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WO1999036105A9 - Immunoglobuline m monoclonale radiomarquee destinee au traitement du cancer et d'une maladie auto-immune - Google Patents

Immunoglobuline m monoclonale radiomarquee destinee au traitement du cancer et d'une maladie auto-immune

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Publication number
WO1999036105A9
WO1999036105A9 PCT/US1999/000857 US9900857W WO9936105A9 WO 1999036105 A9 WO1999036105 A9 WO 1999036105A9 US 9900857 W US9900857 W US 9900857W WO 9936105 A9 WO9936105 A9 WO 9936105A9
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WIPO (PCT)
Prior art keywords
tumor
igm
antibodies
tissue
antibody
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PCT/US1999/000857
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English (en)
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WO1999036105A3 (fr
WO1999036105A2 (fr
Inventor
Paul E Borchardt
Huibert M Vriesendorp
Syed M Quadri
Original Assignee
Mca Dev B V
Paul E Borchardt
Huibert M Vriesendorp
Syed M Quadri
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Publication date
Application filed by Mca Dev B V, Paul E Borchardt, Huibert M Vriesendorp, Syed M Quadri filed Critical Mca Dev B V
Priority to EP99917293A priority Critical patent/EP1047457A2/fr
Priority to CA002318231A priority patent/CA2318231A1/fr
Priority to JP2000539876A priority patent/JP2002509122A/ja
Priority to AU35445/99A priority patent/AU3544599A/en
Publication of WO1999036105A2 publication Critical patent/WO1999036105A2/fr
Publication of WO1999036105A9 publication Critical patent/WO1999036105A9/fr
Publication of WO1999036105A3 publication Critical patent/WO1999036105A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—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 conjugates with carriers being antibodies
    • A61K51/1096—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 conjugates with carriers being antibodies radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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
    • A61K47/6869—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 the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—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 conjugates with carriers being antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention is generally in the area of cancer and autoimmune disease therapy using radiolabeled IgM antibodies specific for tumor or antibody producing cells.
  • Advanced disease which is characterized by large primary tumors and regional lymph node involvement, often requires higher doses of radiation for cure than can be tolerated by the surrounding normal tissues and while bulky disease can be removed by radical surgery, the procedure is often mutilating and accompanied by problems associated with prolonged rehabilitation and residual morbidity.
  • Ovarian carcinoma is the fourth most common cause of death from cancer in women, with 14,200 deaths annually in the U.S. (146Neijt, H . Engl, J- Med, 334, 50-51 (1996); Parker, et al., CA Cancer J. Clin. 199, 47:5- 27 (1997)).
  • the 5-year survival rate ranges from 50% for patients with tumors confined to the ovaries to less than 20% for patients with stage 3 and 4 disease (Teneriello, CA Cancer J. Clin. 45:71-87 (1995).
  • 75- 85%o of all patients have advanced disease (McGuire, Cancer (suppl) 71:1541-150 (1993)).
  • Current first-line therapy for these patients consists of cytoreductive surgery followed by i.v.
  • Intraperitoneal administration of murine IgG radiolabeled with 90 Y has demonstrated efficacy as an adjuvant (Stewart, et al., J. Clin. Oncol. 1941-1950 (1990); Maraveyas, et al., Cancer (suppl) 73:1067-1075 (1994)).
  • myelotoxicity prevented dose escalation beyond 30 mCi of activity.
  • Radioimmunotherapy employs an antibody that recognizes a tumor-associated antigen to selectively deliver therapeutic radiation to a tumor.
  • the specificity and high affinity that antibodies display towards their antigens led to optimistic expectations that radiolabeled antibodies would be "magic bullets" for cancer with very high therapeutic ratios. Pressman D., The Handbook of Cancer Immunology. Waters H (ed), Vol 5, 30-50 (Garland STPM Press, New York, 1978).
  • This initial enthusiasm ignored the pharmaco logic restraints imposed upon delivering such large molecules through the blood stream to tumors. Consequently, clinical RIT trials, particularly those for solid tumors, have been disappointing. Research has continued in order to understand the limitations of these reagents and to develop improved reagents and methodologies.
  • RIT is now on the verge of making a large impact in the systemic treatment of hematologic malignancies, but it remains ineffective in the treatment of solid tumors.
  • Systemic or intravenous RIT Intravenous (i.v.) administration of radiolabeled murine IgG for treatment of solid tumors has been limited by low tumor deposition of radioactivity, myelotoxicity and induction of human anti-mouse antibodies (HAMA). Deposition of radioactivity in tumors following i.v. administration is frequently in the range of between 0.001 and 0.01% injected dose per gram (%LD/g), which rarely produces a tumor response.
  • %LD/g injected dose per gram
  • Myelotoxicity is frequently the dose-limiting normal tissue toxicity after i.v. RIT through irradiation of bone marrow cells by blood borne immunoconjugate. Stein, et al., Br. J. Haematol. 80: 69-76, (1992); Vriesendorp, et al., Exp. Hematol. 24, 1183-1190 (1996). Most monoclonal antibodies used clinically are of murine origin and frequently induce the formation of human anti-mouse antibodies (HAMA) and an anti- antibody response in immunocompetent patients. Radioimmunoconjugates upon readministration can be bound by the anti-antibodies and prevented from reaching tumor.
  • HAMA human anti-mouse antibodies
  • Intracompartmental RIT In view of the low level of tumor response following systemic RIT for patients with solid tumors, intracompartmental RIT has been attempted using radiolabeled IgG or its fragments. Small numbers of human cancer patients with a variety of solid tumors, such as in colon cancer, glioma, and ovarian cancer have been treated through the direct administration to the tumor or physical cavity that contains tumor. The total response rate for the combined quoted studies was 31%. Riva P. et al., Int J Biol Markers 8: 192- 197 (1993). Papanastassiou V. et al., Br I Cancer 67: 144-151 (1993). Finkler NJ. et al, Gvnecol Oncol 34: 339-344 (1989).
  • IgM immunoglobulin-associated antigen
  • linked IgG dimers, trimers, etc.
  • IgG fusion proteins IgG fusion proteins
  • the antibody is IgM and the toxin is a radioisotope, most preferably u 'in- labelled IgM for diagnostic or dosimetric purposes or 90 Y-labelled IgM for therapeutic purposes.
  • mice models demonstrate effectiveness in mice models, and include protocols for treatment of peritoneal carcinomatosis and recurrent or persistent Hodgins' Disease, Kaposi Sarcoma, and head and neck cancer.
  • the antibody conjugates can also be used to treat autoimmune disease characterized by production of antibodies that produce immune complexes at defined surfaces, for example, Rheumatoid arthritis, where the conjugates diffuse into the joints to provide relief from the inflammation associated with the disorder.
  • Figure 1 is a graph of tumor response following i.l. administration of 90 Y-labeled CR4E8, as a function of tumor volume (mm 3 ) and the amount of radioactivity which is administered.
  • Figure 2 is a graph of the occurence of moist desquamation in mice treated with 90 Y-labeled CR4E8, as a function of initial tumor volume (mm 3 ) and the amount of radioactivity which is administered.
  • Figure 3 is a graph of the incidence of tumor recurrence in mice treated with i.l. 90 Y-labeled CR4E8, as a function of initial tumor volume (mm 3 ) and the amount of radioactivity which is administered.
  • IgG Intralesional or intracompartmental administration of antibody and (2) selection of an antibody that delivers a high dose of toxin, preferably radioisotope, and, due to the antibody's large size and affinity for tumor cells, stays in the general vicinity of where it is administered, as well as is targeted to the tumor.
  • toxin preferably radioisotope
  • Unmodified IgM in circulation has a half-life of only five days, less than two half-lives of yttrium-90. The decrease in IgM catabolism by antigen binding allows for the delivery of more radiation and higher clinical efficacy.
  • the tumor binding of IgM also keeps the radiation limited to the location where it is needed.
  • the best way to achieve high tumor deposition of radioactivity and avoid systemic toxicity, is to directly administer the radioimmunoconjugate at the disease site.
  • This route of administration is typically applied to diseases that stay confined over their clinical history to a compartment.
  • a compartment is defined as either a physical space like the peritoneal or pleural cavities or a large primary tumor.
  • the intralesional (i.l.) or intraperitoneal (i.p.) administration of radiolabeled IgM provides much higher levels of radioactivity in tumors. Myelotoxicity is avoided since the intralesionally or intracompartmentally administered radioimmunoconjugate remains localized in the tumor or compartment.
  • the reactivity of the antibody with tumor cell antigens coupled with the high avidity (10 antigen binding sites) and large mass of IgM (900 kDa) keeps the radioimmunoconjugate within the tumor. Because IgM is soluble, the radioimmunoconjugate should diffuse through the tumor over time. Thus, more selective high-dose radiation of irregular tumor volumes is possible with i.l. RIT than with conventional radiation therapy. RIT can result in a mixture of immunological and radiation effects.
  • RIT has an effect based on and eqiuvalent to external beam radiation but in another form. Any pathological process in the body which is susceptible to radiation but cannot be treated successfully with it due to toxicity concerns, can therefore be treated with RIT, which by definition has a higher therapeutic ratio than other non specific delivery systems of radiation.
  • rheumatoid arthritis examples include rheumatoid arthritis and restenois, as discussed in more detail below.
  • Components of RIT In vitro Several components determine the design and synthesis of a therapeutic radioimmunoconjugate: tumor antigen, an antibody that is immunoreactive with the tumor antigen, a cytotoxic agent such as a radioisotope and means for coupling the cytotoxic agent to the antibody which does not alter immunoreactivity of the antibody. These in vitro components are discussed below, followed by a discussion of the variables that influence the in vivo distribution of the radioimmunoconjugate after administration.
  • Antibodies There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Of these, only IgG and IgM have been used as radioimmunoconjugates. This is, in part, due to technical reasons since splenic B-cells from mice generate hybridomas that usually secrete IgGi and IgG 2a (Keenan, et al., J. Nucl. Med. 26:531-537 (1985)) while hybridomas produced from human lymphocytes frequently secrete IgM. IgG and IgM are very similar in structure and function. IgG is a monomer, while IgM is a pentamer composed of five identical monomers.
  • Each monomer is composed of two identical heavy chains and two identical light chains.
  • the light chains are composed of two domains: the variable domain on the amino terminus, which contains the antigen-binding site, and a constant domain on the carboxy terminus.
  • the heavy chain has a similar arrangement with one variable domain on the amino terminus end followed by three constant domains for IgG and four constant domains for IgM.
  • Each heavy chain is bound to a light chain through disulfide bonds. This association produces a more effective antigen- binding site that gives the antibody its specificity.
  • the last two constant domains of the heavy chain contain the Fc region, which regulates effector functions of the antibody.
  • Two strands composed of a light chain and a heavy chain are linked together through the heavy chains by disulfide bonds to form the monomer.
  • IgM possesses an additional polypeptide chain, the J chain, that is thought to assist the polymerization of the pentamer.
  • IgM immunoglobulin fusion proteins
  • recombinant fragments or humanized IgM antibodies can be delivered to a site or compartment where the antibody molecules will remain localized. These are collectively referred to herein as "IgM" unless specifically noted.
  • IgG and IgM can be isolated from the sera of immunized animals. These antibodies are termed polyclonal in that they arise from many different clones of antigen-stimulated B-cells. The percentage of the polyclonal antibodies that is reactive with the antigen used for immunization is low, (less than 20%>). Affinity purification is sometimes used to increase this percentage. Polyclonal antibodies are now used infrequently since it takes several months of immunizations to generate antibodies of sufficient specificity and titer, and there is lot-to-lot variability. Monoclonal Antibodies The introduction of hybridoma technology allowed the large-scale production, through cell culture techniques, of monoclonal antibodies with defined specificity and very high reactivity.
  • the resulting antibodies are termed chimeric antibodies and can still be immunogenic (Khazaeli, et al., Cancer Res. 51:5461-5466 (1991)). Further refinements through the grafting of the hypervariable regions of a mouse antibody onto the variable domains of a human antibody have produced what are termed humanized antibodies that may possess reduced immunogenicity (Jones, et al., Nature 321:522-525 (1986)).
  • Human antibodies are the preferred choice as the least immunogenic antibody for human patients (De Jager, et al. Semin. Nucl. Med. 23:165-179 (1993)). Additionally, human antibodies may be less reactive with normal human tissue antigens and be more specific for human tumor antigens. The realization that patients with malignancies may have stimulated B-cells in their tumor-draining lymph nodes and in their peripheral blood that produce immunoglobulins against their cancer has provided an important source of human B-cells (Freedman, et al., Hybridoman 10:21-33 (1991); Chen, et al., Hum.
  • tumor antigens used to target radioimmunoconjugates are almost exclusively tumor-associated antigens. These are not specific markers for tumor cells in most cases; rather, they are overexpressed on tumor cells compared with normal tissue, or they are found in association with normal fetal tissue, such as CEA (Gold, et al., J. Exp. Med. 122, 467-481 (1965)), AFP (Abelev, Adv. Cancer Res. 14, 295-350 (1971)) or with normal progenitor cells of that organ in the adult (CEA).
  • Tumor antigens can be localized in the tumor interstitium, on the tumor cell membrane, or in the tumor cell cytoplasm or nucleus.
  • the location of the tumor antigen directs the selection of the route of administration of the radioimmunoconjugate and the type of therapeutic radioisotope needed for effective tumor sterilization.
  • Antigens that are found on leukemia cells in circulation and antigens expressed on tumor neovasculature are readily accessible to i.v. admimstered reagents.
  • Antigens that are expressed on the surface of tumor cells are readily accessible to i.l. or i.p. administered radioimmunoconjugates.
  • Antigens secreted into the tumor interstitium are most accessible to i.l. administration.
  • the preferred anti-tumor antibodies are human IgM that recognize tumor associated antigens that are either expressed on the tumor cell membrane or secreted into the tumor interstitium. Table 1 lists the preferred antibodies, disease applications, and sources.
  • AC6C3-2B12 1 human 32 kDa cell membrane Adenoma (SKOV3, human (breast, colon, ovarian cell line) ovarian)
  • CR4E8' human 55 kDa cell membrane Squamous cell SW756, human cervical (cervical, head carcinoma cell line) carcinoma and neck) prostate
  • ACC 0101 murine ferritin (440 kDa), Hodgkin's disease, interstitium head and neck, hepatoma,
  • Freedman RS Ioannides CG, Tomasovic B, et al. Development of a cell surface reacting human monoclonal antibody recognizing ovarian and certain other malignancies. Hybridoma, 1991, 10: 21-33. Chen P-F, Freedman RS, Chernajovsky Y, et al. Amplification of immunoglobulin transcripts by the non-palindromic adapter polymerase chain reaction (NPA_PCR). Nucleotide sequence analysis of two human monoclonal antibodies recognizing two cell surface antigens expressed in ovarian, cervix, breast, colon and other carcinomas. Hum Antibod Hybridomas, 1994; 5: 131-142. Katano M, Jien M, Irie Reiko. Human monoclonal antibody to ganglioside GD2-inhibited human melanoma xenograft. 1984; Eur J Cancer Clin Oncol, 20; 1053-1059.
  • Radioisotopes Although described primarily with reference to radioisotopes, especially indium (“In”), which is useful for diagnostic purposes, and yttrium (“Y”), which is cytotoxic, other substances which kill cells can be substituted for the radioisotope.
  • the radioisotopes are preferred since they are small and well characterized, and can be used as diagnostics and followed after administration using standard non-invasive radioimaging techniques.
  • Non- radioisotopes can include toxins and substances which elicit the host to attack the tumor cells, as well as synthetic or natural chemotherapeutic drugs (Halpern, et al., J. Nucl. Med. 29:1688-1696 (1988); Quadri, et al., Nucl. Med. Biol. 20:559-570 (1993); Wang, et al., Radiat. Res. 141:292-302 (1995)), oligonucleotides (Mujoo, et al., Oncogene 12:1617-1623 (1996)), cytokines (Markman, Semin. Oncol. 18:248-254 (1991); Dedrick, et al., Cancer. Treat Rep.
  • radioactive colloids (Rowlinson, et al, Cancer Res. 47:6528-6531 (1987)). These can be conjugated to the antibody using standard chemical techniques, or in some cases, using recombinant technology, for example, fusion proteins.
  • Radioisotopes As radioisotopes decay, they emit characteristic photons or particles or both. Photons, commonly referred to as gamma rays, are penetrating. If their energy level is high enough, they can travel through the body and be detected by diagnostic instrumentation. Radioisotopes that emit photons can be attached to an antibody and used for diagnostic imaging. This application is termed radioimmunoscintigraphy (RIS).
  • RIS radioimmunoscintigraphy
  • Auger electrons have a very short path length (5-10 nm) and need to be internalized to be cytotoxic (Adelstein, et al., Nucl. Med. Biol. 14:165-169 (1987)). Only antibodies that are internalized after binding to a cell should be considered for radioisotopes that emit Auger electrons. Alpha particles need to be close to a cell ( within 3-4 cell diameters) to be effective
  • the radiometals ⁇ n In and 90 Y are, respectively, pure ⁇ - and pure ⁇ - emitters.
  • Iodine- 125 the most commonly used emitter of Auger electrons, has a half-life of 60 days and frequently is released by the immunoconjugate in vivo (dehalogenation) (Vriesendorp, et al., 1992).
  • the most commonly considered alpha emitters for clinical use, astatine-211 and bismuth-212 have short half-lives (7.2 h and 1.0 h, respectively) and decay into radioactive isotopes, that may not be retained by the immunoconjugate after the first alpha emission (Wilbur, Antibiot. Immunoconjug. Radiopharm. 4:85-97 (1991)).
  • Beta-emitting isotopes for RIT can be either mixed beta and gamma emitters like 131 I, with 90% gamma, and 67 Cu, with 60%> gamma (Schubiger, et al., Bioconjug. Chem. 7:165-179 (1996)) or pure beta-emitters like 90 Y and 32 P. Both types of radioisotopes have their supporters. Advocates of radioisotopes with mixed emissions like the simplicity of using the same radioimmunoconjugate at a low activity to demonstrate tumor targeting with gamma camera imaging and, by escalating the activity, administer therapy in the next treatment.
  • the pharmacokinetics of the radio-immunoconjugate should be identical for both administrations, and the first administration should be predictive for the second administration if the protein dose is held constant.
  • this simplicity comes at the cost of exposing patients to high doses of gamma rays when therapeutic activities are employed.
  • elaborate inpatient management becomes necessary to prevent the exposure of medical personal and the general public to unacceptable levels of radiation.
  • Radioisotopes can be attached directly to the antibody; others require an indirect form of attachment.
  • the radioisotopes 125 1, 131 1, 99m Tc, 186 Re and 188 Re can be covalently bound to proteins (including antibodies) through amino acid functional groups.
  • radioactive iodine it is usually through the phenolic group found on tyrosine. There are numerous methods to accomplish this: chloramine-T (Greenwood, et al. Biochem J. 89: 114-123 (1963)); and Iodogen (Salacinski, et al. Anal. Biochem. 117: 136-146
  • Tc and Re can be covalently bound through the sulfhydryl group of cysteine (Griffiths, et al. Cancer Res. 51: 4594-4602 (1991)).
  • the problem with most of the techniques is that the body has efficient methods to break these covalent bonds, releasing the radioisotopes back into the circulatory system. Generally, these methods are acceptable for imaging purposes ( 99m Tc), but not for therapeutic purposes.
  • cytotoxic compounds can be joined to proteins through the use of a reactive group on the cytotoxic compound or through the use of a cross-linking agent.
  • a common reactive group that will form a stable covalent bond in vivo with an amine is isothiocyanate (Means, et al. Chemical modifications of proteins (Holden-Day, San Francisco 1971) pp. 105-110). This group preferentially reacts with the ⁇ -amine group of lysine.
  • Maleimide is a commonly used reactive group to form a stable in vivo covalent bond with the sulfhydryl group on cysteine (Ji. Methods Enzymol 91: 580-609 (1983)).
  • Monoclonal antibodies are incapable of forming covalent bonds with radiometal ions, but they can be attached to the antibody indirectly through the use of chelating agents that are covalently linked to the antibodies.
  • Chelating agents can be attached through amines (Meares, et al, Anal. Biochem. 142:68-78 (1984)) and sulfhydral groups (Koyama Chem. Abstr. 120:217262t (1994)) of amino acid residues and also through carbohydrate groups (Rodwell, et al., Proc. Natl. Acad. Sci. 83:2632-2636 (1986); Quadri, et al, Nucl. Med. Biol. 20:559-570 (1993)).
  • chelating agents contain two types of functional groups, one to bind metal ions and the other to joining the chelate to the antibody, they are commonly referred as bifunctional chelating agents (Sundberg, et al., Nature 250:587- 588 (1974)).
  • Crosslinking agents have two reactive functional groups and are classified as being homo or heterobifunctional.
  • homobifunctional crosslinking agents include bismaleimidohexane (BMH) which is reactive with sulfhydryl groups (Chen, et al. J Biol Chem 266: 18237-18243 (1991) and ethylene glycolbis[succinimidylsucciate] EGS which is reactive with amino groups (Browning, et al., J. Immunol. 143: 1859-1867 (1989)).
  • BMH bismaleimidohexane
  • EGS ethylene glycolbis[succinimidylsucciate] EGS which is reactive with amino groups
  • An example of a heterobifunctional crosslinker is m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (Myers, et al. L Immunol.
  • In and Y are available as trivalent ions in aqueous hydrochloric acid (pH 1). They precipitate as insoluble hydroxides at pH greater than 3.4 for In and greater than 7 for Y (Brunner, et al., Radiometals and their chelates. In: Principles of Nuclear Medicine. Wagner, et al., (eds.) (2nd ed., Saunders, Philadelphia 1995)). Trivalent indium can be stabilized at neutral pH if a chelate with at least 6 donor ligands is used to saturate the coordination sites of the ion.
  • the ionic radius of trivalent yttrium is 15% larger than that of indium and requires a chelating group that can donate at least 8 ligands.
  • the polyamino-polycarboxylic ligand, diethylenetriaminepentaacetic acid (DTP A) has 8 ligands and will strongly chelate both indium and yttrium.
  • the structure of DTP A is shown below. Initially, DTP A, as a cyclic anhydride, was bound to antibody by an amide bond formed between one of DTPA's carboxylic acids and an amine on the antibody (Krejcarek, et al., Biochem. Biophys. Res. Commun.
  • the methyl group on carbon 3 provides additional stability to the coordination complex (Margerum, et al., Kinetics and mechanisms of complex formation and ligand exchange. In: Coordination Chemistry, monograph 174, Martell (ed) vol. 2 (American Chemical Society, Washington, D.C. 1978) pp. 1-220).
  • the linker is stable in vivo and, depending on the application of the radioimmunoconjugate, may increase normal tissue toxicity.
  • Linker-chelate structures that are more labile in normal tissue than tumor have been developed to decrease normal tissue toxicity (Deshpande, et al., Nncl. Med. Biol. 16:587-597 (1989); Quadri, et al., J. Nucl. Med. 34:938-945 (1993)).
  • the location of chelating groups on the immunoglobulin depends on the location and accessibility of the functional groups with which the reactive group on the linker can form a covalent bond.
  • the isothiocyanato group reacts preferentially with the epsilon amino groups of lysine.
  • the number of chelating groups attached to an antibody is controlled by the stoichiometric ratio between the chelating group and the antibody when they are reacted together.
  • Low numbers of chelating groups are desirable, typically one group for IgG and four groups for IgM, in order to lessen the chance of blocking the antigen-binding sites and keep the specific activity, once the immunoconjugate is radiolabeled, within a reasonable range (10 mCi/mg for 90 Y). Higher activities increase radiolysis, i.e. breakage of the bond between the chelate and the protein by beta emissions.
  • bifunctional chelating structures have been developed including hinge-specific (AH, et al., Bioconjug. Chem. 7:576-583 (1996)) and macrocyclic structures (Li, et al., Bioconjug. Chem. 4:275-283 (1993); Cox, et al., J. Chem. Soc. Perkin Trans. 1:2567-2576 (1990); Moi, et al., L Am. Chem. Soc. 110:6266 (1988)).
  • Hinge-specific bifunctional chelators are attached to antibodies through the sulfhydryl groups and form a single covalent bridge between two heavy chains in the hinge region.
  • the macrocyclic chelates are cyclic polyamino-polycarboxylates as opposed to an open chain like DTP A.
  • the chelation complexes formed with the macrocyclics have higher fhermodynamic stability than those formed with DTPA (Harrison, et al., Nucl. Med. Biol. 18:469-476 (1991)), but they suffer from low specific activities, awkward labeling procedures and immunogenicity (Kosmas, et al., Cancer Res. 52:904-911 (1992)). Methods and Dosages Required for In vivo Treatment
  • the targeted cytotoxic molecules can be administered intralesionally or intraregionally, so that the molecules remain in the targeted area for a time sufficient for binding to the tissue to be killed.
  • tissue which it may be desirable to kill, such as the overproliferative tissue characteristic of endometriosis, or joint linings in the case of RA or other disorders characterized by immune complex deposition, inflammation, and overproliferation of tissue.
  • the targeting molecule is designed to have two characteristics: selective binding to the targeted tissue, and large size, low diffusibility out of the region or tissue into which it is administered. Intralesional administration will typically be into the tissue to be killed, usually a solid tumor.
  • Intraregional administration will be into a cavity such as the peritoneum or lungs.
  • Types of cancer which can be treated include adenocarcinomas, squamous cell cancers, sarcomas, gschreiblastomas, melanomas, neuralblastomas, and lymphomas ( recurrent or persistent Hodgins' Disease; other than Hodgkin's lymphoma).
  • Regions or compartments to be treated include intrapleural compartments, head and neck cancer, breast, ovary, peritoneum (peritoneal carcinomatosis), brain, prostrate, as well as other solid tumors and overproliferative tissue such as endometriosis in the uterus and peritoneum.
  • administration variables must be considered, such as route of administration, administered activity, single or fractionated administration, protein dose, specific activity, predosing with unlabeled antibody.
  • the administered activity level can be based on those used in previous studies or dosimetric calculations performed using the data acquired with the diagnostic radioisotope in the RIS phase of RIT.
  • a radioimmunoconjugate can be administered once or divided into smaller activities over a short (1-2 week) period of time (i.e., fractionated). Fractionation can increase the therapeutic index by decreasing normal tissue toxicity (Schlom, et al, I. Natl.
  • High protein doses can activate complement, which will in turn produce side effects such as fevers, chills and shortness of breath.
  • tumors cells may be killed through immunologic mechanisms like complement-dependent tumor cytolysis or antibody-mediated cellular cytolysis.
  • the analysis of RIT effects on tumor becomes more complicated if the tumor response is due to a combination of immunologic and radiation effects. Higher levels of protein are also more immunogenic.
  • the specific activity of a radioimmunoconjugate relates to its immunoreactivity, stability and therapeutic efficacy. If the specific activity is too high, the large number of chelates required per immunoglobulin molecule might reduce immunoreactivity.
  • a high specific activity increases radiolysis.
  • too low of a specific activity may result in undertreatment of a tumor since unlabeled immunoconjugates will compete with radiolabeled immunoconjugates for antigen binding. This is particularly important with an antigen that is expressed at a low density on a tumor cell.
  • Predosing with cold antibody has increased tumor deposition of radioactivity in some clinical trials using i.v. administration (DeNardo, et al. Cancer 73:1023-1032 (1994)). This may occur through the saturation of receptor sites that are involved in the metabolism of immunoglobulins or through immunologic mechanisms by which the tumor becomes more accessible to the radioimmunoconjugate.
  • the demonstration of biodistribution and tumor targeting of a radio- immunoconjugate through gamma camera imaging is a major strength of RIT that is not available for other cancer treatment modalities. Imaging can be used to screen patients so that only those patients expected to benefit from therapy receive the immunoconjugate labeled with a high activity of the therapeutic radioisotope.
  • the diagnostic part of RIT can be separated from the therapeutic part of RIT by employing the same immunoconjugate twice but radiolabeled with a different radioisotope for each administration.
  • the immunoconjugate would be radiolabeled with a pure gamma-emitting radioisotope like indium-111 ("'in) or technetium-99m ( 99m Tc).
  • Both of these isotopes emit gamma rays within the appropriate energy range for imaging, (100-250 keV). Energys below this range are not penetrating enough to reach an external imaging device. Higher energy levels are difficult to collimate and provide diagnostic images with poor resolution. The short-half life of 99m Tc restricts its use to immunoconjugates with rapid tumor uptake.
  • the use of ' "in-labeled immunoconjugate has been proposed to predict the in vivo behavior of an immunoconjugate radiolabeled with 90 Y, a pure beta-emitter, since they have similar half-lives and comparable chelation chemistry (Vriesendorp, et al., Cancer.
  • An additional advantage of using two separate radioisotopes, one for imaging and one for therapy, is that it allows for outpatient treatment.
  • the low amount of radioactivity used diagnostically does not represent a radiation hazard, while the radiation emitted by a therapeutic pure beta- emitter will largely be absorbed in the vicinity of the tumor.
  • This treatment scheme is dependent on similar pharmacokinetics for both radiolabeled reagents and requires a stable means of attaching both radiometals to the antibody, which is discussed below.
  • Implicit in a treatment program that uses tumor targeting as a selection criterion for treatment is the presence of measurable disease, i.e. tumor with a diameter greater than 1 cm. Tumors smaller than 1 cm are usually not measurable by CT, by MRI or with an '"in-labeled radioimmunoconjugate.
  • measurable disease greater than 1 cm
  • 90 Y with its long-range beta emission, has the advantage of providing a higher and more homogenous tumor dose than beta-emitters with a shorter range (Leichner, et al., Med. Phys. 20:529-534 (1993)).
  • an indwelling catheter will be inserted transcutaneously. Free flow within the peritoneal cavity needs to be demonstrated with a radiographic contrast medium. If there are no serious hindrances to the dispersion of the contrast media, like adhesions, then the radioimmunoconjugate will be administered following the infusion of 1 to 1.5 1. of D5-1/4 NS (dextrose- saline solution).
  • IgM For '"in-labeled IgM, approximately 0.5 mg of IgM labeled with 1 mCi will be used.
  • the initial dose will be 1.0 mg of IgM labeled with 5 mCi.
  • the next set of patients will be administered 1.0 mg of IgM labeled with 10 mCi.
  • the last set of patients will be admimstered 2 x 1.0 mg of IgM labeled with 5 mCi.
  • Other effective dosage ranges can be determined in the same manner using routine testing. I.l. administration:
  • one or more injections will be made.
  • the initial activity will be approximately 0.01 mg of IgM labeled with 0.1 mCi of 90 Y per 1 cc 3 of tumor volume.
  • the next activity to be tested will be 0.2 mCi per 1 cc 3 of tumor volume. If this is well tolerated then the final activity will be 0.4 mCi per 1 cc 3 of tumor volume.
  • Other effective dosage ranges can be determined in the same manner using routine testing.
  • the conjugates can also be administered for treatment of disorders induced by deposition of immune complexes, such as in RA, or overproliferation of tissue.
  • RA is a chronic inflammatory arthropathy of unknown cause, but with a suspected autoimmune component in its pathogenesis.
  • RA is a serious invalidating and life shortening disease with a major socio-economic impact due to its high incidence and prevalence throughout the Western World. It is most common in women over 60 years of age, but can occur even in adolescents. Curative therapy is not available at this time. Chronic palliative therapies are used with serious iatrogenic side effects. For the painful inflammation of larger joints surgical intervention (synoviectomy) is ineffective and has been abandoned in most Centers.
  • normal human IgM which can be obtained, for example, from Centra processing blood products, that usually discard this protein for lack of clinical use
  • Y-90 and Y-90 labeled IgM instillations are used in place of colloidal Y-90.
  • the advantages of the IgM are that it is a soluble product leading to better diffusion in the joint fluids and better dose distribution for all synovial membranes.
  • the size of the IgM prevents it from entering blood vessels and leaving the intra-articular space.
  • Some radioactive IgM might translocate to lymph nodes draining the joint due to the insertion of IgM in lymphvessels through the fenestrations common to those vessels.
  • Example 1 Preparation of Therapeutic Radiolabeled anti-tumor IgMs.
  • a human monoclonal antibody secreted by the AC6C3 heterohybridoma was used in these experiments (Freedman, et al., HybridQPia 10:21-33 (1991); Chen, et al, Hum. Antibodies Hybridomas 5:131-142 (1994)). It is of the IgM isotype and is reactive with the cell membranes of human ovarian, breast and colon carcinomas, and certain other malignancies. At approximately annual intervals, the hybridoma is subjected to recloning and the secreted antibody tested for reactivity against an ovarian tumor cell line (SKOV3, American Type Culture Collection, Rockville, MD) by fluorescence-activated cell sorter (FACS) analysis. The experiments presented here were conducted with the antibody secreted by the recloned cell line, AC6C3-2B12, hereafter referred to as 2B12. An isotype matched human monoclonal IgM (CH-1B9) was utilized as an irrelevant control.
  • CH-1B9
  • the human monoclonal IgM CR4E8 (Chen, et al. Hum. Antibodies Hybridomas 5:131-142 (1994)) recognizes a 55 kDa cell surface membrane protein found on a human cervical carcinoma cell line (SW756). It is reactive with squamous cell carcinomas of both cervical and head and neck origin along with breast and colon malignancies.
  • An isotype matched human monoclonal IgM (CH-1B9) served as a specificity control.
  • hybridomas were cultured in either tissue culture flasks or spin culture flasks in Iscove's Modified Dulbecco's Medium (IMDM) with 20% v/v fetal bovine serum (FBS), 10% NCTC 109 (Life Technologies,
  • the growth media was removed by centrifuging the cultures in sterile centrifuge bottles. The media was poured off and then the cells were resuspended in the production media and placed back into sterile culture flasks.
  • the production media contains only IMDM and insulin (5 ⁇ g/ml) so it is devoid of serum immunoglobulins that copurify with the monoclonal antibodies.
  • the cell culture supernate was harvested by centrifuging the culture in sterile 500 ml centrifuge tubes.
  • the supernate was poured off and saved and then sterile filtered using a 0.22 m filter. Sodium azide, 0.005% w/v, was added to the supernate which was then stored at 4°C.
  • Antibody Purification The supernate (1-3 L) was concentrated on a CH2RS spiral cartridge concentrator (Amicon, Beverly, MA) equipped with 1 ft 2 , 30 K or 100 K MW cutoff membrane. The volume of liquid was concentrated down to less than 200 ml by running the apparatus at a pump speed of 7-8 and a back pressure of 25-30 psi. Next 1 L of 0.1 M PBS, pH 7.3, with 1 mM EDTA was added and the solution reconcentrated back down to less than 200 ml.
  • the filtration apparatus was then washed twice with approximately 200 ml of 0.1 M PBS which was collected.
  • the concentrated supernatant solution was then further concentrated using a 8200 stirred cell (Amicon, Beverly, MA) equipped with a 100 K MW cutoff membrane and charged with 10-15 psi of nitrogen gas.
  • the solution was reduced to approximately 30 ml and then the first wash from above was added.
  • the solution was reconcentrated and then the second wash was added.
  • the solution was reconcentrated to approximately 25 ml and then collected.
  • the apparatus was washed with 10 ml of PBS which was then added to the concentrated solution.
  • the solution at this point has been concentrated approximately 100- fold.
  • the metal ions were removed through chelation with the EDTA and most of the proteins and other molecules below 100 kDa in size were filtered out. Large proteins were still present which were then removed by size- exclusion chromatography using a 2.5 x 100 cm column packed with Sephacryl S-300 HR (Pharmacia Biotech, Piscataway, NJ). Approximately 5-6 ml of the concentrated antibody solution was loaded on the column and the antibodies were eluted with 0.1 M PBS containing 0.001% NaN 3 .
  • FACS Fluorescence-Activated Cell Sorter Analysis The reactivity of the immunoglobulins with the cell surface of their respective cell lines used in these experiments was demonstrated by FACS.
  • a human ovarian carcinoma cell line (SKOV3) was used for 2B12. These cells were cultured in LI 5 media containing 10%(v/v) fetal bovine serum (FBS) in the presence of 5% CO 2 at 37°C.
  • FBS fetal bovine serum
  • Cells were detached from the tissue culture flasks with Versene and washed with phosphate buffered saline (PBS) pH 7.3 containing 2% FBS and 0.02% NaN 3 (w/v). They were then incubated with 100 ⁇ l (100 ⁇ g) of human IgM for 60 min at 4°C, washed, and then incubated in a 1 :100 dilution of FITC-conjugated goat antihuman F(ab') 2 for 30 min at 4°C. The cells were then washed and resuspended in PBS with 2% FCS and 0.02% NaN followed by fixation in 2.0% (v/v) paraformaldehyde.
  • PBS phosphate buffered saline
  • the immunoconjugate was purified from unreacted ITC-2B3M-DTPA by filtration in a Centricon-30 (Amicon Corp., Beverly, MA) and washed with 0.1 M PBS, pH 7.3. Purity was ascertained by size-exclusion HPLC using a Bio-Silect SEC 250-5 column with 0.2 M sodium phosphate buffer, pH 7.2, as the mobile phase. The average number of DTP A molecules per IgM was then determined (Quadri, et al, J. Nucl. Med. 34:938-945 (1993)). Radiolabeling of IgM-2B3M-DTPA Conjugates In-Labeled Immunoconjugates
  • the '"in-labeled immunoconjugates were prepared by mixing equal volumes of 0.6 M sodium acetate buffer, pH 5.3, with 0.06 M sodium citrate buffer, pH 5.5, and the immunoconjugate solution (10 mg/ml). Typically volumes of 100 ⁇ l were used. Next pure "'lnCl 3 solution (typically 5 ⁇ 1, 1 mCi; New England Nuclear, Boston, MA) was added to the buffered immunoconjugate, mixed well, and allowed to incubate for 40 min. The radioimmunoconjugates were challenged with excess chelator by incubating the labeling mixture with 0.01 M diethylenetriamine pentaacetic acid (DTP A), pH 6.5, for 10 min.
  • DTP A diethylenetriamine pentaacetic acid
  • the radiolabeled IgM was purified on a Sephadex G50 gel column (1.5 x 20 cm) using 0.1 M PBS as the elutant. Fractions were collected and assayed with a CRC-15R dose calibrator (Capintec, Ramsey, NJ). The degree of radiometal incorporation and the purity of the radioimmunoconjugate was assessed at each step by instant thin-layer chromatography (ITLC) with saline as the mobile phase and thin- layer chromatography (TLC) using a 1 : 1 ratio of methanol and 10% (w/v) ammonium acetate in water as the mobile phase. The strips were cut in half and counted in a Cobra II gamma counter (Packard Instrument Co., Meriden, CT).
  • the 90 Y-labeled radioimmunoconjugates were prepared by mixing equal volumes of 2.0 M sodium acetate, pH 6.0, with the immunoconjugate solution (10 mg/ml). The combined volumes ranged from 100-200 1 for the biodistribution studies to 300-600 ⁇ l for the therapeutic studies. To this was added pure 90 YC1 3 solution (Battelle Pacific Northwest Laboratories, Richland, WA). Volumes and activities ranged from 1-5 ⁇ l, 2.0-5.0 mCi for the biodistribution studies and 10-20 ⁇ 1, 20-50 mCi for the therapeutic studies. The solution was mixed well and allowed to incubate for 60 min. The reaction was quenched with a hundred- fold excess of chelator using 0.01 M DTP A, pH 6.5. After a 10 min incubation, the solution was loaded onto a
  • radioimmunoconjugate solutions were sterile filtered and then diluted to the appropriate activities using sterile 0.1 M PBS, pH 7.3.
  • the radioimmunoconjugates had greater than 95% purity and were used within 2 h of their preparation.
  • Example 2 Administration of Radioisotope-labeled Antibodies into Tumors to Determine Biodistribution.
  • the purified antibodies demonstrated cell-surface reactivity by FACS of 86%) for CR4E8 versus 12% for CH-1B9.
  • Size-exclusion HPLC analysis demonstrated a purity of 98% for the immunoconjugates after derivatization. An average of four 2B3M-DTPA molecules were conjugated to each immunoglobulin.
  • after purification of the radioimmunoconjugates at least 96% of the radioactivity was antibody bound and more than 90% of the radioactivity remained bound when incubated for up to 72 h at 37°C in human serum.
  • YC1 3 Five microliters of YC1 3 (3.3-7.7 mCi) was mixed with 50 ⁇ 1 of 0.05 N NaOH. The solution was vortexed vigorously. One hundred microliters of a 12.5% solution of human serum albumin in PBS, pH 7.3, was added. The mixture was vortexed again and then allowed to incubate for 1 h. A gelatinous substance formed. An aliquot of the aggregate was resuspended with sterile 0.1 M PBS, pH 7.3, for injection into mice. The suspension was checked by ITLC and TLC to ensure that the presence of radioactivity was limited to the aggregate phase. The 90 Y-aggregate preparations were greater than 99%> pure and were administered within 1 h of their preparation. Tumor Inoculation
  • I.P. Model A human epithelial ovarian carcinoma cell line, SKOV3-NMP2 (Mujoo, et al., Oncogene 12:1617-1623 (1996)), was obtained from Dr. Kalpana Mujoo (The University of Texas M. D. Anderson Cancer Center). It was grown in MEM supplemented with 10% fetal bovine serum and 5% CO 2 at 37°C. Cells were detached with 0.25% trypsin at 37°C for 2 min. The trypsin was neutralized with media, and the cells were then centrifuged at 800 x g. The cell pellet was then resuspended in media at the required cell density.
  • mice Female athymic nude mice, 6-10 weeks old, received 0.2 ml of the cell suspension through i.p. administration using a 30-gauge needle. For the biodistribution, therapy and tumor burden experiments, mice received 2.5 x 10 6 cells. For the study of the effect of cell number on survival, mice received either 5 x 10 4 , 5 x 10 5 , 2.5 x 10 6 or 10 7 cells.
  • the human head and neck squamous cell carcinoma cell line, 886 was cultured as above. Cells were detached with 0.1 % trypsin at 37°C for 2 minutes. The trypsin was neutralized with media and the cells were centrifuged at 800 x g. The cell pellet was resuspended in media to a density of 8 x 10 7 cells per ml. Male athymic nude mice, 6-10 weeks old, were injected with 0.1 ml of cell suspension subcutaneously using a 30-gauge needle.
  • mice were housed in filter-top cages and provided with sterile food and water. Animal studies were conducted in compliance with the USDA and Animal Welfare Act. Animal protocols were approved by the Animal Care and Use Committee at the M.D. Anderson Cancer Center. Biodistribution Studies I.P. Model The reagents were administered to mice that received the tumor cells inoculum 12 days earlier. Both the i.p. and i.v. treated mice received approximately 200 ⁇ 1 of the radioimmunoconjugate. The '"in-treated mice received 10-15 ⁇ Ci, except for the mice treated for the measurement of whole-body retention of radioactivity. They received 55 ⁇ Ci. The 90 Y-IgM- treated mice received 40 ⁇ Ci. The 90 Y-aggregate was administered i.p. to the mice. They received 30 ⁇ Ci in approximately 200 ⁇ 1.
  • mice treated i.p. were euthanized at 3, 24, 48, and 96 h post injection.
  • Mice treated i.v. were euthanized at 24 and 48 h posttreatment.
  • Blood was drawn by cardiac puncture and weighed. Normal tissue and tumor nodules were excised, rinsed in PBS, blotted dry, weighed, and then counted in a gamma counter along with the blood samples. The results were corrected for radioactive decay and were expressed as percent injected dose per gram of tissue (% ID/g).
  • Tumors sizes ranged from 8-12 mm in diameter at the time of administration. All the i.l. treated mice received a single injection into the center of their tumor with approximately 10-20 ⁇ l of the radioimmunoconjugate. The i.v. treated mice received approximately 200 ⁇ l of the radioimmunoconjugate. The ' ' ' In-IgM treated mice received approximately 5 -10 ⁇ Ci of activity and the 90 Y-IgM treated mice received approximately 20 ⁇ Ci of activity. The 90 Y-aggregate treated mice received approximately 5-10 ⁇ l at an activity range of 10-20 ⁇ Ci.
  • mice treated i.l. with a radioimmunoconjugate were euthanized at 3, 24, 48, 96 and 144 h post injection.
  • Mice treated by i.v. administration were euthanized at 24 and 48 h posttreatment.
  • Mice treated i.l. with 90 Y-aggregate were sacrificed at 3, 24, 48 and 96 h post administration.
  • Blood was drawn by cardiac puncture and weighed. Normal tissue and tumors were excised, rinsed in PBS, blotted, weighed, then counted in a gamma counter along with the blood samples. The uptake of radioactivity was expressed as % ID/g. Therapy Studies Intraperitoneal Therapy
  • the volume of the injectate was 200 ⁇ l.
  • mice were screened after each administration in the dose calibrator to ensure that they retained the administered radioactivity.
  • Intralesional Therapy Nude mice bearing s.c. tumors 8-12 mm in diameter received i.l. 10 ⁇ g of the unlabeled immunoconjugate in 10 ⁇ l volume of 0.1 M PBS.
  • the % ID/g of tumors for both '"in-labeled CR4E8 (tumor-reactive) and "'in-labeled CH-1B9 (irrelevant) antibodies were obtained from the i.l. biodistribution experiments and plotted with the % ID/g as the abscissa and time as the ordinate.
  • the area under the curve for both radioimmunoconjugates was obtained by summation of the area of the trapezoids on the graphs. Since measurement of tumor response in the intraperitoneal model was not possible without either performing surgery or sacrificing the animals, an indirect way of measuring tumor response was sought.
  • the number of surviving tumor cells in the mice might be obtained by comparing their mean survival time (S 50 ) to the mean survival time of untreated mice that were inoculated with different numbers of tumor cells. The number of cells needed to produce a given S 50 can be found. This number divided by the initial injected standard number of cells, 2.5 x 10 6 in the treated mice provided the surviving fraction (SF). This demonstrates a log-linear relationship between the surviving fraction of cells in the treated mice and the administered activity.
  • This survival curve lacks a quadratic function that is typically observed after high doses of high dose rate, sparsely ionizing radiation.
  • the model commonly used to describe radiation survival curves produced by sparsely ionizing radiation is the linear-quadratic model
  • RIT allows for repair of single strand breaks.
  • RIT optimizes other modes of cell killing, such as accumulation of tumor cells in the more radiosensitive G 2 phase of the cell cycle (Marin, et al., Tnt. J. Radiat. Oncol. Biol. Phvs. 21:397-402 (1991); Knowx, et al., Radiat. Res. 135:24-31 (1992)), accelerated dose delivery or increased tumor cell kill by a process described as protracted exposure sensitization (PES) (Williams, et al. Int. J. Radiat. Oncol. Biol. Phvs. 24, 699-704 (1992)).
  • PES protracted exposure sensitization
  • a drawback of the peritoneal carcinomatosis model is that tumor volume at the time of administration of the radioimmunoconjugate is not accurately known. A direct calculation of dose (energy deposited in a given volume) is therefore not possible. Indirect estimates of tumor doses from RIT can be made by extrapolation of the observed biologic responses. In the section on surviving fraction of tumor cells, calculations are given for determining the number of surviving tumor cells after i.p. RIT. In this section, another model used to describe the shape of a radiation survival curve, the multi-target single hit model (Johns, et al., The Physics of Radiology. 4th ed. Charles C. Thomas (Springfield, IL 1983) p ⁇ .679-681) will be employed to simplify the calculations for estimating the absorbed radiation dose from i.p. RIT. In the multi-target model,
  • tumor kill is estimated to be 3.5 logs. If external beam radiation and i.p. RIT show similar surviving fractions after similar doses of radiation and if one assumes an n of 3 and a Do of 2.0 Gy as radiation survival curve characteristics of SKOV3 NMP2:, the amount of external beam radiation needed in 2.0 Gy daily fractions to obtain 3.5 log tumor cell kill is approximately 52 Gy. However, the maximum tolerated dose to whole abdomen of human patients by external beam is less than 30 Gy.
  • the current estimate for the dose delivered to tumor by RIT is between 3 and 10 Gy per 100 ⁇ Ci of 0 Y (14-52 Gy divided by 500 ⁇ Ci).
  • Tumor Autoradiography and Histology Male nude mice bearing 8-12 mm s.c. tumors were injected with 5-10 ⁇ C of either 90 Y-labeled CR or 90 Y-aggregate in a volume of 5- 10 ⁇ l. Mice were sacrificed at 3, 24, 48, 96 and 144 h. The tumors were excised, rinsed in PBS, blotted, covered with Tissue-Tek embedding medium (Miles Inc., Elkhart, IN) and then frozen at -20°C. The frozen blocks were later trimmed and mounted on cooled chucks in a refrigerated cryostat. Serial sections, 8 m thick, were taken approximately every 80 m through the entire tumor for the purpose of autoradiography.
  • Adjacent sections 4 m thick, were taken for histology. The sections were collected on slides. The histology specimens were stained with hematoxylin and eosin.
  • the autoradiographic sections were mounted in sequence on stiff paper, covered with plastic film and then placed in a cassette with a phosphorous storage screen (Molecular Dynamics, Sunnyvale, CA). The screen was exposed for 1-2 days and then read on a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). Chords were drawn across the tumor images on the screen and counts were obtained along the length of the chords. Autoradiographs were obtained by placing slides on hyperfilm-ECL (Amersham, Arlington Heights, IL) for several hours to several days to expose the film.
  • Biodistribution of CR4E8 administered either i.l. or i.v. The biodistribution data comparing i.1. and i.v. administration of '"in-labeled CR4E8 showed that intralesional administration resulted in high tumor uptake at 24 h (108% ID/g) and the uptake remained elevated at 48 h (104%) ID/g).
  • the normal organs had low uptake, with kidney (3% ID/g) and liver (2% ID/g) being the highest at 24 h and both organs at 3% ID/g at 48 h. All other organs had ⁇ 2% ID/g uptake at both time points. Blood radioactivity was ⁇ 1% ID/g at both time points. In comparison, i.v.
  • Kidney (3%> ID/g at 24 and 48 h for " 'in and 96 h for 90 Y) and liver (3% ID/g at 48 h for '"in) had the highest uptake.
  • Blood and bone radioactivity were ⁇ 0.7% ID/g at all time points for both radiometals.
  • the tumor uptake ratios of tumor-reactive to irrelevant IgM were 1.2 for 3 h, 1.3 for 24 h, 1.2 for 48 h, 1.7 for 96 h and 1.6 for 144 h.
  • Normal organ uptake of irrelevant IgM was low with kidney having the highest value (2% ID/g) at 48 and 96 h.
  • Blood radioactivity of irrelevant IgM was ⁇ 1 % ID/g at all time points. Biodistribution data comparing the i.l.
  • tumors were calculated to have received 80 Gy per 100 ⁇ Ci of 90 Y-labeled IgM.
  • Tumor regression occurred with and without damage to the overlying skin. Tumor regression was marked by a color change in the tumor from pinkish white of viable tumor to a sallow, yellowish color. Tumor ablation involved either crusting over and scabbing of the entire tumor or the tumor and surrounding normal skin were sloughed off resulting in an area of moist desquamation with a hole in the center where there once was tumor. Tumors frequently changed to a purplish pink color approximately 4-6 days after treatment prior to scabbing over. Other tumors turned whitish and necrosed from the center out.
  • Blood counts in tumor-bearing mice following treatment with a single dose of 90 Y-labeled CR4E8 were measured.
  • segmented neutrophils dropped to 40% and lymphocytes to 21% of their initial values.
  • the RBC count was 92% of the initial value.
  • segmented neutrophils and lymphocytes were both at 58% of their initial value.
  • the RBC count was 90% of the initial value.
  • Blood counts in tumor-bearing mice following treatment with a single dose of 90 Y-aggregate were measured.
  • segmented neutrophils were at 71 > and lymphocytes were at 40% of their initial values.
  • the RBC count was stable at 103% of the initial value.
  • segmented neutrophils continued to drop to 51% of the initial value. Lymphocytes had recovered and were at 98%o of the pretreatment value.
  • the RBC count was at 101% of the initial value.
  • mice treated with low activities ( ⁇ 100 ⁇ Ci) of the radioimmunoconjugate had mild skin damage in the form of reduced or absent adnexa (hair follicles, sebaceous and sweat glands). Some mice treated with higher activities had in addition to reduced or absent adnexa, fibrosis of the dermis, subcutis and skeletal muscle, vasculopathies and necrosis of skeletal muscle. The absence of skin adnexa is not indicative of radiation damage and is probably due to secondary healing of the denuded areas. The fibrosis and vasculopathies were attributed to radiation injury.
  • Fractionated administration of radioimmunoconjugate also prolonged the lives of treated mice in a dose-responsive manner if the fractions were administered within an appropriate time period. For mice receiving a low activity (75-100 ⁇ Ci) per fraction, the fractions could not be administered more than three weeks apart. With medium levels of activity (165-200 ⁇ Ci) per fraction, the interval between fractions could be between 1-3 weeks. Mice treated in this manner demonstrated an increase in survival of 13 days for every 100 ⁇ Ci of activity administered from 150 to 510 ⁇ Ci. Fractionation increased the total activity that could be administered without producing weight loss or early death while at the same time maintaining the therapeutic efficacy. High activities (300 ⁇ Ci and above) administered as a single dose produced overt toxicity. Both weight loss in excess of 10% of initial body weight and early death were noted in groups treated at such doses. In contrast, fractionated administration did not cause overt toxicity even up to 510 Ci. Blood counts were obtained from mice treated with an average of 133
  • Ci as a single dose and from mice that received 75 ⁇ Ci x 2, separated by 2 weeks. Approximately 4 weeks from the initiation of treatment, both sets of mice demonstrated an increase in their segmented neutrophils. Mice treated with a single dose had more than twice as many segmented neutrophils than the fractionated mice. Lymphocytes in both groups ofmice dropped to similar levels, 28% of the initial value. The drop in peripheral blood lymphocyte count was expected as lymphocytes are known to be radiosensitive and to die rapidly from apoptosis after receiving low doses of radiation (Anderson, et al., Adv. Immunol. 24:215-335 (1976)).
  • segmented neutrophils in the peripheral blood was unexpected and may best be explained by the secretion of a growth factor specific for segmented neutrophils by the tumor cells or other cells in the peritoneal cavity. Indeed, various growth factors have been found in malignant human ascitic fluid (Hirte, et al., Proc. Ann. Meet. Am. Assoc. Cancer Res. 35:A258 (Abstract) (1994); Hirte, et al., Proc. Ann. Meet. Am. Assoc. Cancer Res.
  • G- CSF granulocyte-colony stimulating factor
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • mice from the tumor burden study had their blood sampled at different time points. There was an increase in the segmented neutrophil count over time. In one mouse that fortuitously did not have any tumor deposits, no increase in segmented neutrophils was observed.
  • lymphocytes, granulocytes and erythrocytes after tumor inoculation and subsequent RIT indicate that the tumor has specific effects on the granulocytic line of hemopoiesis and not on hemopoietic stem cells.
  • i.p. RIT does not depress erythrocytes or lymphocytes to the same degree as granulocytes. Again, this indicates that decreased tumor burden leads to less granulocyte growth factor production and that relatively little stem cell damage was inflicted by i.p. RIT. Discussion
  • the primary goal of this study was to test the concept of intracompartmental RIT with human monoclonal tumor-reactive IgM.
  • Intralesional 90 Y-labeled CR4E8 was an effective therapeutic agent in a single administration.
  • the only significant acute normal tissue side effect noticed was moist desquamation of the skin overlying and surrounding the tumor. Fractionation experiments to decrease this toxicity were not performed.
  • the 90 Y-aggregate remained localized in the center of the tumor nodule and was a less effective therapeutic agent. It also caused less moist desquamation presumably due to the larger distance between the radioisotope and the skin, leading to a lower radiation dose to skin.
  • the dose-limiting normal tissue which remains to be defined, will probably be in the vicinity of the treated tumor. This tissue may vary, depending upon the location of the tumor and it may include vasculature, nervous, or interstitial tissue. Bone radioactivity was less than 0.7% ID/g for the tumor-reactive radioimmunoconjugates at all time point. The clearance of radioactivity from bone was faster for ' "in-labeled IgM. This may account for the higher kidney activity seen for ' "in-labeled IgM at both 24 and 48 h. A small amount of 90 Y inco ⁇ orated into bone could explain the slower bone clearance of 90 Y. The low level of bone radioactivity (0.5-0.6% ID/g) is not expected to have significant biologic effects (Lewis, et al., Hybridoma 14:115-120 (1995)).
  • the long biologic half-lives of the radioimmunoconjugates in tumor indicates that greater effective half-lives of the radioimmunoconjugates and hence radiation doses could be achieved through the use of therapeutic radioisotopes with half- lives longer than 0 Y (2.7 days). With such isotopes, the observed difference in tumor retention between tumor-reactive and irrelevant IgM, which became statistically significant at 144 h, would be of greater therapeutic significance.
  • the i.l. administration of 90 Y-aggregate produced high tumor deposition of radioactivity which remained elevated longer than 90 Y-labeled tumor-reactive IgM and resulted in a biologic half-life of 20 days in tumor. While this longer half-life suggests that higher radiation doses to tumor could be administered with 90 Y-aggregate, comparison of whole-body half- lives with tumor half-lives and examination of tumor autoradiographs predict that overall 90Y-labeled IgM will be the superior therapeutic agent.
  • the different tumor and whole body half-lives of the radioimmunoconjugates compared to the 90 Y-aggregate suggest fundamental differences in the metabolism of these reagents.
  • the tumor half-lives of the radioimmunoconjugates are longer than their whole-body half-lives. Radioactive fragments produced from the metabolism of the radioimmunoconjugates appear to be rapidly eliminated from the body. This is in contrast to what is observed with the 90 Y-aggregate where free 90 Y from 90 Y-aggregate appears to be retained in the body by transchelation to the bone matrix. Autoradiographs from sectional tumors demonstrate that tumor- reactive IgM diffuses throughout the tumor, whereas 90 Y-aggregate remains localized at the injection site. Analysis of the distribution of the counts per pixel across lines drawn on the tumor images confirmed the greater distribution of radioactivity with the radioimmunoconjugate.
  • the radioimmunoconjugate provides a more uniform dose distribution within the tumor than the 90 Y-aggregate.
  • the combined advantages of more uniform dose distribution within the tumor and rapid elimination of metabolized radioimmunoconjugate should provide a therapeutic advantage to the radioimmunoconj ugate.
  • Serial gamma camera imaging corroborated the results of the biodistribution studies. The images show that high initial tumor deposition of radioactivity is achieved with i.l. administration of radiolabeled IgM and that the radioactivity remains localized. Furthermore, the activity appeared to become more homogenous over time. Very little normal tissue uptake of radioactivity was evident with either tumor-reactive or irrelevant radiolabeled IgM.
  • Example 3 Protocol for Intraperitoneal IgM for the treatment of peritoneal carcinomatosis.
  • Patient Eligibility 1. Histologically or cytologically confirmed epithelial ovarian carcinoma with extra ovarian peritoneal involvement - colon, breast, or pancreatic cancer in the peritoneal cavity.
  • CBC with differential, platelets, PT PTT.
  • Renal function tests including BUN, creatinine and urinalysis.
  • the main objectives of this study are to establish safety and to obtain information on potential utility. Data to be obtained from this study may be used in the design of future trails on a larger number of patients.
  • the pilot trial will be stopped if tumor localization is not shown in the first 6 patients or if grade III or higher toxicity occurs in equal or more than one-third of patients.
  • the first 6 patients will receive 5 mCi of 90 Y, the next 6 patients will receive 10 mCi of 90 Y, the last 6 patients will receive 5 mCi of 90 Y 3 times at 1 week intervals.
  • Reagents
  • Antibodies AC6C3-H8 is a new human tumor cell surface reactive monoclonal antibody (HMAB) obtained by fusing lymph node lymphocytes from a patient with epithelial ovarian carcinoma (EOC) with cells of the SPAZ 4 heterohybridoma line (Freedman, et al., Hybridoma 10:21-33 (1991)).
  • This antibody is an IgM2 and recognizes a determinant expressed on the cell surface of an ovarian carcinoma cell line that can be detected by fluorescent activated cell sorter analysis (FACS) (Quadri, et al., J. Nucl. Med. 34:938- 945 (1993)).
  • FACS fluorescent activated cell sorter analysis
  • the AC6C3 does not stain nylon wool purified nonadherent peripheral blood lymphocytes or red blood cells from normal donors.
  • AC6C3 stains nonpermeabilized tissue sections of ovarian carcinoma and ovarian tumor xenografts using indirect immunoperoxidase. Most normal tissues including brain, liver, heart and kidney give negative or weak reactions to AC6C3.
  • the antibody also reacts with other adenocarcinomas including breast and colon and may therefore be described as "pan-reactive”.
  • the antibody mediates complement-dependent cytotoxicity and precipitates a 32Kd band by Western blotting immonoprecipitation experiments conducted on SKOV 3 cultured ovarian cancer cells.
  • AC6C3 may have certain advantages in climcal trials in patients with peritoneal carcinomatosis: (i) The human mAb may be used in repeated dosing schedules assuming anti-human antibodies are not produced, (ii) Although IgG mAbs may be preferred for clinical trials, IgMs may also be attractive for intracavitary treatments because of their large size and likelihood of a prolonged residence time. Prolonged residence time of the mAb in the peritoneal cavity may promote greater concentrations of the mAb on the tumor cells.
  • AC6C3-H8 is a novel entity with a potential role for in vivo diagnosis and therapy of ovarian carcinoma.
  • the media supernatant was concentrated by using Amicon Stirred Cell unit mounted with PM30 membrane at 4°C in a cold room.
  • the retentate containing IgM was purified on Sephacryl S-300 column (2.5 x 50cm) to isolate the pure IgM fraction.
  • the column was eluted with 0.05 M PBS pH 7.4 at a flow rate of 1.5 ml per minute.
  • Purity of IgM was analyze by size-exclusion HPLC using two gel filtration columns (GF 250 & GF 450; 25 x 0.94 cm i.d.) connected in tandem, and SDS-PAGE techniques. Protein concentration was measured by UV spectrophotometer at 280nm absorbence.
  • the IgM protein was sterile filtered through 0.2 ⁇ m Acrodisc and stored in aliquots of 4.0 mg/ml in PBS at 4°C in sterile tubes.
  • Human IgM AC6C3-H8 was purified and conjugated with an average of two ITCB-DTPA chelating agents per IgM molecule (Leichner, et al., Antibody Immunoconjugates and Radiopharmaceuticals 2:125-144 (1989)).
  • the purified immunoconjugates showed no loss of activity to the ovarian carcinoma cell line (SKOV 3 ) in vitro.
  • the '"in labeled immunoconjugates were injected intravenously into nude mice bearing a human ovarian carcinoma (SKOV ) xenograft.
  • the human IgM conjugate cleared rapidly from the circulation with a Tl/2 (half-life) in the blood of less than 10 hours.
  • ITCB-DTPA isothiocyanatobenzyl-DTPA derivative
  • TLC strips are monitored by a radio chromatography scanner, the percent activity bound to the antibody fraction is determined by counting TLC strip in a gamma counter.
  • HPLC Analysis Size exclusion HPLC analysis with a Bio-Sil SEC 250 column (300 x 7.8 mm i.d.) is used to determined the molecular weight and purity of radioimmunoconj ugates .
  • the column was eluted with buffer (containing 50 mM NaH 2 P0 , 50 mM Na 2 HPO 4 , 150 mM NaCl, pH 6.9) at a flow rate of 1 ml/min.
  • buffer containing 50 mM NaH 2 P0 , 50 mM Na 2 HPO 4 , 150 mM NaCl, pH 6.9
  • the radioimmunoconjugate in PBS solution is filtered through 0.2 micron Acrodisc filter and tested for lack of pyrogens by Limulus Amebocyte Lysate assay (LAL).
  • a small aliquot of the antibody batch (0.25 ml) is diluted to a final volume of 5 ml with 0.9% sodium chloride and tested for sterility using the protocol outlined by the FDA Code of Federal Regulation CFR 21, Part 210.
  • 90 Y labeled AC6C3-H8 Five, ten or 3x five mCi of 90 Y labeled AC6C3-H8 will be administered following identical methods as described for the 11 Hn labeled product.
  • 90 Y labeled IgM will only be administered if the '"in labeled IgM stays confined to the peritoneal cavity ()75% of injected dose) and tumor targeting is observed.
  • Dose ofMoAb Starting dose will be 0.5 mg. If no, or poor, imaging is observed in first six patients next dose level will be 5 mg.
  • Radioimmunoconjugate Uptake and elimination of the radioimmunoconjugate will be studied for: (1) peritoneal cavity; (2) tumor nodules; (3) blood; (4) liver and spleen; (5) kidney; (6) urine. In vivo decay of radioactivity can be determined by planar views of the gamma camera. Tumor volumes and radioactive content of tumors and smaller volume normal tissues will require SPECT. The ideal radioimmunoconjugate will target tumor, have a long residence time in the peritoneal cavity with little or no spill-over into the blood. Normal tissue uptake should be low. The data points to be obtained will allow for calculating the relative biodistribution of the radioimmunoconjugate over time in the listed compartments.
  • Tumor Dosimetry Cumulative radioactivity in tumor volumes will be calculated by area under the curve techniques. Translation/extrapolation from Indium radioactivity to Yttrium dosimetry will be made for the planned administration of 5, 10 or 3 x 5 mCi of Yttrium labeled radioimmunoconjugate per kilogram, retrospectively when the appropriate computer programs have been installed and verified. Criteria for Removal from Study:
  • Example 4 Protocol for Intralesional Radiolabeled Antiferritin IgM of recurrent or persistent Hodgkin's Disease, Kaposi Sarcoma, and Head & Neck Cancer.
  • Second line treatment with salvage chemotherapy is curative in a small group of patients.
  • Second or third line treatment with high dose chemotherapy followed by autologous bone marrow transplantation may be curative in certain patients. Patients who fail second or "higher" line treatment and those who are not eligible for bone marrow transplantation are rarely cured and are candidates for Phase I studies. Phase I-II studies with 90 Y labeled polyclonal antiferritin have been completed.
  • Kaposi's Sarcoma is a vascular tumor arising from the mid- dermis. It consists of interweaving bands of spindle cells and irregular slit- like vascular channels embedded in reticular and collagen fibers, infiltrations with mononuclear cells, and plasma cells. It was first described by the Austrian dermatologist Kaposi. Kaposi's Sarcoma (KS) occurs frequently in immunodeficient patients, such as HIV positive patients or allograft recipients. The skin, oral mucosa, lymph nodes and visceral organs such as the submucosa of the gastrointestinal tract, lung, liver and spleen may be involved.
  • KS tumors are considered radiosensitive and palliation can be obtained with doses as low as 16 Gy.
  • HNSCCa high-density squamous cell carcinoma
  • HNSCCa present with large (> 6 cm) primaries and/or large lymph nodes metastases.
  • Combination of radical surgery and high dose external beam radiation can control the disease in a proportion of the patients.
  • disease recurs in at least 30%) of patients, who then become candidates for phase I chemotherapy.
  • Distant metastases are relatively uncommon in such patients and the use of intralesional IgM could provide a new treatment option for such patients after prior high dose radiation.
  • Up to 50% of squamous cell carcinoma's of the Head and Neck appear to contain significant amounts of ferritin. Abstract
  • Treatment Plan 0.3 mCi of indium- 111 labeled antiferritin is deposited in the tumor mass.
  • Whole body gamma camera imaging will be performed at 1, 20, 40, 120, and 165 hours after administration.
  • SPECT scans will be performed of tumor containing areas at 40 and 165 hours after administration.
  • Blood and urine samples will be taken at the same time points as for whole body gamma camera imaging. All urine will be collected for the first 48 hour after administration.
  • Patients will receive 0.1 mCi per gram of tumor of yttrium-90 labeled antiferritin which will be divided in three equal volume (0.5 cc) directly into three different parts of the tumor (intralesionaly).
  • the yttrium-90 labeled antiferritin will only be given to the patients if the studies after indium- 111 administration show that 90 percent or more of the deposited radioimmunoconjugates remains localized in tumor and draining lymph nodes.
  • Blood and urine sample will be collected at 1, 20, 40, 120 and 165 hours after the yttrium-90 administration. The first 48 hours urine will also be collected. Bremsstrahlung scans will be performed at the same time points.
  • Procedures will be repeated twice with a week interval, and patients will receive a total activity of 0.3 mCi of yttrium-90 per gram of the tumor in three treatments over two weeks.
  • the yttrium-90 activity will be escalated to 0.2 mCi per gram of tumor. If an additional five patients do not show acute side effects at the 0.2 mCi per gram of tumor level, an additional five patients will be done at the 0.4 mCi per gram of tumor level. Patients with Kaposi's Sarcoma will not receive more than 0.2 mCi of yttrium-90 per gram of tumor.
  • the radioimmunoconjugate is prepared in four consecutive phases: Preparation of antiferritin antibody; Synthesis of immunoconjugate; Radiolabeling of immunoconjugate; Quality control of radioimmunoconjugate prior to clinical administration.
  • Preparation of Monoclonal Antibody is prepared in four consecutive phases: Preparation of antiferritin antibody; Synthesis of immunoconjugate; Radiolabeling of immunoconjugate; Quality control of radioimmunoconjugate prior to clinical administration.
  • the isothiocyanatobenzyl - DTPA chelate is synthesized as described above.
  • Radiolabeling of the immunoconjugates The chelate conjugated antibody is maintained at 4°C until used. Radiolabeling is accomplished by adding to an (2 mg/ml) aliquot of immunoconjugate at room temperature purified carrier-free, freshly prepared [11 InCl 3 that is dissolved in a mixture of acetate and citrate buffers at pH 5.5. Chelation of [1] In occurs within 30 minutes. The chelate is challenged by free DTPA in greater than 100 fold excess. The indium labeled immunoconjugates are purified on Sephadex G50 gel column chromatography (1.5 X 20 cm) using 0.05 M phosphate buffered saline.
  • 90 Y radiolabeling is achieved by using a acetate buffer (2.0 M), pH 6.0 at room temperature similar to the [1] In chelation.
  • 90 Y labeled immunoconjugates are purified on Sephadex G-100 column (1.5 X 30 cm) chromatography and eluted with 0.05 M PBS.
  • Radioimmunoconjugates The radioimmunoconjugate is filtered through 0.22 micron sterile filter and tested for lack of pyrogens by a Limulus Amebocyte Lysate (LAL) assay.
  • LAL Limulus Amebocyte Lysate
  • the purified antibody is tested for the presence of unconjugated [ ' ] In- DTPA by thin layer chromatography using a solvent mixture containing 10% ammonium acetate and methanol in 1 : 1 ratio (TLC silica) or 0.9% saline (ITLC silica). These solvent systems leave the labeled antibody at the origin and unconjugated [1 ] In-DTPA at R f 1.0.
  • TLC silica 10% ammonium acetate and methanol in 1 : 1 ratio
  • ITLC silica 0.9% saline
  • the radiolabeled immunoconjugates are also analyzed by size- exclusion HPLC to determine any colloid formation, and tested for radiochemical purity.
  • the integrity of the radiolabeled antibody, prior to administration, is examined using HPLC.
  • Analyses are performed using size-exclusion filtration Bio-silect SEC 250-5 (0.78 X 30 cm) column equilibrated in 100 mM sodium phosphate containing 150 mM sodium chloride, pH 6.8.
  • Antibody samples (25 ⁇ l) are applied and the column runs at a flow rate 1.0 ml/min.
  • the protein is detected by absorbance at 280 nm; fractions are collected at 1.0 min intervals and the radioactivity is measured in a gamma scintillation counter.
  • Patient will be at least 10 years of age, with a life expectancy of over 3 months, and not pregnant. Patients will be advised to practice effective birth control during study.
  • Indium-Ill labeled antiferritin IgM Under local anesthesia, three different needles (23 G) will be placed in three different parts of the tumor mass. Positioning of needle tip will be verified by two orthogonal X-rays films, real time ultrasound or CT. The selection of verification methods will depend on the anatomical location of the tumor. A volume of 0.5 cc containing 0.1 mg of antiferritin IgM labeled with 0.1 mCi of indium- 111 will be utilized per injection site. Whole body scans will be obtained by gamma camera at 1, 20, 40, 120, and 165 hours after administration. A medium energy collimator will be utilized with collection centered at the peaks of indium- 111 (173, 247 KeV).
  • SPECT scans of tumor bearing area will be performed at 40 and 165 hours. Blood and urine samples will be obtained at 1, 20, 40, 120 and 165 hours and checked for radioactivity levels. The first and second 24 hours after administration, total urine will be collected for radioactivity testing. The percent injected dose in first and second twenty four hour urine, will be determined. The amount of radiation activity present in the tumor in one hour will be determined. Effective and biological half-life of the radioimmunoconjugate in the tumor will be determined, then elimination of blood radioactivity will be determined: monophasic, biphasic T ⁇ , T ⁇ and ⁇ / ⁇ .
  • Yttrium-90 labeled antiferritin IgM Yttrium-90 labeled antiferritin IgM
  • the volume of the tumor mass will be determined by summation of all transverse CT slices encompassing the tumor.
  • the activity for administration will be 0.1 mCi per gram of the tumor (assuming the density of the tumor is 1).
  • Administration will be performed as described under 5.1.
  • the same studies will be performed after administration as given under 5.1., with the exception of using a low energy collimator and peaking of counts below 147 KeV.
  • the results obtained will be compared to the results under 5.1 to verify as well as possible that the indium- 111 labeled IgM has the same biodistribution and pharmacokinetics as the yttruim-90 labeled IgM.
  • the yttrium administration will be repeated twice, with one week intervals if the tumor remains detectable.
  • New activity will be 0.2 mCI of yttrium-90 labeled antiferritin per gram of tumor per administration X 3.
  • New activity will be 0.2 mCI of yttrium-90 labeled antiferritin per gram of tumor per administration X 3.
  • a third higher activity 0.4 mCi of yttrium- 90 labeled antiferritin per gram of tumor X 3 be utilized if the second level (0.2 mCi X 3) does not produce acute side effects in five patients.
  • the radiation dose per tumor is anticipated to be 30, 60 and 120 Gy for the 0.1, 0.2 and 0.4 mCi activity levels respectively.
  • Serum sickness or immune complex disease has not been observed in previously studied Hodgkin's Disease patients (n>80).
  • the radiation dose received by normal tissues or Hodgkin's Disease tissues from indium labeled immunoconjugate is within the diagnostic X-ray/nuclear medicine range (less than 2 rad) and will not lead to biological effects.
  • the yttrium labeled immunoconjugate is anticipated to cause tumoricidal effects and possibly some acute or delayed side effects. Side effect evaluation is described under section 8.0 - Evaluation of Toxicity.
  • Tumor response Responses will be evaluated by repeated physical examinations, CT scans, gallium-67 scans and/or indium- 111 antiferritin scans.
  • CR Complete response
  • PR partial response
  • SD stable disease
  • PD progressive disease
  • Toxicity grades will be assigned according to common toxicity grading criteria. After completion of treatment cycle, patients will be reevaluated in follow up once a month for two months. Thereafter follow up will be every two months X 2, followed by every three months X 2. Thereafter patients will be off study. Criteria for Tumor response: Acute and late side effects will be assessed.
  • Tumor responses and duration will be determined by restaging methods, that will be selected for each patient and consist of physical exam and a selection of repeat diagnostic radiology studies (chest X-ray, CT scan, head and neck, chest, abdomen, pelvis, gallium scan, bone scan, indium- 111 antiferritin scan). Only those diagnostic studies will be repeated that can provide unique information, instead of only duplicating information already known by other tests. In addition, the cheapest possible diagnostic test will be selected first.
  • Stopping rules are for toxicity. If toxicity exceeds grade 2 for two patients per group of five, that part of the study will be stopped.
  • Criteria for Removal From The Study Patients will be removed from study, in the case of progressive disease or toxicity of grade 3 or more. Patients removed from study will be followed for survival and late toxicity.

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Abstract

On a trouvé que l'on pouvait utiliser de grands agrégats d'anticorps ou des molécules telles que l'immunoglobuline M, l'immunoglobuline G conjuguée ou les protéines de fusion de l'immunoglobuline G pour traiter des tumeurs par administration intra-compartimentale ou intratumorale d'un anticorps antitumoral couplé à une toxine. Le traitement convient également contre certains troubles caractérisés par le dépôt d'un complexe immun, et notamment la polyarthrite rhumatoïde. Selon la réalisation préférée, l'anticorps est l'immunoglobuline M, et la toxine est un radio-isotope, et de façon plus préférentielle une immunoglobuline M radiomarquée par le 111In ou le 90Y. Les exemples mettent en évidence l'efficacité sur des modèles murins.
PCT/US1999/000857 1998-01-16 1999-01-15 Immunoglobuline m monoclonale radiomarquee destinee au traitement du cancer et d'une maladie auto-immune WO1999036105A2 (fr)

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JP2000539876A JP2002509122A (ja) 1998-01-16 1999-01-15 癌および自己免疫疾患のための治療における放射性標識化モノクローナルＩｇＭの使用
AU35445/99A AU3544599A (en) 1998-01-16 1999-01-15 Use of radiolabeled monoclonal igm in therapy for cancer and autoimmune disease

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