US20020102208A1 - Radiolabeling kit and binding assay - Google Patents

Radiolabeling kit and binding assay Download PDF

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US20020102208A1
US20020102208A1 US09/259,337 US25933799A US2002102208A1 US 20020102208 A1 US20020102208 A1 US 20020102208A1 US 25933799 A US25933799 A US 25933799A US 2002102208 A1 US2002102208 A1 US 2002102208A1
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Prior art keywords
antibody
cells
dtpa
kit
radiolabeling
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Paul Chinn
Ronald Morena
Michael Labarre
John E. Leonard
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Biogen Inc
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Idec Pharmaceuticals Corp
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Priority to US09/259,337 priority Critical patent/US20020102208A1/en
Assigned to IDEC PHARMACEUTICALS CORPORATION reassignment IDEC PHARMACEUTICALS CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LABARRE, MICHAEL, CHINN, PAUL, LEONARD, JOHN E., MORENA, RONALD
Priority to MYPI20000754A priority patent/MY138674A/en
Priority to NZ530868A priority patent/NZ530868A/en
Priority to SI200031085T priority patent/SI2112512T1/sl
Priority to NZ513788A priority patent/NZ513788A/xx
Priority to AT00913645T priority patent/ATE433108T1/de
Priority to HU0202522A priority patent/HU229627B1/hu
Priority to HU1300275A priority patent/HU230516B1/hu
Priority to ES09158514.1T priority patent/ES2526723T3/es
Priority to MXPA01008837A priority patent/MXPA01008837A/es
Priority to EEP200100465A priority patent/EE05650B1/xx
Priority to HK02107258.0A priority patent/HK1045730B/zh
Priority to EP09158514.1A priority patent/EP2112512B1/en
Priority to PL352531A priority patent/PL205780B1/pl
Priority to CZ20013126A priority patent/CZ303262B6/cs
Priority to AU35052/00A priority patent/AU776747B2/en
Priority to CA2742153A priority patent/CA2742153C/en
Priority to SK1216-2001A priority patent/SK287745B6/sk
Priority to SK91-2010A priority patent/SK288096B6/sk
Priority to SI200031036T priority patent/SI1192463T1/sl
Priority to IL14510900A priority patent/IL145109A0/xx
Priority to CZ2010-746A priority patent/CZ306959B6/cs
Priority to PT91585141T priority patent/PT2112512E/pt
Priority to RU2001126400/15A priority patent/RU2251110C2/ru
Priority to PT00913645T priority patent/PT1192463E/pt
Priority to BRPI0017596A priority patent/BRPI0017596B8/pt
Priority to MEP-2009-21A priority patent/ME00782B/me
Priority to DK09158514.1T priority patent/DK2112512T3/en
Priority to YU61601A priority patent/RS51778B/sr
Priority to CNB008057397A priority patent/CN1235639C/zh
Priority to KR1020017011093A priority patent/KR100729247B1/ko
Priority to ES00913645T priority patent/ES2328100T3/es
Priority to DK00913645T priority patent/DK1192463T3/da
Priority to UA2001096636A priority patent/UA78484C2/uk
Priority to BRPI0008719 priority patent/BRPI0008719B8/pt
Priority to DE60042326T priority patent/DE60042326D1/de
Priority to JP2000602634A priority patent/JP4704570B2/ja
Priority to HR20010714A priority patent/HRP20010714B1/xx
Priority to PCT/US2000/005061 priority patent/WO2000052473A2/en
Priority to CA2362119A priority patent/CA2362119C/en
Priority to EP00913645A priority patent/EP1192463B1/en
Priority to TW089103582A priority patent/TWI294969B/zh
Priority to ZA200106943A priority patent/ZA200106943B/xx
Priority to NO20014245A priority patent/NO329142B1/no
Priority to BG105957A priority patent/BG65690B1/bg
Publication of US20020102208A1 publication Critical patent/US20020102208A1/en
Assigned to BIOGEN IDEC INC. reassignment BIOGEN IDEC INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: IDEC PHARMACEUTICALS CORPORATION
Priority to US11/033,439 priority patent/US7608241B2/en
Priority to CY20091100912T priority patent/CY1109335T1/el
Priority to HK09110560.0A priority patent/HK1131208B/en
Priority to NO20100717A priority patent/NO330705B1/no
Priority to CY20151100160T priority patent/CY1116265T1/el
Abandoned legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances

Definitions

  • the present invention relates to antibody binding assays and radiolabeling kits, lyophilized cell preparations, reagents and protocols for testing the clinical efficacy of therapeutic antibodies for the treatment/imaging of tumors and tumor cells.
  • the kits of the present invention are used for making and evaluating radiolabeled antibody conjugates that will be used for the treatment and imaging of B-cell lymphoma tumors by targeting the B cell surface antigen BP35 (“CD20”).
  • the immune system of vertebrates (for example, primates, which include humans, apes, monkeys, etc.) consists of a number of organs and cell types which have evolved to: accurately and specifically recognize foreign microorganisms (“antigen”) which invade the vertebrate-host; specifically bind to such foreign microorganisms; and, eliminate/destroy such foreign microorganisms.
  • Lymphocytes as well as other types of cells, are critical to the immune system. Lymphocytes are produced in the thymus, spleen and bone marrow (adult) and represent about 30% of the total white blood cells present in the circulatory system of humans (adult).
  • T cells are responsible for cell mediated immunity, while B cells are responsible for antibody production (humoral immunity).
  • B cells are responsible for antibody production (humoral immunity).
  • T cells and B cells can be considered as interdependent—in a typical immune response, T cells are activated when the T cell receptor binds to fragments of an antigen that are bound to major histocompatability complex (“MHC”) glycoproteins on the surface of an antigen presenting cell; such activation causes release of biological mediators (“interleukins”) which, in essence, stimulate B cells to differentiate and produce antibody (immunoglobulins”) against the antigen.
  • MHC major histocompatability complex
  • Each B cell within the host expresses a different antibody on its surface—thus one B cell will express antibody specific for one antigen, while another B cell will express antibody specific for a different antigen. Accordingly, B cells are quite diverse, and this diversity is critical to the immune system. In humans, each B cell can produce an enormous number of antibody molecules (i.e., about 10 7 to 10 8 ). Such antibody production most typically ceases (or substantially decreases) when the foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B cell will continue unabated; such proliferation can result in a cancer referred to as “B cell lymphoma.”
  • T cells and B cells both comprise cell surface proteins which can be utilized as “markers” for differentiation and identification.
  • One such human B cell marker is the human B lymphocyte-restricted differentiation antigen Bp35, referred to as “CD20.”
  • CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation. Specifically, the CD20 molecule may regulate a step in the activation process which is required for cell cycle initiation and differentiation and is usually expressed at very high levels on neoplastic (“tumor”) B cells.
  • CD20 by definition, is present on both “normal” B cells as well as “malignant” B cells, i.e., those B cells whose unabated proliferation can lead to B cell lymphoma.
  • the CD20 surface antigen has the potential of serving as a candidate for “targeting” of B cell lymphomas.
  • such targeting can be generalized as follows: antibodies specific to the CD20 surface antigen of B cells are, e.g., injected into a patient. These anti-CD20 antibodies specifically bind to the CD20 cell surface antigen of (ostensibly) both normal and malignant B cells; the anti-CD20 antibody bound to the CD20 surface antigen may lead to the destruction and depletion of neoplastic B cells. Additionally, chemical agents or radioactive labels having the potential to destroy the tumor can be conjugated to the anti-CD20 antibody such that the agent is specifically “delivered” to, e.g., the neoplastic B cells. Irrespective of the approach, a primary goal is to destroy the tumor: the specific approach can be determined by the particular anti-CD20 antibody which is utilized and, thus, the available approaches to targeting the CD20 antigen can vary considerably.
  • Murine (mouse) monoclonal antibody 1F5 an anti-CD20 antibody
  • 1F5 an anti-CD20 antibody
  • Extremely high levels (>2 grams) of 1F5 were reportedly required to deplete circulating tumor cells, and the results were described as being “transient.” Press et al., “Monoclonal Antibody 1F5 (Anti-CD20) Serotherapy of Human B-Cell Lymphomas,” Blood 69/2:584-591 (1987).
  • non-human monoclonal antibodies typically lack human effector functionality, i.e., they are unable to, inter alia, mediate complement dependent lysis or lyse human target cells through antibody dependent cellular toxicity or Fc-receptor mediated phagocytosis.
  • non-human monoclonal antibodies can be recognized by the human host as a foreign protein; therefore, repeated injections of such foreign antibodies can lead to the induction of immune responses leading to harmful hypersensitivity reactions.
  • HAMA Human Anti-Mouse Antibody response
  • these “foreign” antibodies can be attacked by the immune system of the host such that they are, in effect, neutralized before they reach their target site.
  • lymphoma cells are inherently sensitive to radiotherapy. Therefore, B cell malignancies are attractive targets for radioimmunotherapy (RIT) for several reasons: the local emission of ionizing radiation of radiolabeled antibodies may kill cells with or without the target antigen (e.g., CD20) in close proximity to antibody bound to the antigen; penetrating radiation, i.e., beta emitters, may obviate the problem of limited access to the antibody in bulky or poly vascularized tumors; and, the total amount of antibody required may be reduced.
  • target antigen e.g., CD20
  • penetrating radiation i.e., beta emitters
  • Radiolabeled antibodies include the use of a radioactive substance which may require the need for precautions for both the patient (i.e., possible bone marrow transplantation) as well as the health care provider (i.e., the need to exercise a high degree of caution when working with radioactivity).
  • “Chimeric” antibodies i.e., antibodies which comprise portions from two or more different species (e.g., mouse and human) have been developed as an alternative to “conjugated” antibodies.
  • Mouse/human chimeric antibodies have been created, and shown to exhibit the binding characteristics of the parental mouse antibody, and effector functions associated with the human constant region. See e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,745; Beavers et al., U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. No.
  • chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from pre-existing murine hybridomas. Nishimura et al. (1987) Cancer Research 47: 999. The library is then screened for variable region genes from both heavy and light chains exhibiting the correct antibody fragment rearrangement patterns. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes are then expressed in a cell line of choice, usually a murine myeloma line.
  • Liu, A. Y., et al. “Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20 with Potent Fc-Dependent Biologic Activity”, J. Immun. 139/10:3521-3526 (1987), describes a mouse/human chimeric antibody directed against the CD20 antigen. See also, PCT Publication No. WO 88/04936. However, no information is provided as to the ability, efficacy or practicality of using Liu's chimeric antibodies for the treatment of B cell disorders in the reference.
  • “In2B8” conjugate comprises a murine monoclonal antibody, 2B8, specific to human CD20 antigen, that is attached to Indium[111] ( 111 In) via a bifunctional chelator, i.e., MX-DTPA (diethylenetriaminepentaacetic acid), which comprises a 1: 1 mixture of 1-isothiocyanatobenzyl-3-methyl-DTPA and 1-methyl-3-isothiocyanatobenzyl-DTPA.
  • MX-DTPA diethylenetriaminepentaacetic acid
  • Indium-[111] is selected as a diagnostic radionuclide because it emits gamma radiation and finds prior usage as an imaging agent.
  • Patents relating to chelators and chelator conjugates are known in the art.
  • U.S. Pat. No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation.
  • U.S. Pat. Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein in their entirety.
  • the specific bifunctional chelator used to facilitate chelation in application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967 was selected as it possesses high affinity for trivalent metals, and provides for increased tumor-to-non-tumor ratios, decreased bone uptake, and 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 are known in the art and may also be beneficial in tumor therapy.
  • the Y2B8 conjugate comprises the same anti-human CD20 murine monoclonal antibody, 2B8, attached to yttrium-[90] ( 90 Y) via the same bifunctional chelator.
  • This radionuclide was selected for therapy for several reasons. The 64 hour half-life of 90 Y is long enough to allow antibody accumulation by the tumor and, unlike e.g.
  • 131 I is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range of 100 to 1000 cell diameters.
  • the minimal amount of penetrating radiation allows for outpatient administration of 90 Y-labeled antibodies.
  • internalization of labeled antibodies is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target antigen.
  • the Y2B8 conjugate possesses the same advantages discussed above, e.g., increased retention of radionuclide at a target site (tumor).
  • a diagnostic “imaging” radionuclide such as 111 In, can be used for determining the location and relative size of a tumor prior to and/or following administration of therapeutic chimeric or 90 Y-labeled antibodies for the purpose of tumor reduction.
  • indium-labeled antibody enables dosimetric assessment to be made.
  • radionuclides which have been used in clinical diagnosis include 131 I, 125 I, 123 I, 99 Tc, 67 Ga, as well as 111 In.
  • Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. (1987) The use of monoclonal antibody conjugates for the diagnosis and treatment of cancer. Immunol. Cell Biol. 65: 111-125).
  • radionuclides include 188 Re and 186 Re as well as 90 Y, and to a lesser extent 199 Au and 67 Cu. I-[131] has also been used for therapeutic purposes.
  • U.S. Pat. No. 5,460,785 provides a listing of such radioisotopes and is herein incorparted by reference.
  • murine antibodies may find particular utility as diagnostic reagents.
  • a therapeutically effective dosage of the yttrium-labeled anti-CD20 antibody following administration of Rituxan® is sufficient to (a) clear any remaining peripheral blood B cells not cleared by the chimeric anti-CD20 antibody; (b) begin B cell depletion from lymph nodes; or (c) begin B cell depletion from other tissues.
  • conjugation of radiolabels to cancer therapeutic antibodies provides a valuable clinical tool which may be used to assess the potential therapeutic efficacy of such antibodies, create diagnostic reagents to monitor the progress of treatment, and devise additional therapeutic reagents which may be used to enhance the initial tumor-killing potential of the chimeric antibody.
  • additional therapeutic reagents which may be used to enhance the initial tumor-killing potential of the chimeric antibody.
  • radiolabeled proteins may be inherently unstable, particularly those labeled with radiolytic isotopes such as 90 Y, which have the tendency to cause damage to the antibody the longer they are attached to it in close proximity.
  • radiolysis causes unreliable efficiency of the therapeutic due to loss of radiolabel and/or reduced binding to the target antigen, and may lead to undesired immune responses directed at denatured protein.
  • clinicians have had no choice but to order therapeutic antibodies already labeled, or have them labeled off site at a related facility and transported in following labeling for administration to the patient. All such manipulations add precious time to the period between labeling and administration, thereby contributing to the instability of the therapeutic, while in effect decreasing the utility of radiolabeling kits in the clinical setting.
  • kits of the present invention allow rapid labeling which may be affected in approximately a half an hour or as little as five minutes depending on the label. Moreover, the kit protocols of the present invention have a labeling efficiency of over 95% thereby foregoing the need for further purification. By foregoing the need for further purification, the half-life of the radiolabel and the integrity of the antibody is reserved for the therapeutic purpose for which it is labeled.
  • kits and methods whereby diagnostic and therapeutic antibodies may be radiolabeled and administered to a patient in a reproducible, reliable and convenient manner.
  • the kits of the present invention transform the process of radiolabeling antibodies into a hassle-free, worry-free standardized process, which greatly facilitates patient treatment protocols.
  • the present kits provide advantages over the prior art in that the optimum parameters for labeling and administering therapeutic or diagnostic have been determined, thereby reducing the cost of goods. Since the kits described herein provide the optimum parameters according to the particular label, use of a kit designed for a particular label will also minimize cannibalization, i.e., which occurs when an inappropriate kit is used for a particular label. Avoiding cannibalization in turn also provides for optimum labeling efficiency. Moreover, the protocols and sterile, pyrogen-free ingredients included with each kit make for a more user-friendly process, since sterility, pyrogen testing and post-labeling purification of the reagents are obviated.
  • the present invention includes a kit for radiolabeling a diagnostic or therapeutic antibody before administration to a patient comprising at least (i) a vial containing a chelator-conjugated antibody, (ii) a vial containing formulation buffer for stabilizing and administering the radiolabeled antibody, and (iii) instructions for radiolabeling the antibody, wherein said vial components are supplied in such an amount and at such a concentration that when they are combined with a radiolabel of sufficient purity and activity according to the kit instructions, no further purification of the labeled antibody is required before administration to said patient.
  • such isotope incorporation may reach levels higher than 95%, and even as high as 98% or higher.
  • the antibody included in the kit is most preferably an anti-CD20 antibody.
  • the antibody is supplied in a form whereby it is attached to a bifunctional chelator.
  • the antibody is conjugated to MX-DTPA, but other chelators such as phenyl- or benzyl-conjugated DTPA, cyclohexyl-DTPA, EDTA derivatives and DOTA may be used.
  • a chelator according to the present invention may be any chelator that is at least bifunctional, i.e., which possesses at least two binding sites (at least one site for chelating a metallic ion and at least one site for coupling to a protein ligand).
  • the conjugated antibody is typically supplied at a concentration of 0.5 to 30 mg/ml, more preferably 2 mg/ml.
  • the volume of conjugated antibody will depend on the concentration and the amount required for optimum labeling depending on the radiolabel.
  • the conjugated antibody is to be supplied in such a volume and concentration that the entire volume will be added to the reaction vial using a sterile syringe and aseptic technique. This will allow for increased reproducibility and ease of use. All reagents of the kits disclosed herein are sterile and pyrogen-free, and specifically designed for simplicity and speed in advancing directly from antibody testing to administration. With some labels, the need for testing labeling efficiency may not be required.
  • a particularly advantageous component of the kit is the formulation buffer for stabilizing against the effects of radiolysis and administering the radiolabeled conjugated antibody to a patient.
  • the formulation buffer is a pharmaceutically acceptable carrier which serves as both a diluent for the labeled antibody and an administration buffer.
  • any pharmaceutically acceptable diluent may be used for administering therapeutic or diagnostic antibodies to patient, the formulation buffer of the present invention is particularly suited for administering radiolabeled antibodies.
  • the formulation buffer of the present invention comprises a radioprotectant such as human serum albumin (HSA) or ascorbate, which minimize radiolysis due to yttrium, and to a lesser degree, indium.
  • a radioprotectant such as human serum albumin (HSA) or ascorbate
  • HSA human serum albumin
  • Other radioprotectants are known in the art and could also be used in the formulation buffer of the present invention, i.e., free radical scavengers (phenol, sulfites, glutathione, cysteine, gentisic acid, nicotinic acid, ascorbyl palmitate, HOP(:O)H 2 , glycerol, sodium formaldehyde sulfoxylate, Na 2 S 2 O 5 , Na 2 S 2 O 3 , and SO 2 , etc.).
  • free radical scavengers phenol, sulfites, glutathione, cysteine, gentisic acid, nicotinic acid,
  • radioprotectants are generally employed in the formulation buffer to protect the antibody from radiolysis, it may be possible to affect further protection by including the radioprotectant in the reaction buffer as well. This has generally not been done before, i.e., with HSA, due to the presence of metals which would interfere with the labeling process. However, it may be possible to “clean” the HSA using a chelating resin such that it could be included in the reaction buffer as well. Ascorbate or other radioprotectants may also need to be treated to remove contaminating metals.
  • the formulation buffer of the present invention also comprises excess unconjugated chelator.
  • the purpose for including unconjugated chelator is that this chelator serves to scavenge any non-protein bound radiolabel in the patient, and effects excretion of the radiolabel thereby reducing uptake of “bone-seeking” isotopes, i.e., 90 Y, by the bones of the patient.
  • excess DTPA or any other chelator may be included in the formulation buffer.
  • the formation buffer is also preferably supplied in a volume such that the entire contents are transferred to the reaction vial. As discussed above, this results in increased ease of use and reproducibility because exact volumes do not have to be measured and transferred.
  • a preferred formulation buffer comprises phosphate buffered or physiological saline, human serum albumin and DTPA.
  • the human serum albumin is preferably at a concentration of between about 1 to 25% (w/v), and more preferably at a concentration of about 7.5% (w/v).
  • the concentration of DTPA is preferably about 1 mM.
  • Ascorbate may be used as an alternative to human serum albumin, and is typically used at a concentration of about 1 to 100 mg/ml. Although a wider range of concentrations may be used without compromising patient safety.
  • the antibody of the radiolabeling kit is readily labeled with a radioisotope of choice via a bifunctional chelator according to the methods of the present invention.
  • the kit of the present invention may also include a vial containing a buffer for adjusting the pH of the radioisotope solution, and a sterile glass reaction vial for performing the labeling and subsequently for resuspending the final radiolabeled antibody in formulation buffer.
  • a 10 ml reaction vial is typically sufficient, but vials capable of holding 5 to 20 mls may also be used.
  • the buffer is preferably a low-metal sodium acetate solution at a concentration of 10 to 1000 mM, most preferably 50 mM.
  • a specific kit of the present invention comprises the MX-DTPA conjugated antibody, 2B8-MX-DTPA.
  • 2B8 is an anti-CD20 antibody shown to affect B cell depletion upon administration to lymphoma patients.
  • the radiolabeling kit of the present invention may be optimized for the radiolabeling of other anti-CD20 antibodies, or any other antibody which has been conjugated to DTPA or other polyvalent chelator.
  • the preferred kit of the present invention may comprise at least (i) a vial containing the MX-DTPA conjugated 2B8 antibody, either in solution or lyophilized (requiring reconstitution); and (ii) a vial containing formulation buffer for administering the radiolabeled antibody to a patient.
  • the preferred kit will also contain (iii) a buffer for adjusting isotope pH, and (iv) a reaction vial.
  • the buffer is supplied in the reaction vial, thereby eliminating the steps of measuring and transferring the buffer and increasing the simplicity, consistency and sterility of the kit components.
  • the buffer is added to the isotope vial first, and the buffered isotope is then transferred to the reaction vial.
  • the reaction vial could be supplied with the required antibody volume.
  • the isotope/buffer vial could be made large enough to accommodate addition of the antibody conjugate, i.e., directly to the supplier's vial. This would eliminate the need for the reaction vial.
  • the reaction vial itself contains the required volume of conjugated antibody (i.e., 1 or 1.5 mL for 111 In and 90 Y, respectively).
  • the antibody may be supplied in a buffer that provides the appropriate radiolabeling pH according to the specific desired isotope (i.e., pH 3-6 for 111 In, pH 3-5 for 90Y). Different buffers may be used, depending on the isotope (i.e., sodium acetate for 90 Y, sodium citrate for 111 In).
  • the pH and composition of the buffer may also vary depending on the nature of the binding ligand to be labeled (i.e., labeling peptides may permit ⁇ pH 3 to be used).
  • the isotope would be transferred directly to the reaction vial, as would the formulation buffer. Limiting use of the kit to two transfer steps would further increase reproducibility and simplicity, and further decrease the chance for contamination of sterility during manipulation of the kit components.
  • the radiolabeling kits of the present invention may further comprise a vial of radioisotope, or radioisotope may be ordered separately from an appropriate supplier.
  • Preferred radioisotopes of the present invention are 111 In chloride and 90 Y chloride in HCl although the disclosed methods are not limited to these isotopes.
  • Other radionuclides that have been used for imaging applications are known in the art, i.e., as described in U.S. Pat. Nos. 4,634,586, 5,460,785 and 5,766,571, which are herein incorporated by reference.
  • Indium-[111] is particularly advantageous for imaging B cell tumors and beta emitters such as 90 Y are particularly useful as radiotherapeutic agents.
  • other radioisotopes suitable for these or other purposes i.e., alpha emitters, may be used depending on the chelator used for antibody conjugation.
  • kits embodiment will also include a separate vial of chimeric antibody, i.e., Rituxan®, to be administered before or after the radiolabeled anti-CD20 antibody.
  • chimeric antibody i.e., Rituxan®
  • the HAMA response which might generally occur in response to administration of a murine anti-CD20 antibody may be significantly decreased, thereby increasing the therapeutic utility of radiolabeled murine antibodies.
  • chimeric anti-CD20 is employed to clear circulating B cells, subsequent diagnostic images achieved with 111 In-labeled antibodies may be much clearer.
  • kits of the present invention may include separate, perhaps color-coded, buffer vials specifically formulated according to the optimum pH requirements for radiolabeling antibodies with the specific radioisotopes to be used. Such a system would ensure that the appropriate buffer was used for each label, and would allow the clinician the same ease in radiolabeling the two antibodies as if two kits had been purchased. Such a kit in effect combines the components from two radiolabeling kits into one.
  • the components of the radiolabeling kit of the present invention are supplied at the appropriate concentration and pH so that sterility is readily maintained before antibody administration and there is little need for additional buffers or media.
  • some of the reagents can be prepared, sterilized and tested for sterility on site.
  • variations of the kit of the invention are envisioned depending on the budget and preference of the consumer.
  • the radiolabeling kit of the present invention may be used in a method for radiolabeling a chelator-conjugated antibody for administration to a patient.
  • a method for radiolabeling a chelator-conjugated antibody for administration to a patient comprises, in general, (i) mixing a chelator-conjugated antibody with a solution containing a radioisotope; (ii) incubating the mixture for an appropriate amount of time at appropriate temperature; and (iii) diluting the labeled antibody to an appropriate concentration in formulation buffer, such that the radiolabeled antibody may be administered directly to a patient without further purification.
  • the antibody is an anti-CD20 antibody, and in particular, the anti-CD20 antibody may be 2B8.
  • the antibody may be conjugated to any appropriate chelator, i.e., MX-DTPA, CHX-DTPA, phenyl- or benzyl-DTPA, DOTA, EDTA derivatives, etc. MX-DTPA is preferred. Methods for affecting antibody conjugation are known in the art (Kozak et al. (1989); Mirzadeh et al. (1990), Brachbiel et al. (1986)).
  • the present inventors have found that the method of radiolabeling a chelator-conjugated antibody works best wherein the solution containing the radiolabel is adjusted to a pH of between about 3.0 and 6.0, and more preferably to about 4.2 before it is mixed with the chelator-conjugated antibody.
  • Low-metal sodium acetate is particularly preferred for adjusting the pH, although other buffers may be used.
  • the sodium acetate is at a concentration of between about 10 and 1000 mM, and more preferably 50 mM.
  • the volume quantity of 111 In chloride which should be used to prepare a single administrative dose is typically about 5.5 mCi divided by the radioactivity concentration at the time of labeling.
  • a typical diagnostic dose of 111 In is about 2 to 10 mCi.
  • the quantity of sodium acetate used for adjusting the pH varies depending on the sodium acetate concentration and the isotope carrier solution, and may therefor be quite broad.
  • the concentration of sodium acetate is 50 mM
  • the amount required for adjusting the pH is typically about 1.2 times the volume quantity of 111 In chloride used although larger volumes may be used.
  • the ratio of sodium acetate to HCl is what is important, and the amount of sodium acetate used would change depending on the amount and concentration of HCl in the buffer.
  • About 1 ml of a chelator-conjugated antibody at a concentration of about 2 mg/ml is then mixed with the radiolabel acetate solution, and the mixture is incubated for about 30 minutes, or for a time sufficient to achieve optimal labeling of the antibody. Such time may range from about 30 seconds to about 60 minutes.
  • Formulation buffer is then added in an amount necessary to achieve a total final volume of about 10 ml.
  • the optimum time required for labeling the antibody may vary depending on the antibody, the particular radiolabel and the particular conjugate employed.
  • An underlying factor in the optimization of the time allotted for radiolabeling is the chelator to antibody ratio of the reagent which is to be labeled.
  • the chelator to antibody ratio must be high enough to achieve a therapeutically useful level of incorporation, i.e., 90 to 95% depending on the radioisotope, but must also not be too high such that the structural integrity or immunoreactivity of the antibody is compromised. This requires a certain balancing process that in some cases may lead to a lower level of conjugated chelator and longer labeling time.
  • the volume quantity of 90 Y chloride used for preparing a single administrative dose typically ranges from about 10 to 50 mCi, and is preferably about 45 mCi, divided by the radioactivity concentration at the time of labeling.
  • the quantity of sodium acetate used for adjusting the pH varies depending on the sodium acetate concentration and the concentration of isotope carrier, and may therefor be quite broad.
  • the concentration of sodium acetate is 50 mM and the 90 Y is supplied in 50 mM HCl
  • the amount required for adjusting the pH is typically about 1.2 times the volume quantity of 90Y chloride used.
  • a chelator-conjugated antibody at a concentration of about 2 mg/ml is then mixed with the radiolabel acetate solution, and incubated for about 5 minutes, or for a time sufficient to achieve optimal labeling of the antibody. Such time may range from about 30 seconds to about 60 minutes.
  • Formulation buffer is added in an amount necessary to achieve a total final volume of about 10 ml.
  • the radiolabeling method of the invention is performed using the radiolabeling kit described herein.
  • the preferred components and conditions are merely acceptable guidelines for practicing the method of the invention, and may be altered to some degree with appropriate optimization. Conditions which depart from those preferred but still accomplish the purpose of the method are considered to be within the scope of the invention.
  • the radiolabeling kit of the present invention may also be supplied with reagents suitable for conveniently verifying the binding affinity of the antibody following radiolabeling.
  • the kit of the invention may also be used for determining the percent binding of a radiolabeled antibody to its target cell before administering the antibody to a patient.
  • the present inventors have also found that the particular binding assay kit disclosed may be useful for testing the affinity of any antibody generally for which purified antigen is not available. Accordingly, the binding assay components may also be sold as a separate kit.
  • a binding assay and radiolabeling kit comprises (i) at least one vial of lyophilized cells which express the antigen which is recognized by the antibody in the kit; (ii) a vial containing chelator-conjugated antibody; (iii) a vial containing formulation buffer, and (iv) instructions for radiolabeling the antibody such that the radiolabeled antibody may be administered directly to a patient without the need for subsequent purification.
  • this kit may also comprise a vial containing a buffer for adjusting the pH of the radioisotope, and a sterile glass reaction vial.
  • the buffer is a low-metal sodium acetate solution at a concentration of between about 10 and 1000 mM, and the glass reaction vial holds a volume of at least 5 ml.
  • the antibody is preferably an anti-CD20 antibody, and the chelator is preferably MX-DTPA. Other chelators may be used as described previously.
  • the preferred conjugated antibody is 2B8-MX-DTPA, although any chelator-conjugated antibody may be labeled and its affinity assessed.
  • the formulation buffer is phosphate buffered saline comprising a radioprotectant and unconjugated chelator as described above, and radioisotope may or may not be included and is preferably 111 In chloride or 90 Y chloride. Other radioisotopes may be used depending on the chelator.
  • the difference between the binding assay/radiolabeling kit and the radiolabeling kit described above is the inclusion of antigen-positive cells to serve as a substrate target for testing antibody affinity.
  • antigen is CD20
  • preferred CD20-positive cells are SB cells (ATCC # CCL 120) but any CD20-positive cells may be used.
  • the binding assay and radiolabeling kit may further include antigen-negative cells for use as a negative control.
  • Preferred CD20-negative cells are HSB cells (ATCC # CCL 120.1) but any CD20-negative cells may be used.
  • the combined radiolabeling and binding assay kit may further comprise a vial of chimeric anti-CD20 antibody in addition to the antibody to be labeled for the purposes of affecting a combined therapeutic regimen, or for clearing peripheral B cells prior to diagnostic imagery.
  • a separate antibody is preferably Rituxan®, but may be any antibody shown to effectuate tumor cell killing.
  • two different types of antibodies may be combined in one kit, i.e., antibodies directed to two different B cell antigens, so long as the combined therapeutic regimen serves to target the same type of cell, i.e., the B cell lymphoma.
  • the binding assay and radiolabeling kit of the present invention is particularly suited for the commercial setting in that the target cells are provided in lyophilized form. This allows the verification of antibody efficacy to proceed simply and systematically, and negates the hassle and expense involved in maintaining tissue culture facilities.
  • the lyophilized cells are generally supplied in aliquots of between 0.5 and 500 ⁇ 10 6 cells per vial according to the methods of the invention.
  • the present invention also provides for a binding assay kit for determining the percent binding of a radiolabeled antibody to its target cell.
  • a binding assay kit for determining the percent binding of a radiolabeled antibody to its target cell.
  • Such a kit includes at least one vial of fixed and/or lyophilized antigen-positive cells, and may optionally contain antigen-negative cells as described above for the binding assay and radiolabeling kit.
  • variations of such a kit may include an unlabeled control antibody for verifying the binding specificity of the consumer's antibody via a competitive assay.
  • the CD20-positive cells are preferably SB cells (ATCC # CCL 120) and the CD20-negative cells are preferably HSB cells (ATCC # CCL120. 1), which are supplied in lyophilized form in aliquots of between 0.5 and 50 ⁇ 10 6 cells.
  • the antibody is preferably an MX-DTPA conjugate of 2B8 labeled with 111 In or 90 Y.
  • the present inventors have also found that the method used for preparing fixed and lyophilized cells for the binding assay kits of the present invention is particularly suitable for preparing cells for commercial kits.
  • Cells may be fixed prior to lyophilization to improve structure/stability.
  • the cells of the present invention demonstrate high reproducibility when used for antibody binding assays.
  • the present invention includes a method of preparing lyophilized cells comprising (i) harvesting cells at a cell density of 0.5 to 2 ⁇ 10 6 cells per ml by centrifugation; (ii) washing cells at least one time in a balanced salt solution, i.e., HBSS; (iii) resuspending pelleted cells in a lyophilization buffer comprising a balanced salt solution containing carrier protein and at least one type of sugar; (iv) dispensing an aliquot of resuspended cells into a microfuge tube or a glass vial; and (v) lyophilizing the cells 12-96 h and more preferably 24-72 h at about 30-60 millitor.
  • the method is particularly suitable for preparing lyophilized cells wherein said cells are SB cells (ATCC # CCL 120) or HSB cells (ATCC # CCL 120.1), but is likely applicable to other cell types as well.
  • the buffer generally contains bovine serum albumin as the carrier protein at a concentration of 1% (w/v) and mannitol at a concentration of 10%.
  • carrier proteins i.e., HSA, and other sugars may be used.
  • the cells are harvested by centrifugation at a speed of about 1300 rpm, and the salt solution HBSS (Hank's balanced salt solution) is added.
  • the cells are generally resuspended at a concentration of 50 ⁇ 10 6 cells per ml.
  • the above conditions may be modified slightly without significantly compromising cell viability.
  • the above conditions may be supplemented by additional procedures designed to optimize the process for larger quantities of cells, e.g., tangential flow diafiltration to exchange cells into the lyophilization buffer.
  • the binding assay kits of the present invention may be used in an assay for assessing the binding affinity of a radiolabeled antibody. Such an assay is also a subject of the present invention.
  • a binding assay for determining the percent binding of a radiolabeled antibody to its target cell comprises in general the following steps: (i) mixing at least one aliquot of a radiolabeled antibody with at least one aliquot of antigen positive cells; (ii) mixing at least one aliquot of a radiolabeled antibody identical to the aliquot of step (i) with at least one aliquot of dilution buffer of the same volume as the aliquot of antigen-positive cells in step (i) as a control; (iii) pelleting the cells by centrifugation; (iv) measuring the radioactivity in the supernatant of the pelleted cells and the control; and (v) comparing the quantity of radioactivity in the cell supernatant to the quantity of radioactivity in the control.
  • the binding assay of the present invention is typically performed with antibodies labeled with 111 In or 90 Y.
  • 111 In is the radiolabel
  • radioactivity in the assay tubes is measure using a gamma counter.
  • 90 Y is the label, radioactivity is measured using a scintillation counter, although a gamma counter could be used.
  • the preferred antibody is an anti-CD20 antibody
  • the anti-CD20 antibody is preferably 2B8, wherein the 2B8 antibody is labeled using the radiolabeling kit of the present invention.
  • any radiolabeled antibody may be tested provided cells expressing the particular antigen are available.
  • CD20 is the antigen
  • the preferred cells for performing the assay are SB cells (ATCC # CCL 120), however, the assay may also be optimized and performed with any radiolabeled antibody and appropriate target cell.
  • the dilution buffer used for the assay should maintain binding of the antibody, i.e., physiological buffer, possibly containing a carrier protein, e.g. BSA, to minimize non-specific binding to cells.
  • a carrier protein e.g. BSA
  • a further control may be included in the assay by using antigen-negative cells.
  • the binding assay further comprises the following steps: (i) mixing at least one aliquot of a radiolabeled antibody with at least one aliquot of antigen-negative cells; (ii) pelleting the antigen-negative cells by centrifugation; (iv) measuring the radioactivity in the supernatant of the antigen-negative pelleted cells; and (v) comparing the quantity of radioactivity in the antigen-negative cell supernatant to the quantity of radioactivity in the supernatant of the antigen-positive cell supernatant and the control. Comparing the radioactivity obtained from this tube to the dilution buffer control will serve as a measure of the amount of non-specific binding to antigen-positive cells.
  • CD20 negative cells are preferably HSB cells (ATCC # CCL 120.1).
  • the lyophilized cells of the present invention provide a simple, efficient and reproducible standard for testing the binding efficacy of a radiolabeled antibody. Therefore, the binding assay of the present invention is preferably performed using the lyophilized cells included in the binding assay kits of the present invention.
  • the radiolabeling assays of the present invention may be combined with the binding assays of the present invention, wherein the antibody is first labeled by the method of labeling an chelator-conjugated antibody as described in the present invention. Most preferably, the binding assay of the present invention is performed using one of the binding assay and radiolabeling kits described herein.
  • the present invention also encompasses a competitive binding assay for assessing affinity of a test antibody to a target cell, comprising (i) preparing a ruthenium-labeled control antibody using a known antibody specific for the same antigen; (ii) incubating increasing amounts of test antibody and increasing amounts of unlabeled control antibody with a fixed concentration of target cells and a trace amount of ruthenium-labeled antibody wherein each separate concentration of test antibody and each separate concentration of control antibody are in separate tubes, respectively; (iii) determining the quantity of binding in each reaction tube based on relative electrochemiluminescence (ECL) using ORIGEN instrumentation; and (iv) calculating the average affinity value of the test antibody.
  • ECL electrochemiluminescence
  • the average affinity value may be calculated from the EC50 values and the known concentration of trace antibody using the method of Muller (J. Immunological Methods (1980) 34:345) or any other appropriate method. It should be noted that this assay may also be used to test the affinity of radiolabeled antibodies, or any antibody for which antigen cannot be purified and cells are required as an antigen source.
  • the fixed, lyophilized cells of the present invention may be used as target cells.
  • the control antibody may be 2B8, or any other unconjugated anti-CD20 antibody.
  • the control antibody may be a chelator-conjugated antibody.
  • the test antibody may also be a chelator-conjugate of the control antibody.
  • the test antibody may be another anti-CD20 antibody whose binding affinity to CD20 as compared to 2B8 is of interest.
  • the assay may be adapted for use with antibodies having other specificities so long as an appropriate target cell is available.
  • the preferred target cells are CD20-positive cells, more preferably SB cells (ATCC # CCL 120), and are more preferably resuspended lyophilized SB cells prepared according to the method of the present invention. Cells lyophilized using other methods or fixed cells may also be used.
  • the ruthenium-labeled antibody is typically prepared by a process comprising incubating the control antibody with N-hydroxysuccinimide ester of ruthenium (II) tris-bypyridine chelator (TAG-NHS), although other known method of labeling antibodies are also envisioned.
  • TAG-NHS N-hydroxysuccinimide ester of ruthenium (II) tris-bypyridine chelator
  • the control antibody and TAG-NHS are preferably incubated at about a 1:15 molar ratio.
  • FIG. 1 Immunoreactivity of native 2B8 was compared to commercially available anti-CD20 antibodies Bi (Coulter) and Leu 16 (Becton Dickinson) by direct competition in a radioimmunoassay using 125 I-labeled B1.
  • Antigen-positive SB cells 100,000 were added to each well of V&P filter plates; 10 ng of radiolabeled B1 was mixed with various concentrations of unlabeled competition and the mixture added to the cells.
  • the antibodies were incubated with the cells for one hour at ambient temperature; determinations were performed in triplicate. Subsequently, the wells were washed, dried and the filter-associated radioactivity determined. The data shown were corrected for background radioactivity and are the means of triplicate determinations.
  • FIG. 2 Increasing amounts of unconjugated 2B8 were analyzed for binding to human B-cells (SB) using FACS analysis. Comparisons were made with a commercially available anti-CD20 monoclonal antibody (B1) and with two irrelevant isotype antibodies. Goat anti-mouse IgG-FITC F(ab)′ 2 was used as the secondary reagent. The results show that 2B8 is specific for the CD20 antigen and that it exhibits greater binding than B1.
  • FIG. 3 Human B-cells (SB) were incubated with increasing amounts of 125 I-labeled 2B8. Triplicate samples were incubated for one hour and cell-bound radioactivity was determined after filtration to collect cells. Scatchard analysis allowed calculation of an apparent affinity constant of 4.3 ⁇ 10 ⁇ 9 M.
  • FIG. 4 Immunoreactivity of native 2B8, 2B8-MX-DTPA, and B1.
  • the B1 antibody was radiolabeled as described in the Methods section. Ten nanograms of radiolabeled B1 were mixed with increasing concentrations of the competitor and the mixture added to wells of V&P filter plates containing 100,000 antigen-positive SB cells each; all determinations were performed in triplicate. Following a one hour incubation at ambient temperature, the wells were washed extensively. Subsequently, the filters were dried and the associated radioactivity determined by gamma counting; all values were corrected for background. Values shown are the means of triplicate determinations.
  • FIG. 5 Antibody 2B8 was formulated at a final concentration of 10 mg/mL in normal saline or normal saline containing 10 mM glycine-HCl, pH 6.8. Duplicate sets of samples were then placed in screw-capped vials, the vials purged with nitrogen, and then capped. The samples were then incubated at 4° C. or 30° C. for 12 weeks; the immunoreactivity of the samples was evaluated weekly. No loss of immunoreactivity was observed with any of the 2B8 samples throughout the 12-week study. Immunoreactivities at week 1 (FIG. 5A), week 6 (FIG. 5B) and week 12 (FIG. 5C) are depicted.
  • FIG. 6 Binding assay for determination of immunoreactivity of 111 In-labeled 2B8-MX-DTPA incubated in PBS, pH 7.4 containing 50 mg/mL human serum albumin (48 h incubation).
  • FIG. 6A A constant amount of radiolabeled antibody (5 ng/mL) was incubated with increasing volumes of SB cells (20 ⁇ 106 cells/mL). The amount of radioactivity (cpm) bound to cells was plotted against the volume of cell suspension added.
  • FIG. 6B Total applied radioactivity over bound radioactivity (AT/B) was plotted. Linear extrapolation allowed calculation of the y-intercept (0.997). The reciprocal of the y-intercept ⁇ 100 yielded an immunoreactivity value of 100% at infinite antigen excess.
  • FIG. 7 Autoradiograms obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 75 mg/mL human serum albumin and lmM DTPA. At the indicated times, samples were electrophoresed on 4-20% Tris-glycine gels using non-reducing conditions, denaturing conditions (SDS). The samples were loaded at 5 ⁇ L (lanes 1,2), 10 ⁇ L (lanes 5,6). The gels were exposed to x-ray film for approximately 15 min at ambient temperature and photographed.
  • SDS denaturing conditions
  • FIG. 8 Densitometric scan of time zero autoradiogram obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 75 mg/mL human serum albumin and 1 mM DTPA. The sample was electrophoresed on a 4-20% Trib-glycine gel using non-reducing conditions. Samples were loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. The gel was exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#2) was 96.2%.
  • FIG. 9 Densitometric scan of 48 h autoradiogram obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 75 mg/mL human serum albumin and 1 mM DTPA. The sample was electrophoresed on a 4-20% Tris-glycine gel using non-reducing conditions. Samples were loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. The gel was exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#2) was 95.5%.
  • FIG. 10 Autoradiograms obtained from SDS-PAGE analysis of 111 In-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 50 mg/mL human serum albumin. At the indicated times, samples were electrophoresed on 4-20% Tris-glycine gels using non-reducing conditions. The samples were loaded at 5 ⁇ L (lanes 1, 2), 10 ⁇ L (lanes 3, 4), and 20 ⁇ L (lanes 5, 6). The gels were exposed to x-ray film for approximately 15 min at ambient temperature and photographed. (Note: The 48 h autoradiogram was obtained using intensifying screens resulting in a more intense signal compared to the time zero autoradiogram).
  • FIG. 11 Densitometry scan of time zero autoradiogram obtained from SDS-PAGE analysis of 111 In-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 50 mg/mL human serum albumin. The sample was electrophoresed on a 4-20% Tris-glycine gel under non-reducing conditions. The sample was loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. The gel was exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#3) was 95.9%.
  • FIG. 12 Densitometry scan of 48 h autoradiogram obtained from SDS-PAGE analysis of 111 In-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 50 mg/mL human serum albumin. The sample was electrophoresed on a 4-20% Tris-glycine gel under non-reducing conditions. The sample was loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. The gel was exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes scanned using a densitometer. The relative area of the radiolabeled conjugate was 97.0% (combined areas of peaks #2, 3, and 4).
  • FIG. 13 Autoradiograms obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 37° C. in human serum. At the indicated times, samples were electrophoresed on 4-20% Tris-glycine gels using non- reducing conditions. The samples were loaded at 5 ⁇ L (lanes 1, 2), 10 ⁇ L (lanes 3, 4), and 20 ⁇ L (lanes 5, 6). The gels were exposed to x-ray film for approximately 15 min at ambient temperature and photographed.
  • FIG. 14 Densitometric scan of time zero autoradiogram obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 37° C. in human serum. The sample was electrophoresed on a 4-20% Tris-glycine gel using non-reducing conditions. The sample was loaded at 5 ⁇ L, 10 ,L, and 20 ⁇ L in duplicate wells. Gels were exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#2) was 97.9%.
  • FIG. 15 Densitometric scan of 98 h autoradiogram obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 37° C. in human serum. The sample was electrophoresed on a 4-20% Tris-glycine gel using non-reducing conditions. The sample was loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. Gels were exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#2) was 94.7%.
  • FIG. 16 Autoradiograms obtained from SDS-PAGE analysis of 111 In-labeled 2B8-MX-DTPA incubate at 37° C. in human serum. At the indicated times, samples were electrophoresed on 4-20% Tris-glycine gels using non-reducing conditions. The samples were loaded at 5 ⁇ L (lanes 1, 2), 10 ⁇ L (lanes 3, 4), and 20 ⁇ L (lanes 5, 6). The gels were exposed to x-ray film for approximately 16-20 h at ambient temperature and photographed.
  • FIG. 17 Densitometric scan of time zero autoradiogram obtained from SDS-PAGE analysis of 111 In-labeled 2B8-MX-DTPA incubated at 37° C. in human serum. The sample was electrophoresed on a 4-20% Tris-glycine gel using non-reducing conditions. The sample was loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. The gel was exposed to x-ray film for approximately 16-20 h at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#3) was 95.3%.
  • FIG. 18 Densitometric scan of the 96 h autoradiogram obtained from SDS-PAGE analysis of 111 In-labeled 2B8-MX-DTPA incubated at 37° C. in human serum. The sample was electrophoresed on a 4-20% Tris-glycine gel using non-reducing conditions. The sample was loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. The gel was exposed to x-ray film for approximately 16-20 h at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#3) was 94.0%.
  • FIG. 19 Cynomolgus monkeys were injected intravenously every 48 hours for a total of seven injections; the amounts administered are shown. Circulating T- and B-cell levels were determined by FACS analysis using anti-CD2 (T-cell), anti-Mo-IgG (2B8), anti-CD20 (Leu 16), and anti-human-IgG (B-cell). No effect was observed on circulating T-cell levels. (Group V animals were given a single dose).
  • FIG. 20 The recovery of circulating B-cell levels in animals receiving 2B8 was followed by FACS analysis using the fluorescently-labeled antibodies described in the brief description of FIG. 19. The animals in Groups III and IV were not monitored as they were sacrificed on day 13.
  • FIG. 21 Cynomolgus monkeys were injected intravenously with 89y 2B8-MX-DTPA which had been prepared using clinical-grade 2B8-MX-DTPA. The animals were dosed every 48 hours with the amounts shown above for a total of seven doses. On days 0, 2, 7, 10 and 14 the monkeys were bled and evaluated for serum chemistries hematology and circulating B-cell levels (day 10 sera were not analyzed for B-cell content). Other than decreased total lymphocyte count in all animals, except one individual in groups II, no significant abnormalities were noted during the course of the study.
  • FIG. 22 The clearance of murine anti-CD20 antibody 2B8 from cynomolgus monkeys was determined by ELISA following a single injection of 10 mg/kg on day zero. As shown in panel A, the antibody exhibited at ⁇ t 1 ⁇ 2 value of approximately 4.5 days. The clearance of the 2B8 antibody and its MX-DTPA conjugate from the circulation of BALB/c mice are shown in panel B. Mice were injected intravenously with 25 ⁇ g of native or conjugated 2B8 and blood samples taken at various times up to 264 hours following injection; sera was subsequently analyzed by enzyme immunoassay using SB cells as the capture agent. Both the native and conjugated antibodies exhibited clearance values of 8.75 days.
  • FIG. 23 Twenty BALB/c mice were each injected with 1.1 ⁇ Ci of radiolabeled conjugate (100 ⁇ L) formulated in PBS, pH 7.4, containing 50 mg/mL HSA. Groups of five mice each were sacrificed at 1, 24, 48, and 72 hours and then blood and various tissues prepared and analyzed for associated radioactivity.
  • FIG. 24 Twenty BALB/c mice were each injected intravenously with approximately 1.0 ⁇ Ci (in 100 ⁇ l) of radiolabeled conjugate formulated in 1 ⁇ PBS, pH 7.4, containing 75 mg/mL human serum albumin and 1 m MDPA. Groups of five mice each were sacrificed at 1, 24, 48 and 72 hours and their blood and various tissues prepared and analyzed for associated radioactivity.
  • FIG. 25 Athymic mice bearing Ramos B-cell tumors were injected intravenously with 24 ⁇ Ci of 111 -In-2B8-MX-DTPA and groups of three mice each were sacrificed at 0, 24, 48 and 72 hours. Following tissue preparation and determination of associated radioactivity, the percent injected dose per gram tissue values were determined and plotted as shown.
  • FIG. 26 Binding assay for determination of immunoreactivity of “mix-&-shoot” 90 Y-labeled 2B8-MX-DTPA incubated in PBS, pH 7.4 containing 50-75 mg/mL human serum albumin (48 h incubation).
  • Panel A A constant amount of 90 Y-labeled antibody (approximately 1 ng/ml) was incubated with increasing amounts of SB cells. The amount of radioactivity (cpm) bound to cells was plotted against the cell concentration.
  • Panel B Total applied 90 Y radioactivity over bound radioactivity (AT/B) was plotted. Linear extrapolation allowed calculation of the y-intercept (1.139). The reciprocal of the y-intercept ⁇ 100 yielded an immunoreactivity value of 87.9% at infinite antigen excess. No binding was observed with CD20-negative cells (HSB).
  • FIG. 27 Autoradiograms obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 75 mg/mL human serum albumin and 1 mM DTPA. At the indicated times, samples were electrophoresed on 4-20% Tris-glycine gels using non-reducing conditions, denaturing conditions (SDS). The samples were loaded at 5 ⁇ L (lanes 1,2), 10 ⁇ L (lanes 5,6). The gels were exposed to x-ray film for approximately 15 min at ambient temperature and photographed.
  • FIG. 28 Densitometric scan of time zero autoradiogram obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 75 mg/mL human serum albumin and 1 mM DTPA. The sample was electrophoresed on a 4-20% Trib-glycine gel using non-reducing conditions. Samples were loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. The gel was exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#2) was 96.1%.
  • FIG. 29 Densitometric scan of 48 h autoradiogram obtained from SDS-PAGE analysis of 90 Y-labeled 2B8-MX-DTPA incubated at 4° C. in PBS, pH 7.4 containing 75 mg/mL human serum albumin and 1 mM DTPA. The sample was electrophoresed on a 4-20% Tris-glycine gel using non-reducing conditions. Samples were loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. The gel was exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#2) was 94.1%.
  • FIG. 30 Autoradiograms obtained from SDS-PAGE analysis of “mix-&-shoot” 90 Y-labeled 2B8-MX-DTPA incubated at 37° C. in human serum. At the indicated times, samples were electrophoresed on 4-20% Tris-glycine gels using non-reducing conditions. The samples were loaded at 5 ⁇ L (lanes 1, 2), 10 ⁇ L (lanes 3, 4), and 20 ⁇ L (lanes 5, 6). The gels were exposed to x-ray film for approximately 15 min at ambient temperature and photographed.
  • FIG. 31 Densitometric scan of time zero autoradiogram obtained from SDS-PAGE analysis of “mix-&-shoot” 90 Y-labeled 2B8-MX-DTPA incubated at 37° C. in human serum. The sample was electrophoresed on a 4-20% Tris-glycine gel using non-reducing conditions. The sample was loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. Gels were exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#2) was 89.1%.
  • FIG. 32 Densitometric scan of 72 h autoradiogram obtained from SDS-PAGE analysis of “mix-&-shoot” 90 Y-labeled 2B8-MX-DTPA incubated at 37° C. in human serum. The sample was electrophoresed on a 4-20% Tris-glycine gel using non-reducing conditions. The sample was loaded at 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L in duplicate wells. Gels were exposed to x-ray film for approximately 15 min at ambient temperature and one of the lanes was scanned using a densitometer. The relative area of the radiolabeled conjugate peak (#2) was 88.8%.
  • FIG. 33 Twenty BALB/c mice were each injected intravenously with 5 ⁇ Ci 90 Y-labeled 2B8-MX-DTPA formulated in 1 ⁇ PBS, pH 7.4, containing 75 mg/mL human serum albumin and 1 mM DTPA. Groups of five mice each were sacrificed at 1, 24, 48 and 72 hours and their blood and various tissues prepared and analyzed for associated radioactivity.
  • FIG. 34 Increasing amounts of CHO-derived 2B8 antibody labeled were incubated with a fixed concentration of freshly harvested CD20-positive B-cells (SB) or CD20-negative T-cells (HSB). Antibody binding to cells was quantified using FACS analysis using goat anti-mouse IgG-FITC F(ab)′ 2 as described herein. Comparison was made to an irrelevant isotype antibody (S004). Only the CHO-derived 2B8 antibody showed any appreciable binding to CD20-positive SB cells.
  • FIG. 35 The immunoreactivity of CHO-derived 2B8 was compared to the 2B8-49 parent antibody produced in a hybridoma cell line by direct competition in an ORIGEN assay. Increasing amounts of antibody was incubated with a fixed concentration of CD20-positive B-cells (SB) and a trace amount of ruthenium-labeled CHO 2B8. After incubation for three hours at ambient temperature, binding, expressed as relative electrochemiluminescence (ECL), was determined using the ORIGEN instrument as described in the Materials and Methods. Values represent the means of duplicate determinations. Average affinity constants for CHO 2B8 and 2B8-49 were calculated to be 1.3 ⁇ 10 ⁇ 10 M and 2.5 ⁇ 10 ⁇ 10 M, respectively. An irrelevant isotype antibody (S004), was included for comparison.
  • S004 CD20-positive B-cells
  • FIG. 36 The binding of 2B8-MX-DTPA conjugates prepared from CHO-derived 2B8 was compared to the unconjugated antibody by direct competition in an ORIGEN assay.
  • Conjugates were prepared by incubation of 2B8 with MX-DTPA for 8, 17, and 24 h before removal of unreacted chelate.
  • antibodies were incubated with a fixed concentration of CD20-positive B-cells (SB) and a trace amount of ruthenium-labeled CHO 2B8. After incubation for three hours at ambient temperature, binding, expressed as relative electrochemiluminescence (ECL), was determined using the ORIGEN instrument as described in the Materials and Methods. Values represent the means of duplicate determinations.
  • Conjugate preparations exhibited similar binding compared to unconjugated 2B8 antibody.
  • FIG. 37 A) SB cells were washed and resuspended to 90 ⁇ 10 6 cells/mL with dilution buffer (1 ⁇ PBS, pH 7.4 containing 1% (w/v) bovine serum albumin. Increasing concentrations of cells were incubated for 3 h with 7.5 ng/mL In2B8 prepared using 2B8-MX-DTPA lot 0165A. B) Double-inverse plot of cell concentration vs. bound radioactivity/total radioactivity (B/AT). Immunoreactivity was calculated as 1/y-intercept ⁇ 100. Immunoreactivity and correlation coefficient (R) values were 80.6% and 0.981, respectively.
  • FIG. 38 A) SB cells were washed and resuspended to 90 ⁇ 10 6 cells/mL with dilution buffer (1 ⁇ PBS, pH 7.4 containing 1% (w/v) bovine serum albumin. Increasing concentrations of cells were incubated for 3 h with 2 ng/mL Y2B8 prepared using 2B8-MX-DTPA lot # 0165A. B) Double-inverse plot of cell concentration vs. bound radioactivity/total radioactivity (B/AT). Immunoreactivity was calculated as 1/y-intercept x 100. Immunoreactivity and correlation coefficient (R) values were 72.2% and 0.999, respectively.
  • low metal refers to reagents treated to reduce metal contamination to a level which does not impact radioincorporation
  • antigen positive means expresses antigen that is recognized by particular antibody of the invention in such a way that the antibody is capable of binding.
  • % radioincorporation refers to the amount of radiolabel from a radiolabeling reaction that is conjugated to the antibody relative to the total amount of radiolabel initially added to the reaction.
  • % binding refers to the amount of antibody from a sample which binds to the target antigen, with or without specificity.
  • % immunoreactivity or binding specificity refers to that amount of an antibody sample which binds to the target antigen with specificity.
  • diagnostic antibody refers to an antibody conjugated to a radiolabel such as 111 In which can effect diagnostic imaging of tumors and antigen positive cells.
  • therapeutic antibody refers to an antibody conjugated to a alpha or beta emitting radiolabel (such as 90 Y) which can effect cell killing when bound to the targeted antigen.
  • a alpha or beta emitting radiolabel such as 90 Y
  • the human cell lines SB and HSB were obtained from the American Type Culture Collection and cultured in RPMI-1640 containing 10% fetal bovine serum.
  • the CD20-positive SB cell line is a B lymphoblastoid cell line derived from the peripheral blood buffy coat of a patient with acute lymphoblastic leukemia (1).
  • the antigen-negative cell line HSB is a T lymphoblastoid cell line developed from tumors induced in newborn Syrian hamsters (2).
  • the murine myeloma cell line SP2/0 was similarly maintained in RPMI-1640 containing 10% fetal bovine serum.
  • the anti-CD20 antibodies Bi and Leu 16 were purchased from Coulter Immunology and Becton/Dickinson, respectively.
  • the 125 I-labeled goat anti-mouse IgG and goat anti-human IgG antibodies were obtained from ICN.
  • Goat F(ab′)2 anti-mouse IgG was obtained from Cappel.
  • Freund's complete and incomplete adjuvants were purchased from Sigma Chemical Company. Polyethylene glycol, HAT concentrate, and HT concentrate were all obtained from Boehringer Mannheim. Fluorescein isothiocyanate (FITC) was purchased from Sigma Chemical Company. Indium-[ 111 ] chloride and 90 Y chloride were obtained from Amersham or NEN Dupont. Yttrium-[89] chloride was purchased from Aldrich Chemical Company. All other reagents were obtained from standard sources.
  • FITC Fluorescein isothiocyanate
  • Reagents used for conjugation and radiolabeling protocols were processed to remove contaminating heavy metal ions which could compete with the radioisotopes during the radiolabeling step.
  • Reagents were typically processed by passing the solutions through a column of Chelex 100 ion exchange resin (BioRad Industries) or batch processing by addition of Chelex 100 to a prepared solution.
  • Low metal-containing water, either Milli-Q-purified or Water for Irrigation (WFIr) was used for all preparations and dilutions.
  • the metal-free solutions were sterile-filtered and collected in sterile plastic containers.
  • mice Ten BALB/c mice were immunized with 20 million SB cells suspended in PBS containing Freunds complete adjuvant. The cells were injected both s.c and i.p at multiple sites on the animal. After a 2 week rest period the mice were injected a second time with SB cells emulsified in Freund′ s incomplete adjuvant. Subsequent immunization boosters were performed on a weekly schedule with SB cells suspended in PBS. Mice were immunized for a period of 6 weeks to 4 months.
  • Radiolabeled antibody was placed in each well of a previously blocked filter assay plate (blocking buffer: dilution buffer containing 10% FBS) along with 50 ⁇ L of hybridoma supernatant from test wells and 100,000 SB cells suspended in 50 ⁇ L dilution buffer. The suspension was incubated for one hour at ambient temperature. The plates were washed thoroughly with wash buffer (PBS, pH 7.4, containing 0.2% gelatin and 0.02% sodium azide) on a V&P Scientific vacuum manifold and filter bottoms containing trapped SB cells were transferred to a gamma counter. Wells containing only HAT media and labeled B1 antibody were used as background controls and identical wells containing SB cells were used as positive controls. Inhibition controls contained radiolabeled B1 and various amounts of unlabeled B1 antibody ranging from 2 ⁇ g to 2 ng.
  • blocking buffer dilution buffer containing 10% FBS
  • fluorescein was coupled to purified 2B8 antibody through the reaction of amino groups with fluorescein isothiocyanate (FITC). Briefly, 2B8 antibody (1.2 mg/mL) was incubated in pH 9.5, 0. 1M sodium carbonate buffer with 150-200 ⁇ g FITC per mg protein. The solution was incubated at room temperature for 2 hours and the resulting 2B8-FITC conjugate was purified on a Sephadex G-25 column. Other reagents used in these studies such as B1 and Leu 16 were purchased as fluorescein conjugates directly from Coulter or Becton Dickinson.
  • FITC fluorescein isothiocyanate
  • Cells to be analyzed were harvested and washed three times with PBS containing 0.2% BSA and 0.1% sodium azide. Viability was determined by trypan blue exclusion with a viability requirement of >90%. Cell concentrations were adjusted to 3 million per ml with 50 ⁇ L added per well into 96 well U-bottom plates. Primary antibody (50 ⁇ L) was added to appropriate wells and the mixture incubated for 30 min. to 1 h. at ambient temperature; subsequently the cells were washed 5 times with 200 ⁇ L/well of PBS containing 0.2% BSA and 0.02% sodium azide. Cells were centrifuged in the plates at 1300 RPM for 1 min.
  • the cells were washed twice by centrifugation and resuspension in HBSS. Fetal bovine serum (2 mL) was added and the cells resuspended. One hundred microliters of the resuspended cells were then distributed to each of 6, 15 ml conical centrifuge tubes.
  • Tube #1 Murine anti-CD2-FITC (AMAC), 2.5 ⁇ g/mL, 5 ⁇ g; Tube #2: Goat anti-Human IGM-FITC (Fisher) 2.5 ⁇ g/mL, 5 ⁇ g; Tube#3: Goat anti-mouse IgG-RPE (Fisher) 2.5 ⁇ g/mL, 5 ⁇ g; Tube #4: Goat anti-Human IgM-FITC + Goat anti-mouse IgG-RPE (absorbed), 2.5 ⁇ g/mL, 5 ⁇ g; Tube #5: anti-human CD20-FITC (anti-Leu 16, Becton Dickinson), 5 ⁇ g; Tube #6: Cells only (auto-fluorescence).
  • AMAC Murine anti-CD2-FITC
  • Tube #2 Goat anti-Human IGM-FITC (Fisher) 2.5 ⁇ g/mL, 5 ⁇ g
  • Tube#3 Goat anti-mouse IgG-RPE (Fisher) 2.5 ⁇
  • pre-study lymphocyte levels were determined for each cynomolgus monkey and used as baseline values. The percentage of T- and B-cells and T:B ratios were calculated and used as depletion references. The pre-study B cell population was enumerated with Leu 16 and anti-human IgM antibodies.
  • the extract was centrifuged in a microfuge at high speed for 30 min and the supernatants were transferred to new tubes.
  • Protein A-Sepharose 300 ⁇ L was added to each tube and the resin pelleted by centrifugation. The protein A-Sepharose was then washed 20 times to remove non specifically bound iodinated protein. When the bead-to-supernatant radioactivity ratio reached a value of 100, the pellet was extracted with SDS PAGE sample buffer and heated to boiling. After cooling, approximately 15,000 cpm of each of the extracts were added to wells of a 10% polyacrylamide gel. A low molecular weight pre-stained standard (BioRad Inc.) was added to a separate well and used for molecular weight estimation. The proteins were resolved by electrophoresis and the gel was dried and exposed to a sheet of X-ray film for 24 hours at ⁇ 70° C.; subsequently the film was developed and analyzed.
  • Radiolabeled 2B8 was prepared by reaction with 125 I in the presence of lodobeads. Following removal of free iodine the radiolabeled antibody was incubated in various concentrations, in duplicate, ranging from 5000 ng per well to 35 ng/well with 10,000 SB cells. The amount of antibody binding to cells was calculated from the specific activity of the 125 1-labeled 2B8. The ratio of bound/free antibody was plotted against the molar concentration of bound antibody and the apparent affinity constant was determined from the ratio of the X and Y axis intercepts.
  • MX-DTPA carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid
  • the murine anti-CD20 antibody 2B8 was purified initially from ascites by Protein A and QAE chromatography. For later experiments 2B8 was purified from hollow-fiber bioreactor supernatants using the same purification process. The hollow-fiber-derived antibody has now been replaced for commercialization purposes with the CHO-derived antibody described in Example 2.
  • the antibody was prepared for conjugation by transferring it into metal-free 50 mM bicine-NaOH, pH 8.6, containing 150 mM NaCl, using dialysis or repetitive buffer exchange.
  • buffer exchange was effected using repetitive ultrafiltration with Centricon 30 (Amicon) spin filters (30,OOOD MWCO).
  • 50-200 ⁇ L of protein (10 mg/mL) was added to the filter unit and 2 mL of bicine buffer added.
  • the filter was centrifuged at 4° C. in a Sorval SS-34 rotor at 6,000 rpm for 45 min. Retentate volume was approximately 50-100 ⁇ L. This process was repeated twice with the same filter.
  • Retentate was transferred to a polypropylene 1.5 mL screw cap tube, assayed for protein, diluted to 10.0 mg/mL and stored at 4° C. until used for conjugation.
  • the protein was transferred into 50 mM sodium citrate, pH 5.5 containing l5OmM NaCl and 0.05% sodium azide using the same protocol described above.
  • Conjugation of 2B8 with MX-DTPA was performed in polypropylene tubes at ambient temperature. Frozen stock solutions of MX-DTPA were thawed immediately before use. Typically, 50-200 ⁇ L of antibody at 10 mg/mL were reacted with chelate at a molar ratio of chelate-to-protein of 4:1. Reactions were initiated by adding the chelate stock solution and gently mixing; the conjugation was allowed to proceed overnight, generally for 14 to 20 h, at ambient temperature. Unreacted chelate was removed from the conjugate by dialysis or repetitive ultrafiltration, as described above, into metal-free normal saline (0.9% w/v) containing 0.05% sodium azide. The protein concentration was adjusted to 10 mg/mL and stored at 4° C. in a polypropylene tube until radiolabeled.
  • Chelate incorporation was determined by scintillation counting and comparing the value obtained with the purified conjugate to the specific activity of the carbon-[14]-labeled chelate. For later studies, in which non-radioactive chelate obtained from Coulter Immunology was used, chelate incorporation was assessed by incubating the conjugate with an excess of a radioactive carrier solution of 90 Y of known concentration and specific activity.
  • a stock solution of yttrium chloride of known concentration was prepared in metal-free 0.05 N HC1 to which carrier-free 90 Y (chloride salt) was added. An aliquot of this solution was analyzed by liquid scintillation counting to determine an accurate specific activity for this reagent.
  • a volume of the yttrium chloride reagent equal to 3-times the number of mols of chelate expected to be attached to the antibody, typically 2 mol/mol antibody, was added to a polypropylene tube, and the pH adjusted to 4.0-4.5 with 2 M sodium acetate. Conjugated antibody was subsequently added and the mixture incubated 15-30 min at ambient temperature. The reaction was quenched by adding 20 mM EDTA to a final concentration of 1 mM and the pH of the solution adjusted to approximately pH 6 with 2M sodium acetate.
  • Samples for assay were diluted in 1 ⁇ PBS/1% BSA, applied to plates and serially diluted (1:2) into the same buffer. After incubating plates for 1 h at ambient temperature, the plates were washed three times with 1 ⁇ PBS. Secondary antibody (goat anti-mouse IgGl-specific HRP conjugate) (50 ⁇ L) was added to wells (1:1500 dilution in 1 ⁇ PBS/1% BSA) and incubated 1 h at ambient temperature. Plates were washed four times with 1 ⁇ PBS followed by the addition of ABTS substrate solution (50 mM sodium citrate, pH 4.5 containing 0.01% ATBS and 0.001% H202). Plates were read at 405 nm after 15-30 min incubation.
  • ABTS substrate solution 50 mM sodium citrate, pH 4.5 containing 0.01% ATBS and 0.001% H202
  • Antigen-negative HSB cells were included in assays to monitor non-specific binding. Immunoreactivity of the conjugate was calculated by plotting the absorbance values vs. the respective dilution factor and comparing these to values obtained using native antibody (representing 100% immunoreactivity) tested on the same plate. Several values on the linear portion of the titration profile were compared and a mean value determined.
  • the radiolabeled conjugate was diluted to a final antibody concentration of 1-5 ng/mL with dilution buffer and 0.35 mL was added to each tube. Following a 75-90 min incubation period at ambient temperature the cells were pelleted by centrifugation and the supernatants collected. Radioactivity remaining in the supernatant fraction was determined with a gamma or scintillation counter. The data were plotted as the quotient of the total radioactivity added divided by the cell-associated radioactivity, versus the inverse of the cell number per tube. The y axis intercept thus represents the immunoreactive fraction.
  • radiolabeled conjugates were diluted 1:10 with normal human serum (non-heat-inactivated) and aliquots placed aseptically into sterile capped tubes; these tubes were then incubated at 37° C. for periods up to 96 hours. At selected times conjugate samples were removed and analyzed by non-reducing SDS-PAGE in 4-20% gradient gels followed by autoradiography, and by instant thin layer chromatography.
  • the 2B8-MX-DTPA conjugate was radiolabeled with 111 In and used without HPLC purification (“mix-and-shoot” protocol).
  • the radiolabeled antibody was diluted into PBS and human serum albumin (HSA) added to a final concentration of 50 mg/mL.
  • HSA human serum albumin
  • the specific activity of the formulated radiolabeled conjugate was 2.2 mCi/mg.
  • the formulated conjugate was subsequently incubated at 4° C. for 48 hours and aliquots analyzed at time 0, 24 h and 48 hours using non-reducing SDS-PAGE in 4-20% gradient gels followed by autoradiography, and by instant thin layer chromatography.
  • the immunoreactivity at each time point was assessed using the whole-cell suspension assay described in section 1 above.
  • the 2B8-MX-DTPA conjugate was radiolabeled with 90 Y and purified by size-exclusion chromatography on HPLC using 1 ⁇ PBS as an elution buffer.
  • the radiolabeled conjugate fractions were pooled and human serum albumin and DTPA were added to final concentrations of 75 mg/mL and 1 mM, respectively.
  • the specific activity of the formulated radiolabeled conjugate was 14.6 mCi/mg.
  • the formulated conjugate was subsequently incubated at 4° C. for 48 hours and aliquots analyzed at time 0, 24 h and 48 hours using non-reducing SDS-PAGE in 4-20% gradient gels followed by autoradiography, and instant thin layer chromatography. Immunoreactivity at each time point was assessed using the whole-cell suspension assay described in section 1 above.
  • Antibody 2B8 was evaluated in a high-dose pharmacology study performed under GLP regulations at White Sands Research Center (Study Number 920111).
  • Adult Macaca fascicularis (cynomolgus) monkeys were used; study groups each consisted of one male and one female.
  • the antibody was injected intravenously every 48 hours for a total of seven injections.
  • the study consisted of five groups: Group I (saline); Group II (0.6 mg/kg); Group III (2.5 mg/kg); Group IV (10 mg/kg); and, Group V (10 mg/kg on day 0 only).
  • the mean serum beta half life of 2B8 in cynomolgus monkeys was determined using Group V animals (above).
  • Goat anti-mouse IgGl (Fisher Scientific) was diluted to 2.0 ⁇ g per ml in 10 mM borate buffer, pH 9.6, and 50 ⁇ L was added to each well of a 96-well plate.
  • the antibody was allowed to bind to the plate during an overnight incubation at 4° C., or for 2 h at ambient temperature. Each plate was blocked for 30 min. at ambient temperature with 150 ⁇ L per well of PBS containing 1% BSA.
  • the plates were washed with distilled water and serum or plasma samples were applied in triplicate to individual wells at 1: 100 initial dilution followed by serial 1:2 dilutions.
  • Purified 2B8 was added to pre-bleed sera and diluted for use as a standard curve beginning with 0.5 mg/mL; samples were diluted 1:100 and then serially diluted as with the other samples.
  • the plates were incubated for 1 h at ambient temperature and washed 4 times with distilled water.
  • the secondary reagent (goat anti-mouse IgGl-HRPO) was then added at 1:4000 dilution and incubated at ambient temperature for an additional hour.
  • the plates were washed again in distilled water and 0.1 mL peroxidase substrate was added containing hydrogen peroxide. Color was allowed to develop from the reaction for 20 min.; the absorbance was subsequently determined at 405 nm using a microplate ELISA reader. The results were plotted in ⁇ g antibody per mL serum.
  • ⁇ t 1 ⁇ 2 values of 2B8 and 2B8-MX-DTPA were determined in BALB/c mice.
  • Unconjugated 2B8 stored at ⁇ 70° C. in 1 ⁇ PBS, pH 7.4/10% glycerol was thawed, diluted to 0.5 mg/mL and sterile filtered.
  • Conjugated antibody was prepared following standard protocols but with carbon-[14]-labeled chelate; chelate incorporation was 1.5 mol/mol antibody.
  • the purified conjugate was diluted to 0.5 mg/mL in normal saline (0.9%), sterile filtered, and stored at 4° C. with the native antibody until used.
  • mice Six-to-eight week old mice were injected with 100 ⁇ L of purified 2B8 antibody at a concentration of 250 ⁇ g/mL. Mice were subsequently bled by retro-orbital puncture at various times ranging from 0 to 264 hours and their sera analyzed for the presence of the native and conjugated 2B8 antibody by whole-cell enzyme immunoassay using the antigen-positive B-cell line SB as the capture. The resulting data were plotted as the concentration of 2B8 or 2B8-MX-DTPA versus time; from these results a linear regression plot was generated and the slope used to determine the ⁇ t 1 ⁇ 2 values.
  • Yttrium-[89]-bearing 2B8-MX-DTPA was prepared using the protocol described for insertion of 90 Y, except that HPLC purification was not used.
  • the non-radioactive, metal-bearing conjugate was formulated in 1 ⁇ PBS containing 75 mg/mL HSA and 1 mM DTPA and evaluated in GLP study number 920611 at White Sands Research Center.
  • One male and one female monkey were included in each of four groups. The animals were injected intravenously every 48 hours for a total of 7 injections with the following amounts of drug: group 1 (saline); group II (0.003 mg/kg); group III (0.03 mg/kg); and, group IV (0.3 mg/kg).
  • mice were evaluated during the study by determining body weights and temperatures, food and water consumption, elimination, serum chemistries, hematology, urinalysis, and physical examinations. Animals in groups I through IV were bled prior to infusion on days 0, 2, 7, 10 and 14 and the blood analyzed for circulating B-cell levels by FACS analyses.
  • 2B8-MX-DTPA was radiolabeled with 111 In to a specific activity of 2.3 mCi/mg and approximately 1.1 ⁇ Ci was injected into each of 20 BALB/c mice. Subsequently, groups of five mice each were sacrificed at 1, 24, 48 and 72 hours and their organs removed and prepared for analysis. In addition, portions of the skin, muscle and bone were removed and processed for analysis; the urine and feces were also collected and analyzed for the 24-72 hour time points.
  • 2B8-MX-DTPA was also radiolabeled with 90 Y and its biological distribution evaluated in BALB/c mice over a 72-hour time period.
  • four groups of five mice each were injected intravenously with approximately 1 ⁇ Ci of clinically-formulated conjugate (specific activity: 12.2 mCi/mg); groups were subsequently sacrificed at 1, 24, 48 and 72 hours and their organs and tissues analyzed as described above. Radioactivity associated with each tissue specimen was determined by measuring bremstrahlung energy with a gamma scintillation counter. Activity values were subsequently expressed as percent injected dose per gram tissue or percent injected dose per organ. While organs and other tissues were rinsed repeatedly to remove superficial blood, the organs were not perfused. Thus, organ activity values were not discounted for the activity contribution represented by internally associated blood.
  • B1-FITC Coulter Immunology, (IgG2a,k) 11 Leu 16-FITC (Becton Dickinson, IgGl,k) 12 2B8-FITC (EDEC, IgGl,k) 14 B72.3-FITC (IgGl,k irrelevant control) 4 anti-CD4-FITC (Coulter Immunology 37 anti-CD3-FITC (Becton Dickinson) 59 anti-CD19-RPE (Becton Dickinson) 11 anti-CD19-FITC (Becton Dickinson) 14 B.
  • Double Staining B1-FITC/anti CD19-RPE 10 Leu 16-FITC/anti CD19-RPE 10 2B8 FITC/anti CD19-RPE 9 anti-CD19 FITC/anti CD19-RPE 13 B1-FITC/anti Hu Ig RPE 10 2B8-FITC/anti Hu Ig RPE 10 B72.3-FITC/anti Hu Ig RPE 2 Leucogate Simultest 99
  • the 2B8-MX-DTPA conjugate was produced by reacting the antibody with a 4:1 molar excess of isothiocyanatobenzyl-3-methyldiethylene-triaminepentaacetic acid (4). Typically, 1-2 mol of MX-DTPA chelate were introduced per mol of 2B8 antibody. As shown by the results presented in FIG. 4, the 2B8-MX-DTPA conjugate exhibited no apparent loss in immunoreactivity, vis a vis native 2B8, as both the native and conjugated 2B8 antibodies exhibited virtually identical Bi inhibition profiles; the IC50 values for 2B8 and 2B8-MX-DTPA were approximately 3 and 4 ⁇ g/mL, respectively.
  • the headings represent: 2B8 4 SAL, 2B8 incubated at 4° C. in saline; 2B8 30 SAL, 2B8 incubated at 30° C. in saline; 2B8 4 GLY, 2B8 incubated at 4° C. in normal saline containing 10 mM glycine; 2B8 30 GLY, 2B8 incubated at 30° C. in normal saline containing 10 mM glycine; 2B8-MX 4 SAL; 2B8-MX-DTPA (conjugate) incubated at 4° C. in saline; 2B8-MX 30 SAL, # conjugate incubated at 30° C.
  • 2B8-MX 4′ GLY conjugate incubated at 4° C. in normal saline containing 10 mM glycine
  • 2B8-MX 30 GLY conjugate incubated at 30° C. in normal saline containing 10 mM glycine.
  • Desensitometric Scans of Non-Reducing SDS Gels Reference 0 100.00 0 12 wk/4° C./saline 0 95.42 4.58 12 wk/4° C./glycine 0 100.00 0 12 wk/30° C./saline 7.63 83.34 9.03 12 wk/30° C./glycine 16.70 72.11 11.18
  • Desensitometric Scans of Reducing SDS Gels Reference 0 100.00 0 12 wk/30° C./saline 0 100.00 0 12 wk/30° C./glycine 0 10.00 0
  • the radiolabeling protocols for both 111 In and 90 Y were found to be reproducible. Typically, radioincorporations of >95% for 111 In and >90% for 90 Y were obtained. Specific activities for 111 I- and 90 Y-labeled conjugates were routinely in the range of 2-3 and 10-15 mCi/mg antibody, respectively.
  • uncomplexed radioisotopes were removed from the radiolabeled 2B8-MX-DTPA using HPLC gel permeation chromatography. In later experiments, HPLC purification of the indium-labeled conjugate was eliminated because of the high radioincorporations obtained (>95%) with this isotope.
  • the 90 Y-labeled antibody was formulated in 1 ⁇ PBS, pH 7.4 containing 75 mg/mL HSA; diethylenetriaminepentaacetic acid (DTPA) was also added to a final concentration of 1 mM to insure that any 90 Y which may be lost from the antibody would be chelated.
  • Degradation of 2B8-MX-DTPA, radiolabeled to a specific activity of 14.6 mCi/mg was evaluated at 0 and 48 hours using SDS-PAGE and autoradiography.
  • FIGS. 8 and 9 show that the radiolabeled antibody exhibited no significant degradation over a period of 48 h when incubated at 4° C.
  • FIG. 10 shows photographs of the autoradiograms for time zero and 48 h incubation samples; densitometric analysis of the autoradiograms indicate that there was no degradation of the radiolabeled antibody over the course of the study (FIGS. 11, 12).
  • Instant thin-layer chromatography analysis of the samples demonstrated no loss of 125 In (Table 25); moreover, immunoreactivity was maintained at approximately 100% (Table 25).
  • the study duration was 14 days and the animals were evaluated during the study in the following categories: clinical observations, body weights, body temperature, food and water intake, fecal elimination, serum chemistries, hematology, urinalysis, and physical examinations. Additionally, the animals in each group were bled on days 0, 1, 7, and 13 and the blood analyzed for serum antibody (2B8) levels and for T- and B-cell levels. On day 13 the animals in Groups III and IV were sacrificed and selected tissues examined by light microscopy following specimen preparation. The tissues evaluated were: heart, spleen, liver, kidney, lung, cerebral cortex, spinal cord, lymph node, stomach, ileum, colon, skeletal muscle, testis/ovary, pancreas, and bone marrow.
  • the 89 Y-labeled 2B8-MX-DTPA had little, if any, effect on circulating B-cells in these animals, regardless of the dose administered.
  • the 89 Y-labeled 2B8-MX-DTPA had little, if any, effect on circulating B-cells in these animals, regardless of the dose administered.
  • other than a general depletion of lymphocytes (20-43%) no significant abnormalities were found in any clinical parameter evaluated, including serum chemistry, urinalysis, body weights and temperatures.
  • conjugated 2B8 was radiolabeled with 111 In to a specific activity of 2.3 mCi/mg and roughly 1.1 ⁇ Ci was injected into each of twenty BALB/c mice to determine biodistribution of the radiolabeled material. Subsequently, groups of five mice each were sacrificed at 1, 24, 48 and 72 hours and their organs and a portion of the skin, muscle and bone were removed and processed for analysis. In addition, the urine and feces were collected and analyzed for the 24-72 hour time-points.
  • 2B8-MX-DTPA was prepared and radiolabeled with 111 In to a specific activity of 2.7 mCi/mg.
  • One hundred microliters of labeled conjugate (approximately 24 ⁇ Ci) were subsequently injected into each of 12 athymic mice bearing Ramos B-cell tumors. Tumors ranged in weight from 0.1 to 1.0 grams.
  • 50 ⁇ L of blood was removed by retro-orbital puncture, the mice sacrificed by cervical dislocation, and the tail, heart, lungs, liver, kidney, spleen, muscle, femur, and tumor removed.
  • the radioactivity associated with each tissue specimen was determined using a gamma counter and the values expressed as percent injected dose per gram.
  • mice and guinea pigs were administered a single intra peritoneal dose of 2B8 (0.5 mL or 5.0 mL, respectively) and observed for seven days. No overt signs of toxicity were detected.
  • tissue reactivity of murine monoclonal antibody 2B8 was evaluated using a panel of 32 different human tissues fixed with acetone.
  • Antibody 2B8 reacts with the anti-CD20 antigen which had a very restricted pattern of tissue distribution, being observed only in a subset of cells in lymphoid tissues including those of hematopoietic origin.
  • lymph node In the lymph node, immunoreactivity was observed in a population of mature cortical B-lymphocytes as well as proliferating cells in the germinal centers. Positive reactivity was also observed in the peripheral blood, B-cell areas of the tonsils, white pulp of the spleen, and with 40-70% of the medullary lymphocytes found in the thymus. Positive reactivity was also seen in the follicles of the lamina limbal (Peyer's Patches) of the large intestines.
  • aggregates or scattered lymphoid cells in the stroma of various organs including the bladder, breast, cervix, esophagus, lung, parotid, prostate, small intestine, and stomach, were also positive with antibody 2B8.
  • the tissue reactivity of the 2B8-MX-DTPA conjugate was evaluated using a panel of sixteen human tissues which had been fixed with acetone. As previously demonstrated with the native antibody, the 2B8-MX-DTPA conjugate recognized the CD20 antigen which exhibited a highly restricted pattern of distribution, being found only on a subset of cells of lymphoid origin. In the lymph node, immunoreactivity was observed in the B-cell population. Strong reactivity was seen in the white pulp of the spleen and in the medullary lymphocytes of the thymus.
  • Immunoreactivity was also observed in scattered lymphocytes in the bladder, heart, large intestines, liver, lung, and uterus, and was attributed to the presence of inflammatory cells present in these tissues. As described with the native antibody (above), no reactivity was observed with neuroectodermal cells or with mesenchymal elements.
  • the murine monoclonal anti-CD20 antibody 2B8 produced by a clone with the same designation, exhibits an affinity for the B-cell CD20 antigen which may be higher than that observed for the Bi antibody, as determined by competition with antibodies of known specificity for the CD20 antigen, and by Scatchard analysis. Further, immunoprecipitation data suggest that the antigen precipitated by 2B8 appears to be the same antigen as the one precipitated by B1, as both antibodies precipitated a doublet with relative molecular weights of 33 and 35 KD.
  • a 90 Y-labeled murine monoclonal anti-CD20 antibody (2B8) has been evaluated in a Phase I clinical trial for the treatment of relapsed B-cell lymphoma.
  • the original protocol used for the preparation of the yttrium-labeled antibody used a high performance liquid chromatographic (HPLC) step for removal of non-protein bound radioisotope prior to formulation and administration to patients.
  • HPLC high performance liquid chromatographic
  • this process is particularly time consuming, resulting in a longer exposure of the antibody to radioisotope in an unprotected state. This results in increased radiolysis of the antibody with a concomitant decrease in immunoreactivity.
  • the laborious aspect of the process makes it difficult to prepare more than one dose per day at the radiopharmacy. Simplification of the process would expedite implementation at the clinical site as an alternative to using NIPI Pharmacy Services as a radiopharmacy.
  • the human lymphoblastic cell lines SB (CD20 positive) and HSB (CD20 negative; ) were obtained from the American Type Culture Collection and maintained in RPMI-1640 containing 10% fetal bovine serum and supplemented with glutamine.
  • the 2B8 antibody was purified by the Manufacturing department from hollow-fiber bioreactor supernatant using protocols previously described in the IND (BB-IND 4850/4851).
  • Yttrium-[90] chloride was obtained from Amersham. All other reagents were obtained from sources described in the appended reports cited below. Reagents used for radiolabeling protocols were processed to remove contaminating heavy metal ions which could compete with-the radioisotopes during the radiolabeling step (see Methods section). Reagents were made under GMP conditions by IDEC's—Manufacturing department following established Batch Production Records.
  • Clinical-grade MX-DTPA was obtained from Coulter Immunology as the disodium salt in water and stored at ⁇ 70° C.
  • Conjugate (2B8-MX-DTPA) was prepared by the Manufacturing department. Two different lots of conjugate were used in these studies; both were provided in normal saline at 10 mg/mL. The conjugates were filled in sterile 2 mL polypropylene syringes and stored at 2-8° C.
  • Radioincorporation was determined using instant thin-layer chromatography (ITLC) in triplicate according to SOP SP-13-008.
  • ILC instant thin-layer chromatography
  • the protocol was as follows: radiolabeled conjugate was diluted 1:20 in 1 ⁇ PBS containing 1 mM DTPA or 5 mM EDTA, then 1 ⁇ -L spotted 1.5 cm from one end of a 1 ⁇ 5 cm strip of ITLC SG paper (Gehnan Sciences). The paper was developed using 10% ammonium acetate in methanol:water (1: 1;v/v). The strips were dried, cut in half crosswise, and the radioactivity associated with each section determined by scintillation counting. The radioactivity associated with the bottom half of the strip (protein-associated radioactivity) was expressed as a percentage of the total radioactivity determined by summing the values for both top and bottom halves.
  • Antibodies were radiolabeled with carrier-free 90 Y chloride provided by Amersham in 0.04 M HCl. An aliquot of radioisotope (10-20 mCi/mg antibody) was transferred to a polypropylene tube and 0.02 ⁇ volume of metalfree 2 M sodium acetate was added to adjust the solution to pH 3.6. 2B8-NaDTPA (0.3 mg; 10.0 mg/mL in normal saline) was added immediately and the solution gently mixed. The solution was checked with pH paper to verify a pH of 3.8-4.1 and incubated for 5 min.
  • the reaction was quenched by transferring the reaction mixture to a separate polypropylene tube containing 1 ⁇ PBS with 75 mg/mL human serum albumin (HSA) and 1 mM diethylenetriaminepentaacetic acid (DTPA) and gently mixed.
  • HSA human serum albumin
  • DTPA diethylenetriaminepentaacetic acid
  • Radioactivity remaining in the supernatant fraction was determined with a scintillation counter. Data were plotted as the quotient of the total radioactivity added divided by the cell-associated radioactivity versus the inverse of the cell number per tube. The y axis intercept represents the immunoreactive fraction.
  • the 2B8-MX-DTPA conjugate was radiolabeled with 90 Y and formulated as described in the “mix & shoot” protocol provided above. Two lots of radiolabeled conjugate were prepared; one lot was used for assessing radioincorporation stability and the other lot used to assess retention of immunoreactivity. The formulated conjugates were incubated at 4° C. for 48 hours and aliquots analyzed at time 0, 24 h and 48 hours using non-reducing SDS-PAGE and autoradiography. Immunoreactivity at each time point was assessed using the assay described above.
  • the stability of 90 Y-labeled 2B8-MX-DTPA was assessed by incubation in human serum at 37° C. for up to 72 hours.
  • the conjugated antibody was radiolabeled with yttrium-[90] and formulated as described above.
  • the radiolabeled conjugate was diluted 1:10 with normal human serum (non-heatinactivated) and aliquots incubated in plastic tubes at 37° C. At selected times, samples were removed and analyzed by non-reducing SDS-PAGE and autoradiography.
  • Yttrium-[90]-labeled 2B8-MX-DTPA was evaluated for tissue biodistribution in eight to ten week old BALB/c mice.
  • the radiolabeled conjugate was prepared and formulated as described above. Mice were injected intravenously with 5 ⁇ Ci of 90 Y-labeled 2B8-MX-DTPA and groups of five mice were sacrificed at 1, 24, 48, and 72 hours. After sacrifice, the tail, heart, lungs, liver, kidney, spleen, muscle, femur were removed, washed, weighed; a sample of blood and skin were also removed for analysis. Radioactivity associated with each tissue sample was determined by measuring bremstrahlung radiation using a gamma counter and the percent injected dose per gram tissue and percent injected dose per organ determined.
  • FIGS. 2, 3, and 4 show that the radiolabeled conjugate exhibited no significant degradation over a period of 48 h when incubated at 4° C.
  • Instant thin-layer chromatography analysis showed no loss of 90 Y during the 48 h incubation; these results were corroborated by SDS-PAGE/autoradiographic analysis (Table 28).
  • the immunoreactivity also was relatively constant at >88% (Table 29).
  • radioactivity decreased from 10-12% at 1 h to 8%-10% at 72 h.
  • radioactivity was relatively constant at approximately 3% from 24 h through 72 h.
  • the radioactivity in the gastrointestinal tract was constant at 0.5-1% from 24 h to 72 h.
  • Radioactivity for muscle remained approximately 0.6% throughout the course of the study.
  • the uptake of radioactivity by femur (bone) remained less than 4% at all time points indicating that the amount free yttrium in the conjugate preparation was negligible and that little free radiometal was released during the course of the study.
  • Ref A Schema for Absorbed-dose Calculation for Biologically Distributed Radionuclides, MIRD J. of Nucl. Med./Suppl. #1, 2/68 Calculations Performed Using a Spreadsheet Template in Symphony (Lotus Development Corporation) and Created by Phillip L. Hagan, MS, Nuclear Medicine Service, VA Hospital, San Diego, Calif. 92161
  • the original radiolabeling protocol for preparing 90 Y-labeled 2B8-MX-DTPA utilized a particularly laborious and time-consuming HPLC purification step for removing non-protein bound 90 Y from the preparation.
  • HPLC step in favor of what has been termed a “mix-and-shoot” protocol.
  • the goal was to identify radiolabeling conditions which would result in a very high radioincorporation of isotope into the conjugate, thereby obviating the need for the purification step. It was discovered that >95% radioincorporation could be-obtained at pH 3.6 with a five to ten minute incubation.
  • the dosimetry results are comparable to results obtained for patients enrolled in an on-going clinical trial (IDEC study #1315), when compared per millicurie of injected dose.
  • mean values (rads +SD) for whole body, heart, liver, and spleen were 1.40 ⁇ 0.57, 10.50 ⁇ 4.68, 9.89 ⁇ 8.91, and 9.75 ⁇ 6.00, respectively.
  • the radiolabeled conjugate was stable when incubated in human serum at 37° C. for 72 hours.
  • Biodistribution studies in BALB/c mice demonstrated no unusual tissue deposition, including bone.
  • Estimates of radiation absorbed doses to a “standard” 70 Kg human were comparable to those obtained in an on-going clinical trial using 90 Y-labeled 2B8-MX-DTPA.
  • the results of these studies showed that 90 Y-labeled 2B8-MX-DTPA produced using the “mix-and-shoot” protocol was comparable to that prepared using the conventional HPLC process.
  • Validation of the scale-up protocol for preparing clinical-grade radiolabeled conjugate showed that the method was reproducible and that the product was comparable to that produced using the current HPLC method.
  • the results of these pre-clinical studies indicate that this new “mix-&-shoot” protocol can be used to prepare 90 Y-labeled 2B8-MX-DTPA suitable for use in clinical trials.
  • Radiolabeling kit protocols for preparation of 111 In and 90 Y-labeled 2B8-MX-DTPA (In2B8 and Y2B8, respectively) and to establish release specifications for clinical products.
  • the radiolabeling kit protocols are reproducible with respect to radioincorporation and binding to antigen-positive SB cells and indicate the suitability of the radiolabeling kit for use in the clinical trials. It is recommended that In2B8 and Y2B8 release specifications for radioincorporation and binding be established at ⁇ 95% and ⁇ 70%, respectively.
  • a 90 Y-labeled murine monoclonal anti-CD20 antibody (Y2B8) is currently being evaluated in clinical trials for the treatment of relapsed B-cell lymphoma.
  • the yttrium isotope lacks a gamma component making it unsuitable for imaging systems. Therefore, 111 In-labeled 2B8-MX-DTPA (In2B8) will be used to assess tumor localization and dosimetry in patients prior to or after treatment with the yttrium-labeled therapeutic.
  • the protocols used currently for the preparation of Y2B8 and In2B8, referred to as the “mix-&-shoot” methods produce radiolabeled antibodies suitable for clinical studies. However, simplification of the labeling process would expedite dose preparation in a clinical setting.
  • the new radiolabeling kit is preferably comprised of four components: 1.) 2B8-MX-DTPA in low-metal normal saline at 2 mg/mL, 2.) 50 mM sodium acetate used to adjust radioisotope solution to appropriate labeling pH, 3.) formulation buffer (1 ⁇ PBS, pH 7.4 containing 7.5% human serum albumin and 1 mM DTPA), 4.) empty 10 mL glass vial (reaction vial). All components are tested to be sterile and pyrogen-free.
  • Radiolabeling kit components should be allowed to come to room temperature before use.
  • C 0 Radioactivity concentration at time of calibration (see manufacturer's Certificate of Analysis).
  • Radiolabeling kit components should be allowed to come to room temperature before use.
  • C 0 Radioactivity concentration at time of calibration (see manufacturer's Certificate of Analysis).
  • Step 3 The septa of the reaction vial and the sodium acetate vial were wiped with alcohol. Using a 1 cc syringe, the calculated volume (Step 1a or 1b) of 50 mM sodium acetate (Step 2) was transferred to the reaction vial. The vial was mixed by inverting several times.
  • reaction mixture was calculated by adding the amount of Y-90 chloride added (Step 4), plus the amount of 50 mM sodium acetate added (Step 3), plus the amount of 2B8-MX-DTPA added (Step 5).
  • radiolabeled antibodies were formulated with 1 ⁇ PBS containing 7.5% (w/v) human serum albumin (HSA; clinical-grade; Baxter-Hyland) and 1 mM DTPA. Results of the release tests performed on each validation lot are described below.
  • HSA human serum albumin
  • the human cell lines SB (CD20-positive) and HSB (CD20-negative) were obtained from American Type Culture Collection and cultured in T-flasks using RPMI-1640 containing 10% fetal bovine serum supplemented with 2% glutamine. Cultures were maintained at 37° C. and 5% CO 2 . Cells were typically split 1:2 every other day and harvested at 0.5-2.5 ⁇ 10 6 cells/mL and viability's >80%. Cell concentrations were determined using a hemacytometer and viability determined by trypan blue exclusion.
  • Percent binding was assessed by each operator using lyophilized CD20 positive SB cells according to the following protocols for In2B8 and Y2B8, respectively. These assays provide for a fast and efficient method of confirming that the radiolabeled antibody still recognizes CD20 as an antigen. At one clinical site, CD20-negative HSB cells were also evaluated. Lyophilized cells were prepared and stored according to the above method, “Preparation of Lyophilized SB and HSB Cells”.
  • Dilution buffer (1 ⁇ PBS, pH 7.2-7.4 containing 1% Bovine Serum Albumin (BSA), and 0.02% Sodium Azide 0.2 ⁇ m filtered and stored at room temperature.
  • BSA Bovine Serum Albumin
  • SWFI sterile water for injection
  • kits were developed. The concentrations of sodium acetate and 2B8-MX-DTPA were reduced to 50 mM and 2 mg/mL, respectively, to allow accurate volume transfers using syringes. All kit components were preferably filled in glass septum vials and tested for sterility and pyrogenicity by IDEC before release, thus eliminating the need for these tests to be performed at the clinical sites. At the site, all reagent manipulations are performed using sterile syringes and needles. Therefore, adherence to aseptic technique customarily found in a radiopharmacy environment insures that the radiolabeled and formulated anti-bodies are suitable for patient administration.
  • the murine anti-CD20 monoclonal antibody designated 2B8 has been cloned in CHO cells to yield a high expression cell line. Specificity of the CHO-derived antibody for CD20-positive human cells was demonstrated by FACS analysis and competitive binding. Negligible binding was observed to human T-cells. The affinity of the antibody for CD20-positive cells was determined to be 1.3 ⁇ 10 ⁇ 10 M using a competitive binding assay. The antibody was reacted with the chelating agent MX-DTPA to form a conjugate, 2B8-MX-DTPA, with negligible loss of immunoreactivity (affinity value was 4.4 ⁇ 10 ⁇ 10 M.
  • Optimal chelator conjugation as determined by measuring radioincorporation of 111 In, was achieved after eight hours reaction. Radiolabeling protocols for 2B8-MX-DTPA were optimized for 90 Y or 111 In with respect to pH and incubation time to insure maximal radioincorporation (>95%) and retention of immunoreactivity (>70%). Release specifications for In2B8 and Y2B8 prepared using CHO-derived 2B8-MX-DTPA in clinical trials were recommended for radioincorporation (>95%) and binding to lyophilized and reconstituted CD20-positive human cells (>70%). Taken together, these results indicate the suitability of CHO-derived 2B8-MX-DTPA for use in clinical trials.
  • the 2B8 antibody previously used was produced in hollow-fiber bioreactors. To reduce the manufacturing costs of this antibody, it has been cloned and expressed in CHO cells to yield a high-expression production cell line.
  • This example describes results of the in vitro characterization of the CHO-derived 2B8 antibody, the conjugated antibody (2B8-MX-DTPA), and the 90 Y and 111 In-labeled antibody products prepared using the clinical radiolabeling kit protocols.
  • the human cell lines SB (CD20-positive) and HSB (CD20-negative) were obtained from American Type Culture Collection and cultured in T-flasks using RPMI-1640 containing 10% fetal bovine serum supplemented with 2% glutamine. Cultures were maintained at 37° C. and 5% CO 2 . Cells were typically split 1:2 every other day and harvested at 0.5-2.5 ⁇ 10 6 cells/mL and viability's >80%. Cell concentrations were determined using a hemacytometer and viability determined by trypan blue exclusion. Specific information on cell lots is recorded in Notebook# 1553 and in the binder titled “Cell Activity Logbook 1995 & 1996” authored by Ron Morena.
  • CHO-derived 2B8 was produced under GMP conditions in IDEC's manufacturing facility. The antibody was formulated in low-metal normal saline at 11.5 mg/ml. Antibodies were determined to be homogeneous by SDS-PAGE. 2B8-MX-DTPA was produced under GMP conditions according to PSBR-043 from CHO-derived 2B8 and formulated in low-metal saline at 2 mg/mL (Lot #'s 0165A and 0165B).
  • Ruthenium-labeled tracer antibody was prepared by incubating CHO-derived 2B8 (lot #165) in 1 ⁇ PBS, pH 7.2 with the N-hydroxysuccinimide ester of ruthenium (II) tris-bipyridine chelator (TAG-NHS) at a 15:1 molar ratio of TAG-NHS to antibody. After 1 h incubation at ambient temperature, protected from light, the reaction was quenched with glycine for 10 min. Unreacted TAG was removed by size exclusion chromatography using a Pharmacia PD-10 column equilibrated with 1 ⁇ PBS. Protein concentration was determined using the Bradford protein assay. TAG incorporation was determined by measuring absorbance at 455 nm. The molar ratio of TAG to protein was calculated to be 3.0.
  • Assays were performed in 12 ⁇ 75 mm polypropylene tubes. Varying amounts of competing antibody (0.002-17 ug/mL) were incubated in 1 ⁇ PBS, pH 7.2, containing 1% (w/v) BSA with 0.08 ug/mL TAG-labeled CHO 2B8, 0.08 mg/mL anti-CD19 beads, and 167,000 cells/mL. After incubation at ambient temperature with orbital mixing for 3 h, relative electrochemiluminescence (ECL) was determined using the ORIGEN instrument. Mean ECL values were determined for duplicated samples and plotted vs. competing antibody concentration using Kaleidagraph software. For some experiments, per cent inhibition was plotted. Competition curves were fitted and EC 50 values (antibody concentration giving 50% maximal binding) determined using the following 4-parameter program:
  • m4 maximum signal response in relative ECL units Average affinity values were calculated from EC50 values and the known concentration of trace antibody using the method of Muller.
  • the chelating agent, 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) was provided as a dry powder (free-acid) and stored desiccated at ⁇ 20° or ⁇ 70° C.
  • Approximately 3 mg of CHO 2B8 antibody in low-metal normal saline were adjusted to pH 8.6 by adding one-tenth volume of 50 mM sodium borate, pH 8.6.
  • Antibody at 10-11 mg/mL was incubated at a 4: 1 molar ratio of MX-DTPA to protein by adding MX-DTPA dissolved in 50 mM sodium borate, pH 8.6. After incubation at ambient temperature (2 to 24 h), unreacted chelator was removed from the conjugate by repetitive diafiltration in low-metal normal saline using Centricon 30 spin-filters.
  • In2B8 was prepared using the radiolabeling kit protocol as described herein. Antibody was labeled at a specific activity of 3 mCi/mg and formulated to 0.2 mg/mL. Briefly, 0.5 to 2 mCi of 111 In chloride was transferred to a metal-free microfuge tube and adjusted to approximately pH 4.2 using a 1.2 ⁇ volume of low-metal 50 mM sodium acetate.
  • 2B8-MX-DTPA at 2 mg/mL was added to the indium acetate solution and after incubation at ambient temperature for 30 min., the labeled antibody was formulated to 0.2 mg/mL in 1 ⁇ PBS, pH 7.2 containing 7.5% (w/v) human serum albumin and 1 mM DTPA (4% to 6% final concentration of HSA). All samples were tested for radioincorporation in triplicate; values were >95%.
  • Y2B8 was also prepared using a small-scale version of the radiolabeling kit protocol described in Example 1. Antibody was labeled at a specific activity of 15 mCi/mg and formulated to 0.3 mg/mL. Briefly, 0.5 to 2 mCi of 90Y chloride was transferred to a metal-free microfuge tube and adjusted to approximately pH 4.2 using a 1.2 ⁇ volume of low-metal 50 mM sodium acetate.
  • 2B8-MX-DTPA at 2 mg/mL was added to the 90 Y acetate solution and after incubation at ambient temperature for 5 min., the labeled antibody was formulated to 0.3 mg/mL in 1 ⁇ PBS, pH 7.2 containing 7.5% (w/v) human serum albumin and 1 mM DTPA (final concentration of HSA, 4% to 6%). All samples were tested for radioincorporation in triplicate; values were >95%.
  • radioactivity concentrations of the final radiolabeled products were calculated based on the amount of radioactivity added to the reaction mixture and by reference to the Certificate of Analysis for the radioisotope.
  • Antibody concentration of the quenched reaction mixtures were calculated from the known amount of antibody added.
  • the amount of radioactivity associated with the conjugates (radioincorporation) in the final products or incubation samples was determined using a commercially available kit manufactured by Biodex (Tec-Control Radiochromatographic Kit; see Example 1). In general, 1 ⁇ L of the test samples were applied in duplicate or triplicate using a micropipetter and developed according to the Biodex instructional insert. Strip halves were counted for radioactivity in glass tubes using an Isodata gamma counter or a Packard Top Count scintillation counter as described below. The radiolabel incorporation was calculated by dividing the amount of radioactivity in the top half of the strip by the total radioactivity found in both top and bottom halves. This value was expressed as a percentage and the mean value determined.
  • In2B8 and Y2B8 were prepared and formulated as described above.
  • Assay In2B8 or Y2B8 samples were diluted with assay dilution buffer (LXPBS, pH 7.2, containing 1% (w/v) bovine serum albumin (BSA) to 40 ng/mL and 11 ng/mL, respectively.
  • LXPBS assay dilution buffer
  • BSA bovine serum albumin
  • Antigen-positive (SB) and antigen-negative (HSB) cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum at 37° C. and 5% CO 2 .
  • Cells (viability >90% as determined by trypan blue exclusion) were harvested at ambient temperature at a cell density of 0.5-2 ⁇ 10 6 cells/mL by centrifugation (1300 rpm in a Sorvall centrifuge) and washed twice with 50 mL 1 ⁇ HBSS.
  • Pelleted cells were resuspended to 50 ⁇ 10 6 cells/mL in prechilled 1 ⁇ HBSS containing 1% (w/v) bovine serum albumin (BSA) and 10% (w/v/) mannitol (lyophilization buffer).
  • BSA bovine serum albumin
  • mannitol lyophilization buffer
  • Cell suspensions were dispensed into 1.5 mL polypropylene microfuge tubes with o-ring gaskets at 50 ⁇ 10 6 cells/mL (0.5 mL per tube) and lyophilized overnight at 30 to 60 millitorr. Lyophilized cells were stored desiccated at 2-8° C. and reconstituted in sterile water for assays.
  • Lyophilized SB and HSB cells in 1.5 mL polypropylene tubes were reconstituted to 50 ⁇ 10 6 cells/mL using sterile water. Diluted In2B8 or Y2B8 was added to cells, in triplicate, and incubated for 45 to 60 min with end-over-end mixing at ambient temperature, respectively. After incubation, cells were pelleted by centrifugation and cell-bound radioactivity determined by counting samples in an Isodata Gamma Counter or a Packard Top Count scintillation counter as described below. Radioactivity bound (B) to cells was calculated by subtracting the unbound radioactivity (supernatant) from the total radioactivity added. Total radioactivity was determined from the radioactivity counted in tubes without cells. Percent binding was calculated by expressing the bound counts as a percentage of the total counts.
  • Radioincorporation samples were counted for 1 min. using an Isodata gamma counter.
  • the counter was set for energy windows of 100-500 KeV and the background adjusted to zero immediately before use for samples using 111 In.
  • the Isodata gamma counter was also used for counting ITLC strips having 90 Y spotted on them.
  • the energy windows for detection of the bremstrulung radiation were 100-1000 KeV.
  • Release specifications for radioincorporation and binding to CD20-positive cells were established by preparing six doses each of In2B8 and Y2B8 using two lots of clinical-grade 2B8-MX-DTPA (lot #'s 0219 and 0220) prepared according to the present invention and filled under GMP conditions. Release assays were performed as described above.
  • Binding of CHO 2B8 to CD20-positive cells was evaluated in competition assays using the ORIGEN chemiluminescent detection system. Lyophilized and reconstituted antigen-positive SB cells were incubated with increasing amounts bf antibody in the presence of ruthenium-labeled CHO 2B8 tracer. Results showed that CHO 2B8 inhibits binding to CD20-positive cells to the same extent as the antibody derived from hollow-fiber bioreactors (2B8-49) (FIG. 35). The EC50 values were determined graphically and the method of Muller (1980) used to calculate average affinity values.
  • the affinity for CHO 2B8 was determined to be 1.3 ⁇ 10 ⁇ 10 M; the 2B8 antibody derived from hollow-fiber bioreactors gave an affinity value of 2.5 10 ⁇ 10 M. Non-specific binding was negligible as demonstrated by the lack of competition with the irrelevant isotype antibody, S004.
  • the 2B8 conjugate (2B8-MX-DTPA) was prepared using a protocol similar to that used for the previously characterized 2B8-49. Reactions were performed using approximately 3 mg of antibody and a 4:1 molar ratio of chelator to antibody. Incubations times of 2, 4, 8, 17, and 24 h were evaluated to determine the reaction time giving acceptable retention of binding to CD20 positive cells and high radioincorporation with 111 In.
  • Competitive binding curves comparing CHO 2B8 to CHO 2B8-MX-DTPA conjugate reacted for 8-24 h were similar, indicating that the conjugation process did not significantly alter the binding of the antibody to the CD20 antigen (FIG. 36). Using EC 50 values determined graphically (FIG.
  • Indium-[111]-labeled CHO 2B8-MX-DTPA (In2B8) was prepared using the small-scale radiolabeling kit protocol previously described for the hollow-fiber bioreactor-derived antibody (Example 1). Briefly, conjugated antibody (CHO-derived 2B8-MX-DTPA; lot # 0165A) was incubated with 111 In acetate at the indicated pH for 30 minutes at ambient temperature. Reaction mixtures were formulated with PBS, pH 7.2, containing 7.5% (w/v) human serum albumin and 1 mM DTPA. Formulated samples of In2B8 were assayed for radioincorporation using instant thin-layer chromatography.
  • Binding of In2B8 to CD20-positive cells was determined using lyophilized and reconstituted SB cells.
  • the conjugate prepared from hybridoma-produced antibody(2B8-49) was incubated with 111 In acetate for 30 min. at pH 4.2 (conditions previously established for this antibody).
  • Yttrium-[90]-labeled antibody was prepared by incubating conjugated antibody (CHO-derived 2B8-MX-DTPA) with 90 Y acetate at the indicated pH for 5 minutes at ambient temperature. Reaction mixtures were formulate in PBS, pH 7.2 containing 7.5%(w/v) human serum albumin and 1 mM DTPA. Formulated samples of Y2B8 were assayed for radioincorporation using instant thin-layer chromatography. Binding of Y2B8 to CD20-positive cells was determined using lyophilized and reconstituted SB cells. For comparison, the conjugate prepared from hybridoma-produced antibody (2B8-49) was incubated with 90Y acetate for 5 min. at pH 4.2 (conditions previously established for this antibody).
  • conjugated antibody CHO-derived 2B8-MX-DTPA
  • Immunoreactivities for In2B8 and Y2B8 prepared from CHO 2B8 were determined using the method of Lindmo et al. Increasing amounts of freshly harvested CD20-positive SB cells were incubated with a fixed amount of In2B8 or Y2B8 under conditions of antigen excess. Reciprocal plot analysis of the binding data allowed immunoreactivities of 80.6% and 72.2% for In2B8 and Y2B8, respectively, to be determined (FIGS. 37 and 38).
  • Binding to CD20-positive cells ranged from 81.9% to 85.1% with a mean of 83.6%; binding to CD20-negative cells was ⁇ 4%.
  • radioincorporation ranged from 97.4% to 98.7% with a mean of 98.2%.
  • Binding to CD20-positive cells ranged from 81.4% to 82.7% with a mean of 81.9%; binding to CD20-negative cells was ⁇ 8%.
  • the anti-CD20 murine monoclonal antibody (2B8) cloned and expressed in CHO cells maintains specificity for CD20-positive human cells as shown by FACS and competitive binding analysis. Binding to human T-cells was minimal. The affinity of the antibody for human CD20-positive cells was determined to be 1.3 ⁇ 10 ⁇ 10 M using a competitive binding assay. Using the same assay, the 2B8 antibody derived from hollow-fiber bioreactors gave an affinity value of 2.5 ⁇ 10 ⁇ 10 M.
  • the CHO 2B8 antibody was reacted with MX-DTPA to form a conjugate, 2B8-MX-DTPA, while maintaining suitable retention of immunoreactivity.
  • Optimal chelator incorporation was determined by measuring radioincorporation with 111 In and was achieved after eight hours incubation at ambient temperature. Radiolabeling protocols for the 2B8-MX-DTPA conjugate were optimized for 90 Y or 111 In with respect to pH and incubation time to insure maximal radioincorporation and retention of immunoreactivity.
  • the present invention discloses a labeling procedure, referred to as the “mix-and-shoot” method, for the preparation of clinical doses of radiolabeled antibodies which obviates the need for the currently used high performance liquid chromatographic (HPLC) step for removal of non-protein bound radioisotope.
  • HPLC high performance liquid chromatographic
  • the simplified protocol eliminates this laborious purification step while maintaining a high level of radioisotope incorporation (>95%) and improved retention of immunoreactivity (>70%).
  • the clinically-formulated radiolabeled conjugate was found to be stable in vitro when incubated at 4° C. for 48 hours based on retention of radioisotope and immunoreactivity. Additionally, the radiolabeled conjugate was stable when incubated in human serum at 37° C.

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US09/259,337 1999-03-01 1999-03-01 Radiolabeling kit and binding assay Abandoned US20020102208A1 (en)

Priority Applications (50)

Application Number Priority Date Filing Date Title
US09/259,337 US20020102208A1 (en) 1999-03-01 1999-03-01 Radiolabeling kit and binding assay
MYPI20000754A MY138674A (en) 1999-03-01 2000-02-28 Radiolabeling kit and binding assay
NZ530868A NZ530868A (en) 1999-03-01 2000-02-29 Radiolabeling kit and binding assay
SI200031085T SI2112512T1 (sl) 1999-03-01 2000-02-29 Komplet za radioaktivno označevanje in test vezave
NZ513788A NZ513788A (en) 1999-03-01 2000-02-29 Radiolabeling kit and binding assay
AT00913645T ATE433108T1 (de) 1999-03-01 2000-02-29 Radiomarkierungssatz und bindungstest
HU0202522A HU229627B1 (en) 1999-03-01 2000-02-29 Radiolabeling kit and binding assay
HU1300275A HU230516B1 (hu) 1999-03-01 2000-02-29 Radioaktív jelölő készlet és kötési mérőeljárás
ES09158514.1T ES2526723T3 (es) 1999-03-01 2000-02-29 Kit de radiomarcaje y ensayo de unión
MXPA01008837A MXPA01008837A (es) 1999-03-01 2000-02-29 Equipo de radiomarcado y ensayo de union.
EEP200100465A EE05650B1 (et) 1999-03-01 2000-02-29 Antikehade radioaktiivse m„rgistamise komplekt ja seostamistest
HK02107258.0A HK1045730B (zh) 1999-03-01 2000-02-29 放射性标记试剂盒和结合测定试验
EP09158514.1A EP2112512B1 (en) 1999-03-01 2000-02-29 Radiolabeling kit and binding assay
PL352531A PL205780B1 (pl) 1999-03-01 2000-02-29 Sposób radioaktywnego znakowania skoniugowanego z chelatorem przeciwciała anty-CD20, test wiązania i test wiązania kompetytywnego
CZ20013126A CZ303262B6 (cs) 1999-03-01 2000-02-29 Zpusob radionuklidového znacení protilátky konjugované s chelatotvorným prostredkem a vazebný test
AU35052/00A AU776747B2 (en) 1999-03-01 2000-02-29 Radiolabeling kit and binding assay
CA2742153A CA2742153C (en) 1999-03-01 2000-02-29 Method for radiolabeling a chelator-conjugated antibody with 111in
SK1216-2001A SK287745B6 (sk) 1999-03-01 2000-02-29 Spôsob rádionuklidového značenia protilátky konjugovanej s chelatotvorným prostriedkom a testovanie viazania
SK91-2010A SK288096B6 (sk) 1999-03-01 2000-02-29 Method for radiolabeling chelator-conjugated antibody and binding assay
SI200031036T SI1192463T1 (sl) 1999-03-01 2000-02-29 Komplet za radiooznačevanje in vezavni preizkus
IL14510900A IL145109A0 (en) 1999-03-01 2000-02-29 A radiolabeling kit and binding assay for antibodies
CZ2010-746A CZ306959B6 (cs) 1999-03-01 2000-02-29 Souprava pro značení radionuklidem a imunoesej
PT91585141T PT2112512E (pt) 1999-03-01 2000-02-29 Kit de radiomarcação e análise de ligação
RU2001126400/15A RU2251110C2 (ru) 1999-03-01 2000-02-29 Набор для радиоактивного мечения и анализ связывания
PT00913645T PT1192463E (pt) 1999-03-01 2000-02-29 Kits de radiomarcação e análises de ligação de anticorpos
BRPI0017596A BRPI0017596B8 (pt) 1999-03-01 2000-02-29 método para radiomarcação de um anticorpo conjugado a um quelante com 111in para administração a um paciente
MEP-2009-21A ME00782B (me) 1999-03-01 2000-02-29 Postupak za radioobilježavanje antitijela i postupak za određivanje imunoreaktivnosti radioobilježenih antitijela, ćelijskim vezivanjem
DK09158514.1T DK2112512T3 (en) 1999-03-01 2000-02-29 Kit for radiolabeling and binding assay
YU61601A RS51778B (sr) 1999-03-01 2000-02-29 POSTUPAK ZA RADIOAKTIVNO OBELEŽAVANJE ANTITELA KONJUGOVANOG ZA HELATOR POMOĆU 111In
CNB008057397A CN1235639C (zh) 1999-03-01 2000-02-29 放射性标记试剂盒和结合测定试验
KR1020017011093A KR100729247B1 (ko) 1999-03-01 2000-02-29 방사선표지 키트 및 결합 분석
ES00913645T ES2328100T3 (es) 1999-03-01 2000-02-29 Kit de radiomarcaje y ensayo de union.
DK00913645T DK1192463T3 (da) 1999-03-01 2000-02-29 Kit- og bindingsassay to radioaktiv mærkning
UA2001096636A UA78484C2 (en) 1999-03-01 2000-02-29 Method of radioactive marking of cd 20 antibody
BRPI0008719 BRPI0008719B8 (pt) 1999-03-01 2000-02-29 Método para radiomarcação de um anticorpo conjugado a um quelante com 90y e ensaio de ligação para determinação da ligação percentual de um anticorpo radiomarcado à sua célula alvo
DE60042326T DE60042326D1 (de) 1999-03-01 2000-02-29 Radiomarkierungssatz und bindungstest
JP2000602634A JP4704570B2 (ja) 1999-03-01 2000-02-29 放射標識キット及び結合アッセイ
HR20010714A HRP20010714B1 (hr) 1999-03-01 2000-02-29 Kit za radio-obilježavanje i ispitivanje vezivanja
PCT/US2000/005061 WO2000052473A2 (en) 1999-03-01 2000-02-29 Radiolabeling kit and binding assay
CA2362119A CA2362119C (en) 1999-03-01 2000-02-29 Radiolabeling kit and binding assay
EP00913645A EP1192463B1 (en) 1999-03-01 2000-02-29 Radiolabeling kit and binding assay
TW089103582A TWI294969B (enExample) 1999-03-01 2000-03-01
ZA200106943A ZA200106943B (en) 1999-03-01 2001-08-22 Radiolabeling kit and binding assay.
NO20014245A NO329142B1 (no) 1999-03-01 2001-08-31 Fremgangsmåte for radiomerking av et chelator-konjugert antistoff med 90Y samt bindingsassay
BG105957A BG65690B1 (bg) 1999-03-01 2001-09-27 Радиомаркиран кит и метод за изследване на свързването
US11/033,439 US7608241B2 (en) 1999-03-01 2005-01-12 Radiolabeling method
CY20091100912T CY1109335T1 (el) 1999-03-01 2009-09-01 Κιτ ραδιοεπισημανσης και δοκιμασια δεσμευσης
HK09110560.0A HK1131208B (en) 1999-03-01 2009-11-12 Radiolabeling kit and binding assay
NO20100717A NO330705B1 (no) 1999-03-01 2010-05-18 Fremgangsmåte for radiomerking av antistoff med 111In og bindingsassay for bestemmelse av slikt antistoff
CY20151100160T CY1116265T1 (el) 1999-03-01 2015-02-16 Kit ραδιοεπισημανσης και προσδιορισμος δεσμευσης

Applications Claiming Priority (1)

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EP (2) EP1192463B1 (enExample)
JP (1) JP4704570B2 (enExample)
KR (1) KR100729247B1 (enExample)
CN (1) CN1235639C (enExample)
AT (1) ATE433108T1 (enExample)
AU (1) AU776747B2 (enExample)
BG (1) BG65690B1 (enExample)
BR (2) BRPI0017596B8 (enExample)
CA (2) CA2362119C (enExample)
CY (2) CY1109335T1 (enExample)
CZ (2) CZ306959B6 (enExample)
DE (1) DE60042326D1 (enExample)
DK (2) DK1192463T3 (enExample)
EE (1) EE05650B1 (enExample)
ES (2) ES2526723T3 (enExample)
HK (1) HK1045730B (enExample)
HR (1) HRP20010714B1 (enExample)
HU (2) HU230516B1 (enExample)
IL (1) IL145109A0 (enExample)
ME (1) ME00782B (enExample)
MX (1) MXPA01008837A (enExample)
MY (1) MY138674A (enExample)
NO (2) NO329142B1 (enExample)
NZ (2) NZ513788A (enExample)
PL (1) PL205780B1 (enExample)
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Publication number Priority date Publication date Assignee Title
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US20030003097A1 (en) * 2001-04-02 2003-01-02 Idec Pharmaceutical Corporation Recombinant antibodies coexpressed with GnTIII
US20050123474A1 (en) * 2003-06-25 2005-06-09 Peregrine Pharmaceuticals Methods and apparatus for continuous large-scale radiolabeling of proteins
US20080089885A1 (en) * 2006-10-10 2008-04-17 Vaccinex, Inc. Anti-cd20 antibodies and methods of use
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WO2015131153A1 (en) * 2014-02-27 2015-09-03 Board Of Regents, The University Of Texas System Methods and compositions for isolating exosomes
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US9427478B2 (en) 2013-06-21 2016-08-30 Innate Pharma Enzymatic conjugation of polypeptides
WO2017019858A1 (en) 2015-07-30 2017-02-02 The Trustees Of The University Of Pennsylvania Single nucleotide polymorphic alleles of human dp-2 gene for detection of susceptibility to hair growth inhibition by pgd2
WO2017048902A1 (en) 2015-09-15 2017-03-23 Board Of Regents, The University Of Texas System T-cell receptor (tcr)-binding antibodies and uses thereof
US9717803B2 (en) 2011-12-23 2017-08-01 Innate Pharma Enzymatic conjugation of polypeptides
US9845363B2 (en) 2013-08-13 2017-12-19 Sanofi Antibodies to plasminogen activator inhibitor-1 (PAI-1) and uses thereof
US9856468B2 (en) 2010-07-09 2018-01-02 Bioverativ Therapeutics Inc. Processable single chain molecules and polypeptides made using same
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US10036010B2 (en) 2012-11-09 2018-07-31 Innate Pharma Recognition tags for TGase-mediated conjugation
US10064952B2 (en) 2014-10-09 2018-09-04 Genzyme Corporation Glycoengineered antibody drug conjugates
US10071169B2 (en) 2013-06-20 2018-09-11 Innate Pharma Enzymatic conjugation of polypeptides
US10183995B2 (en) 2012-05-23 2019-01-22 Argen-X N.V. IL-6 binding molecules
WO2019018633A1 (en) 2017-07-19 2019-01-24 Hoffman Technologies, Inc. COMPOSITIONS FOR THE TREATMENT OF STRESS-RELATED DISORDERS
US10214589B2 (en) 2013-03-11 2019-02-26 Genzyme Corporation Site-specific antibody-drug conjugation through glycoengineering
US10278438B2 (en) 2015-08-31 2019-05-07 Grabbies Media, Llc Consumer usable device with redeemable member
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WO2019042555A1 (en) 2017-08-31 2019-03-07 Humabs Biomed Sa MULTISPECIFIC ANTIBODIES SPECIFICALLY BINDING TO ZIKA VIRUS EPITOPES AND USES THEREOF
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US12304946B2 (en) 2018-12-19 2025-05-20 Humabs Biomed Sa Antibodies that neutralize hepatitis B virus and uses thereof
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US20240092872A1 (en) 2021-01-26 2024-03-21 Vir Biotechnology, Inc. Compositions and methods for treating hepatitis b virus infection

Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3994966A (en) * 1972-09-28 1976-11-30 The Board Of Trustees Of The Leland Stanford Junior University Chelating agents
US4043998A (en) * 1974-10-09 1977-08-23 The Board Of Trustees Of The Leland Stanford Junior University 1-(P-benzenediazonium)-ethylenediamine tetraacetic acid
US4315851A (en) * 1978-12-29 1982-02-16 Kureha Kagaku Kogyo Kabushiki Kaisha Pharmaceutical composition having antitumor activity
US4331647A (en) * 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US4348376A (en) * 1980-03-03 1982-09-07 Goldenberg Milton David Tumor localization and therapy with labeled anti-CEA antibody
US4361544A (en) * 1980-03-03 1982-11-30 Goldenberg Milton David Tumor localization and therapy with labeled antibodies specific to intracellular tumor-associated markers
US4444744A (en) * 1980-03-03 1984-04-24 Goldenberg Milton David Tumor localization and therapy with labeled antibodies to cell surface antigens
US4454106A (en) * 1982-06-07 1984-06-12 Gansow Otto A Use of metal chelate conjugated monoclonal antibodies
US4460561A (en) * 1980-03-03 1984-07-17 Goldenberg M David Tumor localization and therapy with labeled antibodies specific to intracellular tumor-associated markers
US4460559A (en) * 1980-03-03 1984-07-17 Goldenberg Milton David Tumor localization and therapy with labeled antibodies specific to intracellular tumor-associated markers
US4472509A (en) * 1982-06-07 1984-09-18 Gansow Otto A Metal chelate conjugated monoclonal antibodies
US4622420A (en) * 1980-03-18 1986-11-11 The Regents Of The University Of California Chelating agents and method
US4634586A (en) * 1984-05-21 1987-01-06 The Board Of Trustees Of The Leland Stanford Junior University Reagent and method for radioimaging leukocytes
US4676609A (en) * 1982-11-26 1987-06-30 Saburo Matsui Adjustable bows for loupe frame
US4707352A (en) * 1984-01-30 1987-11-17 Enzo Biochem, Inc. Method of radioactively labeling diagnostic and therapeutic agents containing a chelating group
US4722892A (en) * 1984-08-31 1988-02-02 Meares Claude F Monoclonal antibodies against metal chelates
US4735210A (en) * 1985-07-05 1988-04-05 Immunomedics, Inc. Lymphographic and organ imaging method and kit
US4824986A (en) * 1985-04-26 1989-04-25 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Metal chelate protein conjugate
US4831175A (en) * 1986-09-05 1989-05-16 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Backbone polysubstituted chelates for forming a metal chelate-protein conjugate
US4855353A (en) * 1986-02-14 1989-08-08 Nihon Medi-Physics Co., Ltd. High molecular compounds having amino groups, and their utilization
US4861579A (en) * 1988-03-17 1989-08-29 American Cyanamid Company Suppression of B-lymphocytes in mammals by administration of anti-B-lymphocyte antibodies
US4921690A (en) * 1986-12-29 1990-05-01 City Of Hope Method of enhancing the biodistribution of antibody for localization in lesions
US4923985A (en) * 1988-05-25 1990-05-08 The United States Of America As Represented By The Department Of Health & Human Services Process for synthesizing macrocyclic chelates
US5009069A (en) * 1990-08-24 1991-04-23 Molini Alberto E Method of recovering energy from ocean water
US5034223A (en) * 1986-10-09 1991-07-23 Neorx Corporation Methods for improved targeting of antibody, antibody fragments, hormones and other targeting agents, and conjugates thereof
US5059518A (en) * 1988-10-20 1991-10-22 Coulter Corporation Stabilized lyophilized mammalian cells and method of making same
US5101827A (en) * 1985-07-05 1992-04-07 Immunomedics, Inc. Lymphographic and organ imaging method and kit
US5124471A (en) * 1990-03-26 1992-06-23 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Bifunctional dtpa-type ligand
US5162115A (en) * 1989-05-09 1992-11-10 Pietronigro Dennis D Antineoplastic solution and method for treating neoplasms
US5208008A (en) * 1990-11-14 1993-05-04 University Of Pittsburgh Regioselective chemical modification of monoclonal antibodies
US5219556A (en) * 1990-07-09 1993-06-15 Mallinckrodt Medical, Inc. Stabilized therapeutic radiopharmaceutical complexes
US5246692A (en) * 1986-09-05 1993-09-21 The United States Of America As Represented By The Secretary Of Health And Human Services Backbone polysubstituted chelates for forming a metal chelate-protein conjugate
US5363846A (en) * 1986-01-16 1994-11-15 The General Hospital Corporation Method for the diagnosis and treatment of inflammation
US5376356A (en) * 1989-03-14 1994-12-27 Neorx Corporation Imaging tissue sites of inflammation
US5403573A (en) * 1992-04-23 1995-04-04 The Curators Of The University Of Missouri Radiolabeled protein composition and method for radiation synovectomy
US5428154A (en) * 1988-05-25 1995-06-27 The United States Of America As Represented By The Department Of Health And Human Services Complexes of functionalized tetraazacyclododecane chelates with bismuth, lead, ytriium, actinium, or lanthanide metal ions
US5500362A (en) * 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5545403A (en) * 1990-10-17 1996-08-13 Burroughs Wellcome Co. Method for treating a mammal by administering a CHO-glycosylated antibody
US5595721A (en) * 1993-09-16 1997-01-21 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20
US5641637A (en) * 1991-01-11 1997-06-24 Cobe Laboratories, Inc. Method of preparing lyophilized and frozen cell standards
US5976492A (en) * 1994-10-05 1999-11-02 Immunomedics, Inc. Radioactive phosphorus labeled proteins for targeted radiotherapy
US6010680A (en) * 1994-06-03 2000-01-04 Immunomedics, Inc. Thiolation of proteins for radionuclide-based radioimmunodetection and radioimmunotherapy

Family Cites Families (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8308235D0 (en) 1983-03-25 1983-05-05 Celltech Ltd Polypeptides
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4636380A (en) 1984-04-23 1987-01-13 Wong Dennis W Novel physiologic chemical method of labeling protein substances with the radionuclides of indium
EP0529645B1 (en) * 1986-09-05 1996-10-23 GANSOW, Otto A. Process for the preparation of backbone polysubstituted chelates
US5099069A (en) 1986-09-05 1992-03-24 Gansow Otto A Backbone polysubstituted chelates for forming a metal chelate-protein conjugate
US5217704A (en) 1987-11-06 1993-06-08 Abbott Laboratories Methods and materials for the preparation of metal labelled antibody solutions
AU619218B2 (en) 1987-11-06 1992-01-23 Abbott Laboratories Methods and materials for the preparation of metal labelled antibody solutions
US5130118A (en) 1987-11-06 1992-07-14 Abbott Laboratories Methods and materials for the preparation of metal labelled antibody solutions
US4978745A (en) 1987-11-23 1990-12-18 Centocor, Inc. Immunoreactive heterochain antibodies
US4975369A (en) 1988-04-21 1990-12-04 Eli Lilly And Company Recombinant and chimeric KS1/4 antibodies directed against a human adenocarcinoma antigen
CA2010511A1 (en) 1989-03-01 1990-09-01 Roberto L. Ceriani Method of enhancing cancer therapy by administration of unsaturated fatty acids
US5460785A (en) 1989-08-09 1995-10-24 Rhomed Incorporated Direct labeling of antibodies and other protein with metal ions
JPH06501705A (ja) 1990-11-05 1994-02-24 ブリストル−マイアーズ　スクイブ　カンパニー 抗−腫瘍抗体及び生物学的活性剤の組合せによる相乗治療
US5541287A (en) 1992-06-09 1996-07-30 Neorx Corporation Pretargeting methods and compounds
US5620675A (en) 1992-06-23 1997-04-15 Diatech, Inc. Radioactive peptides
US5716596A (en) 1992-06-23 1998-02-10 Diatide, Inc. Radioactively labeled somatostatin-derived peptides for imaging and therapeutic uses
US5736137A (en) 1992-11-13 1998-04-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
ES2091684T3 (es) * 1992-11-13 1996-11-01 Idec Pharma Corp Aplicacion terapeutica de anticuerpos quimericos y radiomarcados contra el antigeno de diferenciacion restringida de los linfocitos b humanos para el tratamiento del linfoma de las celulas b.
US5650134A (en) 1993-01-12 1997-07-22 Novartis Ag (Formerly Sandoz Ltd.) Peptides
US5686578A (en) 1994-08-05 1997-11-11 Immunomedics, Inc. Polyspecific immunoconjugates and antibody composites for targeting the multidrug resistant phenotype
US5874540A (en) 1994-10-05 1999-02-23 Immunomedics, Inc. CDR-grafted type III anti-CEA humanized mouse monoclonal antibodies
US5942210A (en) 1994-11-15 1999-08-24 Cytogen Corporation Methods for lyoprotecting a macromolecule using tricine
US5830431A (en) 1995-06-07 1998-11-03 Mallinckrodt Medical, Inc. Radiolabeled peptide compositions for site-specific targeting
US5961955A (en) * 1997-06-03 1999-10-05 Coulter Pharmaceutical, Inc. Radioprotectant for peptides labeled with radioisotope
MY133346A (en) * 1999-03-01 2007-11-30 Biogen Inc Kit for radiolabeling ligands with yttrium-90
US20020102208A1 (en) * 1999-03-01 2002-08-01 Paul Chinn Radiolabeling kit and binding assay

Patent Citations (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3994966A (en) * 1972-09-28 1976-11-30 The Board Of Trustees Of The Leland Stanford Junior University Chelating agents
US4043998A (en) * 1974-10-09 1977-08-23 The Board Of Trustees Of The Leland Stanford Junior University 1-(P-benzenediazonium)-ethylenediamine tetraacetic acid
US4315851A (en) * 1978-12-29 1982-02-16 Kureha Kagaku Kogyo Kabushiki Kaisha Pharmaceutical composition having antitumor activity
US4401592A (en) * 1978-12-29 1983-08-30 Kureha Kagaku Kogyo Kabushiki Kaisha Pharmaceutical composition having antitumor activity
US4460559A (en) * 1980-03-03 1984-07-17 Goldenberg Milton David Tumor localization and therapy with labeled antibodies specific to intracellular tumor-associated markers
US4331647A (en) * 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US4348376A (en) * 1980-03-03 1982-09-07 Goldenberg Milton David Tumor localization and therapy with labeled anti-CEA antibody
US4361544A (en) * 1980-03-03 1982-11-30 Goldenberg Milton David Tumor localization and therapy with labeled antibodies specific to intracellular tumor-associated markers
US4444744A (en) * 1980-03-03 1984-04-24 Goldenberg Milton David Tumor localization and therapy with labeled antibodies to cell surface antigens
US4460561A (en) * 1980-03-03 1984-07-17 Goldenberg M David Tumor localization and therapy with labeled antibodies specific to intracellular tumor-associated markers
US4622420A (en) * 1980-03-18 1986-11-11 The Regents Of The University Of California Chelating agents and method
US4454106A (en) * 1982-06-07 1984-06-12 Gansow Otto A Use of metal chelate conjugated monoclonal antibodies
US4472509A (en) * 1982-06-07 1984-09-18 Gansow Otto A Metal chelate conjugated monoclonal antibodies
US4676609A (en) * 1982-11-26 1987-06-30 Saburo Matsui Adjustable bows for loupe frame
US4707352A (en) * 1984-01-30 1987-11-17 Enzo Biochem, Inc. Method of radioactively labeling diagnostic and therapeutic agents containing a chelating group
US4634586A (en) * 1984-05-21 1987-01-06 The Board Of Trustees Of The Leland Stanford Junior University Reagent and method for radioimaging leukocytes
US4722892A (en) * 1984-08-31 1988-02-02 Meares Claude F Monoclonal antibodies against metal chelates
US4824986A (en) * 1985-04-26 1989-04-25 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Metal chelate protein conjugate
US4735210A (en) * 1985-07-05 1988-04-05 Immunomedics, Inc. Lymphographic and organ imaging method and kit
US5101827A (en) * 1985-07-05 1992-04-07 Immunomedics, Inc. Lymphographic and organ imaging method and kit
US5363846A (en) * 1986-01-16 1994-11-15 The General Hospital Corporation Method for the diagnosis and treatment of inflammation
US4855353A (en) * 1986-02-14 1989-08-08 Nihon Medi-Physics Co., Ltd. High molecular compounds having amino groups, and their utilization
US4831175A (en) * 1986-09-05 1989-05-16 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Backbone polysubstituted chelates for forming a metal chelate-protein conjugate
US5246692A (en) * 1986-09-05 1993-09-21 The United States Of America As Represented By The Secretary Of Health And Human Services Backbone polysubstituted chelates for forming a metal chelate-protein conjugate
US5034223A (en) * 1986-10-09 1991-07-23 Neorx Corporation Methods for improved targeting of antibody, antibody fragments, hormones and other targeting agents, and conjugates thereof
US4921690A (en) * 1986-12-29 1990-05-01 City Of Hope Method of enhancing the biodistribution of antibody for localization in lesions
US5500362A (en) * 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US4861579A (en) * 1988-03-17 1989-08-29 American Cyanamid Company Suppression of B-lymphocytes in mammals by administration of anti-B-lymphocyte antibodies
US4923985A (en) * 1988-05-25 1990-05-08 The United States Of America As Represented By The Department Of Health & Human Services Process for synthesizing macrocyclic chelates
US5428154A (en) * 1988-05-25 1995-06-27 The United States Of America As Represented By The Department Of Health And Human Services Complexes of functionalized tetraazacyclododecane chelates with bismuth, lead, ytriium, actinium, or lanthanide metal ions
US5059518A (en) * 1988-10-20 1991-10-22 Coulter Corporation Stabilized lyophilized mammalian cells and method of making same
US5376356A (en) * 1989-03-14 1994-12-27 Neorx Corporation Imaging tissue sites of inflammation
US5162115A (en) * 1989-05-09 1992-11-10 Pietronigro Dennis D Antineoplastic solution and method for treating neoplasms
US5434287A (en) * 1990-03-26 1995-07-18 The United States Of America As Represented By The Department Of Health And Human Services Bifunctional DTPA-type ligand
US5124471A (en) * 1990-03-26 1992-06-23 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Bifunctional dtpa-type ligand
US5286850A (en) * 1990-03-26 1994-02-15 The United States Of America As Represented By The Department Of Health And Human Services Antibody DTPA-type ligand conjugates
US5219556A (en) * 1990-07-09 1993-06-15 Mallinckrodt Medical, Inc. Stabilized therapeutic radiopharmaceutical complexes
US5009069A (en) * 1990-08-24 1991-04-23 Molini Alberto E Method of recovering energy from ocean water
US5545405A (en) * 1990-10-17 1996-08-13 Burroughs Wellcome Co. Method for treating a mammal suffering from cancer with a cho-glycosylated antibody
US5545403A (en) * 1990-10-17 1996-08-13 Burroughs Wellcome Co. Method for treating a mammal by administering a CHO-glycosylated antibody
US5208008A (en) * 1990-11-14 1993-05-04 University Of Pittsburgh Regioselective chemical modification of monoclonal antibodies
US5641637A (en) * 1991-01-11 1997-06-24 Cobe Laboratories, Inc. Method of preparing lyophilized and frozen cell standards
US5403573A (en) * 1992-04-23 1995-04-04 The Curators Of The University Of Missouri Radiolabeled protein composition and method for radiation synovectomy
US5595721A (en) * 1993-09-16 1997-01-21 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20
US6010680A (en) * 1994-06-03 2000-01-04 Immunomedics, Inc. Thiolation of proteins for radionuclide-based radioimmunodetection and radioimmunotherapy
US5976492A (en) * 1994-10-05 1999-11-02 Immunomedics, Inc. Radioactive phosphorus labeled proteins for targeted radiotherapy

Cited By (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000052031A2 (en) 1999-03-01 2000-09-08 Idec Pharmaceuticals Corporation Kit for radiolabeling proteins with yttrium-90
EP1918305A1 (en) 1999-08-11 2008-05-07 Biogen Idec Inc. New clinical parameters for determining hematologic toxicity prior to radioimmunotherapy
US20030003097A1 (en) * 2001-04-02 2003-01-02 Idec Pharmaceutical Corporation Recombinant antibodies coexpressed with GnTIII
US11168125B2 (en) 2003-05-06 2021-11-09 Bioverativ Therapeutics Inc. Immunoglobulin chimeric monomer-dimer hybrids
AU2004253924B2 (en) * 2003-06-25 2009-07-30 Peregrine Pharmaceuticals, Inc. Method and apparatus for continuous large-scale radiolabeling of proteins
US7591953B2 (en) * 2003-06-25 2009-09-22 Peregrine Pharmaceuticals, Inc. Methods and apparatus for continuous large-scale radiolabeling of proteins
US20090312523A1 (en) * 2003-06-25 2009-12-17 Peregrine Pharmaceuticals, Inc. Methods and apparatus for continuous large-scale radiolabeling
US20050123474A1 (en) * 2003-06-25 2005-06-09 Peregrine Pharmaceuticals Methods and apparatus for continuous large-scale radiolabeling of proteins
US8137540B2 (en) 2003-06-25 2012-03-20 Peregrine Pharmaceuticals, Inc. Methods and apparatus for continuous large-scale radiolabeling
EP2478917A1 (en) 2005-07-08 2012-07-25 Biogen Idec MA Inc. SP35 antibodies and uses thereof
EP2238986A2 (en) 2005-07-08 2010-10-13 Biogen Idec MA Inc. Sp35 antibodies and uses thereof
EP2394661A1 (en) 2005-07-08 2011-12-14 Biogen Idec MA Inc. Sp35 antibodies and uses thereof
US10849841B2 (en) 2006-06-16 2020-12-01 The Trustees Of The University Of Pennsylvania Methods and compositions for inhibiting or reducing hair loss, acne, rosacea, prostate cancer, and BPH
US9889082B2 (en) 2006-06-16 2018-02-13 The Trustees Of The University Of Pennsylvania Methods and compositions for inhibiting or reducing hair loss, acne, rosacea, prostate cancer, and BPH
US20090130089A9 (en) * 2006-10-10 2009-05-21 Vaccinex, Inc. Anti-cd20 antibodies and methods of use
US10301393B2 (en) 2006-10-10 2019-05-28 Vaccinex, Inc. Methods of treating multiple sclerosis using anti-CD20 antibodies
US20080089885A1 (en) * 2006-10-10 2008-04-17 Vaccinex, Inc. Anti-cd20 antibodies and methods of use
US9382327B2 (en) 2006-10-10 2016-07-05 Vaccinex, Inc. Anti-CD20 antibodies and methods of use
EP2740744A2 (en) 2007-01-09 2014-06-11 Biogen Idec MA Inc. SP35 antibodies and uses thereof
EP2982695A1 (en) 2008-07-09 2016-02-10 Biogen MA Inc. Compositions comprising antibodies to lingo or fragments thereof
WO2010078526A1 (en) 2008-12-31 2010-07-08 Biogen Idec Ma Inc. Anti-lymphotoxin antibodies
EP3406141A1 (en) 2010-03-12 2018-11-28 Genzyme Corporation Combination therapy for treating cancer
EP2835053A1 (en) 2010-03-12 2015-02-11 Genzyme Corporation Combination therapy for treating breast cancer
US10927362B2 (en) 2010-07-09 2021-02-23 Bioverativ Therapeutics Inc. Processable single chain molecules and polypeptides made using same
US9856468B2 (en) 2010-07-09 2018-01-02 Bioverativ Therapeutics Inc. Processable single chain molecules and polypeptides made using same
US10968442B2 (en) 2010-07-09 2021-04-06 Bioverativ Therapeutics Inc. Chimeric clotting factors
WO2012019128A1 (en) 2010-08-06 2012-02-09 Genzyme Corporation Vegf antagonist compositions and uses thereof
EP3327032A1 (en) 2010-08-06 2018-05-30 Genzyme Corporation Vegf antagonist compositions and uses thereof
WO2012125408A1 (en) 2011-03-11 2012-09-20 Genzyme Corporation Pegylated apelin and uses thereof
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US11186638B2 (en) 2011-09-12 2021-11-30 Genzyme Corporation Anti-αβTCR antibody
US9717803B2 (en) 2011-12-23 2017-08-01 Innate Pharma Enzymatic conjugation of polypeptides
US9764038B2 (en) 2011-12-23 2017-09-19 Innate Pharma Enzymatic conjugation of antibodies
US10675359B2 (en) 2011-12-23 2020-06-09 Innate Pharma Enzymatic conjugation of antibodies
US11827701B2 (en) 2012-05-23 2023-11-28 argenx BV IL-6 binding molecules
US11117959B2 (en) 2012-05-23 2021-09-14 Argenx Bvba IL-6 binding molecules
US10183995B2 (en) 2012-05-23 2019-01-22 Argen-X N.V. IL-6 binding molecules
US10132799B2 (en) 2012-07-13 2018-11-20 Innate Pharma Screening of conjugated antibodies
WO2014009426A3 (en) * 2012-07-13 2014-04-03 Innate Pharma Screening of conjugated antibodies
US10836813B2 (en) 2012-09-12 2020-11-17 Genzyme Corporation Fc containing polypeptides with altered glycosylation and reduced effector function
US10036010B2 (en) 2012-11-09 2018-07-31 Innate Pharma Recognition tags for TGase-mediated conjugation
US10494439B2 (en) 2013-03-11 2019-12-03 Genzyme Corporation Hyperglycosylated binding polypeptides
US12110338B2 (en) 2013-03-11 2024-10-08 Genzyme Corporation Site-specific antibody-drug conjugation through glycoengineering
US10214589B2 (en) 2013-03-11 2019-02-26 Genzyme Corporation Site-specific antibody-drug conjugation through glycoengineering
US11807690B2 (en) 2013-03-11 2023-11-07 Genzyme Corporation Hyperglycosylated binding polypeptides
US11130816B2 (en) 2013-03-11 2021-09-28 Genzyme Corporation Site-specific antibody-drug conjugation through glycoengineering
US11813338B2 (en) 2013-03-12 2023-11-14 The Trustees Of The University Of Pennsylvania Diagnosing and treating cancer
US10611824B2 (en) 2013-03-15 2020-04-07 Innate Pharma Solid phase TGase-mediated conjugation of antibodies
US10071169B2 (en) 2013-06-20 2018-09-11 Innate Pharma Enzymatic conjugation of polypeptides
US9427478B2 (en) 2013-06-21 2016-08-30 Innate Pharma Enzymatic conjugation of polypeptides
US10434180B2 (en) 2013-06-21 2019-10-08 Innate Pharma Enzymatic conjugation of polypeptides
US9845363B2 (en) 2013-08-13 2017-12-19 Sanofi Antibodies to plasminogen activator inhibitor-1 (PAI-1) and uses thereof
US11052177B2 (en) 2013-09-06 2021-07-06 The Trustees Of The University Of Pennsylvania Antimicrobial polymer layers
US9835626B2 (en) 2014-02-27 2017-12-05 Board Of Regents, The University Of Texas System Methods and compositions for isolating exosomes
KR101738117B1 (ko) 2014-02-27 2017-05-19 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 엑소좀을 단리시키기 위한 방법 및 조성물
WO2015131153A1 (en) * 2014-02-27 2015-09-03 Board Of Regents, The University Of Texas System Methods and compositions for isolating exosomes
CN106030304A (zh) * 2014-02-27 2016-10-12 得克萨斯大学体系董事会 用于分离外来体的方法和组合物
US10995148B2 (en) 2014-03-19 2021-05-04 Genzyme Corporation Site-specific glycoengineering of targeting moieties
US11697690B2 (en) 2014-03-19 2023-07-11 Genzyme Corporation Site-specific glycoengineering of targeting moieties
US10308712B2 (en) 2014-03-27 2019-06-04 Bird Rock Bio, Inc. Antibodies that bind human cannabinoid 1 (CB1) receptor
US11566069B2 (en) 2014-03-27 2023-01-31 Bird Rock Bio, Inc. Treatment of disease responsive to modulation of cannabanoid 1(CB1) receptor signaling
US12214038B2 (en) 2014-07-22 2025-02-04 The Trustees Of The University Of Pennsylvania Compositions and methods for cancer immunotherapy
US11160874B2 (en) 2014-10-09 2021-11-02 Genzyme Corporation Glycoengineered antibody drug conjugates
US10064952B2 (en) 2014-10-09 2018-09-04 Genzyme Corporation Glycoengineered antibody drug conjugates
WO2017019858A1 (en) 2015-07-30 2017-02-02 The Trustees Of The University Of Pennsylvania Single nucleotide polymorphic alleles of human dp-2 gene for detection of susceptibility to hair growth inhibition by pgd2
US10278438B2 (en) 2015-08-31 2019-05-07 Grabbies Media, Llc Consumer usable device with redeemable member
WO2017048902A1 (en) 2015-09-15 2017-03-23 Board Of Regents, The University Of Texas System T-cell receptor (tcr)-binding antibodies and uses thereof
US11421026B2 (en) 2015-09-30 2022-08-23 Bird Rock Bio, Inc. Antibodies that bind human cannabinoid 1 (CB1) receptor
US11427634B2 (en) 2017-05-05 2022-08-30 Vaccinex, Inc. Human anti-semaphorin 4D antibody
US12006365B2 (en) 2017-05-05 2024-06-11 Vaccinex, Inc. Human anti-semaphorin 4D antibody
WO2019018633A1 (en) 2017-07-19 2019-01-24 Hoffman Technologies, Inc. COMPOSITIONS FOR THE TREATMENT OF STRESS-RELATED DISORDERS
US12365734B2 (en) 2019-04-03 2025-07-22 Genzyme Corporation Anti-alpha beta TCR binding polypeptides with reduced fragmentation
US11879004B2 (en) 2020-02-28 2024-01-23 Genzyme Corporation Modified binding polypeptides for optimized drug conjugation
US11702470B2 (en) 2020-06-11 2023-07-18 Imperial College Innovations Limited Use of CXCL13 binding molecules to promote peripheral nerve regeneration
US12503498B2 (en) 2020-10-08 2025-12-23 Genzyme Corporation Fc containing polypeptides with altered glycosylation and reduced effector function
WO2024068944A1 (en) 2022-09-30 2024-04-04 Sanofi Anti-cd28 antibodies
WO2024089609A1 (en) 2022-10-25 2024-05-02 Ablynx N.V. Glycoengineered fc variant polypeptides with enhanced effector function
WO2024133246A1 (en) 2022-12-19 2024-06-27 Sanofi Cd28/ox40 bispecific antibodies

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ZA200106943B (en) 2002-11-22
BRPI0017596A2 (pt) 2011-09-27
NZ513788A (en) 2004-03-26
SK288096B6 (sk) 2013-07-02
YU61601A (sh) 2004-07-15
PT2112512E (pt) 2015-01-13
BRPI0017596B8 (pt) 2023-04-25
CA2362119C (en) 2012-12-11
WO2000052473A2 (en) 2000-09-08
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NO20100717L (no) 2001-10-31
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CY1109335T1 (el) 2014-07-02
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