeptides of the present invention can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions.
  • peptide molecules can be designed which retain the ability to bind the target protein sequence
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e g , Marasco et al , Proc Natl Acad Sci USA, 90 7889-7893 [ 1993])
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other
  • the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine. chemotherapeutic agent, or growth-inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended
  • Therapeutic formulations of the polypeptides identified herein, or agonists thereof are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences. 16th edition, Osol, A ed [ 1980]), in the form of lyophi zed formulations or aqueous solutions
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine preservatives (such as octadecyldimethylbenzy I ammonium chloride, hexamethonium chloride benzalkonium chloride benzethonium chloride, phenol, butyl or benzyl alcohol, alky 1 parabens such as methyl or propyl paraben catechol, resorcinol, cyclohexanol.
  • 3-pentanol, and m-cresol low molecular weight (less than about 10 residues) poh peptides proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvins lpyrrolidone, ammo acids such as glycine, glutamine, asparagine, histidine, argin e or lysine, monosaccha ⁇ des. disaccha ⁇ des, and other carbohydrates including glucose, mannose, or dextrins. chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol. salt-forming counter-ions such as sodium metal complexes (e , Zn-protein complexes), and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG)
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatm-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition Osol, A ed ( 1980)
  • formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution
  • compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle
  • Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g , films, or microcapsules
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacr late), or polv(v ⁇ nylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (mjectable
  • the polypeptides of the present invention and their agonists may be used to treat various tumors, e g , cancers
  • Exemplary conditions or disorders to be treated include benign or malignant tumors (e g renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal prostate, pancreatic, lung vulval, thyroid, hepatic carcinomas, sarcomas, gliobiastomas, and various head and neck tumors), leukemias and iymphoid malignancies, other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc disorders, and inflammatory, angiogenic and immunologic disorders
  • the anti-tumor agents of the present invention including the polypeptides disclosed herein and agonists which mimic their activity, e g , antibodies, peptides
  • chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service ed , M C Perry, Williams & Wilkins, Baltimore, MD (1992)
  • the chemotherapeutic agent may precede, or follow administration of the anti-tumor agent of the present invention, or may be given simultaneously therewith
  • the anti-cancer agents of the present invention may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onap ⁇ stone (see, EP 616812) in dosages known for such molecules
  • the anti-cancer agents herein are co-adm mistered with a growth inhibitory agent
  • the growth inhibitory agent may be administered first, followed by the administration of an anti-cancer agent of the present invention
  • simultaneous administration or administration of the anti-cancer agent of the present invention first is also contemplated Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the antibody herein
  • an anti-tumor agent for the prevention or treatment of disease, the appropriate dosage of an anti-tumor agent herein will depend on the type of disease to be treated as defined above the seventy and course of the disease, whether the agent is administered for preventive or therapeutic purposes previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician
  • the agent is suitably administered to the patient at one time or over a series of treatments
  • Animal experiments provide reliable guidance for the determination of effective doses for human therapy
  • Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J and Chappell, W "The use of interspecies scaling in toxicokinetics" in Toxicokmetics and New Drug Development, Yacobi et al eds , Pergamon Press. New York 1989, pp 42-96
  • an antitumor agent is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above
  • the treatment is sustained until a desired suppression of disease symptoms occurs
  • other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays
  • Guidance as to particular dosages and methods of delivery is provided the literature, see, for example, U S Pat Nos 4 657,760. 5.206,344 or 5,225,212 It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue
  • an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above comprises a container and a label Suitable containers include, for example bottles, vials, syringes, and test tubes
  • the containers may be formed from a variety of materials such as glass or plastic
  • the container holds a composition which is effective for diagnosing or treating the condition and ma ⁇ have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle)
  • the active agent in the composition is an anti-tumor agent of the present invention
  • the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice
  • the article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline. Ringer's solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other
  • RNA for construction of cDNA libraries was then isolated from human small intestine (LIB 99)
  • the cDNA libraries used to isolate the cDNA clones encoding human PR0655 were constructed by standard methods using commercially available reagents such as those from Invitrogen San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science. 253 1278-1280 ( 1991 )) in the unique Xhol and Notl
  • Ohgonucleotides probes based upon the above described EST sequence were then synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0655
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length The probe sequences are typically 40-55 bp in length
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucieotide and one of the primer pairs
  • oligonucieotide probes employed were as follows reverse PCR primer 1
  • the predicted polypeptide precursor is 208 amino acids long, and has a calculated molecular weight of approximately 24 414 daltons and an estimated pi of approximately 8 92
  • Analysis of the full-length PR0655 sequence shown in Figure 2 evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PR0655 sequence ( Figure 2, SEQ ID NO 2) evidences the presence of the following a signal peptide from about amino acid 1 to about am o acid 21 N-glycosy lation sites from about amino acid 95 to about amino acid 99, and from about amino acid 104 to about amino acid 108 a casein kinase II phosphorylation site from about amino acid 181 to about amino acid 185, an N-my ⁇ stoylation site from about amino acid 133 to about amino acid 139, and an interferon alpha, beta and delta family signature from about am o acid 147 to about ammo acid 166 Clone
  • o gonucleotides probes were then synthesized 1) to identify by PCR a cDN A library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0364
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55 bp in length In order to screen several libraries for a full-length clone.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucieotide and one of the primer pairs
  • oligonucieotide probes employed were as follows forward PCR primer 1 5'-CACAGCACGGGGCGATGGG-3' (SEQ ID NO 8) forward PCR primer 2
  • hybridization probe 1 5'-GAGGAGTGCTGTTCCGAGTGGGACTGCATGTGTGTCCAGC-3' (SEQ ID NO 14) hybridization probe 2 5'-AGCCTGGGTCAGCGCCCCACCGGGGGTCCCGGGTGCGGCC-3' (SEQ ID NO 15)
  • RNA for construction of cDNA libraries was then isolated from human small intestine tissue
  • the cDNA libraries used to isolate the cDNA clones encoding human PR0364 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDN A was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikmased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science.
  • a suitable cloning vector such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes
  • N-glycosylation site from about amino acid 146 to about amino acid 150, N-my ⁇ stoylation sites from about amino acid 5 to about amino acid 1 1 , from about am o acid 8 to about amino acid 14, from about ammo acid 25 to about amino acid 31, from about amino acid 30 to about amino acid 36, from about ammo acid 33 to about amino acid 39, from about amino acid 1 18 to about ammo acid 124, from about ammo acid 122 to about amino acid 128, and from about ammo acid 156 to about amino acid 162, a prokaryotic membrane lipoprote lipid attachment site from about amino acid 166 to about amino acid 177, and a leucine zipper pattern from about am o acid 171 to about amino acid 193 Clone DNA47365-1206 has been deposited with ATCC on November 7, 1997 and is assigned ATCC deposit no 209436
  • PR0364 represents the human counterpart to the mouse GITR protein reported by Nocentini et al
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases
  • the EST databases included public EST databases (e g , GenBank), and a proprietary EST database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA)
  • the search was performed using the computer program BLAST or BLAST2 [Altschul et al , Methods in Enzvmologv.
  • DNA34398 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA34398 In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
  • oligonucleotides were synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0344
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55 bp in length
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al . Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucieotide and one of the primer pairs
  • PCR primers (forward and reverse) were synthesized forward PCR primer 1
  • RNA for construction of the cDNA libraries was isolated from human fetal lung tissue
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved w ith Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see
  • In situ Hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis, and aid in chromosome mapping
  • In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision, 1 169-176 ( 1994), using PCR-generated "P-labeled ⁇ boprobes Briefly, formalin-fixed, paraffin- embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37°C, and further processed for in situ hybridization as described by Lu and Gillett, supra A ( j3 -P)UTP-labeled antisense nboprobe was generated from a PCR product and hybridized at 55 °C overnight The slides were dipped in Kodak NTB2TM nuclear track emulsion and exposed for 4 weeks
  • the tubes were incubated at 37 °C for one hour A total of 1 0 ⁇ l RQ1 DNase was added, followed by incubation at 37°C for 15 minutes A total of 90 ⁇ l TE (10 mM T ⁇ s pH 7 6/1 mM EDTA pH 8 0) was added, and the mixture was pipetted onto DE81 paper The remaining solution was loaded in a MICROCON-50TM ultrafiltration unit, and spun using program 10 (6 minutes) The filtration unit was inverted over a second tube and spun using program 2 (3 minutes) After the final recovery spin, a total of 100 ⁇ l TE was added, then 1 ⁇ l of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR IITM The probe was run on a TBE/urea gel A total of 1-3 ⁇ l of the probe or 5 ⁇ l of RNA Mrk III was added to 3 ⁇ l of loading buffer After heating on a 95 °C heat block for three minutes, the gel was immediately placed on ice
  • the slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes The trays were placed in a 55 °C incubator for five minutes to reduce condensation The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0 5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H 2 0) After deprotemation in 0 5 ⁇ g/ml proteinase K for 10 minutes at 37°C (12 5 ⁇ l of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0 5 x SSC for 10 minutes at room temperature The sections were dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each
  • the slides were deparaffinized, placed in SQ H ,0 and rinsed twice in 2 x SSC at room temperature, for
  • the slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper
  • the tissue was covered with 50 ⁇ l of hybridization buffer (3 75 g dextran sulfate + 6 ml SQ H 2 0), vortexed, and heated in the microwave for 2 minutes with the cap loosened After cooling on ice, 18 75 ml formamide, 3 75 ml 20 x SSC, and 9 ml SQ H 2 0 were added and the tissue was vortexed well and incubated at 42°C for 1-4 hours
  • DNA47365-1206 (PRQ364) (novel TNF-receptor Homolog)
  • PRQ364 novel TNF-receptor Homolog
  • the following method describes use of a nucleotide sequence encoding PR0655, PR0364 or PR0344 as a hybridization probe.
  • DNA comprising the coding sequence of full-length or mature PR0655, PR0364 or PR0344 (as shown in Figure 1, 3, and 5, respectively, SEQ ID NOS: 1 , 6, and 16, respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PR0655, PR0364 or PR0344) in human tissue cDNA libraries or human tissue genomic libraries.
  • Hybridization and washing of filters containing either library DNAs is performed under the following high-stringency conditions.
  • Hybridization of radiolabeled probe derived from the gene encoding a PR0655, PR0364 or PR0344 polypeptide to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8. 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1 x SSC and 0.1% SDS at 42°C.
  • DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art.
  • PRQ655, PRQ364 or PRQ344 in £. coli
  • This example illustrates preparation of an unglycosylated form of PR0655, PR0364 or PR0344 by recombinant expression in £. coli.
  • the DNA sequence encoding PR0655, PR0364 or PR0344 is initially amplified using selected PCR primers.
  • the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector.
  • a variety of expression vectors may be employed.
  • An example of a suitable vector is pBR322 (derived from £. coli; see Bolivar et al., Gene, 2:95 ( 1977)) which contains genes for ampicillin and tetracycline resistance.
  • the vector is digested with restriction enzyme and dephosphorylated.
  • the PCR amplified sequences are then ligated into the vector.
  • the vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a poly-His leader (including the first six STII codons. poly-His sequence, and enterokinase cleavage site), the PR0655, PR0364 or PR0344 coding region, lambda transcriptionalterminator, and an argU gene.
  • the ligation mixture is then used to transform a selected £. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing. Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation.
  • the cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PR0655, PR0364 or PR0344 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
  • PR0655, PR0364 or PR0344 may be expressed in £. coli in a poly-His tagged form, using the following procedure.
  • the DNA encoding PR0655, PR0364 or PR0344 is initially amplified using selected PCR primers.
  • the primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase.
  • the PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an £.
  • Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH 4 ) 2 S0 4 , 0.71 g sodium citrate « 2H20, 1.07 g KCI, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 1 10 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgS0 ) and grown for approximately 20-30 hours at 30 °C with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
  • CRAP media prepared by mixing 3.57 g (NH 4 ) 2 S0 4 , 0.71 g sodium citrate « 2H20, 1.07 g KCI, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 1 10 mM M
  • £. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer.
  • Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1 M and 0.02 M, respectively, and the solution is stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization.
  • the solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min.
  • the supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
  • the clarified extract is loaded onto a 5 ml Qiagen Ni 2* -NTA metal chelate column equilibrated in the metal chelate column buffer.
  • the column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4.
  • the protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4°C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
  • the proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine. 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml.
  • the refolding solution is stirred gently at 4°C for 12-36 hours.
  • the refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3).
  • the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2- 10% final concentration.
  • the refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0 1% TFA with elution with a gradient of acetomt ⁇ le from 10 to 80% Aliquots of fractions w ith A :so absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonit ⁇ le since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin Aggregated species are usually eluted at higher acetonit ⁇ le concentrations In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxm from the samples
  • PR0655 and PR0364 were successfully expressed in £ coli in a poly-His tagged form by the above procedure
  • This example illustrates preparation of a potentially glycosylated form of PR0655, PR0364 or PR0344 by recombinant expression in mammalian cells
  • the vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector
  • the PR0655, PR0364 or PR0344 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PR0655, PR0364 or PR0344 DNA using ligation methods such as described in Sambrook etal , supra
  • the resulting vector is called pRK5-PR0655, pRK5-PR0364 or pRK5-PR0344
  • the selected host cells may be 293 cells
  • Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient componentsand/orantibiotics
  • About 10 ⁇ g pRK5-PR0655, pRK5-PR0364 or pRK5-PR0344 DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya et al . Cell.
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 3S S-cyste ⁇ ne and 200 ⁇ O/ml 3 "S-meth ⁇ on ⁇ ne
  • the conditioned medium is collected concentrated on a spin filter, and loaded onto a 15% SDS gel
  • the processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the PR0655, PR0364 or PR0344 polypeptide
  • the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays
  • PR0655 PR0364 or PR0344 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac e/ ⁇ / Proc Natl Acad Sci .
  • PR0655 PR0364 or PR0344 can be expressed in CHO cells
  • the pRK5- PR0655, pRK5-PR0364 or pRK5-PR0344 can be transfected into CHO cells using known reagents such as CaPO, or DEAE-dextran
  • the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as J "S-meth ⁇ on ⁇ ne
  • the culture medium may be replaced with serum free medium
  • the cultures are incubated for about 6 days, and then the conditioned medium is harvested
  • the medium containing the expressed PR0655, PR0364 or PR0344 polypeptide can then be concentrated and purified by any selected method
  • Epitope-tagged PR0655, PR0364 or PR0344 may also be expressed in host CHO cells
  • the PR0655, PR0364 or PR0344 may be subcloned out of the pRK5 vector
  • the subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector
  • the poly-His tagged PR0655, PR0364 or PR0344 insert can then be subcloned into a S V40 driven vector containing a selection marker such as DHFR for selection of stable clones
  • the CHO cells can be transfected (as described above) with the SV40 driven vector Labeling may be performed, as described above, to verify expression
  • the culture medium containing the expressed poly-His tagged PR0655, PR0364 or PR0344 can then be concentrated and purified by any selected method, such as by Nr"-chelate affinity chromatography
  • PR0655, PR0364 or PR0344 may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure
  • the proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e g , extracellular domains) of the respective proteins are fused to an IgG l constant region sequence containing the hinge, CH2 and CH2 domains and/or as a poly-His tagged form
  • CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's
  • the vector used in expression in CHO cells is as described in Lucas et al , Nucl Acids Res , 24 9 ( 1774- 1779 ( 1996) and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihvdrofolate reductase (DHFR) DHFR expression permits selection for stable maintenance of the plasmid following transfection
  • the ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexmg
  • the contents are pipetted into a centrifuge tube containing 10 mis of media and centrifuged at 1000 rpm for 5 minutes
  • the supernatant is aspirated and the cells are resuspended in 10 ml of selective media (0 2 ⁇ m filtered PS20 with 5% 0 2 ⁇ m diafiltered fetal bovine serum)
  • the cells are then aliquoted into a 100 ml spinner containing 90 ml of selective media
  • the cells are transferred into a 250 ml spinner filled with 150 ml selective growth medium and incubated at 37°C After another 2-3 days, 250 ml, 500 ml and 2000 ml spinners are seeded with 3 x 10 5 cells/ml
  • the cell media is exchanged with fresh media by centrifugation and resuspension in production medium
  • any suitable CHO media may
  • the proteins are purified using a Ni 2* -NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni 2+ -NTA column equilibrated in 20 mM Hepes pH 74, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4°C After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 025 M imidazole The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C
  • Immunoadhesin (Fc-containmg) constructs are purified from the conditioned media as follows
  • the conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ l of 1 M Tris buffer, pH 9
  • the highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation
  • PR0364 was stably expressed in CHO cells by the above described method In addition, PR0364 was expressed in CHO cells by the transient expression procedure EXAMPLE 6 Expression of PRQ655 PRQ364 or PRQ344 in Yeast
  • yeast expression vectors are constructed for intracellular production or secretion of PR0655, PR0364 or PR0344 from the ADH2/GAPDH promoter DNA encoding PR0655, PR0364 or PR0344 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR0655, PR0364 or PR0344
  • DNA encoding PR0655, PR0364 or PR0344 can be cloned into the selected plasmid.
  • PR0655, PR0364 or PR0344 together with DNA encoding the ADH2 'GAPDH promoter, a native PR0655, PR0364 or PR0344 signal peptide or other mammalian signal peptide. or, for example, a yeast alpha-factor or mvertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PR0655, PR0364 or PR0344 Yeast cells, such as yeast strain AB1 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media
  • the transformed yeast supernatants can be analyzed by precipitation with 10% t ⁇ chloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain Recombinant PR0655, PR0364 or PR0344 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters
  • sequence coding for PR0655, PR0364 or PR0344 is fused upstream of an epitope tag contained within a baculovirus expression vector
  • epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG)
  • plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PR0655, PR0364 or PR0344 or the desired portion of the coding sequence of PR0655, PR0364 or PR0344 (such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular) is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector
  • Recombinant baculovirus is generated by co-transfecting the above plasmidand BaculoGoldTM virus DNA
  • Expressed poly-His tagged PR0655, PR0364 or PR0344 can then be purified, for example, by Nr * - chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al , Nature, 362 175- 179 ( 1993) Briefly. Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7 9, 12 5 mM MgCI ., 0 1 mM EDTA 10% glycerol. 0 1% NP-40, 0 4 M KC1), and sonicated twice for 20 seconds on ice The sonicates are cleared by centrifugation.
  • sonication buffer 25 ml Hepes, pH 7 9, 12 5 mM MgCI ., 0 1 mM EDTA 10% glycerol. 0 1% NP-40, 0 4 M KC1
  • N ⁇ 2+ -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading buffer The filtered cell extract is loaded onto the column at 0 5 ml per minute The column is washed to baseline A - so with loading buffer, at which point fraction collection is started Next, the column is washed with a secondary wash buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 6 0), which elutes nonspecifically bound protein After reaching A 280 baseline again, the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer One ml fractions are collected and analyzed by SDS
  • the respective coding sequences are subcloned into a baculovirus expression vector(pb PH IgG for IgG fusions and pb PH His c for poly-His tagged proteins), and the vector and Baculogold® baculovirus DNA (Pharmmgen) are co-transfected into 105 Spodoptera friigiperda ("Sf9") cells (ATCC CRL 171 1), using Lipofectin (Gibco BRL) pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmmgen), with modified polyhnker regions to include the His or Fc tag sequences
  • the cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells are incubated for 5 days at 28 °C The supernatant is harvested and subsequently used for the first viral amplification by in
  • the first viral amplification supernatant is used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0 1 Cells are incubated for 3 days at 28 °C The supernatant is harvested and filtered Batch binding and SDS-PAGE analysis is repeated, as necessary, until expression of the spinner culture is confirmed
  • the conditioned medium from the transfected cells (0 5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0 22 micron filters
  • the protein construct is purified using a Ni 2 MTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni -NTA column equilibrated in 20 mM Hepes, pH 7 4 buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of
  • Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows
  • the conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9
  • the highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins
  • the homogeneity of the proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation PR0344 was expressed in baculovirus infected Sf9 insect cells Alternatively, a modified baculovirus procedure may be used incorporating h ⁇ g
  • the conditioned media from the transfected cells (0 5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0 22 micron filters
  • the protein comprising the sequence is purified using a Ni 2* -NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM
  • the conditioned media is pumped onto a 6 ml Ni 2+ -NTA column equilibrated ⁇ n 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at48°C
  • the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole
  • the highly purified protein is then subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mann
  • the conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9
  • the highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins
  • the homogeneity of the sequence is assessed by SDS poiyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary
  • PR0364 and PR0344 were expressed using the above baculovirus procedure employing h ⁇ gh-5 cells
  • This example illustrates preparation of monoclonal antibodies which can specifically bind PR0655, PR0364 or PR0344
  • mice such as Balb/c are immunized with the PR0655, PR0364 or PR0344 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or lntrape ⁇ toneally in an amount from 1 - 100 micrograms
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads
  • MPL-TDM adjuvant Ribi Immunochemical Research, Hamilton, MT
  • the mice may also be boosted with additional immunization injections
  • Serum samples mav be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect ant ⁇ -PR0655, ant ⁇ -PR0364 or ant ⁇ -PR0344 antibodies
  • the animals positive" for antibodies can be injected with a final intravenous injection of PR0655 PR0364 or PR0344
  • the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU
  • hybridoma cells will be screened in an ELISA for reactivity against PR0655, PR0364 or PR0344 Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PR0655, PR0364 or PR0344 is within the skill in the art
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the ant ⁇ -PR0655, ant ⁇ -PR0364 or ant ⁇ -PR0344 monoclonal antibodies
  • the hybridoma cells can be grown in tissue culture flasks or roller bottles Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed
  • Native or recombinant PR0655, PR0364 or PR0344 polypeptides may be purified by a variety of standard techniques in the art of protein purification
  • pro-PR0655, pro-PR0364 or pro-PR0344 polypeptide, mature PR0655, mature PR0364 or mature PR0344 polypeptide, or pre-PR0655, pre-PR0364 or pre-PR0344 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PR0655, PR0364 or PR0344 polypeptide of interest
  • an immunoaffinity column is constructed by covalently coupling the ant ⁇ -PR0655, ant ⁇ -PR0364 or ant ⁇ -PR0344 polypeptide antibody to an activated chromatographic resin
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N J ) Likewise, monoclonal antibodiesare prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB Biotechnology) The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions
  • PR0364 or PR0344 polypeptide by preparing a fraction from cells containing the PR0655 PR0364 or PR0344 polypeptide in a soluble form This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art Alternatively, soluble PR0655 PR0364 or PR0344 polypeptide containing a signal sequence mav be secreted in useful quantity into the medium in which the cells are grown
  • a soluble PR0655, PR0364 or PR0344 polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of the PR0655, PR0364 or PR0344 polypeptide (e g , high ionic strength buffers in the presence of detergent) Then, the column is eluted under conditions that disrupt ant ⁇ body/PR0655 ant ⁇ body/PR0364 or ant ⁇ body/PR0344 polypeptide binding (e g , a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and the PR0655, PR0364 or PR0344 polypeptide is collected
  • PR0655 PR0364 or PR0344 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques
  • the PR0655, PR0364 or PR0344 poiypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PR0655, PR0364 or PR0344 polypeptide or fragment Drugs are screened against such transformed cells in competitive binding assays
  • Such cells either in viable or fixed form, can be used for standard binding assays
  • One may measure, for example, the formation of complexes between a PR0655, PR0364 or PR0344 polypeptide or a fragment and the agent being tested Alternatively, one can examine the diminution in complex formation between the PR0655, PR0364 or PR0344 polypeptide and its target cell
  • the present invention provides methods of screening for drugs or any other agents which can affect a PR0655, PR0364 or PR0344 polypeptide-associated disease or disorder
  • methods comprise contacting such an agent with a PR0655, PR0364 or PR0344 polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the PR0655, PR0364 or PR0344 polypeptide or fragment, or (n) for the presence of a complex between the PR0655, PR0364 or PR0344 polypeptide or fragment and the cell, by methods well know n in the art
  • the PR0655, PR0364 or PR0344 polypeptide or fragment is typically labeled After suitable incubation, the free PR0655, PR0364 or PR0344 polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to the PR0655.
  • PR0364 or PR0344 polypeptide the peptide test compounds are reacted with the PR0655, PR0364 or PR0344 polypeptide and washed Bound PR0655, PR0364 or PR0344 polypeptide is detected by methods well known in the art
  • Purified PR0655 PR0364 or PR0344 polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding a PR0655, PR0364 or PR0344 polypeptide specifically compete with a test compound for binding to the PR0655, PR0364 or PR0344 polypeptide or fragments thereof In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with a PR0655, PR0364 or PR0344 polypeptide
  • the goal of rational drug design is to produce structural analogs of a biologically active polypeptide of interest (/ e , a PR0655, PR0364 or PR0344 polypeptide) or of small molecules with which they interact, e g , agonists, antagonists, or inhibitors Any of these examples can be used to fashion drugs which are more active or stable forms of the PR0655, PR0364 or PR0344 polypeptide or which enhance or interfere with the function of the PR0655, PR0364 or PR0344 polypeptide in vivo (cf Hodgson. Bio/Technology, 9 19-21 ( 1991 ))
  • the three-dimensional structure of the PR0655, PR0364 or PR0344 polypeptide, or of a PR0655, PR0364 or PR0344 polypeptide-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches Both the shape and charges of the PR0655, PR0364 or PR0344 polypeptide must be ascertained to elucidate the structure and to determine active s ⁇ te(s) of the molecule.
  • PR0655, PR0364 or PR0344 polypeptide may be gained by modeling based on the structure of homologous proteins In both cases, relevant structural information is used to design analogous PR0655, PR0364 or PR0344 polypeptide-hke molecules or to identify efficient inhibitors
  • Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wei Is, Biochemistry 3J , 7796-7801 ( 1992) or wh ich act as h lbitors. agonists, or antagonists of native peptides as shown by Athauda et al . J Biochem . U_3_ 742-746 (1993)
  • PR0655, PR0364 or PR0344 polypeptide may be made available to perform such analytical studies as X-ray crystallography
  • knowledge of the PR0655, PR0364 or PR0344 polypeptide am o acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography
  • SRB sulforhodamine B
  • Cells from approximately 60 human tumor cell lines were harvested with trypsin/EDTA (Gibco), washed once, resuspended in IMEM and their viability was determined The cell suspensions were added by pipet (100 ⁇ l volume) into separate 96-well microtiter plates The cell density for the 6-day incubation was less than for the 2-day incubation to prevent overgrowth Inoculates were allowed a preincubation period of 24 hours at 37°C for stabilization Dilutions at twice the intended test concentration were added at time zero in 100 ⁇ l aliquots to the microtiter plate wells (1 2 dilution) Test compounds were evaluated at five half-log dilutions (1000 to 100,000- fold) Incubations took place for two days and six days in a 5% CO . atmosphere and 100% humidity
  • a test sample is considered positive if it shows at least 40% growth inhibitory effect at one or more concentrations

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Abstract

L'invention concerne des méthodes et des compositions permettant d'inhiber la croissance de cellules néoplasiques. Cette invention concerne plus spécialement des compositions antinéoplasiques et des méthodes de traitement des tumeurs. Elle concerne encore des méthodes de criblage permettant d'identifier les inhibiteurs de croissance, par exemple des composés antinéoplasiques. La présente invention a en outre trait à des nouveaux polypeptides et à des molécules d'acide nucléiques codant pour ces polypeptides. L'invention porte encore sur: des vecteurs et des cellules hôtes comprenant ces séquences d'acides nucléiques, des molécules de polypeptides chimériques où les polypeptides obtenus selon l'invention sont fusionnés à des séquences de polypeptides hétérologues, des anticorps qui se lient aux polypeptides obtenus selon l'invention, et des méthodes de préparation de ces polypeptides.
EP99965077A 1998-12-01 1999-11-30 Methodes et compositions permettant d'inhiber la croissance de cellules neoplasiques Withdrawn EP1135491A2 (fr)

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WOPCT/US98/25108 1998-12-01
PCT/US1998/025108 WO1999028462A2 (fr) 1997-12-03 1998-12-01 Polypeptides et acides nucleiques codant ces derniers
US11285098P 1998-12-16 1998-12-16
US112850P 1998-12-16
US11329698P 1998-12-22 1998-12-22
US113296P 1998-12-22
US14475899P 1999-07-20 1999-07-20
US144758P 1999-07-20
US14569899P 1999-07-26 1999-07-26
US145698P 1999-07-26
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CA2301660A1 (fr) 1997-08-26 1999-03-04 Zymogenetics, Inc. Homologues de proteines specifiques aux adipocytes
US6503184B1 (en) 1997-10-21 2003-01-07 Human Genome Sciences, Inc. Human tumor necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2
US6689607B2 (en) 1997-10-21 2004-02-10 Human Genome Sciences, Inc. Human tumor, necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2
EP1692318A4 (fr) * 2003-12-02 2008-04-02 Genzyme Corp Compositions et methodes pour le diagnostic et le traitement du cancer du poumon
TW201605896A (zh) 2013-08-30 2016-02-16 安美基股份有限公司 Gitr抗原結合蛋白
SG10201810507WA (en) 2014-06-06 2018-12-28 Bristol Myers Squibb Co Antibodies against glucocorticoid-induced tumor necrosis factor receptor (gitr) and uses thereof
MA41044A (fr) 2014-10-08 2017-08-15 Novartis Ag Compositions et procédés d'utilisation pour une réponse immunitaire accrue et traitement contre le cancer
CN107743586B (zh) 2015-06-03 2021-07-02 百时美施贵宝公司 用于癌症诊断的抗gitr抗体
ES2926969T3 (es) 2015-11-19 2022-10-31 Bristol Myers Squibb Co Anticuerpos contra el receptor de factor de necrosis de tumor inducido por glucocorticoides (gitr) y usos de los mismos
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US5814307A (en) * 1989-04-10 1998-09-29 Bristol-Myers Squibb Company Method for regulating cell growth, leukocyte differentiation and tumor cell growth using Oncostatin M to stimulate synthesis of IL-6
US5416192A (en) * 1990-04-03 1995-05-16 Bristol-Myers Squibb Company Epithelins: novel cysteine-rich growth modulating proteins
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