WO2007040653A2 - Anticorps diriges contre cd30 se liant a la cd30 intacte mais pas a la cd30 soluble - Google Patents

Anticorps diriges contre cd30 se liant a la cd30 intacte mais pas a la cd30 soluble Download PDF

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WO2007040653A2
WO2007040653A2 PCT/US2006/019128 US2006019128W WO2007040653A2 WO 2007040653 A2 WO2007040653 A2 WO 2007040653A2 US 2006019128 W US2006019128 W US 2006019128W WO 2007040653 A2 WO2007040653 A2 WO 2007040653A2
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seq
antibody
chain
cdrs
group
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WO2007040653A3 (fr
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Ira H. Pastan
Satoshi Nagata
Masanori Onda
Tomoko Ise
Andrew Raubitschek
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The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services National Institutes Of Health
City Of Hope
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A61K51/1039—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants against T-cell receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • CD30 is a promising target for cancer immunotherapy, because CD30 is highly expressed in Hodgkin's disease and anaplastic large cell lymphoma but is expressed on a small subset of normal lymphocytes (Koon, H. B. et al. Curr. Opin. Oncol. 12:588-593 (2000); Stein, H. et al., Int. J. Cancer 30:445-459 (1982); Horie, R. et al., Semin. Immunol. 10:457-470 (1998)).
  • Several different immunotherapy strategies have been tried using CD30 as the target (Wahl, A. F. et al., Cancer Res. 62:3736-3742 (2002); Borchmann, P.
  • CD30 is a 105-120 IcDa type I transmembrane glycoprotein (Durkop, H. et al., Cell 68:421-427 (1992)) containing 6 cysteine-rich domains (CRDs). The first 3 and the last 3 each make up a ligand-binding site (Nagata et al., unpublished data). The extracellular domain of CD30 is released from the cell as a soluble 85-90 IcDa protein, upon cleavage by cell membrane-anchored metalloproteinases (Josimovic-Alasevic, O. et al., Eur. J. Immunol. 19:157-162 (1989); Hooper, N. M.
  • the level of soluble CD30 is elevated according to the progression status of the lymphoma (Horie, R. et al., Semin. Immunol. 10:457-470 (1998); Josimovic-Alasevic, O. et al., Eur. J. Immunol. 19:157-162 (1989); Pizzolo, G. et al., Br. J. Haematol. 75:282-284 (1990); Nadali, G. et al., J. Clin. Oncol. 12:793-797 (1994)).
  • the cleavage site(s) of CD30 has not been precisely identified but it is assumed to be close to the plasma membrane, by analogy with other soluble receptors (Hooper, N. M.
  • IA Soluble CD30 levels measured by a sandwich ELISA in the culture supernatants of various cell lines. Cells were inoculated at 4 X 10 5 /ml and cultured for 36 h. Standard deviations of triplicate measurement are shown as the bars.
  • IB SDS-PAGE and Western blot analysis of the soluble CD30 and cell lysates. Two ⁇ g of affinity-purified soluble CD30 protein were separated in 4-20% gradient SDS- PAGE gel under reducing conditions (left panel). Twenty ng of purified proteins or 40 ⁇ g of total cell lysates were used for the Western blotting (middle and right panels).
  • Figures 2A and B Competitive effects of soluble CD30 on the binding of anti-CD30 MAbs
  • 2B Relative reactivity of each MAb to soluble CD30 compared to CD30-Fc.
  • the soluble CD30 (from L540) concentrations required for 50% inhibition of the ELISA signal were compared to those for CD30-Fc.
  • the soluble CD30 inhibited the binding of EpI, Ep4, Ep5, and Ep6 MAbs (gray bars), partially inhibited the binding of Ep3 and Ep8 MAbs (orange bars), and did not inhibit the Ep2 and Ep7 MAbs (red bars).
  • Hefi-1 (Ep3), T105 (Ep2), T215 (Ep2) and T408 (Ep7) MAbs were labeled with 131 I and incubated with appropriate amounts of CD30-HFc (blue lines), L540 culture supernatant containing soluble CD30 (red lines), an pool of sera of mice that had inoculated L540 cells (green lines) or normal mouse serum (black lines). The mixtures were analyzed with the size by running on a Sepharose® column. The baseline of each chromatogram is offset for clarification. Arrows show each peak and are explained in the text.
  • This Figure presents the amino acid sequences for the variable heavy (“VH”) chain and for the variable light (“VL”) chain of the anti-CD30 antibodies HeFi-I, T105, T405, T408, and T215.
  • the numbering set forth for the amino acid residues of the antibodies is according to the Kabat numbering system.
  • FR Framework region.
  • CDR Complementarity determining region.
  • CD30 was discovered in 1982, and was cloned in 1992.
  • Durkop et al, Cell, 68:421- 427 (1992) identified CD30 as a 595 amino acid protein with an 18 residue leader sequence, a 365 amino acid extracellular domain, a single transmembrane domain of 24 residues, and an intracellular domain of 188 residues.
  • the amino acid sequence of CD30 (SEQ ID NO:1) was identified in the Durkop et al. publication and recently corrected with regard to one residue (SEQ ID NO:1 reflects the correction, the GenBank accession of the correct sequence is set forth in the Definitions section below).
  • transmembrane domain as commencing on the N-terminal side with a proline at residue 380.
  • the C-terminus of the extracellular domain is identified as a lysine at position 379, which may thus be considered as the amino acid of CD30 most proximal to the cell surface.
  • CD30 is known to undergo cleavage and to be released from the cell as soluble CD30 ("sCD30").
  • sCD30 soluble CD30
  • Work from our laboratory has previously revealed that the release of sCD30 leaves a residual, extracellular "stalk" on the side of the cleavage site proximal to the cell surface, and that the stalk provides a useful target for immunotoxins and other agents directed against CD30-expressing cells. That discovery is set forth in International Publication WO 03/104432.
  • the results reported herein are the first example that a conformational change can occur in the soluble form of a membrane protein that destroys selective epitope structures; these epitopes are, therefore, membrane-specific.
  • the present invention provides a new and previously unknown way to target labels, drugs and toxic agents to cells expressing CD30.
  • Ep2 and Ep7 are located near the middle of the extracellular domain of CD30. They almost correspond to CRD3 and CRD6, amino acids 107-153 and 282-338 of the extracellular domain, which contains amino acids 19-383 (Table 1). As noted above, because these epitopes are not accessible to the MAbs after cleavage, it is believed that a major conformational change of CD30 occurs upon shedding of the sCD30 and destroys or buries the structure of the epitopes. Both Ep2 and Ep7 are linear epitopes which are recognized by MAbs after reduction in a Western blot (Table 1).
  • Ep2 Since the sequences of CRD3 and CRD6 are almost identical in the 20 amino acids in the beginning of the domains, Ep2 is believed to be located close to the unique region at the end of the CRD3; for the same reason Ep7 is believed to be located close to the end of the CRD6.
  • a preferred amino acid sequence of Ep2 therefore can be considered to be amino acids 128-153 (HSVCP AGMIVKFPGTAQKNTVCEPASPGVSP, SEQ ID NO.:12), more preferably from about amino acid 130 to 153 (VCPAGMIVKFPGTAQKNTVCE PASPGVSP, SEQ E) NO.: 13), and still more preferably, from about amino acid 135 to amino acid 153 (MIVKFPGTAQKNTVCEP ASPGVSP, SEQ ID NO.: 14), while an amino acid sequence of Ep 7 can be considered to be amino acids 301-338 (VPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDC]SIPT 5 SEQ ID NO.: 15), in some embodiments about amino acid 305 to 338 (ICAAETVTKPQDMAEKDTTFEAPPLGTQ PDCNPT, SEQ ID NO.: 16), and in some embodiments, from about amino acid 310 to 338 (VTKPQDMAEKDTTFEAPP
  • the sequence for Ep7 can be considered to be amino acids 301-328 (VPYPICAAETVTKPQDMAEKDTTFEAPP, SEQ ID NO.: 18), preferably from about amino acid 305 to 328 (ICAAETVTKPQDMAEKDTTFEAPP, SEQ ID NO.: 19), and more preferably, from about amino acid 310 to 328 (VTKPQDMAEKDTTFEAPP, SEQ ID NO.:20).
  • "about” means 1-3 amino acids on either side of the designated residue. More preferably, the sequence for Ep2 or for Ep7 commences with the residue designated.
  • antibodies typically comprise a variable heavy (VH) chain and a variable light (VL) chain, each of which comprises three complementarity determining regions (CDRs).
  • VH variable heavy
  • VL variable light
  • CDRs complementarity determining regions
  • the CDRs of each chain are usually numbered 1, 2, and 3 starting from the amino end of the chain proceeding towards the carboxyl end.
  • the antibodies of the present invention bind Ep7 and linear epitopes of Ep2, provided that the antibodies do not have the CDRs and binding specificity of any of the previously known antibodies which were found to bind to the epitopes defined herein as Ep2 and E ⁇ 7.
  • one of the previously known antibodies is the antibody designated T105.
  • the sequences of CDRs 1, 2, and 3 of the VH chain of T105 and the sequences of CDRs 1, 2, and 3 of the VL chain of T 105 are set forth in Figure 5.
  • the antibodies of the present invention do not include antibodies in which the CDRs of the VL of the antibody have the sequence of the CDRs of the VL of antibody T 105 and in which the CDRs of the VH of the antibody have the sequence of the CDRs of the VH of antibody T 105.
  • the term "corresponding CDR" can be used to compare the CDRs of a variable light or heavy chain to a CDR occupying the same position of the same chain of another antibody of interest.
  • the "corresponding CDR" of antibody T105 is the VH CDR2 of the T105 antibody
  • the corresponding CDR of the VL CDR3 of the antibody of interest is the VL CDR3 of antibody T 105.
  • a reference to the corresponding CDRs of the variable chains of two antibodies therefore compares the VL CDRl of the first antibody to the VL CDRl of the second antibody, the VL CDR2 of the first antibody to the VL CDR2 of the second antibody, and so on.
  • one purpose for employing ligands which bind to CD30 is to target toxic agents to cells expressing CD30.
  • Ligands directed to Ep2 or to Ep7 will not bind to any sCD30 present in the extracellular fluid, and more will therefore be available to bind to membrane-associated intact CD30.
  • the antibodies or other ligands are then available for internalization into the cell and delivery of drugs or cytotoxins attached to the ligand.
  • ligands binding to sCD30 free in the extracellular fluid cannot be internalized into the intended target cells.
  • the invention provides antibodies and other ligands that bind specifically to Ep2 or to Ep7 of CD30.
  • the antibodies and other ligands of the invention bind to intact CD30 without binding in significant amounts to sCD30.
  • Ep2 and Ep7 are linear epitopes; that is, they are created by the primary sequence of the amino acids, as opposed to epitopes created by the three- dimensional conformation of CD30 as a molecule.
  • references to antibodies which bind to a particular amino acid sequence refer to a linear epitope, while references to an antibody mapping to a particular epitope refer to a conformational epitope.
  • Ep2 can be divided into two subgroups, one of which is conformational (including the epitopes to which the antibodies known as T214, T411, and T426 bind) and the other is linear (including the epitopes to which the antibodies known as T 105 and T215).
  • the conformational epitopes cannot be detected by, for example, a Western blot analysis, while the linear epitopes can.
  • the present invention provides antibodies to the linear epitope represented by the Ep2 amino acid residue sequence (SEQ ID NO.:12).
  • amino acid sequences corresponding to Ep2 (SEQ ID NO. : 12) or portions thereof (SEQ ID NOs.:13 and 14) and to Ep7 (SEQ ID NO.: 15) or portions thereof (SEQ ID NOs.: 16-20), respectively, can be used to raise antibodies that will recognize and bind to those epitopes on intact CD30.
  • the binding specificity of the antibodies for CD30 can be readily determined by standard assays, such as those set forth in the Examples, below.
  • Antibodies raised by this method can, for example be coupled to a suitable toxic moiety, such as PE38 (see discussion below) and tested to see if they form immunotoxins that display cytotoxicity to CD30+ cells comparable to that of immunotoxins that are in clinical trials for inhibition of other cancers, typically showing an IC 50 at amounts ranging from 0.2 to 20 ng/mL, more preferably having IC50 values of less than 15 ng/mL, and still more preferably having IC50 values below 10 ng/mL.
  • a suitable toxic moiety such as PE38 (see discussion below) and tested to see if they form immunotoxins that display cytotoxicity to CD30+ cells comparable to that of immunotoxins that are in clinical trials for inhibition of other cancers, typically showing an IC 50 at amounts ranging from 0.2 to 20 ng/mL, more preferably having IC50 values of less than 15 ng/mL, and still more preferably having IC50 values below 10 ng/mL.
  • antibodies can, however, be used in competition studies, as set forth in the Examples, to determine whether that any particular antibody (such as antibodies raised by a peptide as described above or by immunizing an animal with a nucleic acid sequence encoding such a peptide.
  • the ligands are antibodies to CD30, or fragments thereof which retain their antigen recognition capability.
  • Particularly preferred antibody fragments include single chain Fv fragments ("scFv”) and disulfide stabilized Fv fragments ("dsFv”), with dsFv being especially preferred because of their stability.
  • scFv single chain Fv fragments
  • dsFv disulfide stabilized Fv fragments
  • reference herein to "antibody” includes, as appropriate, reference to fragments of the antibody which retain antigen recognition.
  • immunotoxins of the invention can be used to purge a blood sample or culture of CD30-expressing cancer cells from a patient.
  • the purged sample can then be readministered to the patient to boost the functional white-blood cell population.
  • the purged cells can be transduced or used in other manipulations.
  • immunotoxins made with the antibodies or antibody fragments of the invention can be used to inhibit the growth and proliferation of cancer cells bearing the CD30 antigen.
  • the properties of the antibodies and antibody fragments of the invention and of the resulting immunotoxins means that smaller amounts of the immunotoxins can be administered, thereby achieving the same therapeutic effect while reducing the chance of side effects.
  • the results reported in the Examples show that the activity of the immunotoxins varies in different cell lines. In some embodiments, use of the immunotoxins of the invention is less preferred with respect to Hodgkin's Disease.
  • the antibody is a scFv or a dsFv.
  • Many of the recombinant immunotoxins produced from constructs of scFv are one-third the size of IgG- toxin chemical conjugates and are homogeneous in composition. Elimination of the constant portion of the IgG molecule from the scFv results in faster clearance of the iminunotoxin after injection into animals, including primates, and the smaller size of the conjugates improves drug penetration in solid tumors. Together, these properties lessen the side effects associated with the toxic moiety by reducing the time in which the immunotoxin interacts with non-target tissues and tissues that express very low levels of antigen.
  • dsFvs disulfide stabilized Fvs
  • the antibodies of the invention can also activate complement-dependent cytotoxicity (or "antibody-dependent cell-mediated cytotoxicity").
  • complement-dependent cytotoxicity or "antibody-dependent cell-mediated cytotoxicity”
  • intact immunoglobulin molecules are used to bind to CD30-expressing cells, activating a complement cascade that results in the destruction of the cell.
  • CDRs or Fvs of anti-Ep2 or Ep7 antibodies can be grafted or engineered into an antibody of choice to confer CD30-specificity upon that antibody.
  • CDRs of an anti-Ep2 or anti-Ep7 antibody can be grafted onto a human antibody framework of known three dimensional structure (see, e.g., WO98/45322; WO 87/02671; U.S. Patent Nos. 5,859,205; 5,585,089; and 4,816,567; EP Patent Application 0173494; Jones, et al.
  • the constant regions of the antibodies can be engineered by replacing residues found in non-human animals, such as mice, with residues typically found in humans.
  • Antibodies engineered in this way are referred to as "humanized antibodies” and are preferred, since they have a lower risk of inducing side effects and can remain in the circulation longer.
  • Methods of humanizing antibodies are known in the art and are set forth in, for example, U.S. Patent Nos. 6,180,377; 6,407,213; 5,693,762; 5,585,089; and 5,530,101.
  • CD30 also known as "Ki-I” is a human lymphocyte activation marker that is a member of the tumor necrosis factor receptor family highly overexpressed on cells of Hodgkin lymphoma and anaplastic large-cell lymphoma.
  • Human CD30 was first identified in 1982 and was cloned in 1992. Durkop et al., Cell, 68:421-427 (1992). Durkop et al. identified CD30 as a 595 amino acid protein. The sequence was previously set forth by the National Center for Biotechnology Information (NCBI) under accession number XP_001744; the corrected sequence (SEQ ID NO.:1) is available on the NCBI website under the accession number P28908.
  • NCBI National Center for Biotechnology Information
  • CD30 refers to the activation marker and "Ki-I” refers to a specific antibody known in the art which binds the marker.
  • anti-CD30 in reference to an antibody or fragment retaining antigen recognition capability, refers to an antibody that specifically binds human CD30, and includes reference to an antibody which is generated against CD30.
  • sCD30 or "soluble CD30” refers to that extracellular portion of CD30 which is proteolytically cleaved from intact CD30 and released into the extracellular fluid.
  • epitopope is used herein in two senses. When in lower case, the term has the meaning usually understood in the art as the portion of an antigen that is recognized and bound by an antibody. When capitalized and followed by a Roman or Arabic number (e.g., "Epitope VI" or “Epitope 2”), it refers to a portion of CD30 defined by the competitive binding of various antibodies, as described in the Examples.
  • stalk is meant the residual extracellular component of CD30 that corresponds to that region of CD30 that is bound to the cell following the proteolytic cleavage of CD30 that results in the release of sCD30. The stalk is present regardless of whether the segment of CD30 which corresponds to sCD30 is cleaved or uncleaved from CD30.
  • cell membrane proximal or “cell surface proximal” is meant next to or nearer the cell membrane.
  • cell membrane distal or “cell surface distal” is meant farther away from the cell membrane.
  • extracellular is meant the region extending outward from the lipid bilayer encompassing a cell.
  • bind is meant that no more than 15% of a ligand which specifically binds to a target molecule is bound to a particular non-target molecule. More preferably, no more than 10% is bound to the non-target molecule, even more preferably less than 5%, and most preferably less than 1%.
  • physiological conditions an extracellular milieu having conditions (e.g., temperature, pH, and osmolality) which allows for the sustenance or growth of a cell of interest.
  • PE Pseudomonas exotoxin
  • PE Native Pseudomonas exotoxin A
  • the native PE sequence is provided in U.S. Patent No. 5,602,095. Modified forms of PE are discussed in detail herein in the section on Toxins, infra.
  • Cells are generally understood in the art to be bounded by a plasma membrane (herein referred to as the "cell membrane") comprising a lipid bilayer, in which proteins such as the ABC transporters are situated. See, generally, Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3 rd Ed., 1994), Chapter 10.
  • the cell membrane maybe considered to have a surface facing on the cytosol, or the interior of the cell, and a surface facing to the exterior of the cell, or the extracellular space.
  • Transmembrane proteins such as CD30 are amphipathic, that is, they have regions that are hydrophobic and regions that are hydrophilic.
  • Regions that pass through the membrane are hydrophobic and interact with the hydrophobic tails of the lipid molecules comprising the bilayer. Regions that are hydrophilic are exposed to water on either the cytosolic or the extracellular side of the membrane. Id.
  • the transmembrane domain of transmembrane proteins are either in an alpha helix or multiple beta strands. Lodish et al., Molecular Cell Biology, W.E. Freeman and Co., New York (4th Ed., 2000), at chapter 3.
  • references herein to amino acid positions of antibody heavy or light chains refer to the numbering of the amino acids under the "Kabat and Wu”" system. See, Kabat, E., et ah, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, U.S. Government Printing Office, NIH Publication No. 91-3242 (1991), which is hereby incorporated by reference (the Kabat and Wu database and numbering system are also referred to herein as the "Kabat” system and numbering).
  • the Kabat and Wu database is the most widely used system in the art for numbering amino acid residues of antibodies. It contains sequence information on of thousands of antibody chains and is now too large to be conveniently printed.
  • an immunoglobulin typically has a heavy and light chain.
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains").
  • Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions” or "CDRs".
  • CDRs complementarity-determining regions
  • the extent of the framework region and CDRs have been defined. See, Kabat and Wu, supra. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDRl, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • a V L CDRl is the CDRl from the variable domain of the light chain of the antibody in which it is found.
  • Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to VH--CH by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') 2 dimer into a Fab' monomer.
  • the Fab 1 monomer is essentially a Fab with part of the hinge region ⁇ see, W. E.
  • the term “antibody” includes whole (sometimes referred to herein as “intact”) antibodies, antibody fragments that retain antigen recognition and binding capability, whether produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies, monoclonal antibodies, polyclonal antibodies, and antibody mimics, unless otherwise required by context.
  • the antibody may be an IgM, IgG (e.g. IgGi, IgG 2 , IgG 3 or IgG 4 ), IgD, IgA or IgE.
  • antibody fragments means molecules that comprise a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, domain antibody (dAb), and Fv fragments; helix-stabilized antibodies ⁇ see, e.g., Arndt et al., J MoI Biol 312:221-228 (2001); diabodies (see below); single-chain antibody molecules ("scFvs,” see, e.g., U.S. Patent No. 5,888,773); disulfide stabilized antibodies ("dsFvs", see, e.g., U.S. Patent No.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain ("V H " or "VH”) connected to a variable light domain ("VL” or “VL”) in the same polypeptide chain (V H -V L ).
  • V H variable heavy domain
  • VL variable light domain
  • Diabodies and their production are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
  • references to "VH” or a “VH” refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv , dsFv or Fab.
  • References to "V L “ or a “VL” refer to the variable region of an immunoglobulin light chain, including of an Fv, scFv , dsFv or Fab
  • single chain Fv or “scFv” refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.
  • a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
  • linker peptide includes reference to a peptide within an antibody binding fragment (e.g., Fv fragment) which serves to indirectly bond the variable domain of the heavy chain to the variable domain of the light chain.
  • Fv fragment an antibody binding fragment
  • parental antibody means any antibody of interest which is to be mutated or varied to obtain antibodies or fragments thereof which bind to the same epitope as the parental antibody, but with higher affinity.
  • hotspot means a portion of a nucleotide sequence of a CDR or of a framework region of a variable domain which is a site of particularly high natural variation.
  • CDRs are themselves considered to be regions of hypervariability, it has been learned that mutations are not evenly distributed throughout the CDRs. Particular sites, or hotspots, have been identified as these locations which undergo concentrated mutations.
  • the hotspots are characterized by a number of structural features and sequences. These "hotspot motifs" can be used to identify hotspots.
  • a "targeting moiety” is the portion of an immunoconjugate intended to target the immunoconjugate to a cell of interest.
  • the targeting moiety is an antibody, or a fragment of an antibody that retains antigen recognition capability, such as a scFv, a dsFv, an Fab, or an F(ab') 2 .
  • a "toxic moiety" is the portion of a immunotoxin which renders the immunotoxin cytotoxic to cells of interest.
  • a "therapeutic moiety” is the portion of an immunoconjugate intended to act as a therapeutic agent.
  • therapeutic agent includes any number of compounds currently known or later developed to act as anti-neoplasties, antiinflammatories, cytokines, anti-infectives, enzyme activators or inhibitors, allosteric modifiers, antibiotics or other agents administered to induce a desired therapeutic effect in a patient.
  • the therapeutic agent may also be a toxin or a radioisotope, where the therapeutic effect intended is, for example, the killing of a cancer cell.
  • a "detectable label” means, with respect to an immunoconjugate, a portion of the immunoconjugate which has a property rendering its presence detectable.
  • the immunoconjugate may be labeled with a radioactive isotope which permits cells in which the immunoconjugate is present to be detected in immunohistochemical assays.
  • effector moiety means the portion of an immunoconjugate intended to have an effect on a cell targeted by the targeting moiety or to identify the presence of the immunoconjugate.
  • the effector moiety can be, for example, a therapeutic moiety, such as a toxin, a radiolabel, or a fluorescent label.
  • immunoconjugate includes reference to a covalent linkage of an effector molecule to an antibody (the effector molecule is referred to as an effector moiety upon conjugation to the antibody, which can then be referred to as a targeting moiety).
  • the effector molecule can be, for example, a toxin.
  • the terms "effective amount” or “amount effective to” or “therapeutically effective amount” includes reference to a dosage of a therapeutic agent sufficient to produce a desired result, such as inhibiting cell protein synthesis by at least 50%, or killing the cell.
  • toxin includes reference to abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof.
  • PE and DT are highly toxic compounds that typically bring about death through liver toxicity.
  • PE and DT can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (e.g., domain Ia of PE or the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.
  • native targeting component of the toxin e.g., domain Ia of PE or the B chain of DT
  • contacting includes reference to placement in direct physical association.
  • An "expression plasmid” comprises a nucleotide sequence encoding a molecule or interest, which is operably linked to a promoter.
  • polypeptide As used herein, “polypeptide”, “peptide” and “protein” are used interchangeably and include reference to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms also apply to polymers containing conservative amino acid substitutions such that the protein remains functional.
  • amino acid residue or “amino acid residue” or “amino acid” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “peptide”).
  • the amino acid can be a naturally occurring amino acid and, unless otherwise limited, can encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
  • a “conservative substitution”, when describing a protein refers to a change in the amino acid composition of the protein that does not substantially alter the protein's activity.
  • “conservatively modified variations” of a particular amino acid sequence refers to amino acid substitutions of those amino acids that are not critical for protein activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids do not substantially alter activity.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups in Table B each contain amino acids that are conservative substitutions for one another:
  • substantially similar in the context of a peptide indicates that a peptide comprises a sequence with at least 90%, preferably at least 95% sequence identity to the reference sequence over a comparison window of 10-20 amino acids. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • disulfide bond or "cysteine-cysteine disulfide bond” refers to a covalent interaction between two cysteines in which the sulfur atoms of the cysteines are oxidized to form a disulfide bond.
  • the average bond energy of a disulfide bond is about 60 kcal/mol compared to 1-2 kcal/mol for a hydrogen bond.
  • the cysteines which form the disulfide bond are within the framework regions of the single chain antibody and serve to stabilize the conformation of the antibody.
  • conjugating “joining,” “bonding” or “linking” refer to making two polypeptides into one contiguous polypeptide molecule.
  • the terms include reference to joining an antibody moiety to an effector molecule (EM).
  • the linkage can be either by chemical or recombinant means.
  • Chemical means refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.
  • recombinant includes reference to a protein produced using cells that do not have, in their native state, an endogenous copy of the DNA able to express the protein.
  • the cells produce the recombinant protein because they have been genetically altered by the introduction of the appropriate isolated nucleic acid sequence.
  • the term also includes reference to a cell, or nucleic acid, or vector, that has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid to a form not native to that cell, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, express mutants of genes that are found within the native form, or express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.
  • nucleic acid or “nucleic acid sequence” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof as well as conservative variants, i.e., nucleic acids present in wobble positions of codons and variants that, when translated into a protein, result in a conservative substitution of an amino acid.
  • encoding with respect to a specified nucleic acid, includes reference to nucleic acids which comprise the information for translation into the specified protein. The information is specified by the use of codons.
  • the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.
  • variants of the universal code such as are present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum (Proc. Nat 'I Acad. ScL USA 82:2306- 2309 (1985), or the ciliate Macronucleus, may be used when the nucleic acid is expressed in using the translational machinery of these organisms.
  • the phrase "fusing in frame” refers to joining two or more nucleic acid sequences which encode polypeptides so that the joined nucleic acid sequence translates into a single chain protein which comprises the original polypeptide chains.
  • expressed includes reference to translation of a nucleic acid into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane or be secreted into the extracellular matrix or medium.
  • host cell is meant a cell which can support the replication or expression of the expression vector.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.
  • phage display library refers to a population of bacteriophage, each of which contains a foreign cDNA recombinantly fused in frame to a surface protein.
  • the phage display the foreign protein encoded by the cDNA on its surface. After replication in a bacterial host, typically E. coli, the phage which contain the foreign cDNA of interest are selected by the expression of the foreign protein on the phage surface.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, more preferably 65%, even more preferably 70%, still more preferably 75%, even more preferably 80%, and most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sd. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection ⁇ see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).
  • BLAST and BLAST 2.0 algorithms are described in Altschul et al. (1990) J. MoI. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (and can be found online by entering "http://www.”, followed by "ncbi.nhn.nih.gov/").
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. ScI USA 89:10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences ⁇ see, e.g., Karlin & Altschul, Proc. Nat 7. Acad. ScL USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
  • in vivo includes reference to inside the body of the organism from which the cell was obtained.
  • Ex vivo and zn vitro means outside the body of the organism from which the cell was obtained.
  • malignant cell or “malignancy” refers to tumors or tumor cells that are invasive and/or able to undergo metastasis, i.e., a cancerous cell.
  • mammalian cells includes reference to cells derived from mammals including humans, rats, mice, guinea pigs, chimpanzees, or macaques. The cells may be cultured in vivo or in vitro.
  • selectively reactive refers, with respect to an antigen, the preferential association of an antibody, in whole or part, with a cell or tissue bearing that antigen and not to cells or tissues lacking that antigen. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, selective reactivity, may be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they may do so with low affinity. On the other hand, specific binding results in a much stronger association between the antibody and cells bearing the antigen than between the bound antibody and cells lacking the antigen.
  • Specific binding typically results in greater than 2- fold, preferably greater than 5-fold, more preferably greater than 10-fold and most preferably greater than 100-fold increase in amount of bound antibody (per unit time) to a cell or tissue bearing CD30 as compared to a cell or tissue lacking CD30.
  • Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein.
  • a variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • immunologically reactive conditions includes reference to conditions which allow an antibody generated to a particular epitope to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes.
  • Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, supra, for a description of immunoassay formats and conditions.
  • the immunologically reactive conditions employed in the methods of the present invention are "physiological conditions” which include reference to conditions (e.g., temperature, osmolality, pH) that are typical inside a living mammal or a mammalian cell.
  • ligands such as nucleic acids, proteins or peptides (collectively, "peptides), or antibodies, or small organics or inorganics (e.g., U.S. Pat. No. 5,143,854; WO 90/15070; WO 92/10092; WO 96/11878) having the desired specific binding characteristics are well known in the art.
  • ligands of the present invention will, under physiological conditions, bind to CD30 without substantially binding to sCD30. More preferably, the ligands of the present invention specifically bind only to portions of intact CD30 under physiological conditions, but will not bind to sCD30.
  • ligands may be chosen which bind to bind to an extracellular ligand binding site contained within CD30 epitopes 2 (SEQ ID NO.: 12) or 7 (SEQ ID NO.:15).
  • Antibodies including polyclonal, monoclonal, or recombinant single chain Fv antibodies, can be constructed for use as ligands in the present invention.
  • Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, N. Y.; Stites et al.
  • Suitable techniques for antibody or peptide ligand preparation include selection of libraries of recombinant antibodies/peptides in phage or similar vectors.
  • High affinity antibodies and peptides which bind to Ep2 (SEQ ID NO.:12) or portions thereof (SEQ ID NOs.:13 and 14) or to Ep7 (SEQ ID NO.:15) or portions thereof (SEQ ID NOs.:16- 20) can be rapidly isolated by using phage display methods to express recombinant single chain Fv (scFv) fragments or peptide ligands on the phage surface.
  • scFv single chain Fv
  • genes encoding the surface protein of a phage are altered so as to allow the insertion of an antibody or peptide gene which is expressed as a fusion protein on the surface of the phage that carries the gene.
  • the phage expressing the desired antibody or peptide ligand can be selectively enriched and isolated by virtue of its affinity/avidity for Ep2 (SEQ E) NO.: 12) or portions thereof (SEQ ID NOs.:13 and 14) or to Ep7 (SEQ ID NO.:15) or portions thereof (SEQ ID NOs.:16-20).
  • the DNA encoding the ligand is packaged in the same phage and which allows the gene encoding the ligand to be isolated.
  • DCR Divide, Couple and Recombine
  • oligonucleotide synthesis "degenerate” or “wobble” mixtures of oligonucleotide products can be made by, for example, delivery of equimolar mixtures of monomers to an oligonucleotide polymer at specific steps during synthesis. See, Atkinson and Smith, in "Oligonucleotide Synthesis. A Practical Approach", 1984, IRL Press, Oxford, edited by M. Gait, pp 35-81. These methods of synthesizing peptides or oligonucleotides provide large numbers of compounds for testing which, if active, can be readily identified.
  • ligands will be constructed to minimize immunogenicity in the host as, for example, by maximizing the number of autologous (self) sequences present in the ligand.
  • chimeric antibodies having non-xenogenic variable regions are preferred.
  • Particularly preferred are the use of antibodies in which xenogenic portions are excluded, or are essentially limited to the complementarity determining regions as in humanized antibodies.
  • Binding (i.e., attachment) of the ligand to CD30 may occur prior or subsequent to cleavage of sCD30.
  • Binding affinity of antibodies to a target antigen is typically measured or determined by standard antibody-antigen assays, such as competitive assays, saturation assays, or immunoassays such as ELISA or RIA. Such assays can be used to determine the dissociation constant of the antibody.
  • KD [Ab-Ag]/[Ab][Ag] where [Ab] is the concentration at equilibrium of the antibody, [Ag] is the concentration at equilibrium of the antigen and [Ab-Ag] is the concentration at equilibrium of the antibody-antigen complex.
  • the binding interactions between antigen and antibody include reversible noncovalent associations such as electrostatic attraction, Van der Waals forces and hydrogen bonds.
  • the antibodies can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see also METHODS IN CELL BIOLOGY, VOL. 37, Asai, ed. Academic Press, Inc. New York (1993); BASIC AND CLINICAL IMMUNOLOGY 7TH EDITION, Stites & Terr, eds. (1991).
  • Immunological binding assays typically utilize a ligand (e.g., CD30) to specifically bind to and often immobilize an antibody.
  • the immunoassay is a radioimmunoassay.
  • Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the ligand and the antibody.
  • the labeling agent may itself be one of the moieties comprising the antibody/analyte complex, i.e., an anti-CD30- Ep2 or Ep7 antibody).
  • the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/CD30 Ep2 or Ep7 complex.
  • a competitive assay is contemplated wherein the labeling agent is a second anti-CD30 epitope 2- or 7- antibody bearing a label.
  • the two antibodies then compete for binding to immobilized CD30.
  • the anti-CD30 antibody lacks a label, but a second antibody specific to antibodies of the species from which the anti-CD30 antibody is derived, e.g., murine, and which binds the anti-CD30 antibody, is labeled.
  • proteins capable of specifically binding immunoglobulin constant regions such as Protein A or Protein G may also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non- immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al., J. Immunol. 111:1401-1406 (1973); and Akerstrom, et al., J. Immunol. 135:2589-2542 (1985)).
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antibody, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.
  • the method of detecting anti- CD30 Ep2 and 7 antibodies in a sample containing the antibodies generally comprises the steps of contacting the sample with an antibody which specifically reacts, under immunologically reactive conditions, to the CD30 Ep 2 or Ep 7/antibody complex.
  • Immunoconjugates include, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody.
  • a therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule.
  • therapeutic agents may include various drugs such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or Diphtheria toxin, encapsulating agents, ⁇ e.g., liposomes) which themselves contain pharmacological compositions, radioactive agents such as 125 1, 32 P, 14 C, 3 H and 35 S and other labels, target moieties and ligands. (Such therapeutic agents are sometimes referred to herein as "effector molecules”.)
  • the choice of a particular therapeutic agent depends on the particular target molecule or cell and the biological effect is desired to evoke.
  • the therapeutic agent maybe a cytotoxin which is used to bring about the death of a particular target cell.
  • the therapeutic agent may be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.
  • nucleic acids which differ in sequence but which encode the same effector molecule ("EM") or antibody sequence.
  • EM effector molecule
  • the present invention provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.
  • Nucleic acid sequences encoding the chimeric molecules of the present invention can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al, Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brown, et al, Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage, et al, Tetra. Lett. 22:1859-1862 (1981); the solid phase phosphorarnidite triester method described by Beaucage & Caruthers, Tetra. Letts.
  • the nucleic acid sequences of this invention are prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook, et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc.
  • Nucleic acids encoding anti-CD30 or CD30 Ep2 or Ep7 antibodies can be modified to form the EM, antibodies, or immunoconjugates of the present invention. Modification by site-directed mutagenesis is well known in the art. Nucleic acids encoding EM or CD30 Ep2 or Ep7 antibodies can be amplified by in vitro methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustained sequence replication system
  • immunoconjugates are prepared by inserting the cDNA which encodes an anti-CD30 or CD30 Ep 2 or Ep 7 scFv antibody into a vector which comprises the cDNA encoding the EM. The insertion is made so that the scFv and the EM are read in frame, that is in one continuous polypeptide which contains a functional Fv region and a functional EM region.
  • cDNA encoding a cytotoxin is ligated to a scFv so that the cytotoxin is located at the carboxyl terminus of the scFv.
  • cDNA encoding a Pseudomonas exotoxin A (“PE”), mutated to eliminate or to reduce non-specific binding is ligated to a scFv so that the toxin is located at the amino terminus of the scFv.
  • PE Pseudomonas exotoxin A
  • nucleic acids encoding an EM, anti- CD30 or CD30 Ep2 or Ep7 antibody, or an immunoconjugate of the present invention are isolated and cloned, one may express the desired protein in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells as discussed above in connection with the discussion of expression vectors. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.
  • modifications can be made to a nucleic acid encoding a polypeptide of the present invention ⁇ i.e., anti-CD30 Ep2- or CD30 Ep7- antibody, PE, or an immunoconjugate formed from their combination) without diminishing its biological activity.
  • Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein.
  • Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation site, and additional amino acids placed on either terminus to create conveniently located restriction sites.
  • the immunoconjugates, EM, and antibodies of the present invention can also be constructed in whole or in part using standard peptide synthesis.
  • Solid phase synthesis of the polypeptides of the present invention of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE SYNTHESIS 5 PART A. pp. 3-284; Merrifield, et al. J. Am. Chem.
  • Proteins of greater length maybe synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (e.g., by the use of the coupling reagent N, N'-dicycylohexylcarbodiimide) are known to those of skill.
  • the recombinant immunoconjugates, antibodies, and/or effector molecules of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, PROTEIN PURIFICATION, Springer- Verlag, N.Y. (1982)). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.
  • a reducing agent must be present to separate disulfide bonds.
  • An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 niM EDTA, 0.3 M DTE (dithioerythritol).
  • Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena, et al, Biochemistry 9: 5015-5021 (1970), incorporated by reference herein, and especially as described by Buchner, et al., supra.
  • Renaturation is typically accomplished by dilution (e.g., 100-fold) of the denatured and reduced protein into refolding buffer.
  • An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 niM oxidized glutathione (GSSG), and 2 mM EDTA.
  • the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution.
  • a preferred yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed.
  • Toxins can be employed with antibodies of the present invention to yield chimeric molecules, such as immunotoxins.
  • exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A through F. These toxins are well known in the art and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or to eliminate non-specific toxicity.
  • diphtheria toxin refers as appropriate to native diphtheria toxin or to diphtheria toxin that retains enzymatic activity but which has been modified to reduce nonspecific toxicity.
  • Ricin is the lectin RCA60 from Ricinus communis (Castor bean).
  • the term "ricin” also references toxic variants thereof.
  • Ricinus communis agglutinin (RCA) occurs in two forms designated RCA 60 and RCA 120 according to their molecular weights of approximately 65 and 120 kD, respectively (Nicholson & Blaustein, J Biochim. Biophys. Acta 266:543 (1972)).
  • the A chain is responsible for inactivating protein synthesis and killing cells.
  • the B chain binds ricin to cell-surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes, et al, Nature 249:627-631 (1974) and U.S. Patent No. 3,060,165).
  • Conjugating ribonucleases to targeting molecules for use as immunotoxins is discussed in, e.g., Suzuki et al., Nat Biotech 17:265-70 (1999).
  • Exemplary ribotoxins such as ⁇ -sarcin and restrictocin are discussed in, e .g., Rathore et al., Gene 190:31-5 (1997) and Goyal and Batra, Biochem 345 Pt 2:247-54 (2000).
  • Calicheamicins were first isolated from Micromonospora echinospora and are members of the enediyne antitumor antibiotic family that cause double strand breaks in DNA that lead to apoptosis.
  • the drug is the toxic moiety of an immunotoxin in clinical trials. See, e.g., Gillespie et al., Ann Oncol 11:735-41 (2000).
  • Abrin includes toxic lectins from Abrus precatorius.
  • the toxic principles, abrin a, b, c, and d have a molecular weight of from about 63 and 67 kD and are composed of two disulfide-linked polypeptide chains A and B.
  • the A chain inhibits protein synthesis; the B- chain (abrin-b) binds to D-galactose residues (see, Funatsu, et al., Agr. Biol. Chem. 52:1095 (1988); and Olsnes, Methods Enzymol. 50:330-335 (1978)).
  • the toxin is Pseudomonas exotoxin (PE).
  • PE Native Pseudomonas exotoxin A
  • EF-2 elongation factor 2
  • Domain Ia (amino acids 1-252) mediates cell binding. Domain II (amino acids 253-364) is responsible for translocation into the cytosol and domain III (amino acids 400-613) mediates ADP ribosylation of elongation factor 2.
  • domain Ib (amino acids 365-399) remains undefined, although a large part of it, amino acids 365-380, can be deleted without loss of cytotoxicity. See Siegall, et al, J. Biol. Chem. 264:14256-14261 (1989).
  • PE Pseudomonas exotoxin
  • modifications may include, but are not limited to, elimination of domain Ia, various amino acid deletions in domains Ib, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus, such as KDEL (SEQ ID NO:21)and REDL (SEQ ID NO:22). See Siegall, et al, supra.
  • the cytotoxic fragment of PE retains at least 50%, preferably 75%, more preferably at least 90%, and most preferably 95% of the cytotoxicity of native PE. In a particularly preferred embodiment, the cytotoxic fragment is more toxic than native PE.
  • the toxicity of PE can be increased, for example, by mutating the residue at position 409 of the native sequence from arginine to glycine, alanine, valine, leucine, or isoleucine.
  • the substituent is G, A, or I. Alanine is the most preferred.
  • the mutation of the arginine at position 490 to alanine doubles the toxicity of the PE molecule. The discovery of this method of increasing the toxicity of PE is disclosed in international application PCT/US2004/039617, which is incorporated herein by reference.
  • PE employed in the present invention include cytotoxic fragments of the native sequence and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell (e.g., as a protein or pre-protein). Cytotoxic fragments of PE known in the art include PE40, PE38, and PE35.
  • the PE has been modified to reduce or eliminate nonspecific cell binding, typically by deleting domain Ia, as taught in U.S. Patent 4,892,827, although this can also be achieved, for example, by mutating certain residues of domain Ia.
  • PE40 is a truncated derivative of PE as previously described in the art. See, Pai, et al, Proc. Nat'lAcad. Sd. USA 88:3358-62 (1991); andKondo, et al, J. Biol Chem. 263:9470-9475 (1988).
  • PE35 is a 35 kD carboxyl-terminal fragment of PE in which amino acid residues 1-279 have deleted and the molecule commences with a met at position 280 followed by amino acids 281-364 and 381-613 of native PE.
  • PE35 and PE40 are disclosed, for example, in U.S. Patents 5,602,095 and 4,892,827.
  • the cytotoxic fragment PE38 is employed.
  • PE38 is a truncated PE pro-protein composed of amino acids 253-364 and 381-613 which is activated to its cytotoxic form upon processing within a cell (see e.g., U.S. Patent No. 5,608,039, and Pastan et al, Biochim. Biophys. Acta 1333:C1-C6 (1997)).
  • the PE is PE4E, PE40, or PE38
  • any form of PE in which non-specific cytotoxicity has been eliminated or reduced to levels in which significant toxicity to non-targeted cells does not occur can be used in the immunotoxins of the present invention so long as it remains capable of translocation and EF-2 ribosylation in a targeted cell.
  • PE38 Conservatively modified variants of PE or cytotoxic fragments thereof have at least 80% sequence similarity, preferably at least 85% sequence similarity, more preferably at least 90% sequence similarity, and most preferably at least 95% sequence similarity at the amino acid level, with the PE of interest, such as PE38.
  • conservatively modified variants refer to those nucleic acid sequences which encode identical or essentially identical amino acid sequences, or if the nucleic acid does not encode an amino acid sequence, to essentially identical nucleic acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
  • Pseudomonas exotoxins employed in the invention can be assayed for the desired level of cytotoxicity by assays well known to those of skill in the art.
  • cytotoxic fragments of PE and conservatively modified variants of such fragments can be readily assayed for cytotoxicity.
  • a large number of candidate PE molecules can be assayed simultaneously for cytotoxicity by methods well known in the art.
  • subgroups of the candidate molecules can be assayed for cytotoxicity.
  • Positively reacting subgroups of the candidate molecules can be continually subdivided and reassayed until the desired cytotoxic fragment(s) is identified.
  • Such methods allow rapid screening of large numbers of cytotoxic fragments or conservative variants of PE.
  • compositions that comprise an immunoconjugate of the invention and a pharmaceutically acceptable carrier.
  • the composition may comprise, for example, a chimeric molecule comprising a targeting molecule and a detector molecule to detect cells expressing CD30 Ep2 or Ep7.
  • the compositions of this invention can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes.
  • compositions of the invention are antibody and/or immunoconjugate compositions of this invention (i.e., PE linked to an anti- CD30 Ep2 or Ep7 antibody). These compositions are particularly suited for parenteral administration, such as intravenous administration.
  • compositions for administration will commonly comprise a solution of the antibody and/or immunoconjugate dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable 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 and the like.
  • concentration of fusion protein in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • a typical pharmaceutical imrmniotoxin composition of the present invention for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly if the drug is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as REMINGTON'S PHARMACEUTICAL SCIENCE, 19TH ED., Mack Publishing Company, Easton, Pennsylvania (1995).
  • intact anti-CD30 Ep2 or Ep7 antibodies are administered to induce complement-dependent cytotoxicity of CD30+ cells.
  • antibodies may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% Sodium Chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight.
  • infusion bag containing 0.9% Sodium Chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight.
  • a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.
  • an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.
  • compositions of the present invention can be administered to slow or inhibit the growth of cells of CD30-expressing cancers.
  • compositions are administered to a patient suffering from a disease, in an amount sufficient to inhibit growth of CD30- expressing cells.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient.
  • the composition should provide a sufficient quantity of the immunotoxins or antibodies of this invention to effectively treat the patient.
  • the dosage is administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
  • the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.
  • Controlled release parenteral formulations of the immunoconjugate compositions of the present invention can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core.
  • microspheres the therapeutic is dispersed throughout the particle.
  • Particles, microspheres, and microcapsules smaller than about 1 ⁇ m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 ⁇ m so that only nanoparticles are administered intravenously.
  • Microparticles are typically around 100 ⁇ m in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp.
  • Polymers can be used for ion-controlled release of immunoconjugate compositions of the present invention.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston, et al, Pharm. Res. 9:425-434 (1992); and Pec, et al, J. Parent. Sci. Tech.
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema, et ah, Int. J. Pharm. 112:215-224 (1994)).
  • liposomes are used for controlled release as well as drug targeting of the lipid- capsulated drag (Betageri, et ah, LIPOSOME DRUG DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, PA (1993)).
  • Numerous additional systems for controlled delivery of therapeutic proteins are known. See, e.g., U.S. Pat. No.
  • the immunotoxins and antibodies of the present invention are included a variety of disease conditions caused by specific human cells that may be eliminated by the toxic action of the fusion protein.
  • One preferred application for the immunotoxins and antibodies of the invention is the treatment of malignant cells expressing CD30.
  • kits for the detection of CD30 or an immunoreactive fragment thereof, ⁇ i.e., collectively, a "CD30 protein”) in a biological sample are provided.
  • a "biological sample” as used herein is a sample of biological tissue or fluid that contains CD30. Such samples include, but are not limited to, tissue from biopsy, blood, and blood cells ⁇ e.g., white cells). Preferably, the cells are lymphocytes. Biological samples also include sections of tissues, such as frozen sections taken for histological purposes.
  • a biological sample is typically obtained from a multicellular eukaryote, preferably a mammal such as rat, mouse, cow, dog, guinea pig, or rabbit, and more preferably a primate, such as a macaque, chimpanzee, or human. Most preferably, the sample is from a human.
  • Kits will typically comprise an anti-CD30 Ep2 or Ep7 antibody of the present invention.
  • the anti-CD30 Ep2 or Ep7 antibody will be an anti-CD30 Fv fragment, such as a scFv or dsFv fragment, although intact antibodies may be used for some purposes.
  • kits will typically include instructional materials disclosing means of use of an antibody of the present invention (e.g. for detection of mesothelial cells in a sample).
  • the kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kit may additionally contain means of detecting the label ⁇ e.g. enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a sheep anti-mouse-HRP, or the like).
  • the kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
  • the diagnostic kit comprises an immunoassay.
  • the method of detecting CD30 in a biological sample generally comprises the steps of contacting the biological sample with an antibody of the present invention which specifically reacts, under immunologically reactive conditions, to CD30.
  • the antibody is allowed to bind to CD30 under immunologically reactive conditions, and the presence of the bound antibody is detected directly or indirectly.
  • the antibodies of the invention will be especially useful as diagnostic agents and in in vitro assays to detect the presence of CD30 in biological samples.
  • the antibodies taught herein can be used as the targeting moieties of immunoconjugates in immunohistochemical assays to determine whether a sample contains cells expressing CD30. Detection of CD30 in lymphocytes would indicate either that the patient has a cancer characterized by the presence of CD30-expressing cells, or that a treatment for such a cancer has not yet been successful at eradicating the cancer.
  • immunotoxins targeted by antibodies of the invention can be used to purge targeted cells from a population of cells in a culture.
  • cells cultured from a patient having a cancer expressing CD30 can be purged of cancer cells by contacting the culture with immunotoxins which use the antibodies of the invention as a targeting moiety.
  • Antibodies of the present invention may optionally be covalently or non-covalently linked to a detectable label.
  • Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g.
  • DYNABEADS fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. [0159] Means of detecting such labels are well known to those of skill in the art.
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • A431/CD30 (Rozemuller, H. et al, Int. J. Cancer 92:861-870 (2001)) is a stable transformant of A431 cells that express CD30 on the cell surface.
  • CD30-positive HD lines, L540, KM-H2, and L591 and CD30-positive ALCL lines, Karpas 299 and SU-DHL-I, and CD30-negative (very weak) line, HL60 were cultured in Iscove's modified Dulbeccos' medium (Invitrogen, San Diego, CA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT).
  • FBS fetal bovine serum
  • CD30-human IgGl Fc Fusion Proteins The extracellular domain of CD30 was expressed as a fusion protein with the Fc portion of human IgG (Nagata, S. et al., CHn. Cancer Res. 8:2345-2355 (2002); Nagata, S. et al., J. Immunol. Methods 280:59-72 (2003)).
  • the CD30-Fc maintains the same conformation as membrane- associated CD30 as verified by its reactivity with conformational dependent MAbs.
  • Immunoglobulin superfamily receptor translocation associated 2 (IRTA2)-Fc fusion protein Ise, T. et al., Clin. Cancer Res. 11:87-96 (2005) was used as the control in some assays.
  • the acid eluates (pH 2.7) containing soluble CD30 were immediately neutralized with IM Tris (pH 8.0) and extensively dialyzed against phosphate buffered saline (PBS).
  • the purified soluble CD30s and the CD30-producing cell lysates were analyzed by SDS-PAGE or Western blotting using a mixture of anti-CD30 MAbs Ber-H2, T105 and T405 as previously described (Nagata, S. et al., J. Immunol. Methods 280:59-72 (2003)).
  • the soluble CD30s in the culture supernatants were separated on a size- exclusion column (TSK SW4000, 7.5 mm X 300 mm, TOSOH Bioscience LLC, Montgomeryville, PA) with PBS as the mobile phase and each fraction was analyzed by CD30 enzyme-linked immunosorbent assay (ELISA) (see below).
  • a part of the purified soluble CD30 sample was also in-gel digested with chymotrypsin or trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) to determine the peptide sequences for the protein identification (Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Institutes of Health).
  • LC/MS/MS liquid chromatography/tandem mass spectrometry
  • CD30 ELISA The level of soluble CD30 was measured by a sandwich ELISA with purified CD30-Fc as the standard. Microtiter plates were coated with 200 ng/50 ⁇ l/well of rat anti-mouse IgGl MAb (90-6551, Zymed, San Francisco, CA) in PBS and added with 200 ng/100 ⁇ l/well of anti-CD30 MAb Tl 07 (mouse IgGl). After washing, 100 ⁇ l of samples (soluble CD30) and CD30-Fc standards with 2 ⁇ g/ml of another anti-CD30 MAb T420 (mouse IgG2b) in blocking buffer were added.
  • Microtiter plates were coated with 20 ng/well of CD30-Fc and added with an appropriate dilution of anti-CD30 MAbs (5-20 ng/ml) with various dilutions of CD30-Fc (1-100 ng/ml), soluble CD30 (1-100 ng/ml of soluble CD30s obtained in L540 or Karpas 299 cell culture supernatants), or IRTA2-Fc (1-100 ng/ml) as a negative control and incubated for 2 h at room temperature.
  • the bound MAbs were detected with HRP-labeled rat-anti-mouse K MAb (04-6620, Zymed, 1/500 dilution).
  • Peak fractions were selected and incubated with CD30-human Fc (HFc), soluble CD30 from L540 cells or serum of the mouse inoculated with L540 cells, and then injected to Sepharose® 6 (Amersham Biosciences). The radioactivity of elute was monitored.
  • HFc CD30-human Fc
  • Sepharose® 6 Amersham Biosciences
  • Soluble CD30 is produced in patients with Hodgkin's lymphoma or anaplastic large cell lymphoma as well as by many cell lines derived from them (Josimovic-Alasevic, O. et al., Eur. J. Immunol. 19:157-162 (1989); Hansen, H. P. et al., FASEB J. 18:893-895 (2004); Pfreundschuh, M. et al., Int. J. Cancer 45:869-874 (1990); Hansen, H. P. et al., Int. J. Cancer 63:750-756 (1995)).
  • IA showed the levels of soluble CD30 in the culture supernatants from various cells lines. All CD30-positive cells except KM-H2 produced significant amounts of soluble CD30 (4 X 10 5 cells produced 15-35 ng soluble CD30 in 36 h), whereas CD30-negative HL60 cells produced no soluble CD30. Soluble CD30 from L540 cells and Karpas 299 cells accumulated in the culture medium in a time-dependent manner.
  • Soluble CD30 produced by L540 cells and Karpas 299 cells was purified on an affinity column on which anti-CD30 MAbs, T420 (EpI) and T427 (Ep5), were immobilized. These purified soluble CD30s were analyzed by SDS-PAGE (Fig. IB, left panel) and by Western blotting using a pool of anti-CD30 MAbs (Fig. IB, middle panel). The major top band in the purified soluble CD30 from L540 migrated about 8OkD size (in PAGE panel) consistent with the reported size of soluble CD30.
  • soluble CD30 from L540 and Karpas 299 cells was also analyzed by size exclusion chromatography. As shown in Fig. 1C, soluble CD30 from both cells types eluted in the same fractions with similar shaped peaks, suggesting that the different cell lines produced the same soluble CD30 molecule(s). Soluble CD30s eluted after CD30-Fc that forms a disulfide linked homo-dimer between the two Fc portions. The relative positions of the elution indicate that there is a stable multimer without aggregates.
  • the molecular sizes of the CD30 and CD30-Fc are estimated to be 317kD and 645IcD, respectively, which possibly agreed with the trimer formation of the tumor necrosis factor receptor (TNFR) (Chan, F. K. et al, Science 288:2351-2354 (2000)), a member of the TNFR family to which CD30 belongs.
  • TNFR tumor necrosis factor receptor
  • a control Fc-fusion protein, IRTA2-Fc showed no competition (black lines), indicating that the inhibitory effects are CD30-specific.
  • the cross-reactivity of each anti-CD30 MAb to the soluble CD30 correlated quite well with the topographical epitope, suggesting that the difference in cross-reactivity was based on a structural difference between soluble CD30 and CD30-Fc.
  • the epitopes recognized by MAbs whose relative relativities to soluble CD30 were more than 70% were considered to be conserved epitopes in soluble CD30 (EpI, Ep4 and Ep5 shown in gray, 124% average cross-reactivity); 5-70% are partially altered epitopes in soluble CD30 (Ep3, Ep6 and Ep8 shown in orange, 16.6% average cross-reactivity); less than 5% are considered to be specific to the whole CD30 molecule (Ep2 and Ep7 shown in red, 2.1% average cross-reactivity).
  • CD30-Fc made a larger immune complex (blue arrow) because of the presence of Fc and its homo-dimerization in the Fc portion.
  • the MAbs to the membrane-specific epitopes did not bind to the soluble CD30 but bind to CD30-Fc.
  • CD30 contains two duplicate regions, thus some MAbs reacted with two different fragments.
  • Reference 1 Stein et al., Int. J. Cancer, 30:445-459 (1998) Reference 2: Nagata et al., Clin Cancer Res 8:2345-2355 (2002) Reference 3: Horn-Lohrens et al.,Int J Cancer, 60:539-44 (1995) Reference 4: Hecht et al., J Immunol 134:4231-36 (1985) Reference 5: Engert et al., Cancer Res 50:84-88 (1990) Reference 6: Gruss et al., Blood, 83:2045-2056 (1994) Reference 7: Schwarting et al., Blood 74:1678-89 (1989) Reference 8: Nagata et al., J. Immunol Methods 280:59-72 (2003)
  • CD30 it has previously been reported from the laboratory of some of the present inventors that, after cleavage, CD30 retains an extracellular stalk that can be used as the target of immunotherapeutics. See, International Publication WO 03/104432. To our knowledge, however, the results reported herein are the first example that a conformational change can occur in the soluble form of a membrane protein that destroys selective epitope structures; these epitopes are, therefore, membrane-specific.
  • Soluble CD30 has been extensively investigated as a disease marker especially for Hodgkin's lymphoma (Horie, R. et al., Semin. Immunol. 10:457-470 (1998); Josimovic- Alasevic, O. et al., Eur. J. Immunol. 19:157-162 (1989); Pizzolo, G. et al., Br. J. Haematol. 75:282-284 (1990); Nadali, G. et al., J. CIm. Oncol. 12:793-797 (1994); Pfreundschuh, M. et al., Int. J. Cancer 45:869-874 (1990)).
  • soluble CD30 will be extensively diluted when it enters the circulation, it is likely that the soluble CD30 level in the microenvironment of CD30-positive tumors can reach levels of ⁇ g/ml. This level of soluble CD30 can neutralize anti-CD30 agents. A precise determination of soluble CD30 levels in the tumors of patients needs to be determined. The formation of immune complexes composed of soluble CD30 and anti-CD30 Fv was reported in clinical studies (Schnell, R. et al., Clin. Cancer Res. 8:1779-1786 (2002); Borchmann, P., Blood 100:3101-3107 (2002)).
  • Ep2 and Ep7 are located near the middle of the extracellular domain of CD30. They almost correspond to CRD3 and CRD6, amino acid 107-153 and 282-338 of the extracellular domain, which contains amino acids 19-383 (Table 1). Because these epitopes are not accessible to the MAbs after cleavage, a major conformational change of CD30 likely occurs upon shedding and destroys or buries the structure of the epitopes. Both Ep2 and Ep7 are linear epitopes which are recognized by MAbs after reduction in a Western blot (Table 1).
  • Ep2 should be close to the unique region at the end of the CRD3; for the same reason Ep7 should also be located close to the end of the CRD6.
  • MAbs to these epitopes do not inhibit ligand binding to CD30 (Froese, P. et al. J Immunol. 139:2081-2087 (1987).

Abstract

Etant donné que la CD30 est fortement exprimée dans le lymphome de Hodgkin et le lymphome à grandes cellules anaplasiques, elle constitue une cible prometteuse pour l'immunothérapie. La partie extracellulaire de CD30 est clivée, d'où la libération d'une forme connue sous le nom de CD30 soluble ('sCD30') pouvant réduire les effets des agents ciblant CD30 par liaison compétitive. L'invention concerne en partie la découverte de deux épitopes sur la CD30 associée à la membrane qui sont absents sur la CD30 soluble du fait d'un changement de conformation lors d'une excrétion pendant le clivage de CD30, ce qui rend ces épitopes indisponibles pour une liaison. Des ligands se liant à ces épitopes peuvent être utilisés pour cibler des cellules avec une CD30 intacte sans compétition de sCD30.
PCT/US2006/019128 2005-05-16 2006-05-16 Anticorps diriges contre cd30 se liant a la cd30 intacte mais pas a la cd30 soluble WO2007040653A2 (fr)

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WO2016196344A1 (fr) 2015-05-30 2016-12-08 Molecular Templates, Inc. Supports de sous-unité a de toxine de shiga, déimmunisés, et molécules de ciblage de cellule les comprenant
WO2020135426A1 (fr) * 2018-12-24 2020-07-02 信达生物制药(苏州)有限公司 Anticorps à chaîne unique anti-cd30 entièrement humains et leur utilisation
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WO2016007968A3 (fr) * 2014-07-11 2016-03-17 Expression Pathology, Inc. Dosage srm/mrm de la protéine du membre 8 de la superfamille des récepteurs du facteur de nécrose tumorale (cd30)
WO2016196344A1 (fr) 2015-05-30 2016-12-08 Molecular Templates, Inc. Supports de sous-unité a de toxine de shiga, déimmunisés, et molécules de ciblage de cellule les comprenant
EP3636660A1 (fr) 2015-05-30 2020-04-15 Molecular Templates, Inc. Échafaudages à sous-unités a de shiga-toxines désimmunisés et molécules de ciblage de cellules les comprenant
EP3660035A1 (fr) 2015-05-30 2020-06-03 Molecular Templates, Inc. Supports de sous-unité a de toxine de shiga, déimmunisés, et molécules de ciblage de cellule les comprenant
WO2020135426A1 (fr) * 2018-12-24 2020-07-02 信达生物制药(苏州)有限公司 Anticorps à chaîne unique anti-cd30 entièrement humains et leur utilisation
CN113227147A (zh) * 2018-12-24 2021-08-06 信达生物制药(苏州)有限公司 全人源的抗cd30单链抗体及其应用
WO2023057571A1 (fr) 2021-10-08 2023-04-13 Genmab A/S Anticorps se liant à cd30 et cd3
US11814437B2 (en) 2021-10-08 2023-11-14 Genmab A/S Antibodies binding to CD30 and CD3

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