WO2005007692A1 - Anticorps qui reconnaissent la proteine brms1 et leurs utilisations - Google Patents

Anticorps qui reconnaissent la proteine brms1 et leurs utilisations Download PDF

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Publication number
WO2005007692A1
WO2005007692A1 PCT/US2004/022084 US2004022084W WO2005007692A1 WO 2005007692 A1 WO2005007692 A1 WO 2005007692A1 US 2004022084 W US2004022084 W US 2004022084W WO 2005007692 A1 WO2005007692 A1 WO 2005007692A1
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antibody
brmsl
protein
cancer
antibodies
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PCT/US2004/022084
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English (en)
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Danny R. Welch
Lalita A. Shevde-Samant
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Uab Research Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds

Definitions

  • the present invention relates to an antibody capable of detecting a protein in a cancerous tumor, the presence of the protein in the tumor indicating a decreased metastatic potential of the tumor. Specifically, the invention relates to detecting BRMSl protein in a cancerous tumor, cancerous tissue or cancerous cell line.
  • metastasis suppressors In addition to metastasis-promoting genes, a new class of molecules called metastasis suppressors has been described (reviewed in Steeg 2003; Shevde and Welch 2003). A metastasis suppressor inhibits metastasis without blocking primary tumor growth, presumably by inhibiting one or more steps necessary for metastasis. To date, 13 metastasis suppressor genes have been identified that reduce the metastatic ability of cancer cell lines in vivo without affecting tumorigenicity. An example of a metastasis suppressor protein encoded by a metastasis suppressor gene is BRMSl.
  • a person of skill for example a physician, would want to know whether the tumor had a high or low potential of metastasizing to distant parts of the subject's body. If it could be determined that a cancerous tumor had a low metastatic potential, a physician might choose not to administer chemotherapy and/or radiation therapy to the subject, thereby avoiding unnecessary morbidity. Conversely, if it could be determined that a cancerous tumor had a high metastatic potential, a physician might choose to administer chemotherapy and/or radiation therapy to the subject to increase the likelihood of a cure.
  • needed in the art is a means of predicting metastatic potential of a primary cancerous tumor.
  • the present invention overcomes the previous limitations and shortcomings in the art by providing novel antibodies that can detect the presence of BRMS 1 , a protein whose presence in a primary cancerous tumor indicates a decreased metastatic potential of the cancer, methods for making these antibodies, and methods for using these antibodies for a variety of therapeutic, diagnostic and prognostic applications.
  • This invention in one aspect, relates to an isolated antibody, light or heavy chains of the antibody, or fragments of the antibody or heavy or light chains, wherein the antibody specifically binds breast cancer metastasis suppressor 1 (BRMSl) protein.
  • isolated nucleic acids comprising a nucleotide sequence that encodes an immunoglobulin heavy chain, light chain, or a fragment thereof of the BRMSl antibody.
  • methods of using the antibodies, heavy and light chains, and fragments thereof are also provided.
  • the invention relates to a method for detecting BRMSl protein in a biological sample of a subject, comprising obtaining a sample from the subject, contacting the sample with the antibody of the invention under conditions whereby the antibody can bind BRMS 1 protein in the sample, and detecting antibody bound to BRMSl protein.
  • the invention relates to a method of screening for metastatic potential of a cancer in a subject, comprising detecting in a biological sample of the cancer the presence of an elevated or reduced level of BRMSl protein as compared to a control level, wherein the detection is performed using the antibody of the invention.
  • the presence of elevated level of BRMSl protein indicates a reduced metastatic potential of the cancer in the subject.
  • the absence of BRMSl protein or a reduced elvel of BRMSl protein compared to control indicates an increased metastatic potential of the cancer in the subject.
  • the invention relates to a method of screening for a compound that reduces metastatic potential of a cancer, comprising contacting a cancer cell that expresses BRMSl protein with the compound to be screened, contacting the cancer cell with the antibody of the invention, and detecting bound antibody in the cancer cell contacted with the compound to be screened.
  • An increase in bound antibody as compared to a control indicates a compound that reduces metastatic potential, whereas a reduction in the bound antibody indicates a compound that increases metastatic potential.
  • FIG. 1 shows rGST-BRMSl using ⁇ GEX-2TK.
  • Multiple Cloning Site (MCS) of vector has sites for BamHI: present in cDNA of BRMSl; Smal: overlaps with Eco Rl, hence difficult for double digest; Eco Rl: Not present in BRMSl cDNA. Thus, BRMSl was cloned at Bam HI and Eco Rl sites.
  • Figure 2 shows strategy for cloning BRMSl into pGEX-2TK. Use enzymes with compatible cohesive ends.
  • FIG. 3 shows induction of GST-BRMS1. Following induction with ImM IPTG, an immunoblot using anti-GST antibody confirmed production of GST-BRMS1.
  • Figure 4 shows purification of GST-BRMS1. The induced culture lysate was subject to purification using an immobilized glutathione column followed by elution using reduced glutathione.
  • Figure 5 shows that post-immune serum shows reactivity to GST-BRMS1. Sera from rabbits PSU#87 and #88 were tested by i munoblotting (serum used at 1 :200 dilution).
  • Figure 6 shows that post-immune serum shows reactivity to GST-BRMS1.
  • Sera from chickens PSU#20 and #21 were tested by immunoblotting (serum used at 1 :200 dilution).
  • Figure 7 shows that serum from rabbit PSU#88 shows reactivity to a ⁇ 35 kDa protein from neol 1/435 by immunoblotting. Serum was used at 1 :200 dilution for immunoblotting.
  • Figure 8 shows immunized sera from rabbits PSU#87 and #88 and chickens PSU#20and #21 immunoprecipitate 901-BRMSl from cell lysates (30 ⁇ g protein; sera used at 1:200 dilution).
  • Figure 9 shows sera from rabbits PSU#87 and #88 iir iimoprecipitate 901-BRMSl. Protein A/G-agarose beads alone, without any antibody, were used as control (sera used at 1:200 dilution).
  • Figure 10 shows immunized serum from PSU#87 and #88 recognizes S-BRMS1 on an immunoblot (sera used at 1 :200 dilution).
  • Figure 11 shows immunized serum from PSU#20 and #21 recognizes S-BRMSlon an immunoblot (sera used at 1:200 dilution).
  • Figure 12 shows serum from rabbit PSU#87 does not recognize BRMSl by immunoblotting.
  • Figure 15 shows immunized sera from rabbit PSU#87 did not recognize 901- BRMSl on an immunoblot.
  • Cell lysates (30 ⁇ g protein) were resolved on a 12% polyacrylamide gel and transferred to a PVDF and nitrocellulose membrane.
  • PSU#87 serum was used at a dilution of 1 :200.
  • Figure 16 shows purified IgGs from rabbits PSU#87 and #88 do not recognize
  • BRMSl on a WB.
  • Cell lysates (30 ⁇ g protein) resolved on a 12% polyacrylamide gel were immunoblotted using 1 :200 dilution of serum from rabbits.
  • Figure 17 shows a strategy for cleaving off the GST-tag from GST-BRMS1.
  • Figure 18 shows IgY from PSU#20 recognizes BRMSl (cleaved from GST- BRMSl). All samples were analyzed by Coomassie staining, inmiunoblotting with anti- GST antibody and serum from chicken PSU#20 (1:200 dilution).
  • Figure 19 shows reverse-affinity purified IgY from PSU#20 (dilution 1 :600) recognizes BRMSl from cell lysates (30 ⁇ g protein).
  • Figure 20 shows reverse-affinity purified IgY from PSU#20 recognizes BRMSl from cell lysates (30 ⁇ g protein) at dilutions of 1 :600 and 1 :300.
  • Figure 21 shows reverse-affinity purified IgY from PSU#20 (1:600) immunoprecipitates BRMSl from cell lysates.
  • Figure 22 shows that chicken antiserum immunoprecipitates BRMSl, which is recognized by immunoblotting with antibodies to the epitope tag on BRMSl (anti-901). Deletion mutants of BRMS 1 are also recognized and immunoprecipitated by antiserum.
  • Figure 23 A shows that rabbit antiserum co-immunoprecipitates BRMSl and deletion mutants.
  • Figure 23B shows BRMSl immunoprecipitated by rabbit antiserum is also immunoprecipitated with chicken antiserum.
  • Figures 24A-C show immunoblots demonstrating that antisera to BRMSl effectively immunoprecipitate BRMSl and deletion mutants of BRMSl.
  • Figure 24 A shows BRMSl immunoprecipitated with rabbit antisera (#87 and #88);
  • Figure 24B shows BRMSl immunoprecipitated with chicken antisera (#20 and #21);
  • Figure 24C shows control immunoprecipitation with anti-901 epitope. All immunoblots were done with anti-901 antibody with which BRMSl had been epitope tagged.
  • Figure 25 shows an immunoblot demonstrating that affinity enriched antibody
  • Lane 1 myc-tagged BRMSl
  • Lane 2 COS7
  • Lane 3 MDA-M-231
  • Lane 4 231-BRMS1
  • Lane 5 231 -BRMS 1 ⁇ 204
  • Lane 6 231- BRMS1 ⁇ 164.
  • an antibody includes mixtures of antibodies; reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
  • Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • the phrase “optionally obtained prior to treatment” means obtained before treatment, after treatment, or not at all.
  • subject is meant an individual.
  • the subject is a mammal such as a primate, and, more preferably, a human.
  • subject includes domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).
  • the present invention provides an isolated antibody or a fragment thereof that specifically binds breast cancer metastasis suppressor 1 (BRMSl) protein.
  • the antibody can be a polyclonal antibody or a monoclonal antibody.
  • the antibody is raised to BRMS 1 protein from any species, including, but not limited to human, pig, guinea pig, mouse, rat, dog, rabbit or chicken.
  • the antibody is raised to human BRMSl protein.
  • the antibody of the invention selectively binds BRMSl, and optionally to BRMSl of a specific species.
  • ком ⁇ онент binds or “specifically binds” is meant an antibody binding reaction which is determinative of the presence of the antigen (in the present case, BRMSl) among a heterogeneous population of proteins and other biologies.
  • BRMSl antigen
  • the specified antibodies bind preferentially to a particular peptide and do not bind in a significant amount to other proteins in the sample.
  • Specific binding to BRMSl under such conditions requires an antibody that is selected for its specificity to BRMS 1.
  • the antibody competes for binding to natural BRMSl interactors.
  • the antibody binds BRMSl protein ex vivo or in vivo.
  • the antibody of the invention is labeled with a detectable moiety.
  • the detectable moiety can be selected from the group consisting of a fluorescent moiety, an enzyme-linked moiety, a biotin moiety and a radiolabeled moiety.
  • BRMSl protein includes the full length polypeptide, variants of BRMSl protein, fusion proteins comprising BRMSl protein, and immunogenic fragments of BRMSl protein.
  • the antibody binds full length BRMSl protein, variants of BRMSl protein (e.g., an alternatively spliced variant or a deletion mutant), a fusion protein, or any epitope thereon.
  • the BRMS 1 protein to which the antibody is raised is naturally occurring or recombinant.
  • the antibody can be used in techniques or procedures such as diagnostics, screening, or imaging.
  • Anti-idiotypic antibodies and affinity matured antibodies are also considered to be part of the invention.
  • the term "antibody” encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class.
  • Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between 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.
  • 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.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse or other species.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • immunoglobulin heavy chain or fragments thereof and “immunoglobulin light chain or fragments thereof encompass chimeric peptides and hybrid peptides, with dual or multiple antigen or epitope specificities, and fragments, including hybrid fragments.
  • fragments of the heavy chains and/or fragments of the light chains that retain the ability to bind their specific antigens are provided.
  • fragments of the heavy chains and/or fragments of the light chains that maintain BRMSl protein binding activity are included within the meaning of the terms “immunoglobulin heavy chain or fragments thereof and “immunoglobulin light chain and fragments thereof” respectively.
  • Such heavy chains and light chains and fragments thereof, respectively, can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
  • the term "variable” is used herein to describe certain portions of the variable domains that differ 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 usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • 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 E. A. et al, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1987)).
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab and the like, including hybrid fragments.
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided.
  • antibody or fragment thereof fragments of antibodies which maintain BRMSl protein binding activity are included within the meaning of the term "antibody or fragment thereof.”
  • Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
  • Also included within the meaning of "antibody or fragments thereof are conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by reference.
  • the antibodies are generated in other species and "humanized” for administration in humans, hi one embodiment of the invention, the "humanized” antibody is a human version of the antibody produced by a germ line mutant animal.
  • 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 include 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.
  • the present invention provides a humanized version of an antibody, comprising at least one, two, three, four, or up to all CDRs of a BRMS 1 monoclonal antibody.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of or 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 consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, Nature, 321:522-525 (1986); Riechmami et al, Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct.
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source that 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); Rieehmanii 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. Pat. No.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity.
  • the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al, J.
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al, J. Immunol., 151:2623 (1993)). It is important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences.
  • Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • J(H) antibody heavy chain joining region
  • Human antibodies can also be produced in phage display libraries (Hoogenboom et al, J. Mol. Biol., 227:381 (1991); Marks et al, J. Mol. Biol., 222:581 (1991)).
  • the techniques of Cote et al and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol., 147(l):86-95 (1991)).
  • the present invention further provides a hybridoma cell that produces the monoclonal antibody of the invention.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies 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 activity (See, U.S. Pat. No.
  • Monoclonal antibodies of the invention may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) or Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988).
  • a hybridoma method a mouse 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.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent comprises BRMS 1 protein.
  • the antibodies of the present invention specifically bind BRMS 1 protein.
  • the generation of monoclonal antibodies has depended on the availability of purified protein or peptides for use as the immunogen. More recently DNA based immunizations have shown promise as a way to elicit strong immune responses and generate monoclonal antibodies.
  • DNA-based immunization can be used, wherein DNA encoding a portion of BRMSl protein expressed as a fusion protein with human IgGl is injected into the host animal according to methods known in the art (e.g., Kilpatrick KE, et al Gene gun delivered DNA-based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor. Hybridoma. 1998 Dec; 17(6):569-76; Kilpatrick KE et al. High-affinity monoclonal antibodies to PED/PEA-15 generated using 5 microg of DNA. Hybridoma. 2000 Aug;19(4):297-302, which are incorporated herein by reference in full for the methods of antibody production).
  • methods known in the art e.g., Kilpatrick KE, et al Gene gun delivered DNA-based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor. Hybridoma. 1998 Dec; 17(6):569-76; Kilpat
  • An alternate approach to immunizations with either purified protein or DNA is to use antigen expressed in baculovirus.
  • the advantages to this system include ease of generation, high levels of expression, and post-translational modifications that are highly similar to those seen in mammalian systems.
  • Use of this system involves expressing domains of BRMSl protein antibody as fusion proteins.
  • the antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the BRMSl protein antibody nucleotide sequence. This results in the display of the foreign proteins on the surface of the virion. This method allows immunization with whole virus, eliminating the need for purification of target antigens.
  • peripheral blood lymphocytes are used in methods of producing monoclonal antibodies if cells of human origin are desired, or 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” Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transfo ⁇ ned mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin.
  • 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 culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • 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, Calif, and the American Type Culture Collection, Rockville, Md. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the 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 BRMSl protein.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
  • the hybridoma cells maybe 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, protein G, 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. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g.
  • 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 ovary (CHO) cells, plasmacytoma 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 may 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. Pat. No.
  • non-immunoglobulin polypeptide is substituted for the constant domains of an antibody of the invention or substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for BRMSl protein and another antigen-combining site having specificity for a different : antigen.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • Papain digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994, U.S. Pat. No. 4,342,566, and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988). Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab')2 fragment, that has two antigen combining sites and is still capable of cross-linking antigen. The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain 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 domain including one or more cysteines from the antibody hinge region.
  • the F(ab')2 fragment is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the 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.
  • 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.
  • An isolated immunogenically specific paratope or fragment of the antibody is also provided.
  • a specific immunogenic epitope of the antibody can be isolated from the whole antibody by chemical or mechanical disruption of the molecule.
  • the purified fragments thus obtained are tested to determine their immunogenicity and specificity by the methods taught herein, frrrmunoreactive paratopes of the antibody, optionally, are synthesized directly.
  • An immunoreactive fragment is defined as an amino acid sequence of at least about two to five consecutive amino acids derived from the antibody amino acid sequence.
  • One method of producing proteins comprising the antibodies of the present invention is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
  • Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry Applied Biosystems, Inc., Foster City, CA.
  • a peptide or polypeptide corresponding to the antibody of the present invention can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin, whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abralimsen L et al, Biochemistry, 30:4151 (1991)).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
  • the first step is the chemoselective reaction of an unprotected synthetic peptide-alpha-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
  • IL-8 human interleukin 8
  • the invention also provides fragments of antibodies which have bioactivity.
  • the polypeptide fragments of the present invention can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system.
  • an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system.
  • an adenovirus or baculovirus expression system for example, one can determine the active domain of an antibody from a specific hybridoma that can cause a biological effect associated with the interaction of the antibody with BRMSl protein.
  • amino acids found not to contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity.
  • amino or carboxy-terminal amino acids are sequentially removed from either the native or the modified non- immunoglobulin molecule or the immunoglobulin molecule and the respective activity assayed in one of many available assays
  • a fragment of an antibody comprises a modified antibody wherein at least one amino acid has been substituted for the naturally occurring amino acid at a specific position, and a portion of either amino terminal or carboxy terminal amino acids, or even an internal region of the antibody, has been replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate in the purification of the modified antibody.
  • a modified antibody can be fused to a maltose binding protein, through either peptide chemistry or cloning the respective nucleic acids encoding the two polypeptide fragments into an expression vector such that the expression of the coding region results in a hybrid polypeptide.
  • the hybrid polypeptide can be affinity purified by passing it over an amylose affinity column, and the modified antibody receptor can then be separated from the maltose binding region by cleaving the hybrid polypeptide with the specific protease factor Xa. (See, for example, New England Biolabs Product Catalog, 1996, pg. 164.). Similar purification procedures are available for isolating hybrid proteins from eukaryotic cells as well.
  • the fragments include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. hi any case, the fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc.
  • Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antigen. (Zoller MJ et al. Nucl. Acids Res. 10:6487-500 (1982).
  • a variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein, variant, or fragment.
  • solid-phase ELIS A immunoassays are routinely used to select antibodies selectively immunoreactive with a protein, protein variant, or fragment thereof. See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
  • the binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al, Anal. Biochem., 107:220 (1980).
  • an antibody reagent kit comprising containers of the monoclonal antibody or fragment thereof of the invention and one or more reagents for detecting binding of the antibody or fragment thereof to the BRMSl protein.
  • the reagents can include, for example, fluorescent tags, enzymatic tags, or other tags.
  • the reagents can also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the enzymatic reactions produce a product that can be visualized.
  • a molecular complex comprising the antibody of the invention linked to a therapeutic agent.
  • a therapeutic agent is a chemotherapeutic agent.
  • the present invention provides an isolated nucleic acid, comprising a nucleotide sequence that encodes either the immunoglobulin heavy chain, the immunoglobulin light chain, or a fragment thereof expressed by hybridoma expressing a BRMSl antibody.
  • the isolated nucleic acid comprises a nucleotide sequence that encodes immunoglobulin heavy chain, light chain, or a fragment with one or more conservative amino acid substitutions.
  • the isolated nucleic acid of the invention comprises a nucleotide sequence that encodes an amino acid sequence having 70, 75, 80, 85.
  • homology with an immunoglobulin heavy chain, light chain or a fragment thereof expressed by a hybridoma of the invention that expresses a BRMS 1 antibody.
  • identity mean the same thing as similarity.
  • the word homology is used to refer to two non-natural sequences, it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences.
  • Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related.
  • variants of nucleic acids and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
  • homology can be calculated after aligning the two sequences so that the homology is at its highest level. Another way of calculating homology can be performed by published algorithms.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.
  • the same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M.
  • a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).
  • the present invention provides a purified polypeptide, comprising an amino acid sequence of an immunoglobulin heavy chain, light chain, or a fragment thereof expressed by a hybridoma expressing a BRMSl antibody, optionally with one or more conservative amino acid substitutions, with one or more amino acid deletions.
  • the invention provides a purified polypeptide, comprising an amino acid sequence having 95% homology with an immunoglobulin heavy chain, light chain, or a fragment thereof expressed by a hybridoma that expresses BRMS 1 antibody.
  • the invention provides an antibody directed against BRMSl protein, a humanized BRMSl antibody, heavy and light chains of the antibody, humanized heavy and light chains of the antibody, nucleic acids that encode the antibodies, heavy and light chains, and fragments thereof.
  • Certain truncations of these proteins or genes perform the regulatory or enzymatic functions of the full sequence protein or gene.
  • the nucleic acid sequences coding therefor can be altered by substitutions, additions, deletions or multimeric expression that provide for functionally equivalent proteins or genes. Due to the degeneracy of nucleic acid coding sequences, other sequences which encode substantially the same amino acid sequences as those of the naturally occurring proteins may be used in the practice of the present invention.
  • nucleic acid sequences including all or portions of the nucleic acid sequences encoding the above polypeptides, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • nucleotide sequence of an immunoglobin tolerates sequence homology variations of up to 25% as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127- 149, 1998, Alan R. Liss, Inc.) so long as such a variant forms an operative antibody which recognizes BRMSl protein.
  • one or more amino acid residues within a polypeptide sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acid belongs (i.e., a conservative substitution).
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, tlireonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • proteins or fragments or derivatives thereof which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligands, etc.
  • the recombinant vector encoding nucleic acid sequences of the antibodies of the present invention may be engineered so as to modify processing or expression of a vector. Other modifications can be made in either the nucleic acid or amino acid sequence without reducing or without substantially reducing activity in the antibody.
  • Such modifications can occur in the CDRs or non-CDR regions using techniques routine in the art. See, e.g., Yang et al. (1995), J. Mol. Biol. 254:392-403, which is hereby incorporated by reference in its entirety for methods of CDR walking mutagenesis.
  • an inhibitor encoding nucleic acid sequence can be mutated in vitro or in vivo to create and/or destroy translation, initiation, and/or termination sequences or to create variations in coding regions and/or form new restriction endonuclease sites or destroy pre-existing ones, to facilitate further in vitro modification. Any technique for mutagenesis l ⁇ iown in the art can be used, including but not limited to in vitro site directed mutagenesis, J.
  • the present invention provides vectors comprising the nucleic acids of the invention. Also provided are cultured cells comprising the vectors of the invention.
  • Vectors of the present invention include a nucleic acid sequence encoding an antibody or a fragment thereof, an immunoglobulin heavy chain or a fragment thereof, and/or an immunoglobulin light chain or a fragment thereof directed against BRMSl protein, linked to a regulatory element such as a promoter or enhancer.
  • "Operably linked” refers to an arrangement of nucleotide sequences configured so as to perform their usual function.
  • a regulatory element operably linked to a nucleotide sequence encoding a polypeptide is capable of directing the transcription, replication and/or translation of the polypeptide. It will be recognized by those skilled in the art that a single vector optionally includes coding sequences for both an immunoglobulin heavy chain and a light chain. In one embodiment the vectors encode humanized immunoglobulin light or heavy chains.
  • the present invention provides a method for detecting BRMSl protein in a biological sample of a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample with the antibody of the invention, or fragment thereof, under conditions whereby the antibody, or fragment thereof, can bind BRMS 1 protein in the sample; and c) detecting antibody, or fragment thereof, bound to BRMSl protein, bound antibody, or fragment thereof, indicating BRMSl protein in the sample.
  • the antibody, or fragment thereof can be linked to a detectable label either directly or indirectly through use of a secondary and/or tertiary antibody; thus, bound antibody, fragment or molecular complex can be detected directly in an ELISA or similar assay.
  • the sample can be taken from various tissues of a subject, including, but not limited to breast cells, kidney cells, skin cells, prostate cells, spleen cells, liver cells, pancreas cells, placenta cells, heart cells, blood, urine, cerebrospinal fluid, pleural fluid and ascites fluid.
  • a method of screening for reduced metastatic potential of a cancer in a subject comprising detecting in a biological sample of the cancer the presence of BRMSl protein using the antibody of the invention, the presence of BRMSl protein indicating a reduced metastatic potential of the cancer in the subject.
  • the antibody is used to bind BRMSl protein and the bound antibody is detected.
  • a cancer can be a carcinoma, a sarcoma, a leukemia and/or a lymphoma.
  • a cancer includes, but is not limited to, cancer selected from the group consisting of brain, uterus, ovary, small intestine, gall bladder, larynx, breast, kidney, prostate, testis, bladder, melanoma, skin, lung, liver, esophagus, stomach, pancreas, colon, leukemias, lymphomas, myelomas and sarcomas.
  • the present invention provides a method of screening for reduced metastatic potential of a cancer in a subject, comprising detecting in a biological sample of the cancer an elevated level of BRMSl protein as compared to control level using the antibody of the invention, an elevated level in the sample indicating a reduced metastatic potential of the cancer in the subject. Further provided is a method of screening for increased metastatic potential of a cancer in a subject, comprising detecting in a biological sample of the cancer the absence of BRMSl protein using the antibody of the invention, the absence of BRMSl protein indicating an increased metastatic potential of the cancer in the subject.
  • BRMSl levels correlate with a reduced metastic potential as compared to a control tumor.
  • the higher the level of BRMSl the lower the metastatic potential.
  • the metastatic potential increases.
  • Also provided by the present invention is a method of screening for a compound that reduces metastatic potential of a cancer, comprising: a) contacting a cancer cell that expresses BRMSl protein with the compound to be screened; b) contacting the cancer cell with the antibody of the invention; and c) detecting bound antibody in the cancer cell contacted with the compound to be screened, an increase in bound antibody as compared to a control indicating a compound that reduces metastatic potential.
  • Example 1 Generating the immunogen, BRMSl BRMSl protein was produced in bacteria using the pET expression system (Novagen, Madison, WI) which introduces a defined epitope (designated the S-tag) at the N- terminus.
  • the S-tag allowed for detection of the expressed protein (by immunoblot).
  • An additional histidine (His) tag also allows for purification using Ni2 + affinity columns.
  • the presence of a T7 lac promoter allows the induction of S-BRMS1 by addition of lmM IPTG to the cells.
  • Example 2 Generating BRMSl antibodies Monoclonal antibodies BALB/c mice were immunized with recombinant S-BRMS1. An emulsion (200 ⁇ l) containing 100 ⁇ g S-BRMS1 in phosphate-buffered saline containing complete Freund's adjuvant was injected into 10 BALB/c mice into the peritoneum. Four booster injections were given using incomplete Freund's adjuvant at an interval of 3 wk. Sera were checked for reactivity to S-BRMS1 by immunoblot. Two weeks after the fourth booster injection, mice were injected with the antigen alone (S-BRMS1 in PBS) and were euthanized 3 days later.
  • S-BRMS1 antigen alone
  • Spleens were harvested and processed to collect a single cell population of splenocytes which were fused with the P3x.63.Ag (myeloma cell line syngeneic to the BALB/c mouse). Resultant hybrids (-2000) were screened for reactivity to S-BRMS1 byELISAusing standard techniques. Twenty hybrids produced a positive signal by ELISA. A secondary screen of the positive hybrids utilized an immunoblot loaded with another variant of BRMSl (tagged with the SV40T-901 epitope). Anti-901 was used as apositive control and indicated that the antigenicity was based on the S-tag.
  • Chicken polyclonal antisera S-BRMS1 was injected into chicken. Immunization of chicken yielded immunoglobulins in the egg yolk (isotype IgY) at a concentration of 5-15 mg/ml. Another advantage of chickens is that between booster injections (3 weeks), chickens lay an average of 8-10 eggs.
  • the IgY was purified from the egg yolks by delipidation. inization schedule Day Procedure 0 Initial inoculation 21 Boost 21 Egg collection 31 Test bleed 42 Boost 52 Test bleed 63 Boost Intermediate immuno-reactivity was assessed using serum collected from the chickens. Sera were reactive to S-BRMS1 as were IgY isolated from the yolks. But neither the serum nor the IgY reacted with 901-BRMSl on western blot.
  • GST-tagged BRMSl The ⁇ GEX-2TK expression system (Amersham, Piscataway, NJ) was chosen to introduce a glutathione S-transferase epitope to the N-tenninus ( Figures 1 and 2). GST is known not to be as immunogenic as S-tag.
  • GST-BRMS1 is expressed as a fusion protein from a tac promoter (stronger than Lac but weaker than T7 promoter). The rationale was that production of BRMSl in a "native" configuration would be more likely than with use of stronger promoters. The disadvantage was that lower protein yield would be expected.
  • Protein expression was induced with lmM IPTG.
  • GST-BRMS1 was purified using an immobilized glutathione column and eluted with reduced glutathione ( Figures 3 and 4). Immunizations in two rabbits (PSU# 87 and #88), two chickens (PSU#20 and #21) and 10 rats were initiated. The immunization schedule for rabbits and chickens was as described above. Pre-immune sera were collected prior to beginning immunizations.
  • the strategy was to purify BRMSl using GST-BRMSl followed by cleavage of the GST tag from BRMSl ( Figure 17).
  • the BRMSl was coupled to an activated support over which the sera are eluted.
  • the pGEX-2TK expression vector (Amersham) which has a tlirombin cleavage site between GST and BRMSl was used.
  • Biotinylated thrombin two dilutions, lx and 0.04x units; 2 hours at room temperature
  • Cleavage products were then added to Streptavidin-coated beads (to remove thrombm). Released GST was removed by adding immobilized glutathione (GSH) and incubation for 4 hours at room temperature.
  • GSH resin was then pelleted leaving behind pure BRMSl protein. While the GST-BRMSl cleavage was successful, GSH resin did not completely remove the GST. Furthermore, GSH resin appeared to bind considerable amounts of BRMSl ( Figure 18). While there is significant "noise,” an intense signal at ⁇ 35kDa (where BRMSl migrates) using purified IgY from PSU#20 (chicken) was observed. To reduce noise, it was attempted to remove E. coli reactive antibodies. To do this, immobilized E. coli lysate (Pierce, Cat # 44938; 2.75 ml) was packed into a 5 ml column.
  • IgY from PSU#20 was centrifuged (13,000 rpm; 10 min; 4 C) twice to remove particulates. Fractions were collected in phosphate buffered saline and quantified by spectrophotometer (280 mil). Fractions (500 ⁇ L) having greatest absorbance (Fractions 3-6) indicated highest protein content were pooled. GST-reactive antibodies were eliminated by placing the fractions onto an immobilized GST column (2.0 ml packed). Theoretically, eluate should be free of contaminating anti-GST antibodies. Eluate was tested in an immunoblot having 901- BRMSl and two C-terminal deletion mutants.

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Abstract

La présente invention concerne un anticorps BRMS1, des chaînes lourdes et légères d'immunoglobuline de l'anticorps, et des fragments de l'anticorps et les chaînes légères et lourdes qui en sont tirées. L'invention concerne également des acides nucléiques codant l'anticorps, des chaînes lourdes ou légères d'immunoglobuline de l'anticorps, ou des fragments de l'anticorps et les chaînes légères et lourdes qui en sont tirées. L'invention concerne aussi des vecteurs comprenant les acides nucléiques et des cellules de culture comprenant ces vecteurs. L'invention concerne en outre des procédés de fabrication et d'utilisation de l'anticorps, et des fragments des anticorps ainsi que des chaînes légères et lourdes qui en sont tirées. L'invention concerne notamment des procédés permettant de déterminer le potentiel de métastase d'une tumeur cancéreuse primaire.
PCT/US2004/022084 2003-07-11 2004-07-09 Anticorps qui reconnaissent la proteine brms1 et leurs utilisations WO2005007692A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039504A2 (fr) * 1995-06-06 1996-12-12 Novartis Ag Procede permettant la distinction entre cancers metastatiques et cancers non metastatiques, et polynucleotides et polypeptides utilises dans ce procede
EP1347046A1 (fr) * 2002-03-22 2003-09-24 Research Association for Biotechnology Séquences d'ADN complémentaire entières

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039504A2 (fr) * 1995-06-06 1996-12-12 Novartis Ag Procede permettant la distinction entre cancers metastatiques et cancers non metastatiques, et polynucleotides et polypeptides utilises dans ce procede
EP1347046A1 (fr) * 2002-03-22 2003-09-24 Research Association for Biotechnology Séquences d'ADN complémentaire entières

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Title
DEBIES M T ET AL: "GENETIC BASIS OF HUMAN BREAST CANCER METASTASIS", JOURNAL OF MAMMARY GLAND BIOLOGY AND NEOPLASIA, PLENUM PRESS, NEW YORK, NY,, US, vol. 6, no. 4, October 2001 (2001-10-01), pages 441 - 451, XP001156089, ISSN: 1083-3021 *
MEEHAN WILLIAM J ET AL: "Breast cancer metastasis suppressor 1: Update.", CLINICAL AND EXPERIMENTAL METASTASIS, vol. 20, no. 1, 2003, pages 45 - 50, XP002304108, ISSN: 0262-0898 *
SAMANT R S ET AL: "Analysis of mechanisms underlying BRMS1 suppression of metastasis", CLINICAL AND EXPERIMENTAL METASTASIS, vol. 18, no. 8, 2000, pages 683 - 693, XP002304111, ISSN: 0262-0898 *
SAMANT RAJEEV S ET AL: "Identification and characterization of the murine ortholog (brms1) of breast-cancer metastasis suppressor 1 (BRMS1)", INTERNATIONAL JOURNAL OF CANCER, vol. 97, no. 1, 1 January 2002 (2002-01-01), pages 15 - 20, XP002304110, ISSN: 0020-7136 *
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THE MOLECULAR ROLE OF BRMS1 IN METASTASIS SUPPRESSION, RESEARCH GRANT FROM ANNE MONGIU, 30 April 2002 (2002-04-30), XP002304109, Retrieved from the Internet <URL:http://www.biochem.emory.edu/cl.PDF> [retrieved on 20041103] *

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