WO1994018345A1 - Peptides anti-proliferatifs de fixation de recepteurs - Google Patents

Peptides anti-proliferatifs de fixation de recepteurs Download PDF

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Publication number
WO1994018345A1
WO1994018345A1 PCT/US1994/001319 US9401319W WO9418345A1 WO 1994018345 A1 WO1994018345 A1 WO 1994018345A1 US 9401319 W US9401319 W US 9401319W WO 9418345 A1 WO9418345 A1 WO 9418345A1
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Prior art keywords
peptide
idiotype
antiproliferative
peptides
cells
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PCT/US1994/001319
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English (en)
Inventor
Markus F. Renschler
Ronald Levy
Ramesh R. Bhatt
William J. Dower
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Affymax Technologies N.V.
The Board Of Trustees Of The Leland Stanford Jr. University
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Priority claimed from US08/014,426 external-priority patent/US5512435A/en
Application filed by Affymax Technologies N.V., The Board Of Trustees Of The Leland Stanford Jr. University filed Critical Affymax Technologies N.V.
Priority to AU61711/94A priority Critical patent/AU6171194A/en
Publication of WO1994018345A1 publication Critical patent/WO1994018345A1/fr

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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
    • G01N33/57407—Specifically defined cancers
    • 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
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention provides novel polypeptides which can be used to treat neoplasia, lymphoproliferative diseases, and other pathological states, methods for identifying and administering such peptides or peptidomimetics for therapy and diagnosis of disease, methods of treating neoplastic disease, methods of treating immune diseases, and kits for identifying and producing therapeutic peptides for individual patients.
  • membrane immunoglobulin Signalling by membrane immunoglobulin, T cell receptors, and MHC molecules regulates lymphocyte maturation and development.
  • crosslinking of membrane immunoglobulin by antigen or by anti-immunoglobulin antibodies inactivates immature B cells, eliminating many of the B cells capable of producing autoantibodies; whereas crosslinking of membrane immunoglobulin promotes activation of mature B cells for clonal expansion and antibody production against foreign antigens.
  • Activation of protein kinase activity and growth arrest of B cells by crosslinking of membrane immunoglobulin has been reported (Gold et al. (1990) Nature 345: 810; Campbell and Sefton (1990) EMBQ J. 9 . : 2125) .
  • Anti-membrane immunoglobulin antibodies have been reported to exert inhibitory effects on cell proliferation of immature B cells, possibly by inducing programmed cell death, referred to as apoptosis (Benhamou et al. (1990) Eur. J. Immunol. 20: 1405) .
  • Apoptosis of cytotoxic T lymphocyte clones incubated in the presence of their specific epitopes (i.e., which bind to their surface T cell receptor idiotype) but not in the presence of irrelevant epitopes has also been reported (Moss et al. (1991) J. Ex . Med. 173: 681) .
  • the PD-1 gene which is reportedly a member of the immunoglobulin gene superfamily, has been reported to be induced during programmed cell death of both a murine T cell hybridoma and a murine hematopoietic progenitor cell line (Ishida et al. (1992) EMBQ J. 11: 3887) .
  • the surface Ig receptor of B-cell lymphomas is a tumor specific antigen unique to each patient.
  • Antibodies raised against the Ig receptor idiotype reportedly can arrest the growth of lymphoma cells in vitro (Pennell C and Scott D Eur J Immunol 16: 1577) and reportedly can induce tumor regression in vivo (Maloney et al. (1992) Biologic Therapy of Cancer Updates Vol. 2, No. 6, pp. 1-9; Miller et al. (1982) N Encfl J Med 306:517) .
  • anti-proliferative effect of anti- idiotype antibodies has been reported to involve transmembrane signalling through phosphoinositide hydrolysis, increased intracellular calcium, protein kinase C activation, and tyrosine phosphorylation (DeFranco AL (1992) Eur J. Biochem 210:381; Campbell MA and Sefton BM (1990) EMBQ Journal 9:2125: Gold et al.
  • lymphoproliferative diseases such as lymphomas and lymphocytic leukemias
  • conventional combination chemotherapy remains the principal treatment of choice for these disease states.
  • the efficacy of chemotherapy is limited, especially when patients fail first line drug regimens.
  • Other therapy modalities need to be developed to overcome the limitations of chemotherapy.
  • Anti-idiotype antibodies directed against cell surface immunoglobulins of individual lymphocytic neoplasms requires a lengthy and costly process to generate specific anti-idiotype antibodies for each patient individually, as each individual neoplasm likely has a unique idiotype.
  • anti-idiotype immunoglobulins depends primarily on immunization of nonhuman hosts, such as mice, which yields nonhuman immunoglobulins that are typically immunogenic when administered to humans.
  • the necessity of humanization for therapeutic use of such antibodies would add to the cost and time expenditure, delaying treatment and making individualized anti-idiotype antibody therapy prohibitively expensive.
  • anti-idiotype antibodies theoretically are tools for diagnosis and therapy of B cell lymphomas, practical considerations hinder their widespread use in medicine.
  • the present invention fulfills these and other needs.
  • a predetermined cell population e.g., a neoplastic cell sample
  • a cell surface receptor which is a member of the immunoglobulin superfamily.
  • a predetermined cell population expressing surface immunoglobulin superfamily molecules is isolated from a patient as a cellular sample, such as a lymph node biopsy or blood sample or the like.
  • the predetermined cell population or its clonal progeny propagated in culture are used as a source of specific surface immunoglobulin superfamily molecules for screening a polypeptide library, such as a bacteriophage peptide display library or a spatially defined polypeptide array on a solid substrate, to determine polypeptide sequences which bind the specific surface immunoglobulin superfamily molecules of the predetermined cell population with an affinity of about at least 1 x 10 5 M "1 or more (preferably at least 1 x 10 6 M "1 to 1 x 10 7 M "1 ) , and which substantially lack binding to irrelevant surface immunoglobulin superfamily molecules of other cells, thereby identifying candidate antiproliferative peptide sequences.
  • a polypeptide library such as a bacteriophage peptide display library or a spatially defined polypeptide array on a solid substrate
  • Peptides and/or peptidomimetics comprising sequences corresponding to or substantially identical to the candidate antiproliferative peptide sequences are assayed, either in native form or crosslinked to additional candidate peptides or peptidomimetics, in cell culture.
  • Such assays determine the biological activity(ies) of the peptide or peptido imetic species by detecting at least one of the following functional properties: (1) binding to cell surface immunoglobulin superfamily molecules on the predetermined cell population and lacking substantial binding to irrelevant cell populations, (2) inhibition of cell proliferation of the predetermined cell population, (3) induction of apoptosis of the predetermined cell population, (4) stimulation of protein tyrosine kinase activity in the predetermined cell population treated with the peptide or peptidomimetic, (5) modulation of calcium flux across the plasma membrane, and (6) inhibition of other indicia of lymphocyte proliferation in the predetermined cell population but substantially lacking such inhibition in irrelevant cell populations.
  • Biologically active antiproliferative peptides and peptidomimetics are then formulated for administration to a patient having a pathological condition that is characterized by abnormal proliferation of the predetermined cell population, such as a lymphoproliferative disease (e.g., a B cell lymphoma or leukemia) .
  • a lymphoproliferative disease e.g., a B cell lymphoma or leukemia
  • the invention also provides methods and compositions for identifying and administering anti-idiotype peptides and peptidomimetics for ablating or preventing clonal expansion of lymphocyte subpopulations expressing cell surface immunoglobulin superfamily molecules having specific idiotypes (i.e., antigen or ligand binding clefts) .
  • the anti-idiotype peptides and peptidomimetics are administered therapeutically or prophylactically to modulate immune system function by inactivating, by inducing clonal anergy or apoptosis, lymphocyte subpopulations which bind the anti-idiotype molecules.
  • diagnostic and therapeutic compositions of antiproliferative peptides and peptidomimetics which can be used in therapy and diagnosis of lymphoproliferative diseases in an individual patient.
  • Diagnostic compositions can include imaging conjugates wherein anti-idiotype peptides or peptidomimetics are linked to imaging agents, such as by a covalent linkage or a noncovalent linkage which is substantially nonreleasable under physiological conditions (e.g., a streptavidin-biotin linkage).
  • Therapeutic compositions comprise at least one species of antiproliferative peptide or peptidomimetic for treating lymphoproliferative diseases, other neoplastic conditions, or immune system disorders.
  • Typical therapeutic compositions comprise an effective dosage of a anti-idiotype peptide or peptidomimetic in a pharmaceutically acceptable form, and optionally may include excipients or stabilizers.
  • the present invention provides compositions of peptides and peptidomimetics that have antiproliferative activity against lymphoma and lymphocytic leukemia cells, and more particularly against B cell lymphomas and B cell lymphocytic leukemias.
  • the present invention also provide methods for treating or preventing lymphoma and lymphocytic leukemia, and more particularly against B cell lymphomas and B cell lymphocytic leukemias in humans.
  • a therapeutic method for treating a lymphoma or lymphocytic leukemia comprises delivering a therapeutically effective dosage of an antiproliferative peptide to a patient having a lymphoma or lymphocytic leukemia expressing an idiotype which binds the antiproliferative peptide.
  • the method may be used to treat other lymphoproliferative diseases as well.
  • Antiproliferative peptides are non-immunoglobulin proteins and have advantageous pharmacological properties and are relatively inexpensive to identify and manufacture for a patient as compared to anti-idiotype immunoglobulins.
  • Also provided by the invention is a method for inhibition the proliferation of lymphoma cells and lymphocytic leukemia cells in culture.
  • the method comprises delivering an antiproliferative dosage of an antiproliferative peptide to a cell culture comprising a lymphoma or lymphocytic leukemia expressing an idiotype which binds the antiproliferative peptide.
  • Such methods will allow identification (and isolation) of variants of lymphoma and lymphocytic leukemia cells which have lost a proliferative phenotype and/or have lost expression of the immunoglobulin superfamily molecule to which the antiproliferative peptide has binding affinity for.
  • Such variant cells may be used to screen compounds to identify antineoplastic pharmaceuticals and study the process of neoplastic variability.
  • Also provided in the invention is a method for inhibiting the proliferation of lymphoma cells or lymphocytic leukemia cells, comprising delivering an inhibitory dose of a non-immunoglobulin antiproliferative peptide which specifically binds to an immunoglobulin superfamily molecule idiotype present on the lymphoma cells or lymphocytic leukemia cells.
  • a method for inhibiting the proliferation of lymphoma cells or lymphocytic leukemia cells comprising delivering an inhibitory dose of a non-immunoglobulin antiproliferative peptide which specifically binds to an immunoglobulin superfamily molecule idiotype present on the lymphoma cells or lymphocytic leukemia cells.
  • Fig. 1 shows inhibition of anti-idiotype antibody binding to the relevant immunoglobulin with anti-idiotype peptide 3T802.
  • Fig. 2 shows the antiproliferative effect of biotinylated peptide 3T802 conjugated to streptavidin.
  • Fig. 3 shows a Western blot showing tyrosine phosphorylation in SupB8 (Tab) cells and the effect of peptide tetramers on signal transduction of SUP-B8 cells.
  • Peptide A Intracellular protein tyrosine phosphorylation in SUP-B8 cells stimulated with Peptide A tetramers: SUP-B8 cells were stimulated with medium (Lane 1) , anti-IgM polyclonal serum (lane 2) and 5 ⁇ M control peptide tetramer (Lane 3) for two minutes, or with 5 ⁇ M Peptide A tetramer for 1, 2, 5, 10, or 15 minutes (Lanes 4-8) .
  • Fig. 4 shows binding of SUP-B8 Ig and control immunoglobulins to biotinylated peptides. Biotinylated peptide monomers were immobilized on a streptavidin coated microtiter plate. The binding of purified SUP-B8 Ig, purified Ig from other patients' tumors, purified Ig from the lymphoma cell line SU-DHL4 or purified polyclonal normal IgM was measured in an ELISA. The isotype of each immunoglobulin is shown in the legend ( ⁇ :lambda, K : appa). Data shown is the mean of duplicate samples +/- SD. The data is representative of three experiments with similar results.
  • Fig. 5 shows inhibition of anti-Id antibody binding to SUP-B8 Ig with peptides.
  • Peptide monomers were added at increasing concentrations to inhibit the binding of the anti-id antibody to SUP-B8 Ig, measured in an ELISA assay.
  • Data shown is the mean of duplicate samples +/- SD. The data is representative of five experiments with similar results.
  • Fig. 6 shows the effect of peptide monomers and tetramers on cell proliferation measured by 3 H-thymidine incorporation.
  • Panel A SUP-B8 cells were incubated with peptide monomers or streptavidin at varying concentrations for 72 hours. The streptavidin concentrations used were 1/4 of those shown, to be identical to the concentration of streptavidin used for Panels B and C. • Peptide A, ⁇ Peptide B, ⁇ Peptide C, ⁇ Streptavidin.
  • SUP-B8 cells Panel B or 0CI-Ly8 Cells (Panel C) were incubated with peptide tetramers at varying concentrations for 72 hours • Peptide A tetramer, ⁇ Peptide B tetramer, A Peptide C tetramer, O Control Peptide D tetramer. Data shown are means of raw counts per minute of quadruplicate samples +/- SD. The experiments shown are representative of 3, 8 and 3 experiments with similar results for (Panel A) , (Panel B) and (Panel C) respectively.
  • Fig. 7 shows the effect of peptide dimers on cell proliferation measured by 3 H-thymidine incorporation
  • panel A A variant of peptide A (KPWWVSRVSSC) (•) and a control peptide (YSSVSRVWWPK) (O) which includes the reverse octapeptide sequence of peptide A, were subjected to a dimerization procedure and incubated for 72 hours with SUP-B8 cells.
  • SUP-B8 cells Panel B
  • OCI-Ly8 Cells Panel C
  • Tandem Repeat Peptide C ⁇ Tandem Repeat Scrambled Peptide C.
  • Data shown are means of raw counts per minute of triplicate (Panel A) or quadruplicate (panel B) , (Panel C) samples +/- SD. The experiments shown are representative of 3 experiments with similar results.
  • Fig. 8 shows DNA fragmentation in SUP-B8 cells incubated with peptide tetramers.
  • DNA from SUP-B8 cells incubated for 24 hours with 2.5 ⁇ M peptide tetramers was electrophoresed on an agarose gel and stained with ethidium bromide.
  • Lane 1 Peptide A tetramer
  • lane 2 Peptide B tetramer
  • lane 3 Peptide C tetramer
  • lane 4 Scrambled Peptide C tetramer
  • lane 5 Reverse Peptide C tetramer.
  • the experiment shown is representative of 5 experiments with similar results.
  • naturally-occurring 1 - refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally- occurring.
  • the twenty conventional amino acids and their abbreviations follow conventional usage (Immunology - A Synthesis. 2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Massachusetts (1991) , which is incorporated herein by reference).
  • Stereoisomers e.g., D-amino acids
  • unnatural amino acids such as ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention.
  • Examples of unconventional amino acids include: 4-hydroxyproline, 7-carboxyglutamate, e-N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine, ⁇ -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline) .
  • Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is lie or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
  • antiproliferative peptide refers to a polypeptide that inhibits proliferation or induces apoptosis in a predetermined cell population that expresses a cell surface immunoglobulin superfamily molecule having an idiotype which is substantially absent in other cell populations bearing cell surface immunoglobulin superfamily molecules.
  • Antiproliferative peptides typically have structures and chemical properties defining an epitope which binds to an idiotype- determining binding cleft of an immunoglobulin superfamily molecule, such as a surface IgM or IgD molecule.
  • Antiproliferative peptides are typically polypeptides, although they may comprise D- amino acids in peptide linkage.
  • antiproliferative peptidomimetics comprise polypeptide-like polymers that contain novel backbone structures or unnatural amino acids (Ellman et al. (1992) Science 255: 197, which is incorporated herein by reference) , or other non-peptide chemical constituents, including peptoids (Simon et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89: 9367.
  • Antiproliferative peptides and peptidomimetics generally comprise epitopes which are bound at an idiotype-determining binding cleft (e.g., antigen binding site of an immunoglobulin) and are generally anti- idiotype peptides and peptidomimetics.
  • antiproliferative peptides and peptidomimetics may induce apoptosis, clonal anergy, or proliferative arrest by a mechanism other than binding to an idiotype-determining binding portion of an immunoglobulin superfamily molecule; such antiproliferative peptides may possess the property of inducing apoptosis, clonal anergy, or proliferative arrest in cells bearing immunoglobulin superfamily molecules of various idiotypes.
  • anti-plastic peptide is used herein to refer to peptides that have the functional property of inhibiting development or progression of a neoplasm in a human, particularly a lymphoma, lymphocytic leukemia, or pre-leukemic condition.
  • label refers to incorporation of a detectable marker, e.g.. by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods) .
  • labels for polypeptides include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 32 P, 35 S, 125 I, 131 I) , fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors) , enzymatic labels (e.g., horseradish peroxidase, /3-galactosidase, luciferase, alkaline phosphatase) , biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) .
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition) , and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • normal blood or "normal human blood” refers to blood from a healthy human individual who does not have an active neoplastic disease or other disorder of lymphocytic proliferation, or an identified predisposition for developing a neoplastic disease.
  • normal cells “normal cellular sample”, “normal tissue”, and “normal lymph node” refers to the respective sample obtained from a healthy human individual who does not have an active neoplastic disease or other lymphoproliferative disorder.
  • polypeptide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • substantially similarity or “substantial identity” as used herein denotes a characteristic of a polypeptide sequence or polynucleotide sequence, wherein the polypeptide sequence has at least 50 percent sequence identity compared to a reference sequence, and the nucleic acid sequence has at least 70 percent sequence identity compared to a reference sequence.
  • the percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence.
  • the reference sequence may be a subset of a larger sequence, such as a polypeptide epitope of a larger polypeptide sequence; however, the reference sequence is at least 6 amino residues long in the case of a polypeptide.
  • biological activity As used herein, the terms "biological activity” and
  • an antiproliferative activity are defined as the capacity to inhibit specifically the proliferation, clonal expansion, or immunologic activation (e.g., production of secreted immunoglobulins) of a predetermined cell population.
  • an antiproliferative peptide of the invention has biological activity if a predetermined cell population that has been treated with a suitable concentration of the antiproliferative peptide produces a detectable decrease in cell proliferation (e.g., 3 H- thymidine incorporation, cell number) as compared to a parallel cell culture that is not treated with the antiproliferative peptide and as compared to a cell culture of irrelevant cells (i.e., cells not bearing immunoglobulin superfamily molecules of the same idiotype as the predetermined cell population) .
  • Suitable concentrations (i.e., efficacious dose) of antiproliferative peptides and peptidomimetics can be determined by various methods, including generating an empirical dose- response curve, predicting potency and efficacy of a congener by using QSAR methods or molecular modeling, and other methods used in the pharmaceutical sciences (see Fauchere, J. (1986) Adv. Drug Res. 15: 29, which is incorporated herein by reference).
  • Antiproliferative peptides of the invention also have specificity for inhibiting proliferation of the predetermined cell population.
  • a biologically active peptide of the invention specifically inhibits proliferation of one or more predetermined cell populations without producing significant nonspecific cytotoxicity in irrelevant cells.
  • Significant nonspecific cytotoxicity can be determined by various methods, including, for example, determining that treatment of an irrelevant cell culture with an efficacious dose of an agent reduces cell proliferation by at least about 50 percent as compared to a parallel culture that is not treated with the peptide.
  • Other type of assays that can be used to ascertain nonspecific cytotoxicity in cell cultures include: Trypan Blue dye exclusion, incorporation of radiolabeled amino acids and/or radiolabeled nucleotides, mitotic rate (e.g., doubling time), and other methods known in the art.
  • the peptide backbone of a polypeptide consists of a repeated sequence of three atoms: the amide N, the a C, and the carbonyl C:
  • N which are generally represented as N if C ⁇ , and C ⁇ , respectively, where i is the number of the residue, starting from the amino end.
  • the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • An open hyphen at the lefthand end of a peptide sequence indicates that an amino-terminal peptide extension (including unconventional amino acids) may be attached to the amide nitrogen of the leftmost amino acid residue in the given sequence. If there is no open hyphen at the lefthand end of a peptide sequence, that indicates that the leftmost residue is the amino-terminus of the peptide, although the amide nitrogen of the leftmost residue may be chemically modified, for example with an acetyl or methyl group, but typically the amide nitrogen is bonded to the a carbon and hydrogen substituents only.
  • An open hyphen at the righthand end of a peptide sequence indicates that a carboxy-terminal peptide extension (including unconventional amino acids) may be attached to the carbonyl carbon of the rightmost residue in the given sequence. If there is no open hyphen at the righthand end of a peptide sequence, that indicates that the rightmost residue is the carboxy-terminus of the peptide, although the carbonyl carbon of the rightmost residue may be chemically modified, for example by amidation, but typically the carbonyl carbon is bonded to a hydroxyl group, forming a carboxyl group attached to the a carbon of the rightmost residue.
  • sequence -Lys-Pro-Trp- and the carboxy-terminal sequence -Ser-Arg-Val both occur in the octapeptide Lys-Pro-Trp-Tyr-Val-Ser-Arg-Val.
  • Lys-Pro- Trp designates a tripeptide since there are no righthand or lefthand open hyphens.
  • the recognized immunoglobulin superfamily is described in The Immunoglobulin Gene Superfamily. A.F. Williams and A.N. Barclay, in Immunoglobulin Genes. T. Honjo, F.W. Alt, and T.H. Rabbitts, eds., (1989) Academic Press: San Diego, CA, pp.361-387, which is incorporated herein by reference.
  • a basis of the present invention is the unexpected finding that peptides having defined sequences can inhibit proliferation, induce clonal anergy, mediate a signal that activates the cell through a tyrosine kinase regulated cell proliferation pathway, and/or induce apoptosis in cells, such as lymphocytic cells of a lymphoma or lymphocytic leukemia, by binding to cell surface immunoglobulin superfamily proteins having specific idiotypes which bind the peptides.
  • lymphocytes of any B cell lymphoma generally express a single species of surface immunoglobulin having an idiotype characteristic of that individual B cell lymphoma.
  • lymphoma or lymphocytic leukemia it is generally possible to define, by screening libraries of peptides representing a variety of peptide sequences, specific polypeptide sequences which bind to the cell surface immunoglobulin superfamily molecules (e.g., surface IgM or IgD) having the idiotype characteristic of that individual B cell lymphoma.
  • the peptides identified by such screening methods are tested for the ability to inhibit proliferation, induce clonal anergy, modulate tyrosine kinase activity, and/or induce apoptosis in cultured cells of the individual B cell lymphoma, either as individual peptides (optionally including repeat anti- idiotype motifs) or as complexes of crosslinked peptides.
  • Peptides which inhibit proliferation, induce clonal anergy, modulate tyrosine kinase activity, and/or induce apoptosis in the cultured lymphoma cells are thereby identified as antiproliferative (or anti-idiotype) peptides and may be administered to a patient which has a B cell lymphoma expressing immunoglobulin superfamily molecules with the characteristic idiotype.
  • the tissue penetration of peptides, the ease of synthesis and the ability to modify peptides is superior to anti- idiotype antibodies.
  • the peptides of the present invention tend to be less immunogenic than non-human monoclonal antibodies.
  • the Ig receptor can be targeted with peptides that bind specifically to the individual Ig receptor species as surrogate ligands.
  • the peptides themselves, when made multimeric, inhibit growth by crosslinking Ig receptors, and/or can be conjugated to deliver toxins or radionuclides to neoplastic cells bearing the Ig receptor species to which the peptide(s) bind.
  • anti-idiotype peptide screening methods can define multiple peptide sequences which bind to the same immunoglobulin idiotype.
  • at least one consensus anti-idiotype peptide sequence motif can be generated which can be used as a sequence template for the generation of additional peptide sequence variants having binding affinity for the immunoglobulin superfamily molecule.
  • such peptide sequence variants comprise one or two amino acid variations from the anti-idiotype peptide sequence motif, frequently three amino acid variations, and occasionally four or more amino acid variations from the anti-idiotype peptide sequence motif (which may itself comprise alternative residues at specific amino acid positions) , and may comprise additional amino acid sequences in polypeptide linkage to the portion containing the consensus motif, either amino-terminal extensions, carboxyterminal extensions, or both.
  • the anti-idiotype peptide consensus motif constitutes a convenient description of the structural and conformational characteristics of peptides which bind to the idiotypic binding site (e.g., the antigen binding site of an immunoglobulin) .
  • a consensus motif may form the basis for synthesis of peptidomimetics.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics” or “peptidomimetics” (Fauchere, J. (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem 30: 1229, which are incorporated herein by reference) and are usually developed with the aid of computerized molecular modeling.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • a particularly preferred non-peptide linkage is - CH 2 NH-.
  • Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities) , reduced antigenicity, and others.
  • Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group) , to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure- activity data and/or molecular modeling.
  • Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) (e.g., immunoglobulin superfamily molecules) to which the peptidomimetic binds to produce the therapeutic effect.
  • D ⁇ rivitization (e.g., labelling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may be used to generate more stable peptides.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387, incorporated herein by reference) ; for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • crosslinking anti-idiotype peptides results in enhanced biological activity for inhibiting proliferation, inducing clonal anergy, and/or inducing apoptosis in cells expressing cell surface immunoglobulin superfamily molecules of the characteristic idiotype used for screening the peptide library.
  • Crosslinking is typically accomplished by synthesizing biotinylated anti- idiotype peptides and contacting them with streptavidin under aqueous binding conditions (e.g., buffered physiological saline, optionally including Tween and/or nonspecific blocking protein) to form crosslinked anti-idiotype peptides.
  • aqueous binding conditions e.g., buffered physiological saline, optionally including Tween and/or nonspecific blocking protein
  • anti- idiotype peptides may be crosslinked with a covalent crosslinking agent, such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • Peptides can be crosslinked through carboxy-terminal cysteines (e.g., which may be added carboxyterminal to a consensus sequence or substantially identical variant) , typically using spacers such as bis- maleimidohexane (BMH) (Pierce Chemical Co.) which can covalently link two sulfhydryl groups.
  • spacers such as bis- maleimidohexane (BMH) (Pierce Chemical Co.) which can covalently link two sulfhydryl groups.
  • BMH bis- maleimidohexane
  • crosslinking occurs at non-interfering positions so that substantial biological activity (e.g., antiproliferative activity) is retained.
  • Such non- interfering positions generally are positions that do not form direct contacts with the immunoglobulin superfamily molecule(s) (e.g. , surface IgM) to which the peptide or peptidomimetic binds to produce the therapeutic effect.
  • polypeptides consisting essentially of repeats of a peptide consensus sequence (or substantially identical variants thereof) may be linked in peptidyl linkage, typically including a spacer between the repeats comprising a noninterfering amino acid, such as glycine.
  • Such repeat polypeptides can function as a crosslinked complex in inhibiting proliferation, inducing clonal anergy, mediating a signal that activates the cell through a tyrosine kinase regulated cell proliferation pathway, and/or inducing apoptosis in cells.
  • the crosslinked peptides will form dimers and/or tetramers or higher order polymers for advantageous therapeutic efficacy.
  • neoplastic disease jLn vivo and/or ex vivo e.g., to purge explanted hematopoietic and/or lymphocytic cells of undesired neoplastic cells expressing the relevant immunoglobulin superfamily idiotype
  • antiproliferative peptides can be used in vitro and in vivo as commercial research reagents.
  • antiproliferative peptides to a transplantable human lymphoma model can be sold to researchers for standardizing assays (e.g., screening assays for identifying novel agents having antineoplastic/antiproliferative activity) , for manipulating the proliferative status of the cell line, and/or for inducing a reproducible model of apoptosis, and the like, among many other uses which make the antiproliferative peptides commercially marketable to the research community (e.g. , similar to restriction enzymes, Taq polymerase, and the like) .
  • Antiproliferative peptides to essentially any lymphocytic cell line expressing a surface immunoglobulin superfamily signal transduction molecule can be produced by the methods of the invention; thus antiproliferative peptides can be generated as custom research reagents (e.g., for commercial sale) for essentially any lymphocytic cell line (i.e., in addition to SUP- B8) used in research and diagnostics.
  • the antiproliferative peptides of the invention may be sold in kit form for research and diagnostic usage, either with or without a sample of the cell line(s) which were used to identify the antiproliferative peptides and/or which are induced to undergo apoptosis upon treatment with an efficacious dose of the antiproliferative peptides.
  • kits include vials or ampules of such viable cells which are further capable of propagation in a host animal (e.g., immunodeficient or immunocompromised mouse; SCID mouse) as a model of a lymphoproliferative disease or other hematopoietic disorder (e.g., neoplasia) ; and/or for providing a mechanistic model of lymphocyte apoptosis in vivo, and the like.
  • a host animal e.g., immunodeficient or immunocompromised mouse; SCID mouse
  • SCID mouse hematopoietic disorder
  • a predetermined cell population is a population of cells which is substantially comprised of cells that are the clonal progeny of a precursor cell (e.g., stem cell, neoplastically transformed cell) and which express a surface immunoglobulin superfamily molecule having an identical idiotype.
  • a biopsy sample of a B cell lymphoma comprises a predetermined cell population since a substantial fraction of the cells in the biopsy sample are derived from a common lymphopoietic precursor cell (i.e., presumably the transformed parent cell) and also express a surface immunoglobulin having essentially identical structure and antigen binding properties (i.e., identical idiotype). It is recognized that some degree of somatic mutation may occur so that minor sequence variations in the immunoglobulin may occur yet not alter idiotypic specificity.
  • a predetermined cell population comprising cells which express a cell surface immunoglobulin superfamily molecule, such as a T cell receptor ( ⁇ /j ⁇ ) or a surface IgM or IgD, is obtained, typically by obtaining a blood sample enriched for a clone of lymphoid cells or lymph node biopsy from a patient suspected of having a lymphoproliferative disease, such as a lymphoma or lymphocytic leukemia (ALL or CLL) .
  • the explanted lymphocytic cells may be cultured in a suitable culture medium under growth conditions, may be used directly, or may be used to generate a hybridoma by fusion with an immortalized lymphoid cell line.
  • the explanted cells are B cells, it is generally preferable to obtain secreted immunoglobulin produced by the cells of hybridomas formed by fusion of the cells with a suitable fusion partner.
  • the predetermined cell population may be used directly for screening.
  • the immunoglobulin superfamily molecule(s) can be recovered from cell lysates or extracts made from the cells, generally via immunoaffinity purification using antibodies directed against the immunoglobulin superfamily molecules (e.g., with an anti-human IgM antibody) .
  • a mild detergent such as deoxycholate, may be included to solubilize the molecules.
  • Immunoglobulin produced by the predetermined cell population may be used to screen a peptide library for identifying peptides which bind specifically to the immunoglobulin on the basis of idiotypic identity.
  • the cells of the predetermined cell population may be used to screen a peptide library directly, thereby identifying peptide species which bind to the immunoglobulin superfamily molecule species expressed on the surface of the cells as candidate anti-proliferative peptides.
  • peptide libraries may be screened to identify anti-idiotype peptides.
  • Two preferred peptide library types are: (1) a bacteriophage peptide display library, which can comprise a bacteriophage antibody display library for selecting anti-idiotype antibodies, and (2) a spatially defined array of peptides fixed to a solid support (U.S. Patent 5,143,854, incorporated herein by reference).
  • Alternative peptide libraries suitable for use have been described in the art, including U.S.S.N. 07/963,321; U.S.S.N. 07/778,233; and Cull et al. (1992) Proc. Natl. Acad. Sci. (U.S.A. ) 8_9:1865, incorporated herein by reference.
  • Various bacteriophage peptide display libraries are suitable for screening to identify anti-idiotype peptide sequences, including but not limited to those described in PCT/US91/04384; U.S.S.N. 07/718,577; U.S.S.N. 07/517,659; Cwirla et al. (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87: 6378; de la Cruz et al. (1988) J. Biol. Chem. 263: 4318; Parmley and Smith, Gene 73:305-318 (1988) ; Scott and Smith (1990) Science 249: 386; Devlin et al.
  • Such libraries are screened by a variety of methods, preferably by selective affinity adsorption to a predetermined antigen (e.g. , a relevant immunoglobulin or cell of the predetermined cell population) .
  • a predetermined antigen e.g. , a relevant immunoglobulin or cell of the predetermined cell population
  • Such libraries can be used to find ligands or surrogate ligands to receptors.
  • bacteriophage antibody display libraries and lambda phage expression libraries have been described (Kang et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991) Nature 352: 624; McCafferty et al.
  • Such antibody display libraries may be used to rapidly identify anti-idiotype antibodies by the methods of the invention.
  • a biological molecule such as a peptide, antibody, or other protein (collectively referred to as "(poly)peptide"
  • these methods typically involve establishing a physical or logical connection between each (poly)peptide and the nucleic acid that encodes the (poly)peptide; perhaps the best known method in this category involves the presentation of a (poly)peptide on the surface of a filamentous phage.
  • the phage can be incubated with an immobilized receptor (e.g., a relevant immunoglobulin) of interest, so that phage which present a (poly)peptide that binds to the receptor can be separated from phage that do not.
  • an immobilized receptor e.g., a relevant immunoglobulin
  • Another important recombinant method for the display of (poly)peptide ligands involves the production of a fusion protein composed of a protein that specifically binds to DNA and the potential (poly)peptide ligand.
  • the library of (poly)peptides is produced by transforming recombinant host cells with a vector that encodes a lac repressor/ (poly)peptide fusion protein and contains a lac operator sequence. When the transformed host cells are cultured under conditions that allow for expression of the fusion protein, the fusion protein binds to the vector that encodes the fusion protein.
  • the fusion protein/vector complexes can be screened against a receptor in much the same way the phage are screened in the phage-based display method.
  • in vitro chemical synthesis provides a method for generating libraries of compounds, without the use of living organisms, that can be screened for ability to bind to a receptor.
  • in vitro methods have been used for quite some time in the pharmaceutical industry to identify potential drugs, recently developed methods have focused on rapidly and efficiently generating and screening large numbers of compounds.
  • One early method involves the synthesis of peptides on a set of pins or rods. See PCT patent publication Nos. 84/03506 and 84/03564.
  • Another method involves the use of a synthesis resin or beads and a variety of flow-through containers into which the beads are placed. The containers are then exposed to monomer-coupling solutions and labeled to indicate the monomer coupling reactions to which the container has been exposed (see, U.S. Patent No. 4,631,211).
  • a related method dispenses with the labeling step and separate containers for each peptide to achieve greater diversity at the cost of easy identification of a particular ligand of interest.
  • the synthesis beads are pooled and redistributed after each set of monomer coupling reactions.
  • the ligands on a bead of interest must be identified by removing the ligand from the bead and determining the molecular structure of the ligand (see. PCT patent publication No. 92/00091) .
  • a significant improvement over this latter method involves tagging each bead with an identifier tag, such as an oligonucleotide, so as to facilitate ligand identification.
  • an identifier tag such as an oligonucleotide
  • Another powerful method for generating large collections of compounds addresses the ligand identification problem by forming arrays of different compounds in a manner that places each different compound of the array at a discrete, predefined location. The location identifies each ligand.
  • This method called very large scale immobilized polymer synthesis, is described in U.S. patent No. 5,143,854; PCT patent publication Nos. 90/15070 and 92/10092; Fodor et al. (1991) Science 251: 767; and Dower and Fodor (1991) Ann. Rep. Med. Chem. 26: 271.
  • the peptide library is screened by contacting the library with a predetermined cell population (e.g., lymphoma cells) or the immunoglobulin superfamily molecule from the predetermined cell population under aqueous binding conditions and identifying peptides which bind the cells or immunoglobulin from the predetermined cell type but which substantially do not bind irrelevant cells or immunoglobulin derived from irrelevant cells.
  • a predetermined cell population e.g., lymphoma cells
  • the immunoglobulin superfamily molecule e.g., lymphoma cells
  • Such peptides are identified as candidate antiproliferative peptides which may be further evaluated for antiproliferative activity by in vitro assays using cultured cells of the predetermined cell population or freshly obtained cells (e.g. , can comprise thawed aliquots) from a biopsy specimen.
  • a bacteriophage peptide display library displaying peptide sequences from 6 to 20 amino acids in length (typically as a fusion with a phage coat protein such as pill of a filamentous bacteriophage) is optionally precleared for non-idiotype-specific binding by contacting the library of bacteriophage particles with an irrelevant class- matched immunoglobulin (e.g., a human serum Ig fraction obtained from an individual other than the patient) bound to Sepharose under aqueous binding conditions, and bacteriophage particles bound to the immobilized irrelevant immunoglobulin are removed by separation of the solid phase (e.g., Sepharose).
  • an irrelevant class- matched immunoglobulin e.g., a human serum Ig fraction obtained from an individual other than the patient
  • the remaining precleared library is panned over (i.e, contacted with in aqueous binding conditions) irrelevant immunoglobulin coupled to a solid phase (e.g., a plastic Petri plate) and bacteriophage immobilized by binding to the irrelevant immunoglobulin are removed and discarded with the solid phase.
  • the remaining bacteriophage in the supernatant are panned over relevant immunoglobulin (i.e., immunoglobulin derived from the predetermined cell population) immobilized on a solid support and/or immobilized cells of the predetermined cell population (if cells are used, it is preferred that the library is precleared against an equivalent cell type, e.g., a B lymphocyte population) .
  • preclearance of the bacteriophage peptide display library may be omitted and the phage population screened directly by panning.
  • Bacteriophage which do not bind to the immobilized relevant immunoglobulin or immobilized cells remain unbound in the supernatant and are discarded, whereas bacteriophage which are immobilized by specific binding to the relevant immunoglobulin remain bound and are separated from the supernatant by removal of the supernatant and, optionally, rinsing the immobilized fraction with a mild solution (e.g., PBS) .
  • Phage displaying peptides without the desired specificity are removed by washing. The degree and stringency of washing required will be determined for each relevant immunoglobulin of interest. A certain degree of control can be exerted over the binding characteristics of the anti-idiotype peptides recovered by adjusting the conditions of the binding incubation and the subsequent washing.
  • the temperature, pH, ionic strength, divalent cations concentration, and the volume and duration of the washing will select for anti-idiotype peptides within particular ranges of affinity for the relevant immunoglobulin. Selection based on slow dissociation rate, which is usually predictive of high affinity, is a practical route. This may be done either by continued incubation in the presence of a saturating amount of a known anti-idiotypic peptide or antibody for the relevant immunoglobulin idiotype, or by increasing the volume, number, and length of the washes. In each case, the rebinding of dissociated peptide-displaying phage is prevented, and with increasing time, peptide-displaying phage of higher and higher affinity are recovered.
  • Bound bacteriophage are recovered from the immobilized fraction by elution with a known anti-idiotype peptide ligand or with an acidic solution (e.g., pH 2.0 to 2.5), adjusted to neutral pH, and propagated in bacterial hosts to amplify the library of recovered bacteriophage.
  • the recombinant bacteriophage display vectors comprising the displayed peptide which bound to the relevant immunoglobulin are transformed into bacterial cells (e.g. ⁇ X_ coli) wherein they are expressed and phage particles are assembled to form an enriched bacteriophage antibody phage display library.
  • Such enriched libraries typically are screened further by at least one additional cycle of affinity selection as described above with immobilized relevant immunoglobulin and comprising a low pH (e.g., pH 2.1) elution step.
  • a low pH e.g., pH 2.1
  • at least about 2 to 5 cycles of this enrichment procedure are performed and specific anti-idiotype binding clones are isolated.
  • DNA from the anti-idiotype binding clones is usually prepared and the displayed peptide region sequences are isolated and the nucleotide sequence is determined by sequencing and/or is ligated into a suitable expression vector for high-level expression in bacterial or eukaryotic host cells.
  • the nucleotide sequence of a displayed peptide from a selected clone encodes a putative anti-idiotype peptide.
  • the anti- idiotype peptides thus identified may be used for human therapeutics (e.g., as antiproliferative peptides or toxin conjugates), medical diagnostics (e.g., jLn vivo imaging of peptide-conjugated imaging agents) and in vitro diagnostic assays (e.g. , immunohistochemical staining of histopathological specimens to track recurrence or metastatic disease in a patient) .
  • a variety of techniques can be used in the present invention to diversify a peptide library or to diversify around anti-idiotype peptides found in early rounds of panning to have sufficient binding activity.
  • the positive phage (those identified in an early round of panning) are sequenced to determine the identity of the active peptides.
  • Oligonucleotides are then synthesized based on these peptide sequences, employing a low level of all bases incorporated at each step to produce slight variations of the primary oligonucleotide sequences.
  • This mixture of (slightly) degenerate oligonucleotides is then cloned into the affinity phage as described herein.
  • This method produces systematic, controlled variations of the starting peptide sequences. It requires, however, that individual positive phage be sequenced before mutagenesis, and thus is useful for expanding the diversity of small numbers of recovered phage and selecting variants having higher binding affinity and/or higher binding specificity.
  • peptide display libraries e.g., bacteriophage display libraries
  • other peptide screening methods can be used to select antiproliferative (anti- idiotype) peptides that bind to the relevant immunoglobulin superfamily molecule.
  • solid phase spatially-defined peptide arrays VLSIPS; U.S. Patent No. 5,143,854
  • polysome nascent peptide libraries yeast two-hybrid systems
  • the screening protocol can comprise a yeast two-hybrid system wherein the yeast cells express: (1) a first fusion protein comprising the relevant immunoglobulin superfamily molecule variable region domain (e.g., Ig variable region domains) and a first transcriptional regulatory protein sequence
  • a second fusion protein comprising a random, pseudorandom, or defined sequence peptide sequence and a second transcriptional regulatory protein sequence (e.g., GAL4 DNA-binding domain), and (3) a reporter gene (e.g., -galactosidase) which is transcribed when an intermolecular complex comprising the first fusion protein and the second fusion protein is formed.
  • Yeast cells that express the reporter gene are selected (e.g., on the basis of (3-gal expression and/or growth) and the random, pseudorandom, or defined sequence peptide sequence of the second fusion protein(s) are determined (e.g., by DNA sequencing) .
  • Peptides comprising (or consisting essentially of) the selected random, pseudorandom, or defined sequence peptide sequence(s) are candidate antiproliferative peptides.
  • Anti-idiotype peptides can be used to bind specifically to lymphocytes expressing the relevant immunoglobulin superfamily molecule idiotype, such as to target linked toxins or radionuclides to such cells, or (if labeled or otherwise detectable) to identify such cells in a cellular sample (e.g, a blood sample from a patient undergoing chemotherapy to monitor therapeutic progress and/or recurrence) regardless of whether the anti-idiotype peptide has antiproliferative effects.
  • a cellular sample e.g, a blood sample from a patient undergoing chemotherapy to monitor therapeutic progress and/or recurrence
  • the anti-idiotype peptides identified by screening of a peptide library are thereby identified as candidate antiproliferative peptides.
  • the peptide (usually in dimer or higher multimeric form by crosslinking with a linker) is administered to a cell culture containing the predetermined cell population in growth conditions (e.g., RPMI with fetal bovine serum) and the peptide's activity in producing one or more of the following: (1) inhibition of cell proliferation of the predetermined cell population, (2) induction of apoptosis of the predetermined cell population, (3) stimulation of protein tyrosine kinase activity in the predetermined cell population treated with the peptide or peptidomimetic, (4) modulation of calcium flux across the plasma membrane, and (5) inhibition of other indicia of lymphocyte proliferation (e.g., 3 H-thymidine incorporation) in the predetermined cell
  • growth conditions e.g., RPMI with fetal bovine serum
  • Peptides identified as antiproliferative peptides may be further evaluated by their capacity to reduce tumorigenesis or neoplastic cell burden in nu/nu or SCID mice harboring a transplant of the predetermined cell population, as compared to untreated mice harboring an equivalent transplant of the predetermined cell population.
  • antiproliferative peptides are at least about 4 amino acids in length, typically at least 5 or 6 amino acids in length, often at least seven or eight amino acids long, and frequently 10 to twelve amino acids long or more, and such peptides are often dimerized.
  • Minimal anti-idiotypic and antiproliferative peptides are often less than 13 amino acids in length, and frequently less than 9 amino acids in length.
  • a frequent size range is from 8 to 12 amino acids, with preferred sizes depending upon the specific idiotypic binding characteristics of the subject relevant immunoglobulin superfamily protein.
  • Antiproliferative peptides and peptidomimetics which possess antiproliferative activity towards the predetermined cell population may be formulated for therapeutic and diagnostic administration to a patient having a lymphoproliferative disease (e.g., a B cell lymphoma) comprising a clonal expansion of cells having surface immunoglobulin with substantially the same idiotype as the predetermined cell population.
  • a lymphoproliferative disease e.g., a B cell lymphoma
  • the antiproliferative peptides and peptidomimetics are administered to the patient from whom the predetermined cell population was obtained.
  • Some antiproliferative peptides may comprise N-terminal and C-terminal additions, generally comprising one or more amino acids in peptide linkage, although non-peptide linkage chemistries (e.g., esterification) and/or chemical modification of the N- and/or C-termini (e.g., C-terminal amidation) may be used.
  • the antiproliferative peptides of the invention possess detectable biological activity as inhibitors of proliferation of the predetermined cell population.
  • amino acid sequences of anti-idiotypic peptides identified by the methods of the invention will enable those of skill in the art to produce polypeptides corresponding to the anti-idiotype peptide sequences and sequence variants thereof.
  • Such peptides may be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding an anti-idiotype peptide sequence, frequently as part of a larger polypeptide. Alternatively, such peptides may be synthesized by chemical methods.
  • a sterile composition containing a pharmacologically effective dosage of one or more antiproliferative peptide is administered to a human patient or veterinary non-human patient for treatment of a lymphoproliferative condition.
  • the composition will comprise a anti-idiotype peptide that is identical to or substantially similar to a peptide that binds specifically to the idiotype of a predetermined cell population that is the object of the therapy or prophylaxis.
  • a pharmaceutically acceptable carrier or excipient is often employed in such sterile compositions.
  • Routes of administration are typically intramuscular or intravenous injection or topical application, however some chemical forms of the invention may be effectively administered orally or by other routes.
  • compositions for parenteral administration will commonly comprise a solution of an antiproliferative peptide or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g. , water, buffered water, 0.9% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • the concentration of the antiproliferative peptide(s) in these formulations can vary widely, i.e.. from less than about 0.01%, usually at least about 0.1% to as much as 5% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water, and about 10-1000 mg of antiproliferative peptide.
  • a typical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and about 100-1000 mg of antiproliferative peptide.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science. 15th Ed. , Mack Publishing Company, Easton, Pennsylvania (1980) , which is incorporated herein by reference.
  • Excipients should be chemically compatible with the peptide(s) or peptidomimetic(s) that are the active ingredient(s) of the preparation, and generally should not increase decomposition, denaturation, or aggregation of active ingredient(s) .
  • the antiproliferative peptides of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional peptides and art-known lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of biological activity loss, and that use levels may have to be adjusted to compensate.
  • the compositions containing the present antiproliferative peptides or cocktails thereof can be administered for prophylactic and/or therapeutic treatments. In therapeutic application, compositions are administered to a patient already affected by the particular lymphoproliferative disease, in an amount sufficient to cure or at least partially arrest the condition and its complications.
  • Amounts effective for this use will depend upon the severity of the condition, the general state of the patient, and the route of administration, but generally range from about 1 mg to about 2000 mg of antiproliferative peptide per dose, with dosages of from lOmg to 1000 mg per patient being more commonly used.
  • compositions containing the antiproliferative peptides or cocktails thereof are administered to a patient not presently in a disease state to enhance the patient's resistance to recurrence or to prolong remission time.
  • Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 10 mg to 2000 mg per dose, especially 10 to 1000 mg per patient.
  • Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulations should provide a quantity of the antiproliferative peptide of this invention sufficient to effectively treat the patient.
  • a lymphocyte sample comprising neoplastic lymphocytic cells is obtained from a patient, either as a blood or lymph sample or as a solid tumor biopsy, and used as a source of relevant immunoglobulin (or immunoglobulin superfamily molecule) .
  • the relevant immunoglobulin (or immunoglobulin superfamily molecule) is used to screen a peptide library and identify peptides which specifically bind; such peptides (or their substantially identical variants) are assayed for the ability to inhibit proliferation of the patient's neoplastic cells (obtained from a lymphocyte sample from the patient) in cell culture. Such peptides are identified as antiproliferative peptides and are administered to the patient in therapeutically effective dosage(s) for treating the lymphoma or lymphocytic leukemia.
  • anti-idiotype (antiproliferative) peptide sequences can be linked on a single peptide species for administration. For example and not limitation, ten antiproliferative peptide sequences may be identified for a particular lymphoma; polypeptides can be synthesized wherein each polypeptide comprises multiple copies of an antiproliferative peptide sequence and/or comprises more than one species of antiproliferative peptide sequence. For convenient reference, such peptides comprising multiple antiproliferative peptide sequences are termed "polyvalent peptides" or "repeat peptides”.
  • a polyvalent peptide which is comprised only of multiple copies of a single antiproliferative peptide sequence (plus any additional spacer or terminal amino acid sequences) is termed a homogeneous polyvalent peptide.
  • a polyvalent peptide which is comprised of copies of more than one antiproliferative peptide sequence (plus any additional spacer or terminal amino acid sequences) is termed a heterogeneous polyvalent peptide.
  • Polyvalent peptides typically comprise repeats of the antiproliferative peptide sequence(s) .
  • antiproliferative peptides are dimerized (or form higher order multimers) , either in peptide linkage or by other chemical linkage (e.g. , non-peptide covalent linkage, noncovalent linkage) .
  • a peptide containing two copies of an anti- idiotype peptide sequence are synthesized (with or without a spacer peptide sequence) .
  • two anti- proliferative peptides are linked via a non-peptide linkage.
  • the anti-proliferative peptides can comprise peptide motifs (association peptide sequences) that form tightly associated dimers and can be used to dimerize or otherwise aggregate the antiproliferative peptides.
  • the association peptide sequences can dimerize when present in fusion proteins comprising the association peptide sequence and an antiproliferative peptide sequence added at the amino-terminus or the carboxy-terminus of the resultant fusion peptide.
  • the dimerizable antiproliferative peptides of the invention are very stable, exhibit high binding affinities, and are useful in a wide variety of applications.
  • the association peptide sequence is SKVILF and is fused to the amino-terminus of the anti-proliferative peptide sequence, optionally via a flexible linker or spacer such as GGPP, PPGG, or GGPPGG, or the like.
  • the association peptide sequence is SKVILF, and is fused to the carboxyl terminus of the anti-proliferative peptide sequence by a linker or other attachment means so that a free carboxyl group is located in the resulting fusion peptide complex immediately following the F residue of the association peptide sequence.
  • a pair of association peptide sequences flank one or more copies of an anti-proliferative peptide sequence so that when the pair of association peptide sequences interact intramolecularly, a loop is formed in the fusion peptide.
  • anti-proliferative peptide comprising linked association peptide sequences can be polymerized to make linear polymers or polymeric gels.
  • SKVILF peptide is a preferred association peptide sequence
  • other peptides such as magainin peptide, metenkephalin, neurotensin, substance P, MHC peptide (see, Stagsted et al. (1990) Cell 62: 297) , or the neuropeptide Y, which is a 36 amino acid peptide that can self-dimerize (see Cowley et al. (1992) Eur. J. Biochem. 205: 1099), can be used in the present methods.
  • Heterologous association peptide sequences i.e., two different peptides associate to form a heterodimer, such as "anti-sense" peptide sequences, can also be used to link anti-proliferative peptides (see Blalock and Smith (1984) Biochem. Biophvs. Res. Comm. 121: 203; Shai et al. (1987) Biochem. 26: 669; Goldstein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 42; Shai et al. (1989) Biochem. 28: 8804; and Lu et al. (1991) Proc. Natl. Acad. Sci. USA 88: 3642) .
  • association peptide sequences have one, two, or more copies of the motif defined by +XXX-, where "+” is a positively charged amino acid; “-” is a negatively charged amino acid; and each "X” can independently be any amino acid.
  • an antiproliferative peptide comprises an anti-proliferative peptide sequence in peptide linkage to an association peptide sequence; sometimes it is convenient to refer to these fusion peptides as associable anti-proliferation peptides.
  • anti ⁇ proliferative peptides comprising association peptide sequences will form dimers (or higher multimers) linked by intermolecular bonds via interaction of the association peptide sequences.
  • an anti ⁇ proliferative peptide sequence may be identified as the sequence KPWYVTRV, and an associable anti-proliferative peptide comprising this anti-proliferative peptide sequence could have the sequence KPWYVTRVSKVILF, SKVILFKPWYVTRV, KPWYVTRVGGPPSKVILF, SKVILFGGPPKPWYVTRV, KPWYVTRVPPGGSKVILF, SKVILFPPGGKPWYVTRV, KPWYVTRVGGPPGGSKVILF, SKVILFGGPPGGKPWYVTRV, or the like.
  • a toxin may be conjugated, typically by covalent linkage, to an anti-idiotype peptide to deliver a toxic molecule (e.g., ricin, diphtheria toxin, phospholipase) to cells having surface immunoglobulin with substantially the same idiotype as the predetermined cell population.
  • a toxic molecule e.g., ricin, diphtheria toxin, phospholipase
  • a detection agent such as a label (e.g., biotinyl moieties or FITC) can be linked to an anti-idiotype peptide or peptidomimetic.
  • a label e.g., biotinyl moieties or FITC
  • metallothionein a protein that binds heavy metal atoms, can be linked to, or expressed as a fusion protein with, an anti- idiotype peptide.
  • the resulting product can be used to deliver radionuclides to cells having surface immunoglobulin with substantially the same idiotype as the predetermined cell population for imaging and therapy.
  • Such diagnostic compositions may be used for histopathological diagnosis of neoplasms (e.g., for monitoring chemotherapy efficacy or recurrence of a lymphoma or lymphocytic leukemia) or other applications (e.g., localizing imaging or toxic agents to specific locations in the body for magnetic imaging or radioimaging in vivo or for cytotoxic effect) .
  • agents may include, for example, a linked component comprising: metals, chemotherapeutic drugs, radiosensitizing agents, cellular toxins, radionuclides, and others.
  • Peptides can be labeled with 125 I or 131 I by the Bolton- Hunter method and by chloramine T.
  • the methods of the invention can be used to identify ligands of peptide-binding cell surface receptors other than members of the immunoglobulin gene superfamily.
  • ligands may be antiproliferative peptides, pharmacological antagonists or agonists, or peptides which induce lymphocyte activation.
  • receptors which induce cell activation when antibodies bind to them are candidates for screening peptide libraries to identify peptide ligands which inhibit cell proliferation, induce apoptosis, and/or modulate tyrosine phosphorylation (Ullrich and Schlesinger (1990) Cell 61: 203, incorporated herein by reference) .
  • the proto-oncogene products her-2/neu (Shepard et al. (1991) J.
  • Kits can also be supplied for identifying anti-idiotype peptides which bind a predetermined cell population, such as a lymphoma biopsy sample, obtained from a patient.
  • the kit components generally comprise a peptide library, typically in the form of a bacteriophage peptide display library or solid phase spatially defined peptide array (VLSIPS device) , optionally including suitable host cells for propagating a bacteriophage display library, and a suitable aqueous binding buffer.
  • VLSIPS device solid phase spatially defined peptide array
  • the components for practicing the methods of the present invention may be provided, usually in a packaged form in a container, either alone or in conjunction with additional reagents.
  • the bacteriophage particles or polynucleotide genomes are typically included in the kits with buffers, such as Tris, phosphate, carbonate, etc. , stabilizers, biocides, inert proteins, e.g. , serum albumin, or the like, and a set of instructions for use.
  • buffers such as Tris, phosphate, carbonate, etc.
  • biocides such as Tris, phosphate, carbonate, etc.
  • inert proteins e.g. , serum albumin, or the like
  • the immunoglobulins generally are purified from the hybridoma supernatants and used as the relevant immunoglobulin for screening a bacteriophage peptide display library by antibody panning. After four rounds of panning with relevant immunoglobulin, individual bacteriophage colonies were amplified and used in an ELISA with the relevant immunoglobulin to determine the relative or absolute binding affinity for individual selected bacteriophage clones to the relevant immunoglobulin. DNA from phage exhibiting high affinity binding to relevant immunoglobulin were sequenced to determine the encoded peptide sequence of the displayed peptide.
  • EXAMPLE 1 Antiproliferative Peptides to A Human Lymphoma Purifying the relevant immunoglobulin: A cell suspension obtained from tumor biopsy specimens or from lymphoma cell lines were isolated as sources of predetermined cell populations. The cell suspensions were mixed with HAT-sensitive heterohybridoma B5 cells. B5 is a heterohybridoma that was generated by the fusion of the NS-1 murine fusion partner (American Type Culture Collection, Rockville, MD) with a human lymphoma cell. The B5 clone has lost the ability to secrete Ig spontaneously. A subclone which retains the ability to secrete Ig when fused to with human B cells.
  • This subclone was made HAT- sensitive by growth in medium containing 8-azaguanine.
  • the mixture of lymphoma cells and B5 cells is exposed to 40% polyethylene glycol and washed with PBS.
  • the cells were plated at a density of 2 x 10 5 cells/well in HAT medium into 96-well microtitre plates.
  • the cells were screened by ELISA for secretion of immunoglobulins of the same light chain class as the lymphoma.
  • the highest producing clones are expanded to larger culture volumes.
  • the supernatants of the hybridomas are pooled and run through protein A columns (for IgG-secreting hybridomas) or a Sepharose column coated with a monoclonal anti-IgM antibody (for IgM-secreting hybridomas) .
  • the Ig is eluted from the column with glycine-HCl and dialyzed overnight in PBS.
  • the resultant dialyzed Ig constitutes relevant immunoglobulin with respect to the predetermined cell population used to generate it.
  • a relevant immunoglobulin from a B cell lymphoma of a patient was so prepared and used to screen three bacteriophage peptide display libraries.
  • the cell line was identified and designated Sup B8 which expresses a monoclonal immunoglobulin designated as the Tab idiotype; the Tab monoclonal antibody was used to screen peptide libraries to identify anti-idiotype peptides.
  • Filamentous phage display libraries The vector pAFFl and the libraries used are available from Affymax, Palo Alto, California. A description of the construction of one of the libraries follows: The vector has two BstXI sites at the 5' region of gene III. The vector is cut with BstXI and cut vector is purified.
  • the single stranded gaps are filled in with Klenow DNA polymerase.
  • the DNA is electroporated into E ⁇ _ coli MC1061 in multiple batches.
  • the electrotransformations are pooled and grown in selective media through 10 doublings.
  • the phage are isolated by clearing the supernatant from the bacteria by centrifugation, followed by precipitation of the phage by polyethylene glycol.
  • the phage were plated out on LB- tetracycline plates to determine the titer of infectious particles.
  • the purified Ig's were immobilized on plastic plates or sepharose columns. Approximately 10 11 to 10 12 infectious phage are incubated with the Ig. The plates or the column is washed with a wash buffer. Adherent phage are eluded with glycine-HCl. Eluted phage are amplified by infecting logarithmic E. coli K91 and growing them in selective media overnight. The phage were isolated from the plates, separated from the bacteria, purified by polyethylene glycol precipitation, resuspended, and amplified. The affinity purification step was repeated four times. After the final round, J . coli is infected with the phage and plated at low density.
  • the affinity constant of the peptide ligand is determined with soluble radioimmunoassay using the 32 P labeled variant peptide and the purified Ig.
  • Fig. 1 shows inhibition of anti-idiotype antibody binding to the relevant immunoglobulin with anti-idiotype peptide 3T802 with an IC 50 of between 1 and 10 ⁇ M.
  • Immunoglobulin of the Tab idiotype was plated on 96-well microtitre plates and detected with murine monoclonal anti-idiotype antibody in an ELISA format and the ability of the peptide to inhibit binding of the anti-idiotype monoclonal to Tab was measured. Binding was measured with peptide added prior to the addition of the anti-idiotype antibody.
  • Proliferation assays of lymphoma cells with and without ligand were performed in 96 well tissue culture plates. 5,000 to 10,000 tumor cells per well are plated in 100 ⁇ l complete medium. Peptides, biotinylated peptides, biotinylated peptides with streptavidin were reconstituted in 100 ⁇ l of medium and added to the wells. The cells were incubated for 3 days at 37 degrees Celsius. XTT assays (Jost et al. (1992) J. Immunol. Meth. 147: 153, incorporated herein by reference) were performed to quantitate the number of cells proliferating at that time. Fig.
  • 3 H-thymidine incorporation assays An alternate method for quantitation of proliferation is 3 H-thymidine incorporation assays. 3 H-thymidine is added 24 hours before cell harvest, followed by cell harvest onto glass fiber filters and determination of incorporated radioactivity with a liquid scintillation counter.
  • Immunoblots were used to evaluate the stimulation of protein tyrosine phosphorylation by ligand binding.
  • Cells growing at mid-log phase are washed in fresh medium. About 2 million cells are reconstituted in 1 ml medium.
  • Anti-IgM antibodies or peptides are added for various time periods. Phosphorylation is then inhibited with a buffer containing 1 mM sodium orthovanadate in PBS. The cells are lysed. The lysates are run on an SDS-polyacrylamide gel and subsequently transferred to nitrocellulose. Phosphorylated tyrosine are detected with a monoclonal anti-phosphotyrosine antibody.
  • Antiproliferative peptides The relevant immunoglobulin from a B cell lymphoma of a human patient was used to screen three bacteriophage display libraries. The amino acid sequences of about at least 60 positive phage which bound to the relevant immunoglobulin were identified by sequencing the peptide display portion of the pill gene of the selected phage. Several of the peptides were produced by chemical synthesis. The peptides were determined to bind to immunoglobulin of the predetermined cell population (B cell lymphoma) obtained from the patient but not to those of other B lymphocytic cell types tested.
  • B cell lymphoma predetermined cell population obtained from the patient but not to those of other B lymphocytic cell types tested.
  • the anti- idiotype peptides were biotinylated and linked with streptavidin by allowing 50 ⁇ M streptavidin reconstituted in RPMI to react with 200 ⁇ M biotinylated anti-idiotype peptide in a 1:1 mixture over 30 minutes at room temperature to produce a tetramer of four peptides conjugated to a streptavidin molecule and was shown to inhibit the growth of the predetermined cell population (i.e., a cell line established from the patient's B cell lymphoma cells) but not other B lymphocytic cell lines with different idiotypes.
  • the anti-idiotype peptide streptavidin complex induced tyrosine kinase activity in the cell line established from the patient's B cell lymphoma cells but not in other B lymphocytic cell lines with different idiotypes.
  • Table I shows amino acid sequences of some of the peptides identified and shown to bind the idiotype of the B cell lymphoma SUP-B8.
  • This example further describes peptide ligands found by screening phage display libraries for binding to the purified IgM, ⁇ receptor of the human Burkitt's lymphoma cell line SUP-B8 (Wright et al. (1989) J EXP Med 169:1557; Carroll et al. (1988) Blood 7_1:1068) .
  • the antiproliferative peptides' cytotoxic activity(ies) and effect(s) on signal transduction and cell death in vitro and in vivo is demonstrated.
  • Peptide ligands for the surface immunoglobulin receptor (Ig) of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8 and 12 amino acid peptides. Corresponding synthetic peptides bound specifically to this Ig receptor, and blocked the binding of an anti-idiotype antibody.
  • the ligands when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro at nanomolar concentrations. This effect was associated with the specific stimulation of intracellular protein tyrosine phosphorylation.
  • the B-cell lymphoma cell lines SUP-B8 (Carroll et al. (1988), op.cit) SU-DHL4 (Kaiser-McGaw et al. (1985) Cancer Genetics and Cytogenetics 14 . :205) and OCI-Ly8 (Tweeddale et al. (1987) Blood 9 . : 1307) were cultured as published.
  • a mouse monoclonal anti-idiotype (anti-Id) antibody to the SUP-B8 Ig was produced as published (Carroll et al. (1988), op.cit) .
  • the following E ⁇ coli bacterial strains were used: K91, MC 1061 F, DH5 ⁇ F' . Immunoglobulin receptor purification
  • SUP-B8 Ig was immobilized on 96-well microtiter plates by adding 0.5 ⁇ g Ig to each well. Plates were washed and blocked essentially as described (Scott JK and Smith GP (1990) op.cit; Wright et al. (1989) op.cit) . Transducing units (TU) of bacteriophage in 600 ⁇ l Tris-buffered saline (50mM Tris-HCl, pH 7.5, 150 mM NaCI) were distributed equally among 6 wells and incubated for 2 hours at 4°C. The plates were washed with PBS.
  • Bound phage were eluted with 0.1M glycine HC1, pH 2.2, neutralized with 2M Tris base, amplified in K91 cells, and precipitated for further rounds of panning (Cwirla et al. (1990) op.cit) .
  • 3 and fourth round of panning 1/10 and 1/100 respectively, of the number of phage used in round 1 were applied to the Ig.
  • Individual phage clones were grown, purified, characterized by phage ELISA (see infra) and subjected to DNA sequencing.
  • Mutagenesis phagemid libraries Three independent mutagenesis libraries were constructed in a manner similar to the random libraries (Cwirla et al. (1990) op.cit) except that they were cloned into a pill phagemid vector (pAFF2MBP) (Cull et al. (1992) Proc Natl Acad Sci USA 8_9:1865; Lee, N. (1980) The Qperon pp. 389-409).
  • pAFF2MBP pill phagemid vector
  • recombinant pill expression was regulated by the inducible ara-B promoter.
  • Recombinant pill was incorporated into phage particles along with wild type pill provided by helper phage (Low an et al.
  • the mutagenesis library oligonucleotides were synthesized (A library: 50% C TCT CAC TCC aag ccN tgg gyN NNK agg gtN GGC ACT GTT GAA AGT TGT; 50% C TCT CAC TCC aag ccN tgg tat gyN NNK agg gtN GGC ACT GTT GAA AGT TGT. B library: C TCT CAC TCC ray tgg gsN rtg tgg NNK agg GGC ACT GTT GAA AGT TGT.
  • C library C TCT CAC TCC tat NNK gag gaN wtg tat agg GGC ACT GTT GAA AGT TGT) , gel purified and enzymatically phosphorylated.
  • Each library was constructed by annealing 3 oligonucleotides with BstX I-digested and purified vector as in the random libraries in the following ratios: 1.6 pmoles of PAFF2MBP/4.1 pmoles of library oligo/4.1 pmoles of GGAGTGAGAGTAGA/82 pmoles of CTTTCAACAGT.
  • the gapped plasmid was filled in by the addition of dNTP and T7 DNA polymerase and incubation for 90 minutes at 37°C. The ligated filled plasmid was precipitated and resuspended.
  • the DNA was electroporated into MC1061F cells and grown in LB/Ampicillin (100 ⁇ g/ml)/0.1% MgS0 4 /0.25% KH 2 P0 4 buffered medium containing 0.1% glucose for pill repression.
  • a 600 of 0.3 the cultures were infected with VCSml3 helper phage.
  • Phagemid producing cells were then induced and selected, respectively, by the addition of 0.02% arabinose and kanamycin, (20 ⁇ g/ml) . Phagemid were precipitated with polyethylene glycol/NaCl (Cwirla et al. (1990) op.cit) .
  • a peptide derived from one consensus sequence containing clone from each mutagenesis experiment was synthesized and HPLC purified to 95% purity by the PAN Facility at Stanford University or by Neuros, San Jose, CA. The peptide content was confirmed by amino acid analysis, and the composition was verified by mass spectroscopy. All peptide monomers were synthesized with the C-terminal extension GGK-Biotin, with the e-amino group of lysine biotinylated.
  • amino-acid sequences of the peptides synthesized were: Peptide A: (KPWWVSRVGGK- Biotin) , Peptide B (DWAIWSKRGGK-Biotin) , and Peptide C (YSFEDLYRRGGK-Biotin) .
  • control Peptide D Biotin-KSADWVRDYWS
  • a peptide with a randomly scrambled amino acid sequence of Peptide C Scrambled Peptide C
  • RYSYERLFGGK- Biotin a peptide with the reverse sequence of Peptide C
  • Reverse Peptide C Biotin-KGGRRYLDEFSY
  • Peptide A was also synthesized with the C-terminal extension SSC (KPWWVSRVSSC) for dimerization.
  • a random repeat peptide of Peptide C was synthesized with the amino-acid sequence of Peptide C incorporated twice, separated by 6 glycine residues: Random Repeat Peptide C (YSFEDLYRRGGGGGGYSFEDLYRR) .
  • a control tandem repeat peptide was synthesized incorporating the scrambled sequence of Peptide C: Tandem Repeat Scrambled Peptide C (RDYSYERLFGGGGGGRDYSYERLF) .
  • the variant of peptide A with a C-terminal cysteine was incubated at room temperature for 24 hours in 2M Tris, pH 8.5 to allow the oxidation of the sulfhydryl groups to form dipeptides.
  • 2M Tris, pH 8.5 To form tetramers, equal volumes of 50 ⁇ M streptavidin and 200 ⁇ M biotinylated peptide monomer were mixed and rotated for 30 minutes at room temperature.
  • Peptide ELISA Biotinylated monomer peptides were immobilized on microtiter plates coated with streptavidin: 1 ⁇ g Streptavidin was added to each well of a microtiter plate and incubated overnight at 4°C. After washing and blocking with PBS/5% nonfat dry milk, SUP-B8 Ig or control Ig was added at 5 ⁇ g/ml. Binding of Ig was detected with goat anti-human lambda/horseradish peroxidase (HRP) conjugate or goat anti-human kappa/HRP conjugate (Tago, Burlingame, CA) .
  • HRP goat anti-human lambda/horseradish peroxidase
  • TRP goat anti-human kappa/HRP conjugate
  • Cell lysates were electrophoresed under reducing conditions on an 8% SDS-PAGE gel, electrotransferred to nitrocellulose, and probed with goat anti-mouse IgG-Biotin antibody alone to show equal loading of cell lysates into all wells. After washing, the blot was probed with the monoclonal anti-phosphotyrosine antibody 4G10, detected with goat anti-mouse IgG-Biotin and visualized with ⁇ treptavidin/HRP as published
  • Fig. 3 shows a Western blot showing tyrosine phosphorylation in SupB ⁇ (Tab) cells.
  • Approximately 2 x 10 6 SupB8 (Tab) cells were stimulated with peptides (10 ⁇ M) or antibodies. After 2 minutes or the times shown in Fig. 3, cells were washed in buffer containing orthovanadate to inhibit phosphorylation.
  • Cell lysates were electrophoresed using an 8 % SDS-PAGE and transferred to nitrocellulose.
  • panel (A) phosphorylated tyrosines were detected with a murine anti- phosphotyrosine antibody and visualized with a goat anti-mouse IgG antibody conjugated to horseradish peroxidase.
  • Apoptosis Assay 2.5xl0 6 SUP-B8 cells were cultured with 2.5 ⁇ M peptide tetramers or tandem repeat peptides for 24 hours. Cells were washed in PBS once, resuspended in 50 ⁇ l PBS, and entrapped into agarose by mixing with 100 ⁇ l melted 2% InCert agarose (FMC Bioproducts, Rockland, ME) . The mixture was poured into a plug- forming mold and cooled at 4°C for 30 minutes. The gel plugs were removed from the mold and treated overnight at 50°C with lOOmM EDTA pH 8.0, 1% Sarkosyl, 1 mg/ml proteinase K.
  • RNAse A 0.1 mg/ml in 10 mM Tris, 0.1 mM EDTA, pH 8.0.
  • the gel plugs were inserted into a 1.5% agarose/TBE gel, electrophoresed and stained with ethidiu bromide.
  • phage libraries were subjected to four rounds of panning on the Ig of SUP-B8. Phage from individual clones were purified. Their binding to the SUP- B8 Ig and to control Igs was tested in an ELISA format. Specificity was further tested by inhibiting the binding of the phage with a mouse monoclonal anti-Id antibody. After four rounds of panning, nearly all phage clones bound specifically. Their binding to the SUP-B8 Ig could in most cases be inhibited with anti-Id antibodies, but not with anti-IgM or anti- ⁇ light chain antisera.
  • Those phage clones that bound specifically to SUP-B8 were subjected to DNA sequencing. Amino acid sequences of peptides from these phage are shown in Table I (supra) and in Table II, which groups the amino acid sequences on the basis of sequence identity. The peptide sequences fell into four groups of homology (groups A-D) . Within group A and group C, phage were isolated from two libraries, suggesting that the sequences retrieved were independent of the different linkers used to link the random peptides to pill in the phage constructs. Even though a consensus sequence could be determined for each of the four homology groups, some positions were poorly conserved.
  • phagemid mutagenesis libraries were generated based upon groups A-C of Table II. These libraries were constructed such that the oligonucleotides encoding the pill insert were synthesized as a family of oligonucleotides based upon the DNA sequence of the consensus peptides, varied to a defined degree. The amino acid composition of the resultant libraries differed by approximately 50% from the consensus sequence. The library size for all three libraries was approximately 10 8 TU. After three rounds of affinity selection, phage clones binding to SUP-B8 Ig were identified and subjected to DNA sequencing.
  • Table III summarizes the results, showing the sequences towards which the three libraries were biased (input) and the frequency of amino acids obtained after selection (output) .
  • the consensus sequences determined from screening random libraries were mostly confirmed by screening the mutagenesis libraries. Some exceptions were the emergence of I for V/M in the fourth position of mutagenesis library B, or K for R in the seventh position at the C-terminus of the same group. Other poorly conserved positions from the random library screen were defined better, such as the 6th position of library A. Based on these results, a peptide from each mutagenesis consensus sequence was synthesized.
  • the synthesized peptides A-C bound specifically to the SUP-B8 Ig and not to control Ig receptors from other B-cell lymphomas or to normal polyclonal IgM (Fig. 4) .
  • the control peptides with the scrambled or reverse amino acid sequence of peptide C did not bind to SUP-B8 Ig.
  • Peptides A-C inhibited the binding of the bivalent anti-Id to SUP-B8 Ig (Fig. 5) , while the Control Peptide D did not. None of the peptides inhibited the binding of anti-IgM or anti- ⁇ antisera to SUP-B8 Ig.
  • peptide monomers themselves had little or no effect on the proliferation of SUP-B8 cells in vitro (Fig. 6, panel A) .
  • crosslinking of the surface Ig receptors is required to induce a substantial anti-proliferative effect in B-cell lymphoma cell lines.
  • Monomeric F(ab) fragments of anti-Ig antibodies fail to inhibit cell proliferation (Udhayakumar et al. (1991) J Immunol 146: 4120) .
  • peptide polymers were made by reacting biotinylated peptides with tetravalent streptavidin.
  • the resulting putative peptide ligand tetramers were reproducibly cytotoxic, with an IC 50 of 60 to 200 nM (Fig. 6, panel B) . After three days, only cell fragments were seen in the treated wells.
  • the Control Peptide D tetramer, Scrambled Peptide C tetramer and Reverse Peptide C tetramer had no apparent effect on the cells. Streptavidin alone did not inhibit proliferation (Fig. 6, panel A) .
  • the peptide ligand tetramers had no influence upon the proliferation of the control B-cell lymphoma cell lines 0CI-Ly8 (Fig. 6, panel C) and SU-DHL4, indicating that the cytotoxic effect of the peptides was tumor specific.
  • Peptides containing C-terminal cysteines were oxidized to form dimers through disulfide bridges.
  • the dimer could be reduced with 2-mercaptoethanol to monomers, as confirmed by HPLC analysis.
  • a cytotoxic effect similar to the effect of the tetramers was seen with the peptide ligand dimer (Fig. 7, panel A) .
  • a control peptide subjected to the same dimerization procedure had no effect.
  • a tandem repeat form of Peptide C was synthesized as a 24-mer peptide incorporating the sequence of Peptide C twice, separated by six glycine residues.
  • a control tandem repeat dimer with a randomly scrambled sequence of Peptide C was synthesized as well.
  • the Random Repeat Peptide C was as cytotoxic to SUP-B8 cells as the tetramers (Fig. 7, panel B) , with an IC 50 of about 40 nM.
  • the Tandem Repeat Scrambled Peptide C did not inhibit proliferation of SUP-B8 cells.
  • the Tandem Repeat Peptide C had no effect on the proliferation of the control B-cell lymphoma cell lines OCI-Ly8 (Fig. 7, panel C) and SU-DHL4.
  • panel B shows a non-specific protein with a 76 kDa apparent molecular weight as well as the Ig heavy chain of SUP-B8 cells, detected in the same blot as Fig. 3, panel A with a goat anti-mouse IgG antibody, demonstrating equal loading of all lanes.
  • the SUP-B8 cells underwent programmed cell death when stimulated with the peptide tetramers.
  • DNA fragmentation a characteristic feature of cells undergoing apoptosis, was seen after 24 hours of incubation with Peptide A-C tetramers, but not with the Scrambled Peptide C tetramer or Reverse Peptide C tetramer (Fig. 8) .
  • DNA fragmentation was not seen in the unrelated OCI-Ly8 cells incubated with Peptides A-C tetramers or the control peptide.
  • the Tandem Repeat Peptide C and not the Tandem Repeat Scrambled Peptide C caused DNA fragmentation.
  • This previously undescribed specific cytotoxic activity of peptides provides the basis for a new form of therapy for lymphoma and other lymphoproliferative disorders characterized by an expansion of a clonal population of lymphocytic cells wherein each member of the clonal population bears a surface immunoglobulin superfamily molecule of the same idiotype.
  • the cytotoxic activity of the antiproliferative peptides is mediated through the transmembrane Ig receptor as demonstrated by the rapid and specific increase in tyrosine phosphorylation after exposure to the peptide ligand tetramers, followed by apoptosis.
  • Human lymphomas established in SCID mice (Schmidt-Wolf et al. (1991) J Exp Med 174:139) are known in the art as highly predictive models for studying human lymphoma biology and evaluating therapeutic agents and treatment modalities. As such, human lymphoma lines established in SCID mice provide a suitable in vivo model to test the therapeutic efficacy of synthetic peptide ligands. Indeed, the SUP-B8 tumor can be propagated in SCID mice and afford the in vivo demonstration that Peptides A-C or their derivatives can act as anti-lymphoma drugs. SCID mice were irradiated with 200 cGy whole-body irradiation.
  • mice were injected with 1 x 10 7 SUP-B8 human tumor cells intravenously into the tail veins. Animals were kept under sterile conditions and water was supplemented with Septra for four days of the week. All mice developed detectable neoplastic disease and SUP-B8 immunoglobulin receptor was detected in all mice by Day 37. The mice were split into three groups. One group was not treated with any agent (Control group, 10 mice) . A second group (7 mice) was injected intravenously into tail veins with Tandem Repeat Peptide C (supra) with 200 ⁇ l of a 200 ⁇ M solution in sterile saline on the following day (Day 38), as well as on Days 42, 45, 48, and 52.
  • mice injected with antiproliferative peptide have detectably prolonged survival times and have a significantly reduced susceptibility to death from the transplanted human lymphoma.

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Abstract

L'invention concerne des procédés et une composition qui permettent d'identifier des polypeptides anti-prolifératifs qui inhibent l'expansion clonale et/ou induisent une apoptose chez les cellules d'une population cellulaire prédéterminée (par exemple un échantillon de cellules néoplasiques) exprimant un récepteur de surface cellulaire qui fait partie de la superfamille des immunoglobulines. Une population cellulaire prédéterminée qui exprime des molécules de surface de la superfamille des immunoglobulines, est isolée chez un patient, sous la forme d'un échantillon cellulaire tel qu'une biopsie de ganglion lymphatique ou un prélèvement sanguin contenant des cellules lymphatiques néoplasiques. Les peptides antiprolifératifs identifiés grâce aux procédés inventés peuvent servir d'agents thérapeutiques permettant de traiter des troubles lympho-prolifératifs par une thérapie anti-idiotype.
PCT/US1994/001319 1993-02-05 1994-02-04 Peptides anti-proliferatifs de fixation de recepteurs WO1994018345A1 (fr)

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WO1996014333A1 (fr) * 1994-11-04 1996-05-17 Peptide Therapeutics Limited Peptides utilises dans les traitements anti-allergies
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WO2006113958A3 (fr) * 2005-04-26 2007-05-03 Sandoz Ag Ligands d'affinités
US7247476B2 (en) 1999-10-08 2007-07-24 Arius Research Inc. Individualized anti-cancer antibodies
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US5911995A (en) * 1994-08-19 1999-06-15 Regents Of The University Of Minnesota EGF-genistein conjugates for the treatment of cancer
WO1996006116A1 (fr) * 1994-08-19 1996-02-29 Regents Of The University Of Minnesota Immunoconjugues a base d'inhibiteurs de la tyrosine kinase
WO1996014333A1 (fr) * 1994-11-04 1996-05-17 Peptide Therapeutics Limited Peptides utilises dans les traitements anti-allergies
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WO1999051780A1 (fr) * 1998-04-08 1999-10-14 Robert Massey Ciblage de types de cellules specifiques pour leur suppression par le systeme immunitaire
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WO2000078803A2 (fr) * 1999-06-21 2000-12-28 University Court Of The University Of Dundee Utilisation de peptides
WO2000078803A3 (fr) * 1999-06-21 2001-07-26 Univ Dundee Utilisation de peptides
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US7671258B2 (en) 2001-07-10 2010-03-02 Massachusetts Institute Of Technology Surfactant peptide nanostructures, and uses thereof
WO2004087766A3 (fr) * 2003-04-04 2004-12-16 Univ Lausanne Peptabody pour le traitement du cancer
US7394210B2 (en) 2004-09-29 2008-07-01 Tir Technology Lp System and method for controlling luminaires
WO2006113958A3 (fr) * 2005-04-26 2007-05-03 Sandoz Ag Ligands d'affinités
JP2008539170A (ja) * 2005-04-26 2008-11-13 サンド・アクチエンゲゼルシヤフト 親和性リガンド
EP2348053A3 (fr) * 2005-04-26 2011-11-02 Sandoz AG Ligands oligopeptidiques
US8058410B2 (en) 2005-04-26 2011-11-15 Sandoz Ag Affinity ligands
AU2006239722B2 (en) * 2005-04-26 2012-02-02 Boehringer Ingelheim Rcv Gmbh & Co Kg Affinity ligands
US8163890B2 (en) 2005-04-26 2012-04-24 Sandoz Ag Production of recombinant proteins by autoproteolytic cleavage of a fusion protein
US8372959B2 (en) 2005-04-26 2013-02-12 Sandoz Ag Production of recombinant proteins by autoproteolytic cleavage of a fusion protein

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