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  • 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
  • C07K16/2827—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 against B7 molecules, e.g. CD80, CD86
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  • 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
  • C07K16/2851—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 lectin superfamily, e.g. CD23, CD72
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  • 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
  • C07K16/2875—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/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
• • A—HUMAN NECESSITIES
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

• Hematologic malignancies include lymphomas and leukemias that, in many instances , are more accessible to blood borne chemotherapeutics such as monoclonal antibodies than other types of tumors. While Rituxan has been shown to be effective in the treatment of some of these type of malignancies (particularly non-Hodgkins' lymphoma), there remain a number of hematologic malignancies for which there is no commonly accepted effective treatment. Among these malignancies is chronic lymphocytic leukemia.
• sCD23 In CLL, sCD23 levels in serum correlated with the tumor burden and thus with the clinical stage of the disease (Sarfati and Bran et al. 1988. Blood 71:94).
• the sCD23 particularly the 25 kD species, has been shown to: a) act as an autocrinc factor in some Epstein-Barr virus transformed mature B-ccll lines, b) act as a differentiation factor for prothvmocytes and c) prevent apoptosis (programmed cell death) of germinal center B cells, possibly via the induction o ⁇ bcl-2 expression.
• Rituxan typical treatment for B-cell malignancies is the administration of radiation therapy and chemotherapeutic agents.
• conventional external radiation therapy will be used to destroy malignant cells.
• side effects are a limiting factor in this treatment.
• Another widely used treatment for hematological malignancies is chemotherapy.
• Combination chemotherapy has some success in reaching partial or complete remissions. Unfortunately, these remissions obtained through chemotherapy are often not durable.
• FIG. 11 iss aa ggrraapphhiiccaall rreeppiresentation of the specific binding of Rituxan* and a CD23 antagonist to lymphoma cells in a concentration dependent fashion
• Fig. 2 is a graphical representation of the antibody dependent cellular cytoxicity (ADCC) activity of a CD23 antagonist and Rituxan on lymphoma cells;
• ADCC antibody dependent cellular cytoxicity
• Figs. 3A and 3B show, respectively, the effects of the combination of a CD23 antagonist with Rituxan on ADCC mediated in vitro killing of tumor cells with low concentrations of the antagonist and with high concentrations of the antagonist;
• Fig. 5 illustrates the induction of apoptosis by a CD23 antagonist and Rituxan with cross-linking by a secondary anti-human IgG specific antibody in lymphoma cells;
• Figs. 10A and 10B illustrate, respectively, the induction of apoptosis in CLL cells by a CD23 antagonist alone and the induction of apoptosis in CLL cells by a CD23 antagonist and Rituxan and the combination thereof at various concentrations;
• Fig. 12 shows the anti-tumor activity of a CD23 antagonist as a single agent in a lymphoma/SCID mouse model
• CD23 antagonists may comprise any ligand, polypeptide, peptide. antibody or small molecule that reacts, interacts, binds or associates with the CD23 antigen expressed on B-cells and eliminates, reduces, inhibits or controls the growt of neoplastic cells.
• CD23 refers to the low affinity receptor for IgE expressed by B cells and other cells.
• a CD23 antagonist is a molecule which, upon binding to the CD23 cell surface marker, destroys or depletes CD23 + cells in a mammal and/or interferes with one or more cell functions, e.g. by reducing or preventing a humoral response elicited by the cell.
• the antagonist preferably is able to deplete B cells (i.e. reduce circulating B cell levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis and/or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and/or induction of B cell death (e.g. via apoptosis).
• Antagonists included within the scope of the present invention include antibodies, synthetic or native sequence peptides and polypeptides, ligands and small molecule antagonists which bind to the CD23 cell marker, optionally conjugated with or fused to a cytotoxic agent.
• 1DEC-152 is a primatized monoclonal anti-CD23 antibody (also referred to herein as p5E8) against the CD23 antigen that has been developed for various indications (Nakumura and Kloetzer et al. 2000, 22. 131).
• Monoclonal antibody p5E8 originated from 5E8, a primate anti-human CD23 antibody secreting hybridoma from cynomolgus macaques and was molccularly cloned and expressed as a 150 kDa IgG monomer in CHO cells using proprietary vector technology.
• antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
• Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof.
• antibody fragments include Fab. Fab'. F(ab')Z, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
• Domain deleted antibodies comprising immunoglobulins in which at least part of one or more constant regions have been altered or deleted to provide modified physiological properties (e.g. reduced serum half-life) may also be considered antibody fragments for the memeposes of the instant disclosure.
• the domain deleted antibodies will comprise constant regions that lack the C H 2 domain.
• the relatively short-lived, or mortal, lymphocytes from a mammal which has been injected with antigen arc fused with an immortal tumor cell line (e.g. a myeloma cell line), thus producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
• an immortal tumor cell line e.g. a myeloma cell line
• hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
• the resulting hybrids arc segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They therefore produce antibodies which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed "monoclonal.”
• I lybridoma cells thus prepared arc seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established.
• culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen.
• DNA encoding the desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
• the isolated and subcloned hybridoma cells serve as a preferred source of such DNA.
• the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins.
• DNA encoding antibodies or antibody fragments may also be derived from antibody phage libraries as set forth, for example, in EP 368 684 Bl and U.S.P.N. 5,969,108 each of which is incorporated herein by reference.
• Several publications e.g.. Marks et al. Bio/Technology 10:779-783 (1992) have described the production of high affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries.
• Such procedures provide viable alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies and, as such, are clearly within the purview of the instant invention.
• Yet other embodiments of the present invention comprise the generation of substantially human antibodies in transgenic animals (e.g.. mice) that are incapable of endogenous immunoglobulin production (see e.g.. U.S. Pat. Nos. 6,075,181, 5.939,598, 5,591.669 and 5,589,369 each of which is incorporated herein by reference).
• transgenic animals e.g.. mice
• 6,075,181, 5.939,598, 5,591.669 and 5,589,369 each of which is incorporated herein by reference.
• the homozygous deletion of the antibody heavy-chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array in such germ line mutant mice will result in the production of human antibodies upon antigen challenge.
• Another preferred means of generating human antibodies using SCID mice is disclosed in commonly-owned, co-pending U.S. Pat. No. 5,811.524 which is incorporated herein by reference. It will be appreciated that the
• genetic sequences useful for producing antibody derivatives of the CD23 antagonists of the present invention may be obtained from a number of different sources.
• a variety of human antibody genes arc available in the form of publicly accessible deposits. Many sequences of antibodies and antibody-encoding genes have been published and suitable antibody genes can be synthesized from these sequences much as previously described.
• antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques arc described in a variety of laboratoiy manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan el al, Eds.. Green Publishing Associates and Wiley-lnterscience, John Wiley and Sons. New York (1 91) which is herein incorporated by reference in its entirety, including supplements.
• RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable. mRNA may be isolated from total RNA by standard techniques such as chromatography on oligod F cellulose. Techniques suitable to these purposes are familiar in the art and are described in the foregoing references.
• cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known metliods. It may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
• DNA typically plasmid DNA
• plasmid DNA may be isolated from the cells as described herein, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail in the foregoing references relating to recombinant DNA techniques.
• the DNA may be modified according to the present invention at any point during the isolation process or subsequent analysis.
• Preferred antibody sequences are disclosed herein. Oligonucleotide synthesis techniques compatible with this aspect of the invention are well known to the skilled artisan and may be ca ⁇ ied out using any of several commercially available automated synthesizers. Tn addition, DNA sequences encoding several types of heavy and light chains set forth herein can be obtained through the services of commercial DNA synthesis vendors. The genetic material obtained using any of the foregoing metliods may then be altered or modified to provide antibodies compatible with the present invention. While a variety of different types of antibodies may be obtained and modified according to the instant invention, the modified antibodies of the instant invention will share various common traits.
• immunoglobulin shall be held to refer to a tctramer or aggregate thereof whether or not it possesses any relevant specific immunoreactivity.
• Antibodies refers to such assemblies which have significant known specific immunoreactive activity to an antigen (e.g. a tumor associated antigen), comprising light and heavy chains, with or without covalent linkage between them. The antibodies may be modified to provide beneficial physiological characteristics.
• modified antibodies are held to mean antibodies, or immunoreactive fragments or recombinants thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as increased tumor localization or reduced serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
• immunoreactive single chain antibody constructs having altered or omitted constant region domains may be considered to be modified antibodies.
• Light chains are classified as either kappa or lambda (K, ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain.
• the light and heavy chains arc covalentiy bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages when the immunogobulins are generated either by hybridomas, B cells or genetically engineered host cells. I lowever, if non-covalent association of the chains can be effected in the correct gcometiy. the aggregate of non-disul fide-linked chains will still be capable of reaction with antigen.
• IgGi, IgG 2 , IgG , lgG , IgAj. etc. arc well characterized and arc known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, arc within the purview of the instant invention.
• variable region allows the antibody to selectively recognize and specifically bind epitopes on immunoreactive antigens.
• the Ni, domain and V ⁇ domain of an antibody combine to form the variable region that defines a three dimensional antigen binding site.
• This quaternary antibody structure provides for an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the V ⁇ and Vj chains.
• CDRs complementary determining regions
• variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and. if necessary, by partial framework region replacement and sequence changing.
• the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged thai the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species. It must be emphasized that it may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transler the antigen binding capacity of one variable domain to anotlier. Ratlier, it may only be necessary to transfer those residues that are necessary to maintain the activity of the antigen binding site.
• the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.
• the host cell line used for protein expression is most preferably of mammalian origin: those skilled in the art are credited with ability to preferentially detennine particular host cell lines which are best suited for the desired gene product to be expressed therein.
• Exemplary host cell lines include, but are not limited to, DG44 and DUXB1 1 (Chinese Hamster Ovary lines, DFIFR minus), HELA (human cervical carcinoma).
• CVI monkey kidney line
• COS a derivative of CVI with SV40 T antigen).
• R I 610 Choinese hamster fibroblast
• BALBC/3T3 mouse fibroblast
• H ⁇ K hamster kidney line
• SP2/O mouse myeloma
• CD23 antagonists by incubating pertcchnate, a reducing agent such as SnCL, a buffer solution such as a sodium-potassium phthalate-solution, and the CD23 antagonists.
• a particularly preferred radionuclide for direct labeling is 13 I I covalentiy attached via tyrosine residues.
• CD23 antagonists according to the invention may be derived, for example, with radioactive sodium or potassium iodide and a chemical oxidising agent, such as sodium hypochlorite. chloramine T or the like, or an enzymatic oxidising agent, such as lacloperoxidase. glucose oxidase and glucose.
• the indirect labeling approach is particularly preferred.
• Patents relating to chelators and chelator conjugates are known in the art.
• U.S. Patent No. 4,831.175 of Gansow is directed to polysubstituted diethylenctriaminepentaacctic acid chelates and protein conjugates containing the same, and methods for their preparation.
• U.S. Patent Nos. 5,099,069, 5,246,692, 5,286,850. 5,434.287 and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein in their entirety.
• Compatible chelators including the specific bifunctional chelalor used to facilitate chelation in co-pending application Serial Nos. 08/475,813, 08/475,815 and 08/478,967, are preferably selected to provide high affinity for trivalent metals. ' exhibit increased tumor-to-non-tumor ratios and decreased bone uptake as well as greater in vivo retention of radionuclide at target sites, i.e.. B-cell lymphoma tumor sites.
• target sites i.e.. B-cell lymphoma tumor sites.
• other bifunctional chelators that may or may not possess all of these characteristics are known in the art and may also be beneficial in tumor therapy.
• the CD23 antagonists may be conjugated to different radiolabels for diagnostic and therapeutic purposes.
• ⁇ 1 is a well known radionuclide used for targeted immunothcrapy.
• the clinical usefulness of , 1 l can be limited by several factors including: eight-day physical half-life; dehalogenation of iodinated antibody both in the blood and at tumor sites; and emission characteristics (e.g., large gamma component) which can be suboplimal for localized dose deposition in tumor.
• radiolabels While a great deal of clinical experience has been gained with 13 I I and 90 Y, other radiolabels arc known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging.
• additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to. 123 I, n ⁇ l, P, "Co, M Cu. 67 Cu, "Br, 81 Rb. 8 l Kr. 87 Sr, ' 'V 127 Cs, 129 Cs, ,32 I, ,97 Hg. 203 Pb, 206 Bi, 177 Lu, , 86 Rc, 2l2 Pb, , 2 Bi.
• Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy Peirersz et al. Immunol. Cell Biol. 65: 1 1 1 -125 (1987). These radionuclides include l 88 Re and 186 Re as well as l9 Au and 67 Cu to a lesser extent.
• U.S. Patent No. 5,460,785 provides additional data regarding such radioisotopes and is inco ⁇ orated herein by reference.
• CD23 antagonists may be administered as described immediately above, it must be emphasized that in other embodiments conjugated and unconjugated CD23 antagonists may be administered to otherwise healthy cancer patients as a first line therapeutic agent, in such embodiments the CD23 antagonists may be administered to patients having nonnal or a ⁇ erage red marrow reserves and/or to patients that have not, and are not. undergoing adjunct therapies such as external beam radiation or chemotherapy.
• selected embodiments of die invention comprise the administration of CD23 antagonists to myelosuppressed patients or in combination or conjunction with one or more adjunct therapies such as radiotherapy or chemotherapy (i.e. a combined therapeutic regimen).
• the administration of CD23 antagonists in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed antibodies.
• chemotherapeutic agents could be administered in standard, well known courses of treatment followed within a few weeks by the CD23 antagonists of the present invention.
• cytotoxin associated CD23 antagonists could be administered intravenously followed by tumor localized external beam radiation.
• the antagonists may be administered concurrently with one or more selected chemotherapeutic agents in a single office visit.
• a skilled artisan e.g. an experienced oncologist
• a myelosuppressed patient shall be held to mean any patient exhibiting lowered blood counts.
• blood count parameters conventionally used as clinical indicators of myclosuppresion and one can easily measure the extent to which myelosuppresion is occurring in a patient.
• Examples of art accepted myelosuppression measurements are the Absolute Neutrophil Count (ANC) or platelet count.
• ANC Absolute Neutrophil Count
• Such myelosuppression or partial myeloablation may be a result of various biochemical disorders or diseases or, more likely, as the result of prior chemotherapy or radiotherapy, h this respect, those skilled in the art will appreciate that patients who have undergone traditional chemotherapy typically exhibit reduced red marrow rcscives.
• chemotherapeutic agents that are compatible for use widi the disclosed CD23 antagonists include commercially available antibodies directed to tumor associated antigens such as Rituxan ' , Zevalin , Herceptin ', Lymphocide*, Campath ⁇ . etc.
• antineoplastic antibodies undergoing clinical trials may be used in combination with CD23 antagonists.
• IDEC-1 14 and IDEC-131 I DEC Pharmaceuticals, San Diego CA
• IDEC-131 directed to the B7 antigen and CD40L antigen respectively, may be used with the disclosed antagonists to treat selected neoplasms.
• Chemotherapeutic antibodies that are particularly useful in combination with CD23 antagonists include Y2B8 and C2B8 (ZevalinTM & Rituxan ® ) IDEC-114 and IDEC-131 , (IOEC Pharmaceuticals Corp., San Diego), Lym 1 and Lym 2. LL2 (Immunomedics Co ⁇ ., New Jersey), I IER2 (Herceptin* Gcnentech Inc., South San Francisco), Bl (Bexxar*, Coulter Pharm., San Francisco), MB1, BH3, B4, B72.3 (Cytogen Co .), CC49 (National Cancer Institute) and 5E10 (University of Iowa).
• alkyJating agents such as thiotcpa and cyclosphosphamidc (CYTOXANTM); alkyl sulfonatcs such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturcdopa, and uredopa; ethylenimines and methylamelamines including altrctamine, triethylcnemelaminc, triety encphosphoramide, uiethyienethiophosphaoramide and trimethylolomclamime nitrogen mustards such as chiorambucil, chlomaphazine, cholophosphamide.
• alkyJating agents such as thiotcpa and cyclosphosphamidc (CYTOXANTM)
• alkyl sulfonatcs such as busulfan, improsulfan and piposulfan
• estramustine if sfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mclphalan, novcmbichin, phencsterine, prednimustinc. trofosfamide, uracil mustard; nitrosureas such as carmustinc, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actino ycin, authramycin, azaserine, bleomycins. cactinomycin, calicheamicin, carabiein. carminomycin. carzinophilin, chromomycins, dactinomycin.
• daunorubicin deiorubicin. 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marccllomycin, mitomycins, mycophcnolic acid, nogalamycin, olivomycins, pcplomycin. potfiromycin. puromycin, quelamycin, rodorubicin, st ⁇ eptonigrin. streptozocin, lubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); lblic acid analogues such as denopterin.
• 5-fluorouracil 5-fluorouracil
• methotrexate methotrexate, pteroptcrin, trimetrcxate
• purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine
• pyiimidine analogs such as ancitabine, azacitidine, 6-a/auridine. carmofur. cytarabine, dideoxyuridine, doxifJuridine, enocitabine, floxuridine, 5-FU; androgcns such as calusterone, dromostanolone propionate.
• folic acid replenisher such as Irolinic acid: accglatonc; aldophosphamide glycosidc; aminolevulinic acid: amsacrine; bestrabucil; bisantrenc; edatraxate; del famine; dcmecolcine; diaziquonc; elforniUiine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurca; lcntinan: lonidamine; mitoguazone; miloxantronc; mopidamol; nitracrine; pentostatin; phcnamet; pirarubicin: podophyllinic acid; 2-ethylhydrazidc; procarbazine
• Compatible chemotherapeutic regimens of comprise combinations of drugs.
• MOPP mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazine and prednisone
• MOPP-resi tant patients ABVD (e.g , adriamycin, bleomycin, vinblastine and dacarbazinc), ChlVPP (chlorambucil, vinblastine, procarbazine and prednisone).
• CABS laomusiine, doxorubicin. bleomycin and streptozotocin
• MOPP plus ABVD MOPP plus ABVD.
• MOPP plus ABV doxorubicin, bleomycin and vinblastine
• BCVPP ca ⁇ nustine, cyciophosphamide. vinblastine, procarbazine and prednisone
• These therapies can be used unchanged, or altered as needed for a particular patient, in combination with the CD23 antagonists as described herein.
• CHOP has also been combined with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside and etoposide.
• Other compatible chemotherapeutic agents include, but are not limited to. 2-chlorodeoxyadenosine (2-CDA), 2'-deoxycoformycin and fludarabine.
• 2-CDA 2-chlorodeoxyadenosine
• fludarabine For patients with intermediate- and high-grade NHL. who fail to achieve remission or relapse, salvage therapy is used.
• Salvage therapies employ drugs such as cytosine arabinosidc, cisplatin, etoposide and ifosfamide given alone or in combination.
• IMVP-16 ifosfamide, me ⁇ iotrexate and etoposide.
• MIME methyl-gag, ifosfamide, methotrexate and ctoposide
• DHAP diexamethasone, high dose cytarabine and cisplatin
• ESHAP etoposide. mcthylpredisolone, I ID cytarabine, cisplatin
• CEPP(B) cyciophosphamide, etoposide.
• chemotherapeutic agent to be used in combination with the CD23 antagonists of the instant invention may vary by subject or may be administered according to what is known in the art. See for example, B ⁇ ice A Chabner et al., Antineoplastic Agents, in GOODMAN & OILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 ((Joel G. Ilardman et al, eds., 9 th ed. 1996).
• immunosuppressive agent refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No.
• T-cell receptor fragments (Offner et al, Science. 251 : 430-432 (1991); WO 90/1 1294; lancway, Nature, 341 : 482 (1989); and WO 91/01 133); and T cell receptor antibodies (EP 340.109) such as T10B9.
• cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
• lymphokines lymphokines. monokincs. and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone. N-methionyi human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; rclaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSI I).
• FSH follicle stimulating hormone
• TAI I thyroid stimulating hormone
• LH luteinizing hormone
• hepatic growth factor hepatic growth factor
• fibroblast growth factor prolactin
• placental lactogen tumor necrosis factor- ⁇ and - ⁇
• mullerian-inhibiting substance mouse gonadotropin-associated peptide
• inhibin activin
• vascular endothelial growth factor integrin
• thrombopoietin TPO
• nerve growth factors such as NGF-13
• platelet-growth factor transf ⁇ ning growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇
• insulin-like growth factor-I and -II erythropoietin (EPO); osteoinductive factors
• interferon such as interferon- ⁇ , - ⁇ .
• pha ⁇ naceutical compositions in accordance with the present invention comprise a pha ⁇ naceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
• therapeutic formulations comprising antagonists used in accordance with the present invention are prepared for storage by mixing an antagonist having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the fonn of lyophilized formulations or aqueous solutions.
• the CD23 neoplastic disorder may comprise solid tumors such as melanomas, gliomas, sarcomas, and carcinomas as well as myeloid or hematologic malignancies such as lymphomas and leukemias.
• the disclosed invention may be used to prophylactically or therapeutically treat any neoplasm comprising CD23 antigenic marker that allows for the targeting of the cancerous cells by the antagonist.
• Exemplary cancers that may be treated include, but are not limited to. prostate, colon, skin, breast, ovarian, lung and pancreatic.
• the antagonist may be used to treat More particularly, the antibodies of the instant invention may be used to treat Kaposi's sarcoma, CNS neoplasms (capillary hemangioblastomas. meningiomas and cerebral mctastases), mastocytoma, melanoma, gastrointestinal and renal sarcomas, rhabdomyosarcoma. ghoblastoma (preferably ghoblastoma multiforme), leiomyosarcoma, retinoblastoma, papillaiy cystadenocarcinoma of (lie ovary, Wilm's tumor or small cell lung carcinoma. It will be appreciated that appropriate antagonists may be derived for CD23 as expressed on each of the forgoing neoplasms without undue experimentation in view of the instant disclosure.
• IDEC-152 (p5E8) is a Primatized ® anti-human CD23 M ⁇ b diat contains human gamma 1 heavy chain (Lot // ZC 152-02) and Rituxan ⁇ (riluximab) is an anti-human CD20 specific mouse-human gamma 1 chimeric antibody (Lot E9107A 1; Lot D9097A1).
• the Daudi cell line was isolated from a patient wiU Burkitt ' s lymphoma (CCL-213, ATCC).
• the Raji and Ramos cell lines was also isolated from Burkitt ' s lymphoma patients (CCL-86, CRL-1596. ATCC).
• the DHL-4 was isolated from a patient diagnosed with diffuse histiocytic lymphoma (Epstein et al., Cancer, 1978, 42:2379).
• CD23 In order to demonstrate the clinical applicability of die present invention, the expression of CD23 on several different CLL samples (31 patients) was tested in whole blood by flowcytometry. Using appropriate reagents, flowcytometry was performed as substantially described in Example 1. In this respect, the expression of C 1)20 and CD23 was determined on gated cells that were CD19 + positive. Specifically, PE labeled anti-CD20 (BD Biosciences /Pha ⁇ ningen, Cat # 555623) and anti-CD23 (BD Biosciences /Pharmingen. Cat # 33615X) monoclonal antibodies were used to detect CD20 and CD23 molecules respectively.
• PE labeled anti-CD20 BD Biosciences /Pha ⁇ ningen, Cat # 555623
• anti-CD23 BD Biosciences /Pharmingen. Cat # 33615X
• Rituximab (Lot E9107A l) or IDEC-152 (Lot ZC 152-02) were used as test antibodies.
• Isotype (IgGi) matched CE9.1 (Lot M2CD4156) antibody of i ⁇ elevant specificity was used as the control. All wells were plated in triplicate into a 96 well, round bottom tissue culture plate. The effector cells were harvested, washed once with complete medium, and added at 1 x 10° cells in 100 ⁇ l volume per well to obtain a 50:1 effector to target ratio.
• control wells were also included in triplicate: target cell incubated with l OO ⁇ il complete medium to dcte ⁇ nine spontaneous release and target cell incubated with 100 ⁇ l 0.5% Triton X-100 (Sigma- Aldrich Co ⁇ .) to dete ⁇ nine maximum release.
• the culture was incubated for 4 hours at 37°C and 5%> CO 2 and the ⁇ 'Cr released in the culture supernatant due to cell lysis was dete ⁇ nined by a gamma counter (rSODATA).
• the cytotoxicity was expressed as the percentage of specific lysis and calculated as follows:
• Figure 2 shows the result of this assay and more particularly the ADCC activity of IDEC-152 and Rituxan on CD2 ⁇ 7CD23 + SKW cells.
• IDEC-152 and Rituxan showed a dose-dependent killing of SKW cells with a maximum killing of 75% achieved at 10 ⁇ g/ml and 1 ⁇ g/ml antibody concentrations respectively, indicating that Rituxan is more potent than IDEC-152 in mediating ADCC in this particular cell line.
• the antibody binding activity shown in Figure 1 suggests d at the potency differences between IDEC-152 and Rituxan is not entirely related to the antibody binding efficiency or to the epitope density of CD23 and CD20.
• background levels ( ⁇ 1()%) of ADCC were observed with isotype matched human CE9.1 control antibody (not shown).
• Fig. 3A shows that the combination of the antibodies results in substantially higher levels of cell lysis at all concentrations of Rituxan than either IDEC-152 or Rituxan alone.
• IDEC-l 52 is such an efficient mediator of ADCC that at higher concentrations (i.e. 2.5 ⁇ g/ml) any potential synergistic ' effect is swamped by the anti-CD23 antibody.
• Fig. 3B no change in cytotoxicity was observed at high concentrations of either IDEC-152 or Rituxan.
• This Example graphically illustrates the ability of the present invention to dramatically enhance the effectiveness of proven chemotherapeutic agents such as Rituxan.
• Example 6 IDEC-152 induces apoptosis in CD23 + tumor cells
• 'Table 3 shows apoptosis measured by a caspase-3 activation assay substantially as set forth in Example 1. Percent apoptosis was documented at 4 and 24 hours using mean fluorescent intensity in log scale (MFI).
• cross-linking of the antibodies of the present invention enhances their ability to induce apoptosis in tumor cells.
• One mechanism for inducing apoptosis in vivo could be mediated via the Fc receptors on various effector cells. Accordingly, in this Example cells expressing Fc receptors were used to cross-link IDEC-152 and enhance die induction of apoptosis in vitro.
• IDEC-152 The ability of IDEC-152 to induce apoptosis in CD23 + malignant B cells was further shown in vitro using SKW lymphoma cells. Apoptosis was detected by a caspase-3 assay substantially as set forth in Example 1.
• SKW cells at 0.5x10 6 cells/ml density in culture media (RPMl-2% FBS) were incubated with increasing doses of IDEC- 152 or Rituxan antibodies at 4°C in cell culture tubes on ice. After 1 hour of incubation die unbound antibody in the media was removed by centrifugation.
• the cells were resuspended in growth media in appropriate volumes and added into 24 well tissue culture plates (1.5x10° cells/well) with and without the addition of goat anti-human IgG specific secondary antibody (15 ⁇ g/ml for cross-linking). Following incubation for 18 hours, cells were harvested and analyzed for apoptosis by flowcytometry substantially as described in Example 1.
• Figs. 4A and 4B illustrate that anti-CD23 antibodies may be used to effectively induce apoptosis in 0023 ⁇ tumor cells. More specifically.
• Fig. 4A shows that increasing concentrations of cross-linked IDEC-152 result in increased apoptosis in SKW cells. At concentrations of 5 ⁇ g/mL of IDEC-152 and higher, approximately 60% of the cells underwent apoptosis during the incubation period.
• Fig. 4B serves to illustrate that cross-linking antibodies to both CD20 and CD23 can substantially increase the rate of apoptosis induction in tumor cells.
• SKW cells at lxl 0 6 cells/ml density in culture media were incubated with 10 ⁇ g/ml of p5E8 (IDEC-152) or C2B8 (Rituxan) antibodies at 4 ⁇ C in cell culture tubes. After 1 hour of incubation the unbound antibody in the media was removed by centrifugation. The cells were resuspended in growth media in appropriate volumes and added into 24 well tissue culture plates (2xl 0 6 cells/well) widi and without the addition of goat anti-human IgG specific sccondaiy antibody (50 ⁇ g/ml) to provide cross-linking. Following incubation at different time points, cells were harvested and analyzed for apoptosis by flowcytometry based Tunel assay described in Example 7. The results of this assay are graphically illustrated in Fig. 5.
• Fig. 5 shows that the cross-linking of p5E8 (IDEC-152) and Rituxan with a secondaiy anti-Ig ⁇ -specific antibody substantially enhanced apoptosis of CD23 T SKW cells.
• p5E8 (IDEC-152)
• Rituxan with a secondaiy anti-Ig ⁇ -specific antibody substantially enhanced apoptosis of CD23 T SKW cells.
• the extent of apoptosis appears to drop off over time, it remains significant for a period of two full days.
• substantial apoptosis was not observed in cells incubated with RF2 and the secondary antibody or the secondary antibody alone.
• Example 10 IDEC-152 synergizes with Rituxan to induce apoptosis in CD23 ⁇ cells
• Additional unexpected advantages of the present invention include the ability of anti-CD23 antibodies to enhance the effectiveness of various chemotherapeutic agents including biologies such as Rituxan. This Example serves to illustrate such advantages.
• this Example shows the apoptotic effects of increasing concentrations of an anti-CD23 antibody on SKW cells both alone and in combination widi Rituxan.
• concentrations of both IDEC- 152 and Rituxan were increased and the apoptotic activity of each individual antibod) was determined.
• a second experiment a fixed concentration of IDEC-152 was combined with various concentrations of Ritxuan to elucidate any synergistic effects. ' The experiments are shown in Figs. 6A and 6B respectively.
• FIG. 6A shows that both IDEC-152 and Rituxan induced apoptosis in SKW cells after cross-linking with goat F(ab') 2 anti-human IgG (GaHIg).
• Fig. 6A shows that IDEC-152 induces between 40% and 50% apoptosis at levels of approximately 1 ⁇ g/ml while Rituxan exhibits somewhat less activity.
• Fig. 6B shows that the addition of increasing amounts of Rituxan to a fixed concentration of IDEC-152 (0.1 ⁇ g/ml) enhances apoptotic activity above either of the antibodies individually.
• the addition of Rituxan to IDEC-152 at concentrations of 10 ⁇ g/m] provides apoptotic rales of approximately 45%. This observed synergistic effect dramatically underscores the advantages of the instant invention.
• Example 11 IDEC-152 enhanced Rituxan - mediated apoptosis in CD23 + cells
• Example 10 An additional experiment was performed to confirm the synergistic effects seen in Example 10 with respect to the apoptosis of SKW cells as derived from the combination of an anti-CD23 antibody and an anti-CD20 antibody.
• Fig. 7 unambiguously illustrates thai d e combination of an anti-CD23 antibody such as IDEC-152 with an anti-CD20 antibody such as Rituxan provides for enhanced apoptosis in malignant cell lines. Even at relatively low concentrations of IDEC-152 (i.e. 0.1 ⁇ ig/ml), the apoptotic rate was approximately twice that of cells incubated with Rituxan alone. Fig. 7 further shows that this effect was enhanced at higher concentrations of IDEC-152.
• Adriamycin was used in combination with IDEC-152 ratiier than Rituxan.
• Prescription grade Adriamycin RDF doxorubicin hydrochloride - NDC 0013- 1086-91
• Various concentrations of Adriamycin were combined with three different concentrations of IDEC-152 and the resulting rate of apoptosis in SKW cells was measured using the flow-cytomctry based caspase 3 assay as described in Example I . The results are shown in Fig. 8.
• compositions of the present invention are applicable to a wide range of malignancies including, in preferred embodiments, CLL.
• CLL malignancies
• PBMC Peripheral blood monocytes
• Leukemia cells from CLL patients were phenotyped and incubated with various concentrations of IDEC-152 or IDEC-152 and Rituxan on ice. After 1 hour of incubation, cells were spun down to remove unbound antibodies and resuspended in growth medium (2% FCS-RPMI) and cultured 24 well plates. The cells surface bound antibodies were cross-linked by spiking F(ab * ) fragments of goat anti-human Ig-Fc ⁇ specific antibodies at 15 ⁇ g/ml and die cultures were incubated at 37°C for 18 hours when d ey were assayed for apoptosis using the caspase assay described in Example 1. The results are shown in Figs. 10A and 10B.
• FIG. 10A confirms the results seen in Example 14 in that CD23 antagonists such as IDEC-152 may be used to induce apoptosis in leukemia cells. At 1 ⁇ g/ml IDEC-152 had effectively induced apoptosis in approximately 30% of the CLL ceils.
• Fig. 10B shows that, while IDEC-152 can induce apoptosis on its own in CLL cells, this effect may be enhanced through the addition of Rituxan. More specifically.
• Fig. 10B shows that the addition of varying concentrations of Rituxan substantially increases the percentage of cells undergoing apoptosis at the three concentrations of IDEC-l 52 tested. ' This observed synergy further accentuates the potential clinical efficacy of the present invention.
• mice were injected intravenously with SKW cells (CD2 ⁇ CD23 " ).
• SKW cells (4 x 10° viable in 100 ⁇ l HBSS buffer) were injected (iv) into the tail veins of 6-8 week old female CB17 SCID mice.
• mice were injected (ip) widi IDEC 152 in 200 ⁇ l I IBSS buffer.
• Treatment was repeated every 2 days for a total of 6 M ⁇ b injections (Q2dx6).
• Q2dx6 There were 10 animals irsed for each treatment and control (untreated, injected widi 200 ul HBSS buffer) group. Animals were observed for signs of disease and survival monitored. All mice showing signs of disease developed a paralytic form before death.
• Fig. 12 clearly shows that the CD23 antagonists of the instant invention retarded the growth of tumors in the mice and led to a dramatic increase in survival when compared with the untreated controls. Specifically, anti-tumor activity was evidenced by increased survival of tumor bearing animals over non-treated controls at all doses tested (100, 200 and 400 ⁇ g antibody per injection). At the two higher doses 50% of the animals in the treated groups were still alive at day 46 when ajl of the control animals were dead. Significantly. 30% of the treated animals in these groups were still alive when the experiment concluded twenty days after the last control animal had died (i.e. day 66). These results are significant evidence as to the efficacy of the compounds of die instant invention when used alone.
• CD23 antagonists were extremely effective tumorcidal agents when used alone, experiments were performed to explore the effectiveness of such compounds in conceit with proven chemodierapeutic agents.
• d e CD23 antagonists of the instant invention were tested in combination with Rituxan using the SKW/SCID mouse model as described in Example 17.
• the mice were injected (ip) either with IDEC 152, Rituxan, or IDEC 152 plus Rituxan in 200 ul HBSS buffer at predeteixnined times after tumor inoculation. The results of the experiment are shown in Fig. 13.

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