US20080089898A1 - Immunotoxins directed against CD33 related surface antigens - Google Patents

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  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
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  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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  • 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
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  • 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
  • A61K47/6851—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 determinant of a tumour cell
  • A61K47/6867—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 determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
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  • 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
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  • 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
  • A61K51/1045—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 animal or human tumor cells or tumor cell determinants
  • A61K51/1069—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 animal or human tumor cells or tumor cell determinants the tumor cell being from blood cells, e.g. the cancer being a myeloma
• • A—HUMAN NECESSITIES
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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
  • C07K16/2803—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 immunoglobulin superfamily
• • A—HUMAN NECESSITIES
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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell

Definitions

• the present invention relates generally to the field of treatment of neoplastic disease. More specifically, the present invention relates to novel immunoconjugates and their use in the treatment of neoplastic disease. Even more particularly, the present invention relates to novel immunoconjugates cytotoxic to leukemia cells characterized by expression of the CD33 antigen.
• Neoplastic disease is one of the leading causes of mortality and morbidity in the Western World. All neoplastic diseases or “cancers” share at least one characteristic, i.e., the involvement of defects in the cellular growth regulatory process. Antigens located on the surface of cancer cells have been useful in distinguishing lymphoid from non-lymphoid leukemias, subtyping of acute myelogenous leukemia, predicting therapeutic outcome and in therapy in vivo or via a bone marrow purging ex vivo. Antigens defining acute non-lymphocytic cells also identify normal hematopoietic cells during early stages of their development.
• CD33 provides a useful target antigen for therapy of myelogenous leukemias, as it is expressed in the cell-surface of more than 80% of leukemic isolates from patients with myeloid leukemia with an average density of 10,000 sites/cell. In addition, rapid internalization occurs upon binding of mAb to CD33 both in vitro and in vivo.
• CD33 antigen is a 67 kilodalton glycoprotein found on normal colony forming unit granulocyte-monocyte (CFU-GM), on a fraction of burst-forming unit-erythroid (BFU-E and CFU-granulocyte, erythroid, monocyte, megakaryocyte) CFU-GEMM, and absent from normal pluripotent stem cells.
• Antibodies are proteins normally produced by the immune system of an animal in response to antigenic determinants. Antibodies bind to the specific antigen to which they are directed. The development of specific monoclonal antibodies has provided investigators with a possible means of selectively targeting chemotherapeutic agents to cells which overexpress tumor associated antigens.
• Immunotoxins are hybrid molecules consisting of a monoclonal antibody covalently linked or genetically fused to a toxin molecule and are thus able to direct potent cytotoxicity to particular cells. Immunotoxins have several possible advantages over conventional anti-neoplastic agents including selectivity for tumor cells and potential delivery of extremely potent toxins.
• obstacles to effective therapeutic use of immunotoxins for cancer include (a) lack of suitable tumor-specific targets that are not also found on other vital non-tumor cells; (b) loss of toxin potency or mAb activity after conjugation; (c) unwanted cytotoxicity to nontarget cells and tissues resulting from nonspecific internalization of the immunotoxin; (d) immunogenicity of the immunotoxin; and (e) pharmacological inability to target tumor sites adequately.
• the present invention provides a composition
• a composition comprising a conjugate of an antigen binding region exhibiting binding specificity for the CD33 protein and gelonin, a cell growth modulator derived from plants.
• Such a composition acts as an immunotoxin to specifically kill tumor cells characterized by the expression of the CD33 protein.
• composition comprising a conjugate of an antigen binding region exhibiting binding specificity for the CD33 protein and gelonin or recombinant gelonin or fragments thereof.
• a method of treating neoplastic disease comprising the administration of a cytocidally effective dose of an immunotoxin of the present invention to an individual in need of such treatment.
• a method of killing tumor cells in bone marrow comprising removing bone marrow from an individual having a neoplastic disease, treating the bone marrow with a composition of the present invention and infusing the treated bone marrow back into the individual.
• a method of preventing recurrence of neoplastic disease where the disease is characterized by an expression of CD33 protein. The recurrence is prevented by administration of a cytocidally effective treatment of immunotoxins of the present invention.
• composition of matter comprising a fusion protein formed by the fusion of the CD33 antigen binding region and gelonin or recombinant gelonin or fragments thereof.
• FIG. 1 shows the SDS-gel electrophoresis fractionation of M195 and the immunotoxin M195-IT under non-reducing conditions with a gradient of 5% to 20% acrylamide.
• Lanes purified mouse M195; a reaction mixture containing M195 and gelonin; purified M195-gelonin immunotoxin; purified gelonin.
• FIG. 2 depicts the inhibition of protein synthesis in live cells by gelonin and the M195 immunotoxin on HL60 cells.
• HL60 cells at a final concentration of 1 ⁇ 10 6 cells/ml were incubated for 3 days at 37° C. in the presence of M195-gelonin immunotoxin (solid circle) or gelonin (filled square).
• Levels of protein synthesis were determined by 5 hour incorporation of tritiated amino acids into trichloroacetic-acid-precipitable protein.
• M195 immunotoxin final concentrations ranged from 5 ng/ml to 4 ⁇ g/ml.
• Gelonin final concentration ranged from 0.5 ⁇ g/ml to 50 ⁇ g/ml. The data are representative of four experiments.
• FIG. 3 demonstrates the inhibition of protein synthesis by gelonin and M195 gelonin on SKLY16 and HL60 cell lines.
• HL60 or SKLY16 cells at a final concentration of 5 ⁇ 10 5 cells/ml were incubated for three days at 37° C. in the presence of either gelonin alone or the M195-gelonin immunotoxin.
• Final concentrations of M195-gelonin immunotoxin ranged from 4 mg/ml to 15.2 pg/ml.
• Gelonin final concentrations ranged from 10 mg/ml to 0.1 mg/ml.
• Levels of protein synthesis were determined by a 5 hour incorporation of tritiated amino acids into trichloroacetic acid precipitable protein.
• FIG. 4 illustrates the inhibition of protein synthesis by a three day incubation of M195 immunotoxin with HL60.
• HL60 cells at a final concentration of 1 ⁇ 10 6 were incubated for three days at 37° C. in the presence of the M195-gelonin immunotoxin.
• the final concentration of the immunotoxin ranged from 4 mg/ml to 0.9 ng/ml.
• Levels of protein synthesis were determined by a 5 hour incorporation of tritiated amino acids into trichloroacetic acid precipitable protein.
• FIG. 5 depicts the inhibition of protein synthesis by a five day incubation of M195-IT on HL60 cells.
• HL60 cells at a final concentration of 1 ⁇ 10 6 were incubated for five days at 37° C. in the presence of the M195-gelonin immunotoxin.
• the final concentration of the immunotoxin ranged from 4 mg/ml to 0.9 ng/ml.
• Levels of protein synthesis were determined by a five hour incorporation of tritiated amino acids into trichloroacetic acid precipitable protein.
• FIG. 6 shows the binding titer and specificity of HuM195-gelonin immunotoxin on cell lines.
• HL60, U937 or Molt4 cells at a concentration of 1.5 ⁇ 10 6 cells/ml were incubated on ice for 1 hour with either HuM195 or MuM195-gelonin at a final concentration range of 0.08 to 10 ⁇ g/ml.
• Mean peak fluorescence intensity (y-axis) versus mAb or immunotoxin (IT) concentration (x-axis) was measured using an EPICS Profile flow cytometer.
• Panel A shows HL60 binding by HuM195-gelonin immunotoxin ( ⁇ ) or by HuM195 alone (o).
• Panel B shows U937 binding by HuM195-gelonin immunotoxin ( ⁇ ) or by HuM195 alone (o); Molt4 binding by HuM195-gelonin immunotoxin (filled square) or by HuM195 alone ( ⁇ ).
• FIG. 7 illustrates the inhibition of DNA synthesis in live cells by gelonin and HuM195-gelonin immunotoxin on HL60 cells.
• HL60 cells at a final concentration of 3 ⁇ 10 4 cells/ml were incubated for 5 days at 37° C. in the presence of HuM195-gelonin immunotoxin ( ⁇ ) or gelonin alone (o).
• HuM195-gelonin immunotoxin final concentrations ranged from 0.2 ng/ml to 4 ⁇ g/ml.
• Gelonin final concentration ranged form 0.5 ⁇ g/ml to 50 ⁇ g/ml. The data are representative of five experiments.
• FIG. 8 depicts the competition between the HuM195-gelonin immunotoxin, HuM195 alone or FD79 (an isotype matched control antibody).
• FIG. 9 shows the cytotoxicity and inhibition of protein synthesis in HL60 or RAJI cells by recombinant gelonin (rGel), HuM195 or the HuM195-rGel immunotoxin.
• Panel A shows the inhibition of protein synthesis in HL60 or RAJI cells by rGel, HuM195 and HuM195-rGel.
• HL60 or RAJI cells at a final concentration of 10 5 cells/ml were incubated 3 days at 37° C. in the presence of HuM195-rGel, rGel or HuM195.
• Levels of protein synthesis were determined by 5 hour incorporation of tritiated leucine into trichloroacetic acid precipitable protein. The treatment is shown in parenthesis.
• Panel B shows cell viability determined by typan blue exclusion.
• HL60 or RAJI cells at a final concentration of 10 5 cell/ml were incubated 3 days at 37° C. in the presence of HuM195-rGel. Trypan blue was added and live and dead cells were counted under the microscope.
• FIG. 10 shows the IC 50 of HuM195-rGel for blast cells from 7 patients versus percent of cells positive for CD33 by FACS.
• IC 50 was estimated from the dose-response curves, which were linear in the range of IC 50 when plotted on a log/log scale.
• the open bar shows the mean and standard deviation of IC 50 obtained with 3 patient samples incubated with equimolar HuM195 and free rGel.
• the hatched bar shows the IC 50 for HL60, which is 100% CD33 positive.
• FIG. 11 shows the effect of prolonged exposure on dose-response of OCI/AML5 to the immunotoxin.
• Cells were incubated with the immunotoxin at 0, 0.5, 1, 2 or 4 nM for 48 hours, washed and plated. Cells surviving 4 nM immunotoxin at 48 hours were collected, washed and resuspended with different concentrations of immunotoxin, and this was repeated at 96 and 120 hours.
• Concentration curves are shown for OCI/AML5 incubated 48 hours with unconjugated HuM195 and rGel (open square); immunotoxin without pretreatment (filled square); and immunotoxin following pretreatment with 4 nM immunotoxin for 48 hours (*), 96 hours (X) or 120 hours (X). Mean and SEM of quadruplicate wells is shown.
• FIG. 12 shows the enhanced effect of the immunotoxin when combined with cryopreservation.
• FIG. 13 shows the effect of HL60 concentration upon efficacy of HuM195-gelonin immunotoxin.
• HL60 cells at a final concentration of 3.125 ⁇ 10 4 ⁇ 5 ⁇ 10 5 cells/ml were incubated for 5 days at 37° C. with or without the immunotoxin at a final concentration of 2 ⁇ g/ml.
• DNA synthesis was determined by 5 hour incubation with tritiated thymidine. Percentage inhibition was determined in comparison to control wells without the immunotoxin. The data are representative of 3 experiments.
• FIG. 14 shows the cytotoxicity of HuM195-gelonin immunotoxin on HL60 in the presence of excess irradiated bone marrow.
• HL60 cells at a final concentration of 6.67 ⁇ 10 4 cells/ml in the presence (filled triangle) or absence (filled circle) of normal irradiated bone marrow at 1 ⁇ 10 6 cells/ml were incubated for 6 days at 37° C. in the presence of HuM195-gelonin immunotoxin at a final concentration of 100 pg/ml to 10 ⁇ g/ml.
• Levels of DNA synthesis were determined by measuring 5 hour tritiated thymidine incorporation. The data are representative of two experiments.
• FIG. 15 shows tumor cell surface antigen expression. Expression of CD33 by HL60 cells grown at 4 weeks in a representative tumor mass in vivo as determined by indirect immunofluorescence.
• FIG. 16 shows the specific binding and internalization of 125 I-HuM195 in tumors in vivo.
• Tumor-bearing mice at 4 weeks after transplantation received infusions of 2 or 20 ⁇ g of 125 I-HuM195 shown in parenthesis. Mice were sacrificed at 4 or 24 hours after the infusion. Tumors were excised, weighed and counted. Specific surface-bound and internalized HuM195 were calculated. Standard deviation was less than 10%.
• FIG. 17 shows the treatment of human leukemia cells in vivo by HuM195-rGel immunotoxin.
• Mice were i.p. transplanted with 10 7 HL60 human leukemia cells.
• Panel A shows that at the tenth day, mice were treated by three injections of 100 nM: HuM195-rGel (four mice); rGel (four mice); HuM195 mixed with rGel (five mice); and control saline (five mice).
• tumor surface area was measured.
• Panel B shows that at the 14th or 28th days, mice were treated with 6 injections of 100 nM: HuM195-rGel immunotoxin (four mice at the 14th day; four mice at the 28th day); control saline (five mice). At times indicated by the x-axis, tumor surface area was measured.
• chimeric antibodies or “chimeric peptides” refer to those antibodies or antibody peptides wherein one portion of the peptide has an amino acid sequence that is derived from, or is homologous to, a corresponding sequence in an antibody or peptide derived from a first gene source, while the remaining segment of the chain(s) is homologous to corresponding sequences of another gene source.
• a chimeric heavy chain antibody peptide may comprise a murine variable region and a human constant region.
• the two gene sources will typically be two separate species, but will occasionally involve one species.
• Chimeric antibodies or peptides are typically produced using recombinant molecular and/or cellular techniques. In many cases, chimeric antibodies have variable regions of both light and heavy chains that mimic the variable regions of antibodies derived from one mammalian species, while the constant and/or framework portions are homologous to the sequences in antibodies derived from a second, different mammalian species.
• chimeric antibody is any antibody in which either or both of the heavy or light chains are composed of combinations of sequences mimicking the sequences in antibodies of different sources, whether these sources be from differing classes, differing antigen responses, or differing species of origin, and whether or not the fusion point is at the variable/constant boundary.
• chimeric antibodies can include antibodies where the framework and complementarity-determining regions (CDRs) are from different sources.
• CDRs complementarity-determining regions
• non-human CDRs are integrated into human framework regions linked to a human constant region to make “humanized antibodies.” See, e.g., PCT Application Publication No. WO 87/02671; U.S. Pat. No. 4,816,567; EP Patent Application 0173494; Jones, et al., Nature, 321:522-525 (1986); and Verhoeyen, et al., Science, 239:1534-1536 (1988).
• human-like framework region is a framework region for each antibody chain, and it usually comprises at least about 70 or more amino acid residues, typically 75 to 85 or more residues.
• the amino acid residues of the human-like framework region are at least about 80%, preferably about 80-85%, and most preferably more than 85% homologous with those in a human immunoglobulin. This shared feature with other endergenous antibodies is useful in generating a targeting moiety which introduces only a minor immune reaction, e.g., a mechanism which minimizes response to “self” markers.
• humanized or “human-like immunoglobulin” refers to an immunoglobulin comprising a human-like framework region and a constant region that is substantially homologous to a human immunoglobulin constant region, e.g., having at least about 80% or more, preferably about 85-90% or more and most preferably about 95% or more homology. Hence, most parts of a human-like immunoglobulin, except possibly the CDRs, are substantially homologous to corresponding parts of one or more native human immunoglobulin sequences.
• hybrid antibody refers to an antibody wherein each chain is separately homologous with reference to a mammalian antibody chain, but the combination represents a novel assembly so that two different antigens are recognized by the antibody.
• one heavy and light chain pair is homologous to that found in an antibody raised against one antigen recognition feature, e.g., epitope
• the other heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope.
• Such hybrids may, of course, also be formed using chimeric chains.
• the terms “monoclonal antibody” means an antibody composition recognizing a discrete antigen determinant. It is not intended to be limited as regards the source of the antibody or the manner in which it is made.
• an antibody or other peptide is specific for a CD33 if the antibody or peptide binds or is capable of binding CD33, e.g., protein as measured or determined by standard antibody-antigen or ligand-receptor assays, e.g., competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA.
• This definition of specificity applies to single heavy and/or light chains, CDRs, fusion proteins or fragments of heavy and/or light chains, that are also specific for CD33 if they bind CD33 alone or if, when properly incorporated into immunoglobulin conformation with complementary variable regions and constant regions as appropriate, are then capable of binding CD33 with specificity.
• the ability of an antibody or peptide fragment to bind an antigen can be determined by detecting the ability of the peptide to compete with the binding of a compound known to bind the antigen.
• Numerous types of competitive assays are known and are discussed herein.
• assays that measure binding of a test compound in the absence of an inhibitor may also be used.
• the ability of a molecule or other compound to bind the c-erbB-2 protein can be detected by labelling the molecule of interest directly or it may be unlabelled and detected indirectly using various sandwich assay formats.
• Numerous types of binding assays such as competitive binding assays are known (see, e.g., U.S. Pat. Nos.
• the binding interactions between ligand or peptide and receptor or antigen include reversible noncovalent associations such as electrostatic attraction, Van der Waals forces, and hydrogen bonds.
• assay formats may involve the detection of the presence or absence of various physiological or chemical changes that result from the interaction, such as down modulation, internalization, or an increase in phosphorylation. See Receptor - Effector Coupling—A Practical Approach , ed. Hulme, IRL Press, Oxford (1990).
• Gelonin belongs to a class of potent ribosomal-inactivating plant toxins. Other members of this class of ribosomal-inactivating plant toxins are the chains of abrin, ricin and modeccin. Gelonin, like abrin and ricin, inhibits protein synthesis by damaging the 60S sub-unit of mammalian ribosomes. Gelonin appears to be stable to chemical and physical treatment. Furthermore, gelonin itself does not bind to cells and, therefore, is non-toxic (except in high concentrations) and is safe to manipulate in the laboratory. The inactivation of ribosomes is irreversible, does not appear to involve co-factors and occurs with an efficiency which suggests that gelonin acts enzymatically.
• Gelonin and ricin are among the most active toxins which inhibit protein synthesis on a protein weight basis. Gelonin is 10 to 1000 times more active in inhibiting protein synthesis than ricin A chain. Peptides like ricin and abrin are composed of two chains, an A chain which is the toxic unit and a B chain which acts by binding to cells. Unlike ricin and abrin, gelonin is composed of a single chain, and, because it lacks a B chain for binding to cells, it is itself relatively non-toxic to intact cells. As used herein, gelonin refers to the naturally occurring, purified gelonin.
• Recombinant gelonin, or rGel refers to that gelonin or enzymatically active fragment of gelonin which has been cloned into an expression vector or other suitable vehicle, expressed in Escherichia coli or other suitable organism, and purified accordingly.
• Active fragments and derivatives include any compounds which have the same core structure as the full length structure of gelonin but lack the entire primary sequence. These fragments or derivatives will have the same or improved biological or cytotoxic activity as gelonin. The cytotoxicity of the gelonin fragments or derivatives may be routinely determined by those with skill in the art using the rabbit reticulocyte lysate assay.
• Mammalian cells apparently lack the ability to bind and/or to internalize the native gelonin molecule.
• Conjugates of gelonin with monoclonal antibody, such as M195 directed to a tumor associated antigen present on certain tumor cells provide both a specific method for binding the gelonin to the cell and a route for internalization of the gelonin-antibody complex.
• the M195 antibody may serve as a useful carrier of such drugs providing an efficient means of both delivery to the tumor and enhanced entry into the tumor cells themselves.
• specific antibody delivery of cytotoxic drugs to tumors will provide protection of sensitive sites such as the liver that do not express CD33 and bone marrow stem cells from the deleterious action of the toxin.
• Use of drugs conjugated to the M195 antibody as a delivery system allows lower dosage of the drug itself, since all drug moieties are conjugated to antibodies which concentrate within the tumor or leukemia.
• Conjugates of the monoclonal antibody may be made using a variety of bifunctional protein coupling agents.
• reagents are SPDP, iminothiolane (IT), bifunctional derivatives of imidoesters such as dimethyl adipimidate, HCl, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis(p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such as bis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate, and bis-active fluorine compounds such as a 1,5-difluoro-2,4-dinitrobenzene.
• SPDP iminothiolane
• I iminothiolane
• imidoesters such as dimethyl adipimidate, HCl
• the immunotoxins of the present invention to an individual who has been diagnosed as having a leukemia that is characterized by the expression of CD33 protein will allow targeting and concentration of the cytotoxic agent at the site where it is needed to kill the tumor cells. By so targeting the cytotoxic agent, non-specific toxicity to other organs, tissues and cells will be eliminated or decreased.
• the immunotoxins When used in vivo for therapy, the immunotoxins are administered to the human or animal patient in therapeutically effective amounts, i.e., amounts that eliminate or reduce the tumor burden or in amounts to eliminate residual disease after an earlier treatment with chemotherapy or radiation therapy. They will normally be administered parenterally, preferably intravenously.
• the dose and dosage regimen will depend upon the nature of the leukemia and its population, the characteristics of the particular immunotoxin, e.g., its therapeutic index, the patient, and the patient's history.
• the amount of immunotoxin administered will typically be in the range of about 0.01 to about 10.0 mg/kg of patient weight.
• the immunotoxins will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
• a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers.
• the vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
• the immunotoxin will typically be formulated in such vehicles at concentrations of about 0.1 mg/ml to 10 mg/ml.
• the immunotoxins of the present invention may also be used in a method of killing tumor cells in bone marrow.
• the bone marrow is first removed from an individual having a neoplastic disease such as leukemia. Subsequently, the bone marrow is treated with a cytocidally effective dose of an immunotoxin of the present invention and returned to the individual.
• Recombinant gelonin (rGel) has recently been cloned and expressed in E. coli (Rosenblum et al., J. Interferon Cytokine Res., 15:547 (1995)).
• the chromatographic behavior of recombinant gelonin ( ⁇ 28 kD) appeared to be identical to that of native gelonin with respect to its binding and elution from both CM-52 cellulose and blue Sepharose.
• Functional analysis of the purified recombinant gelonin was performed using inhibition of cell-free protein synthesis in rabbit reticulocyte lysates.
• recombinant gelonin may have an advantage over native gelonin because of the lack of glycosylation of the recombinant molecule and because fusion toxins between HuM195 and recombinant gelonin could result in a more-defined molecule that may avoid inadvertent inactivation of either the toxin or the antibody molecule during the chemical conjugation process or during subsequent reduction during storage.
• the gelonin activity was monitored in a cell-free protein synthesis inhibition assay.
• the cell-free protein synthesis inhibition assay was performed by sequentially adding to 50 ml rabbit reticulocyte lysate, mixing after each addition, the following components: 0.5 ml of 0.2 M Tris-HCl (pH 7.8), 8.9 ml of ethylene glycol, and 0.25 ml of 1 M HCl.
• SAEM salt-amino acid-energy mixture
• [ 14 C]-leucine incorporation was monitored in an aliquot of the mixture by precipitating synthesized protein on glass fiber filters, washing in 10% TCA and acetone, and monitoring the radioactivity in a Beta-counter using Aquasol scintillation fluid.
• Gelonin with a specific activity no lower than 4 ⁇ 10 9 U/mg was used for conjugation with the antibodies.
• a unit of gelonin activity is the amount of gelonin protein which causes 50% inhibition of incorporation of [ 14 C]-leucine into protein in the cell free assay.
• Murine monoclonal antibody M195 was produced from hybridomas resulting from the fusion of NS-1 mouse myeloma cells and the spleen cells of a five week old BALB/c mouse immunized with leukemia cells from a patient with acute non-lymphocytic leukemia (FAB-M2). Supernatant fluids from cloned hybridoma cultures were screened against the panel of leukemia cell lines and the original ANLL leukemia cells using Staphylococcus aureus protein A (PA) erythrocyte resetting. The repeatedly subcloned M195 hybridoma was expanded in the doubly pristane-primed (C57 BL/6 times BALB/c) F1 mice. M195 was purified on a PA-Sepharose by affinity chromatography using sequential PH step dilutions. Purity was determined on SDS-polyacrylamide gels stained with coomassie brilliant blue.
• PA Staphylococcus aureus protein A
• Humanized monoclonal antibody M195 was prepared as described by Co et al., “Chimeric and Humanized Antibodies with Specificity for the CD33 Antigen”, J. Immunol., 148:1149-1154 (1992) and was produced from hybridomas and purified as above.
• Gelonin in phosphate buffered saline was concentrated to approximately 10 mg/ml in a Centriprep 10 concentrator.
• Triethanolamine hydrochloride (TEA/HCl) pH 8.0
• EDTA Triethanolamine hydrochloride
• a 2-iminothiolane stock solution 500 mM in 60 mM TEA/HCl buffer containing 1 mM EDTA, pH 8.0
• M195 linked with 4-succinimidyloxycarbonyl- ⁇ -methyl- ⁇ (2-pyridyldithio)toluene is prepared by coupling 2-IT-modified gelonin with SMPT-modified MAB M195. Briefly, to modify M195 with SMPT, 10 mg of antibody in 1.0 ml of PBS is diluted 1:1 with 2 ⁇ borate buffer (0.05 M sodium borate 1.7% sodium chloride, pH 9.0) and 52 ml of 4 mM SMPT in dry DMF is slowly added to the antibody solution. The reaction is incubated at room temperature for 2 hr with stirring under N 2 .
• 2 ⁇ borate buffer 0.05 M sodium borate 1.7% sodium chloride, pH 9.0
• Excess SMPT is removed by passing the reactions mixture through a Sephadex G-25 column containing phosphate-EDTA buffer (pH 7.5) and antibody positive fractions are evaluated by Bio-Rad assay. The fractions are pooled and stored at 4° C. under N 2 . The cross-link with 2-IT is carried out at 27° C. under N 2 with stirring for 96 hrs. The final product is purified as previously described for SPDP.
• Non-conjugated gelonin and low molecular weight products were removed from the reaction mixtures of Example 6 by gel filtration on a Sephadex S-300 column (1.6 ⁇ 31 cm) pre-equilibrated with PBS. Reaction mixtures from Example 6 were concentrated to approximately 1 ml with a Centricon 30 microconcentrator before loading on the Sephadex column. The column was washed with PBS. One ml fractions were collected and 50 ml aliquots are analyzed for protein by the Bradford assay.
• the high molecular weight peak (fraction 28-40) from the S-300 column was applied to an affinity chromatography column of Blue Sepharose CL-6B (1 ⁇ 24 cm) pre-equilibrated with 10 mM phosphate buffer (pH 7.2) containing 0.1 M NaCl. After sample loading, the column was washed with 30 ml of buffer to completely elute non-conjugated antibody. The column was eluted with a linear salt gradient of 0.1 to 2 M Nacl in 10 mM phosphate buffer (pH 7.2). Protein content of the eluted fractions was determined by the Bradford assay.
• FIG. 1 illustrates the electrophoretic pattern of purified M195, an unpurified reaction mixture of M195 and gelonin, purified M195-gelonin immunotoxin or gelonin alone.
• the flow-through peak (fractions 14-20) contains only free antibody while fractions 50-80, eluted with high salt, contain M195-gelonin conjugate free of unconjugated gelonin or antibody.
• Conjugates with more than two gelonin molecules had a decreased avidity for the antigen, which resulted in a less potent molecule.
• the loss of avidity may be due to steric interference with the antigen-binding site or to instability of the over-conjugated immunotoxin.
• the rabbit reticulocyte in vitro translation system was utilized to estimate the gelonin activity of the essentially pure gelonin-M195 immunotoxin antibody complex.
• One unit of activity in this assay was defined as the amount of protein required to provide 50% inhibition of protein synthesis as compared to untreated controls.
• the specific activity of both the native gelonin and the M195-gelonin conjugate were determined to be 2 ⁇ 10 8 U/mg and 8.2 ⁇ 10 5 U/mg, respectively.
• the essentially pure gelonin M195 antibody is active in the reticulocyte lysate assay.
• a 1:1000 dilution of the original sample caused approximately a 50% inhibition of protein synthesis, i.e., a 50% reduction of the incorporation of 14 C-leucine into protein.
• the activity of the original preparation was 1000 U/ml.
• M195 immunotoxin was tested for its ability to kill HL60 cells in comparison to free gelonin. Inhibition of protein synthesis (using the test of a tritiated mix of amino acid (0.5 mCi/ml; New England Nuclear Corp.) incorporation into trichloroacetic acid (TCA) precipitable protein) was used as a measure of activity of the agent used. Final concentrations of the M195-gelonin immunotoxin ranged from 4 mg/ml to 5 ng/ml. Gelonin final concentrations ranged from 44 mg/ml to 0.6 mg/ml. The M195-gelonin immunotoxin was approximately 600 times more potent than the free gelonin alone.
• ID 50 for the M195-gelonin immunotoxin was approximately 0.4 nM. Inhibition of protein synthesis by the M195-gelonin immunotoxin subsequently leads to either lack of cell division or to cell death. Cell death was confirmed by experiments using trypan blue exclusion to determine total number of live cells and percentages of live cells.
• concentrations of the M195-gelonin immunotoxin inhibited greater than 80% of HL60 protein synthesis, while comparable concentrations of the immunotoxin had no effect on SKLY16 cells.
• concentrations of the M195-gelonin immunotoxin for CD33-expressing cells is apparent.
• the length of time of exposure to the immunotoxin affects its activity.
• an approximate ten-fold increase in potency of the M195-gelonin immunotoxin is seen after a five day incubation with HL60 cells (see FIG. 5 ) as opposed to a three day incubation (see FIG. 4 ).
• FIG. 6 illustrates that the humanized M195-gelonin immunotoxin is capable of binding specifically to target cells.
• the humanized M195-gelonin immunotoxin showed more specific binding to CD33 positive cell lines (HL60 and U937) compared to the humanized M195 antibody alone. It did not bind to the CD33 negative cell line (MOLT4).
• FIG. 7 illustrates the ability of humanized M195-gelonin immunotoxin to kill HL60 cells, as the ID 50 was less than 10 picomoles. This ID 50 for the immunotoxin is more than 4000 times lower than the ID 50 of gelonin alone.
• HL60 cells were incubated for one hour on ice in the presence of the humanized M195 antibody or F79, an isotype matched control antibody.
• Humanized M195-gelonin immunotoxin was added at a concentration of 0.15 mg/ml. Cells were incubated 90 hours at 37° C. The quantity of live cells was determined by the trypan blue exclusion technique. The percent inhibition represents the reduction of cells killed in the presence of the competing antibody as compared to cells killed by the immunotoxin alone.
• the humanized M195 antibody was able to block cytotoxicity of the humanized M195 antibody-gelonin immunotoxin in a dose dependent manner.
• Fd79 a non-specific humanized IgG1
• HuM195-rGel was tested for its ability to kill CD33 positive and CD33 negative cells in comparison to free rGel. Activity and cytotoxicity were determined by inhibition of incorporation of 3 H-leucine into protein and by trypan blue exclusion. Dose-response curves were generated by testing the inhibitory effects of HuM195-rGel on the protein synthesis of HL60 cells (CD33 positive) and RAJI (CD33 negative) in culture ( FIG. 9A ).
• the concentration of HuM195-rGel required to inhibit protein synthesis in HL60 cells by 50% was 0.6 nM, whereas the concentration of rGel alone required to nonspecifically inhibit protein synthesis in both HL60 and RAJI cells by 50% was about 200 nM ( FIG. 9A ).
• the concentration range of 10-100 nM HuM195-rGel protein synthesis in HL60 cells was almost completely inhibited, while no cytotoxicity was observed with the CD33 negative cell lines RAJI ( FIG. 9A ) and DAUDI.
• HuM195 alone did not affect the protein synthesis in CD33 positive HL60 cells ( FIG. 9A ).
• HuM195-rGel The cytotoxicity of HuM195-rGel was directly determined by trypan blue analysis. The concentration of HuM195-rGel required to kill 50% of cells was 0.7 nM ( FIG. 9B ), similar to the concentration of HuM195-rGel required to inhibit protein synthesis by 50% ( FIG. 9A ). However, HuM195-rGel did not kill CD33 negative RAJI cells at the highest concentration of 100 nM, suggesting that it may be used safely for study in vivo.
• the IC 50 of immunotoxin was 1 nM to 30 nM for patient samples and 0.3 nM for HL60 cells (24-hour incubation).
• the IC 50 of unconjugated monoclonal antibody with rGel was 100 nM to 200 nM. Prolonging the incubation time to 72 hours had a variable effect on IC 50 for patient samples, but consistently lowered the IC 50 for unconjugated antibody and rGel.
• Patient samples had varying CD33 expression, measured on bone marrow blasts by flow cytometry as the percent of cells with positive staining.
• the IC90 was determined empirically for HL60 and Patient #2.
• the IC 50 for HL60 cells show a 4 log difference in sensitivity to HuM195-rGel versus free rGel, whereas the IC 90 shows a 2 log difference.
• the patient sample was more sensitive to immunotoxin than to unconjugated antibody and rGel by a factor of 25 at the IC 50 and by a factor of 9 at the IC 90 .
• CD33 expression was also reflected in the does-response curves of OCI/AML5 following preincubation for 0 to 6 days with immunotoxin at an inhibitory concentration. After 2, 4 or 6 days exposure to immunotoxin, OCI/AML5 continued to show sensitivity to HuM195-rGel that is stable, though not as great as that seen with naive cells ( FIG. 11 ). Cells incubated with immunotoxin for 5 days showed only faint staining for CD33 (median fluorescence intensity 5.6 fluorescence units versus 196.1 fluorescence units in the control not treated with immunotoxin).
• cryopreservation itself has some antileukemic effects, to which other purging modalities have been added.
• freezing medium (10% DMSO; 50% FEBS) immediately following a 24 hour exposure to HuM195-rGel (1 nM), frozen at ⁇ 70° C. for a minimum of 24 hours, quickly thawed by immersion in a 37° C. water bath, washed several times and plated on methylcellulose to determine clonogenic cell recovery.
• the data shown in FIG. 12 indicate that there is a greater than additive effect against all but one patient sample.
• Bone marrow aspirates were obtained form normal donors according to Memorial Sloan Kettering Cancer Center IRB protocols. Mononuclear cells were collected by Ficoll-Paque sedimentation, washed, and gamma irradiated with 8 Gy. Marrow cells were divided into aliquots in 96 well plates at a final concentration of 1 ⁇ 106 cells/ml. HL60 cells at a final concentration of 0.667 ⁇ 105 cells/ml and HuM195-gelonin immunotoxin at various concentrations were added to the plates. After a 6 day incubation at 37° C., cells were assayed for [ 3 H]-thymidine incorporation.
• HL60 cells were serially diluted and incubated in the presence of a single concentration of immunotoxin at 2 ⁇ g/ml.
• the immunotoxin was most effective at low cell concentrations ( FIG. 13 ).
• the immunotoxin lost potency. This was not due to an excess of cell surface binding sites over immunotoxin molecules, as even at the highest cell concentrations, there were 100-1000 more molecules of immunotoxin than available binding sites.
• serial dilutions of the immunotoxin and gelonin were incubated with three different concentrations of HL60 cells. Both the immunotoxin and gelonin were more potent at lower cell concentrations.
• the internalization of 125 I-HuM195 into the target cells in vivo was rapid, and similar to the observations in vitro. At 4 hours after infusion of 2 or 20 ⁇ g antibody, 23-26% of bound 125 I-HuM195 was internalized, whereas a higher rate of internalization (38-43%) was seen at 24 hours ( FIG. 16 ).
• the leukemic cell growth in the subcutaneous space and peritoneum of nude mice was substantially reduced by HuM195-rGel.
• tumors At 10 days after transplantation of HL60 cells into the peritoneum of nude mice, tumors of about 2 mm 3 in size were present in the subcutaneous space ( FIG. 17 ).
• HuM195-rGel After three injections of HuM195-rGel at a dose of 36 ⁇ g per mouse beginning at 10 days, two out of four mice did not develop tumors for up to 5 months after transplantation. Tumors grew slowly in the other two immunotoxin-treated mice. Control groups of mice (treatment with saline alone, gelonin alone or HuM195 mixed with rGel at the same final concentrations) did not show significant tumor inhibition or any cures.
• HuM195-rGel was also tested (twice a week for 3 weeks at the same dose of 36 ⁇ g per mouse at 14 days and 28 days after transplantation with HL60 cells). Despite the increase in the number of doses from three to six, the delay in treatment to 28 days caused less inhibition of tumor growth by HuM195-rGel (Table II and FIG. 17B ). After 3 weeks of treatment with HuM195-rGel, two out of four mice had no tumors in the group treated 14 days after transplantation; however, all four mice developed local tumors when treated 28 days after transplantation.
• mice were treated with three injections of equimolar amounts of HuM195 ⁇ rGel, rGel or HuM195 + rGel (administered together but not conjugated) beginning at 10 days after HL60 transplantation. Two other groups of mice were treated with six injections of HuM195 ⁇ rGel beginning at. the 14 or 28 day after HL60 transplantation. Subcutaneous tumor size (cross product in mm 2 ) at 7 weeks is shown. ‘Death’ indicates mice that died in the sixth week.
• compositions of the present invention may include fusion constructs of the M195 monoclonal antibody and a cytotoxic moiety.
• Such fusion constructs of the immunotoxin of the present invention may be prepared by the method of Co et al.
• the Sp2/0-Ag14 cells Prior to use in these studies, the Sp2/0-Ag14 cells will be grown initially in the presence of 0.1 mg/ml of native gelonin. Over several months, the concentration of gelonin will be gradually increased until the cells can be maintained in up to 10 mg/ml. Cells will then be cloned by limiting dilution in the presence of 10 mg/ml gelonin and the resulting colonies resistant to gelonin will be expanded. Gelonin will then be removed from the culture media for two passages and the cells challenged again with gelonin exposure to confirm development of stably-resistant clones.
• gelonin-resistant SP2/0 cell producing antibody After tests to confirm the production and activity of humanized M195, gelonin-resistant SP2/0 cell producing antibody will be grown and the cDNA for the M195 antibody removed from the total DNA by incubation with restriction endonuclease.
• the cDNA from JM105 E. coli expressing optimized gelonin will be removed, purified and the DNA encoding gelonin released after digestion with HindIII and EcoRI.
• the gelonin gene will be ligated into the heavy-chain fragment and the insert replaced into gelonin resistant SP2/0 cells. Cells will then be sub-cloned by limiting dilution and the clones screened for both humanized antibody production and gelonin content.

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