U.S. patent application number 09/259337 was filed with the patent office on 2002-08-01 for radiolabeling kit and binding assay.
Invention is credited to CHINN, PAUL, LABARRE, MICHAEL, LEONARD, JOHN E., MORENA, RONALD.
Application Number | 20020102208 09/259337 |
Document ID | / |
Family ID | 22984524 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102208 |
Kind Code |
A1 |
CHINN, PAUL ; et
al. |
August 1, 2002 |
RADIOLABELING KIT AND BINDING ASSAY
Abstract
Antibody binding assays and radiolabeling kits are disclosed for
radiolabeling and testing therapeutic antibodies in the commercial
setting. In particular, the kits are designed for making and
evaluating radiolabeled anti-CD20 conjugates to be used for the
treatment and imaging of B cell lymphoma tumors. All kit reagents
are sterile and are designed to achieve a high level of antibody
radiolabeling and product stability with results which are highly
reproducible.
Inventors: |
CHINN, PAUL; (VISTA, CA)
; MORENA, RONALD; (EL CAJON, CA) ; LABARRE,
MICHAEL; (SANDIEGO, CA) ; LEONARD, JOHN E.;
(CARLSBAD, CA) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
22984524 |
Appl. No.: |
09/259337 |
Filed: |
March 1, 1999 |
Current U.S.
Class: |
424/1.53 ;
424/1.69; 424/179.1; 530/395 |
Current CPC
Class: |
G01N 33/534 20130101;
A61P 35/00 20180101; G01N 33/60 20130101 |
Class at
Publication: |
424/1.53 ;
424/1.69; 424/179.1; 530/395 |
International
Class: |
A61M 036/14; A61K
039/395; A61K 039/40; A61K 039/42; A61K 039/44; C07K 014/00; C07K
017/00; C07K 001/00; A61K 051/00 |
Claims
What is claimed:
1. A kit for radiolabeling an anti-CD20 antibody before
administration to a patient comprising: (i) a vial containing a
chelator-conjugated antibody, (ii) a vial containing formulation
buffer for stabilizing and administering the radiolabeled antibody
to a patient, 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 according to the kit instructions, no further
purification of the labeled antibody is required before
administration to said patient.
2. The radiolabeling kit of claim 1, wherein the antibody is a
chimeric anti-CD20 antibody.
3. The radiolabeling kit of claim 1, wherein the chelator is
selected from the group consisting of MX-DTPA, phenyl-DTPA,
benzyl-DTPA, CHX-DTPA and DOTA.
4. The radiolabeling kit of claim 1 further comprising a vial
containing a buffer for adjusting the pH of the radioisotope.
5. The radiolabeling kit of claim 4, wherein the buffer is a sodium
acetate solution.
6. The radiolabeling kit of claim 1 further comprising a reaction
vial.
7. The radiolabeling kit of claim 3, wherein the chelator is
MX-DTPA.
8. The radiolabeling kit of claim 1, wherein the antibody is
2B8.
9. The radiolabeling kit of claim 8, wherein the
chelator-conjugated antibody is 2B8-MX-DTPA.
10. The kit of claim 1 wherein the antibody conjugate is supplied
at a concentration of about 0.5 to 30 mg/ml.
11. The radiolabeling kit of claim 5 wherein the sodium acetate
solution is at a concentration of 10 to 1000 mM.
12. The radiolabeling kit of claim 11 wherein the sodium acetate
solution is at a concentration of 50 mM.
13. The radiolabeling kit of claim 1 wherein the formulation buffer
comprises a radioprotectant and non-protein-conjugated
chelator.
14. The radiolabeling kit of claim 13 wherein the radioprotectant
is selected from the group consisting of human serum albumin (HSA),
ascorbate, ascorbic acid, phenol, sulfites, glutathione, cysteine,
gentisic acid, nicotinic acid, ascorbyl palmitate, HOP(:O)H.sub.2,
glycerol, sodium formaldehyde sulfoxylate, Na.sub.2S.sub.2O.sub.5,
Na.sub.2S.sub.2O.sub.3, and SO.sub.2.
15. The radiolabeling kit of claim 14 wherein the radioprotectant
is HSA.
16. The radiolabeling kit of claim 13 wherein the unconjugated
chelator is DTPA or EDTA.
17. The radiolabeling kit of claim 15 wherein the HSA is at a
concentration of about 1 to 25% (w/v).
18. The radiolabeling kit of claim 17 wherein the concentration of
HSA is about 7.5% (w/v).
19. The radiolabeling kit of claim 14 wherein the radioprotectant
is ascorbate.
20. The radiolabeling kit of claim 19 wherein the ascorbate is at a
concentration of about 1 to 100 mg/ml.
21. The radiolabeling kit of claim 1 wherein the radioisotope is
.sup.111In chloride.
22. The radiolabeling kit of claim 1 wherein the radioisotope is
.sup.90Y chloride.
23. A formulation buffer for administering a radiolabeled
chelator-conjugated antibody to a patient comprising: (i) a
physiological salt solution; (ii) a radioprotectant; and (iii)
non-protein conjugated chelator.
24. The formulation buffer of claim 23 wherein the radioprotectant
is selected from the group consisting of HSA, ascorbate, ascorbic
acid, phenol, sulfites, glutathione, cysteine, gentisic acid,
nicotinic acid, ascorbyl palmitate, HOP(:O)H.sub.2, glycerol,
sodium formaldehyde sulfoxylate, Na.sub.2S.sub.2O.sub.5,
Na.sub.2S.sub.2O.sub.3, and SO.sub.2.
25. The formulation buffer of claim 23 wherein the chelator is
DTPA.
26. The formulation buffer of claim 24 wherein the radioprotectant
is HSA.
27. The formulation buffer of claim 24 wherein the radioprotectant
is ascorbate.
28. The formulation buffer of claim 26 wherein the concentration of
human serum albumin is about 1 to 25% (w/v).
29. The formulation buffer of claim 28 wherein the HSA
concentration is about 7.5%.
30. The formulation buffer of claim 25 wherein the concentration of
DTPA is about 1 mM.
31. The formulation buffer of claim 27 wherein the concentration of
ascorbate is about 1 to 100 mg/mL.
32. A method for radiolabeling a chelator-conjugated anti-CD20
antibody for administration to a patient comprising (i) mixing
chelator-conjugated antibody with a solution containing a
radiolabel; (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 said radiolabeled antibody may be administered directly
to the patient without further purification.
33. The method of claim 32 wherein the antibody is a chimeric
anti-CD20 antibody.
34. The method of claim 32 wherein the anti-CD20 antibody is
2B8-MX-DTPA.
35. The method of claim 32 wherein the formulation buffer contains
physiological saline, a radioprotectant, and unconjugated
chelator.
36. The method of claim 35 wherein the radioprotectant is selected
from the group consisting of human serum albumin (HSA), ascorbate,
ascorbic acid, phenol, sulfites, glutathione, cysteine, gentisic
acid, nicotinic acid, ascorbyl palmitate, HOP(:O)H.sub.2, glycerol,
sodium formaldehyde sulfoxylate, Na.sub.2S.sub.2O.sub.5,
Na.sub.2S.sub.2O.sub.3, and SO.sub.2.
37. The method of claim 35 wherein the unconjugated chelator is
DTPAor EDTA.
38. The method of claim 32 wherein the solution containing the
radiolabel is adjusted to a pH of about 3 to 6 before it is mixed
with the chelator-conjugated antibody.
39. The method of claim 38 wherein the pH is adjusted with a low
metal sodium acetate solution.
40. The method of claim 39 wherein the sodium acetate is at a
concentration of about 10 to 1000 mM.
41. The method of claim 32 wherein the radiolabel is .sup.111In
chloride.
42. The method of claim 41 wherein the volume quantity of
.sup.111In chloride used is about 4-6 mCi divided by the
radioactivity concentration at the time of labeling.
43. The method of claim 41 wherein about 1 ml of
chelator-conjugated antibody at a concentration of about 0.5 to 30
mg/ml is mixed with the radiolabel solution.
44. The method of claim 43 wherein the mixture is incubated for
between about 30 seconds and 60 minutes.
45. The method of claim 44 wherein formulation buffer is added in
an amount necessary to achieve a total final volume of about 10 ml
to about 50 ml.
46. The method of claim 32 wherein the radiolabel is .sup.90Y
chloride.
47. The method of claim 46 wherein the volume quantity of .sup.90Y
chloride used is between about 5 to 100 mCi divided by the
radioactivity concentration at the time of labeling.
48. The method of claim 47 wherein the volume quantity of .sup.90Y
chloride used is about 45 mCi divided by the radioactivity
concentration at the time of labeling.
49. The method of claim 46 wherein about 1 to 2 ml of
MX-DTPA-conjugated antibody at a concentration of about 0.5 to 30
mg/ml is mixed with the radiolabel solution.
50. The method of claim 49 wherein the mixture is incubated for a
time between about 30 seconds to 60 minutes.
51. The method of claim 50 wherein formulation buffer is added in
an amount necessary to achieve a total final volume of about 10 ml
to about 50 ml.
52. A binding assay and radiolabeling kit for radiolabeling an
anti-CD20 antibody and determining the percent binding of the
radiolabeled antibody to its target cell before administering the
antibody to a patient, comprising the following components: (i) at
least one vial of fixed or lyophilized antigen-positive cells; (ii)
a vial containing a chelator-conjugated antibody; (iii) a vial
containing formulation buffer; and (iv) 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 according to the kit
instructions, no further purification of the labeled antibody is
required before administration to said patient.
53. The binding assay and radiolabeling kit of claim 52, wherein
said antibody is a chimeric anti-CD20 antibody.
54. The binding assay and radiolabeling kit of claim 52 further
comprising a vial containing a buffer for adjusting the pH of the
radiolabel.
55. The binding assay and radiolabeling kit of claim 52 wherein the
formulation buffer is phosphate buffered saline comprising a
radioprotectant and unconjugated chelator.
56. The binding assay and radiolabeling kit of claim 52, wherein
the antigen positive cells are CD20-positive cells.
57. The binding assay and radiolabeling kit of claim 56 wherein the
CD20-positive cells are SB cells (ATCC # CCL 120).
58. The binding assay and radiolabeling kit of claim 52 further
comprising at least one vial of antigen-negative cells.
59. The binding assay and radiolabeling kit of claim 58, wherein
said antigen-negative cells are CD20-negative cells.
60. The binding assay and radiolabeling kit of claim 59 wherein
said CD20-negative cells are HSB cells (ATCC # CCL120.1).
61. A binding assay kit for determining the percent binding of a
radiolabeled anti-CD20 antibody to its target cell comprising at
least one vial of fixed or lyophilized antigen-positive cells.
62. The binding assay kit of claim 61, further comprising a control
anti-CD20 antibody.
63. The binding assay kit of claim 62 wherein the CD20-positive
cells are SB cells (ATCC # CCL 120).
64. The binding assay kit of claim 61 further comprising
antigen-negative cells.
65. The binding assay kit of claim 64, wherein said cells are
CD20-negative.
66. The binding assay kit of claim 65 wherein said CD20-negative
cells are HSB cells (ATCC # CCL120.1).
67. A binding assay for determining the percent binding of a
radiolabeled antibody to its target cell, comprising: (i) mixing
and incubating at least one aliquot of a radiolabeled antibody with
at least one aliquot of antigen-positive cells; (ii) mixing and
incubating 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.
68. The binding assay of claim 67 wherein said antibody is a CD20
antibody.
69. The binding assay of claim 68 wherein the anti-CD20 antibody is
2B8.
70. The binding assay of claim 67 wherein said antigen is CD20.
71. The binding assay of claim 70 wherein said CD20 positive cells
are SB cells (ATCC # CCL 120).
72. The binding assay of claim 67 further comprising (i) mixing at
least one aliquot of the 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.
73. The binding assay of claim 72 wherein said antigen negative
cells are CD20-negative.
74. The binding assay of claim 73 wherein said CD20 negative cells
are HSB cells (ATCC # CCL 120.1).
75. A binding assay for determining the percent binding of a
radiolabeled antibody to its target cell, comprising: (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 the 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;
wherein said assay is performed using the binding assay and
radiolabeling kit of claim 52.
76. A binding assay for determining the percent binding of a
radiolabeled antibody to its target cell , comprising: (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;
wherein said assay is performed using the binding assay kit of
claim 61.
77. A competitive binding assay for assessing affinity of a test
antibody to a target cell, comprising (i) preparing a
ruthenium-labeled control antibody; (ii) incubating increasing
amounts of test antibody and increasing amounts of unlabeled
control antibody with a fixed concentration of fixed, fresh or
lyophilized antigen-positive 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.
78. The competitive binding assay of claim 77 wherein the control
and test antibodies are anti-CD20 antibodies.
79. A competitive binding assay of claim 77 wherein the
antigen-positive cells as CD20-positive.
80. The competitive binding assay of claim 79 wherein the
antigen-positive cells are SB cells (ATCC # CCL 120).
81. The radiolabeling kit of claim 1 further comprising a vial of
chimeric anti-CD20 antibody to be administered in a combined
therapeutic regimen prior to or subsequent to the radiolabeled
antibody.
82. The binding assay and radiolabeling kit of claim 52, further
comprising a vial of chimeric anti-CD20 antibody to be administered
in a combined therapeutic regimen prior to or subsequent to the
radiolabeled antibody.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] 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.
Specifically, 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").
[0003] 2. Technology Background
[0004] All publications and patent applications herein are
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
[0005] 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).
[0006] There are two major sub-populations of lymphocytes: T cells
and B cells. T cells are responsible for cell mediated immunity,
while B cells are responsible for antibody production (humoral
immunity). However, 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.
[0007] 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.sup.7 to 10.sup.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."
[0008] 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. Thus, the CD20 surface
antigen has the potential of serving as a candidate for "targeting"
of B cell lymphomas.
[0009] In essence, 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.
[0010] For example, attempts at such targeting of CD20 surface
antigen have been reported. Murine (mouse) monoclonal antibody 1F5
(an anti-CD20 antibody) was reportedly administered by continuous
intravenous infusion to B cell lymphoma patients. 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).
[0011] A potential problem with this approach is that non-human
monoclonal antibodies (e.g., murine 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. Furthermore, 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. For murine-based monoclonal
antibodies, this is often referred to as a Human Anti-Mouse
Antibody response, or "HAMA" response. Additionally, 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.
[0012] Lymphocytes and 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. The radionuclide emits
radioactive particles which can damage cellular DNA to the point
where the cellular repair mechanisms are unable to allow the cell
to continue living; therefore, if the target cells are tumors, the
radioactive label beneficially kills the tumor cells. Radiolabeled
antibodies, by definition, 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).
[0013] Therefore, an approach at improving the ability of murine
monoclonal antibodies to effect the treatment of B-cell disorders
has been to conjugate a radioactive label to the antibody such that
the label or toxin is localized at the tumor site. Toxins (i.e.,
chemotherapeutic agents such as doxorubicin or mitomycin C) have
also been conjugated to antibodies. See, for example, PCT published
application WO 92/07466 (published May 14, 1992).
[0014] "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. 4,816,397 all of which
are incorporated by reference herein. Generally these 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.
[0015] For example, 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.
[0016] It is noted that in vitro functional assays (e.g. complement
dependent lysis ("CDC"); antibody dependent cellular cytotoxicity
("ADCC"), etc.) cannot inherently predict the in vivo capability of
any antibody to destroy or deplete target cells expressing the
specific antigen. See, for example, Robinson, R. D., et al.,
"Chimeric mouse-human anti-carcinoma antibodies that mediate
different anti-tumor cell biological activities," Hum. Antibod.
Hybridomas, 2:84-93 (1991) (chimeric mouse-human antibody having
undetectable ADCC activity). Therefore, the potential therapeutic
efficacy of antibodies can only truly be assessed by in vivo
experimentation.
[0017] To this end, copending application Ser. Nos. 08/475,813,
08/475,815 and 08/478,967, herein incorporated by reference in
their entirety, disclose radiolabeled anti-CD20 conjugates for
diagnostic "imaging" of B cell lymphoma tumors before
administration of therapeutic antibody. "In2B8" conjugate comprises
a murine monoclonal antibody, 2B8, specific to human CD20 antigen,
that is attached to Indium[111] (.sup.111In) 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. Indium-[111] is selected
as a diagnostic radionuclide because it emits gamma radiation and
finds prior usage as an imaging agent.
[0018] Patents relating to chelators and chelator conjugates are
known in the art. For instance, 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.
[0019] 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.
However, other bifunctional chelators are known in the art and may
also be beneficial in tumor therapy.
[0020] Also disclosed in application Ser. Nos. 08/475,813,
08/475,815 and 08/478,967 are radiolabeled therapeutic antibodies
for the targeting and destruction of B cell lymphomas and tumor
cells. In particular, the Y2B8 conjugate comprises the same
anti-human CD20 murine monoclonal antibody, 2B8, attached to
yttrium-[90] (.sup.90Y) via the same bifunctional chelator. This
radionuclide was selected for therapy for several reasons. The 64
hour half-life of .sup.90Y is long enough to allow antibody
accumulation by the tumor and, unlike e.g. .sup.131I, it 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 .sup.90Y-labeled antibodies. Furthermore,
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.
[0021] Because the .sup.90Y radionuclide was attached to the 2B8
antibody using the same bifunctional chelator molecule MX-DTPA, the
Y2B8 conjugate possesses the same advantages discussed above, e.g.,
increased retention of radionuclide at a target site (tumor).
However, unlike .sup.111In, it cannot be used for imaging purposes
due to the lack of gamma radiation associated therewith. Thus, a
diagnostic "imaging" radionuclide, such as .sup.111In, can be used
for determining the location and relative size of a tumor prior to
and/or following administration of therapeutic chimeric or
.sup.90Y-labeled antibodies for the purpose of tumor reduction.
Additionally, indium-labeled antibody enables dosimetric assessment
to be made.
[0022] Depending on the intended use of the antibody, i.e., as a
diagnostic or therapeutic reagent, other radiolabels are known in
the art and have been used for similar purposes. For instance,
radionuclides which have been used in clinical diagnosis include
.sup.131I, .sup.125I, .sup.123I, .sup.99Tc, .sup.67Ga, as well as
.sup.111In. 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). These radionuclides include .sup.188Re and .sup.186Re as
well as .sup.90Y, and to a lesser extent .sup.199Au and .sup.67Cu.
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.
[0023] As reported in copending application Ser. Nos. 08/475,813,
08/475,815 and 08/478,967 administration of the radiolabeled Y2B8
conjugate, as well as unlabeled chimeric anti-CD20 antibody,
resulted in significant tumor reduction in mice harboring a B cell
lymphoblastic tumor. Moreover, human clinical trials reported
therein showed significant B cell depletion in lymphoma patients
infused with chimeric anti-CD20 antibody. In fact, chimeric 2B8 has
recently been heralded the nation's first FDA-approved anti-cancer
monoclonal antibody under the name of Rituxan.RTM.. Thus, at least
one chimeric anti-CD20 antibody has been shown to demonstrate
therapeutic efficacy in the treatment of B cell lymphoma.
[0024] In addition, U.S. application Ser. No. 08/475,813, herein
incorporated by reference, discloses sequential administration of
Rituxan.RTM., a chimeric anti-CD20, with both or either
indium-labeled or yttrium-labeled murine monoclonal antibody.
Although the radiolabeled antibodies used in these combined
therapies are murine antibodies, initial treatment with chimeric
anti-CD20 sufficiently depletes the B cell population such that the
HAMA response is decreased, thereby facilitating a combined
therapeutic and diagnostic regimen.
[0025] Thus, in this context of combined immunotherapy, murine
antibodies may find particular utility as diagnostic reagents.
Moreover, it was shown in U.S. application Ser. No. 08/475,813 that
a therapeutically effective dosage of the yttrium-labeled anti-CD20
antibody following administration of Rituxan.RTM. 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.
[0026] Thus, 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. Given the proven efficacy of an anti-CD20 antibody in the
treatment of non-Hodgkin's lymphoma, and the known sensitivity of
lymphocytes to radioactivity, it would be highly advantageous for
such therapeutic antibodies to become commercially available in kit
form whereby they may be readily modified with a radiolabel and
administered directly to the patient in the clinical setting.
[0027] Although there exist many methods and reagents for
accomplishing radiolabeling of antibodies, what is lacking in the
art is a convenient vehicle for placing these reagents in the
clinical setting, in a way that they may be easily produced and
administered to the patient before significant decay of the
radiolabel or significant destruction of the antibody due to the
radiolabel occurs. The lack of such convenient means to
commercialize this valuable technology could be due to the poor
incorporation efficiencies demonstrated by some known labeling
protocols, and the subsequent need to column purify the reagent
following the radiolabeling procedure. The delay in development of
such kits might also in part be due to the previously lack of
accessibility to pure commercial radioisotopes which may be used to
generate efficiently labeled products absent subsequent
purification. Alternatively, perhaps the reason such kits are
generally unavailable is the actual lack of antibodies which have
been able to achieve either the approval or the efficacy that
Rituxan.RTM. has achieved for the treatment of lymphoma in human
patients.
[0028] For instance, as discussed in U.S. Pat. No. 4,636,380,
herein incorporated by reference, it has been generally believed in
the scientific community that for a radiopharmaceutical to find
clinical utility, it must endure a long and tedious separation and
purification process. Indeed, injecting unbound radiolabel into the
patient would not be desirable. The need for additional
purification steps renders the process of radiolabeling antibodies
in the clinical setting an impossibility, particularly for doctors
who have neither the equipment nor the time to purify their own
therapeutics.
[0029] Furthermore, radiolabeled proteins may be inherently
unstable, particularly those labeled with radiolytic isotopes such
as .sup.90Y, which have the tendency to cause damage to the
antibody the longer they are attached to it in close proximity. In
turn, such 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. Yet without the facilities for labeling and
purifying the antibodies on site, 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.
[0030] Others have tried unsuccessfully to develop antibody
radiolabeling kits that would be proficient enough to forego a
separate purification step of the antibody. For instance, Cytogen
has recently launched a commercial kit for radiolabeling a murine
monoclonal antibody directed to tumor-associated glycoprotein
TAG-72. However, Cytogen's antibody is particularly unamenable to a
kit formulation due to the tendency to develop particulates during
storage which must later be removed by a further filtration step.
Moreover, Cytogen's antibody has caused adverse reactions in
patients due to a HAMA responses.
[0031] Others have claimed to have developed radiolabeling
protocols which would be amenable to kit format in that a separate
purification step would not be required (Richardson et al. (1987)
Optimization and batch production of DTPA-labeled antibody kits for
routine use in .sup.111In immunoscintography. Nuc. Med. Commun. 8:
347-356; Chinol and Hnatowich (1987) Generator-produced
yttrium-[90] for radioimmunotherapy. J. Nucl. Med. 28(9):
1465-1470). However, such protocols were not able to achieve the
level of incorporation that the present inventors have achieved
using the protocols disclosed herein, which have resulted in
incorporation efficiencies of at least 95%. Such a level of
incorporation provides the added benefit of increased safety, in
that virtually no unbound label will be injected into the patient
as a result of low radioincorporation.
[0032] The protocols included in the 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.
[0033] The present application discloses convenient 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.
SUMMARY OF THE INVENTION
[0034] 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. Moreover, when labeled according to
the kit instructions and with a radioisotope of sufficient purity
and activity, such isotope incorporation may reach levels higher
than 95%, and even as high as 98% or higher.
[0035] 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. Preferably, 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).
[0036] Depending on the antibody used, 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. However, 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.
[0037] 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. Although 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.
[0038] For instance, 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. 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.sub.2, glycerol, sodium formaldehyde
sulfoxylate, Na.sub.2S.sub.2O.sub.5, Na.sub.2S.sub.2O.sub.3, and
SO.sub.2, etc.).
[0039] It should be noted that, while 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.
[0040] 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., .sup.90Y, by the bones of the patient. For
instance, when the antibody of the kit is conjugated to a DTPA
chelator, 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.
[0041] 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.
[0042] 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. For further simplicity in
this regard, 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.
[0043] 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. However, it should be apparent to those skilled
in the art that 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. Alternatively, and more preferably, 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,
However, other embodiments are also envisioned, i.e., whereby the
buffer is added to the isotope vial first, and the buffered isotope
is then transferred to the reaction vial. In this case, the
reaction vial could be supplied with the required antibody volume.
Alternatively, 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.
[0044] As described above, another preferred kit configuration is
encompassed whereby the reaction vial itself contains the required
volume of conjugated antibody (i.e., 1 or 1.5 mL for .sup.111In and
.sup.90Y, 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 .sup.111In, pH 3-5 for
90Y). Different buffers may be used, depending on the isotope
(i.e., sodium acetate for .sup.90Y, sodium citrate for .sup.111In).
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). Essentially then, 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.
[0045] 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 .sup.111In chloride and .sup.90Y 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
.sup.90Y are particularly useful as radiotherapeutic agents.
Although other radioisotopes suitable for these or other purposes,
i.e., alpha emitters, may be used depending on the chelator used
for antibody conjugation.
[0046] Given the proven efficacy of the combined therapeutic
regimens disclosed in U.S. application Ser. No. 08/475,813, a
further kit embodiment will also include a separate vial of
chimeric antibody, i.e., Rituxan.RTM., to be administered before or
after the radiolabeled anti-CD20 antibody. When the chimeric
antibody is administered before the radiolabeled antibody, 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. Moreover, when chimeric anti-CD20 is employed to clear
circulating B cells, subsequent diagnostic images achieved with
.sup.111In-labeled antibodies may be much clearer.
[0047] It should also be apparent that both a diagnostic
radiolabeled antibody and a therapeutic radiolabeled antibody may
be used together in a combined therapeutic regimen. In this regard,
the diagnostic antibody may be used either before or after the
therapeutic antibody to visualize tumor size before and after
treatment. In this case, the kit 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.
[0048] 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. However,
it should be apparent to those of skill in the art that some of the
reagents can be prepared, sterilized and tested for sterility on
site. Thus, variations of the kit of the invention are envisioned
depending on the budget and preference of the consumer.
[0049] The radiolabeling kit of the present invention may be used
in a method for radiolabeling a chelator-conjugated antibody for
administration to a patient. According to the present invention,
such a method 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.
[0050] Most preferably 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)).
[0051] 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. Preferably, the sodium acetate is at a
concentration of between about 10 and 1000 mM, and more preferably
50 mM.
[0052] When the radioisotope is .sup.111In chloride, the volume
quantity of .sup.111In 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. For
patient administration, a typical diagnostic dose of .sup.111In 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. When 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 .sup.111In chloride used although larger
volumes may be used. It should be appreciated that 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.
[0053] 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. For
instance, 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.
[0054] For instance, for 2B8 and MX-DTPA, it has been discovered
that labeling may be accomplished in under five minutes for
.sup.90Y and in about thirty minutes for .sup.111In to achieve the
desired level of radioincorporation, with only about a 11/2 to 1
molar ratio of chelator to antibody. It was not necessary,
therefor, to increase the chelator to antibody ratio, because a
desirable level of radioincorporation was achieved. Moreover, it
was not advantageous to increase the quantity of conjugated
chelator because this could effect antibody immunoreactivity. Such
parameters could be empirically determined for other antibodies for
the design of kits such as those described in the present
invention.
[0055] When the radioisotope is .sup.90Y chloride, the volume
quantity of .sup.90Y 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. When the concentration of sodium
acetate is 50 mM and the .sup.90Y 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. About 1.5 ml of 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.
[0056] Preferably, the radiolabeling method of the invention is
performed using the radiolabeling kit described herein. However, it
should be apparent to those of skill in the art that 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.
[0057] 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. In such a
case, 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.
[0058] In general, 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.
As described above for the radiolabeling kit, this kit may also
comprise a vial containing a buffer for adjusting the pH of the
radioisotope, and a sterile glass reaction vial. Preferably 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 .sup.111In chloride or
.sup.90Y chloride. Other radioisotopes may be used depending on the
chelator.
[0059] 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. When the 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.
[0060] Of course, 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. Such separate
antibody is preferably Rituxan.RTM., but may be any antibody shown
to effectuate tumor cell killing. In fact, 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.
[0061] Just as the components of the kit may be used to label other
antibodies, other cells for testing antibody affinity may be
prepared depending on the target antigen. However, for anti-CD20
antibodies, 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.times.10.sup.6 cells per vial according to the methods of the
invention.
[0062] It is possible that particular facilities will prefer to
order antibody which has already been radiolabeled, in which case
such a facility might desire the binding assay reagents in order to
ensure that the antibodies retain target affinity. In this case,
the present invention also provides for 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. Moreover, it should be apparent that 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.
[0063] Again, when the antigen is CD20, 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.times.10.sup.6
cells. In this case, the antibody is preferably an MX-DTPA
conjugate of 2B8 labeled with .sup.111In or .sup.90Y.
[0064] In view of the additional kit embodiments disclosed herein,
it should be stressed that one of the advantages of the
radiolabeling kit and method of the present invention is that no
further purification step is necessary, and the radiolabeled
antibody may be administered directly to the patient, thereby
saving valuable time and increasing antibody stability. Therefor,
it is emphasized that, while it might be desirable for the
clinician to test or verify the binding specificity and affinity of
the radiolabeled antibody prior to administration, such test may be
foregone with particular radioisotopes if antibody stability and
the inhibition of radiolysis are particular concerns, i.e., as with
yttrium. By providing kit embodiments whereby the binding affinity
and specificity may be tested, the present inventors are in no way
suggesting that such tests are absolutely required in the methods
or kits of the invention. The option to test such antibody validity
is purely at the option of the clinician.
[0065] 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. In particular, the
cells of the present invention demonstrate high reproducibility
when used for antibody binding assays.
[0066] In particular, the present invention includes a method of
preparing lyophilized cells comprising (i) harvesting cells at a
cell density of 0.5 to 2.times.10.sup.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.
[0067] Preferably, 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%. However, conceivably other
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.times.10.sup.6 cells per ml. However, it should be apparent to
those of skill in the art that the above conditions may be modified
slightly without significantly compromising cell viability.
Moreover, 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.
[0068] 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.
[0069] Just as the radiolabeling kits of the present invention
optionally contain .sup.111In chloride or .sup.90Y chloride, the
binding assay of the present invention is typically performed with
antibodies labeled with .sup.111In or .sup.90Y. When .sup.111In is
the radiolabel, radioactivity in the assay tubes is measure using a
gamma counter. When .sup.90Y is the label, radioactivity is
measured using a scintillation counter, although a gamma counter
could be used.
[0070] For the binding assay of the present invention, the
preferred antibody is an anti-CD20 antibody, and the anti-CD20
antibody is preferably 2B8, wherein the 2B8 antibody is labeled
using the radiolabeling kit of the present invention. However, any
radiolabeled antibody may be tested provided cells expressing the
particular antigen are available. When 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.
[0071] 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. Although the tube with dilution buffer serves as
a control, a further control may be included in the assay by using
antigen-negative cells. In this case, 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. When CD20 is the antigen, and
the CD20-positive cells are SB cells, CD20 negative cells are
preferably HSB cells (ATCC # CCL 120.1).
[0072] As described above, 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. In addition, 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.
[0073] There may be some instances where the affinity of an
antibody should be tested or verified but a radiolabel has not been
attached. For instance, under certain circumstances, i.e.,
trouble-shooting, it may be advantageous to test the binding
affinity of an antibody before radiolabeling. For such a case, 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. 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.
[0074] When the competitive binding assay of the present invention
is performed to test the affinity of anti-CD20 antibodies, 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. Alternatively, the test antibody may be another
anti-CD20 antibody whose binding affinity to CD20 as compared to
2B8 is of interest. However, the assay may be adapted for use with
antibodies having other specificities so long as an appropriate
target cell is available.
[0075] In the competitive binding assay of the present invention,
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. For labeling, the control antibody
and TAG-NHS are preferably incubated at about a 1:15 molar
ratio.
[0076] These and other aspects of the present invention will become
clearly understood by reference to the following figures, examples
and description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] 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 .sup.125I-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.
[0078] 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)'.sub.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.
[0079] FIG. 3. Human B-cells (SB) were incubated with increasing
amounts of .sup.125I-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.times.10.sup.-9 M.
[0080] 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.
[0081] 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.degree. C. or
30.degree. 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.
[0082] FIG. 6. Binding assay for determination of immunoreactivity
of .sup.111In-labeled 2B8-MX-DTPA incubated in PBS, pH 7.4
containing 50 mg/mL human serum albumin (48 h incubation).
[0083] FIG. 6A) A constant amount of radiolabeled antibody (5
ng/mL) was incubated with increasing volumes of SB cells
(20.times.106 cells/mL). The amount of radioactivity (cpm) bound to
cells was plotted against the volume of cell suspension added.
[0084] 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.times.100 yielded an immunoreactivity value of 100% at
infinite antigen excess.
[0085] FIG. 7. Autoradiograms obtained from SDS-PAGE analysis of
.sup.90Y-labeled 2B8-MX-DTPA incubated at 4.degree. 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 .mu.L (lanes 1,2), 10 .mu.L (lanes
5,6). The gels were exposed to x-ray film for approximately 15 min
at ambient temperature and photographed.
[0086] FIG. 8. Densitometric scan of time zero autoradiogram
obtained from SDS-PAGE analysis of .sup.90Y-labeled 2B8-MX-DTPA
incubated at 4.degree. 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 .mu.L, 10 .mu.L, and 20 .mu.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%.
[0087] FIG. 9. Densitometric scan of 48 h autoradiogram obtained
from SDS-PAGE analysis of .sup.90Y-labeled 2B8-MX-DTPA incubated at
4.degree. 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 .mu.L, 10 .mu.L, and 20 .mu.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%.
[0088] FIG. 10. Autoradiograms obtained from SDS-PAGE analysis of
.sup.111In-labeled 2B8-MX-DTPA incubated at 4.degree. 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 .mu.L
(lanes 1, 2), 10 .mu.L (lanes 3, 4), and 20 .mu.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).
[0089] FIG. 11. Densitometry scan of time zero autoradiogram
obtained from SDS-PAGE analysis of .sup.111In-labeled 2B8-MX-DTPA
incubated at 4.degree. 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 .mu.L, 10 .mu.L, and 20 .mu.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%.
[0090] FIG. 12. Densitometry scan of 48 h autoradiogram obtained
from SDS-PAGE analysis of .sup.111In-labeled 2B8-MX-DTPA incubated
at 4.degree. 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 .mu.L, 10
.mu.L, and 20 .mu.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).
[0091] FIG. 13. Autoradiograms obtained from SDS-PAGE analysis of
.sup.90Y-labeled 2B8-MX-DTPA incubated at 37.degree. 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 .mu.L (lanes 1, 2), 10 .mu.L (lanes 3, 4), and 20
.mu.L (lanes 5, 6). The gels were exposed to x-ray film for
approximately 15 min at ambient temperature and photographed.
[0092] FIG. 14. Densitometric scan of time zero autoradiogram
obtained from SDS-PAGE analysis of .sup.90Y-labeled 2B8-MX-DTPA
incubated at 37.degree. 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 .mu.L, 10 ,L, and 20 .mu.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%.
[0093] FIG. 15. Densitometric scan of 98 h autoradiogram obtained
from SDS-PAGE analysis of .sup.90Y-labeled 2B8-MX-DTPA incubated at
37.degree. 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 .mu.L, 10 .mu.L, and 20 .mu.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%.
[0094] FIG. 16. Autoradiograms obtained from SDS-PAGE analysis of
.sup.111In-labeled 2B8-MX-DTPA incubate at 37.degree. 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 .mu.L (lanes 1, 2), 10 .mu.L (lanes 3, 4), and 20
.mu.L (lanes 5, 6). The gels were exposed to x-ray film for
approximately 16-20 h at ambient temperature and photographed.
[0095] FIG. 17. Densitometric scan of time zero autoradiogram
obtained from SDS-PAGE analysis of .sup.111In-labeled 2B8-MX-DTPA
incubated at 37.degree. 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 .mu.L, 10 .mu.L, and 20
.mu.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%.
[0096] FIG. 18. Densitometric scan of the 96 h autoradiogram
obtained from SDS-PAGE analysis of .sup.111In-labeled 2B8-MX-DTPA
incubated at 37.degree. 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 .mu.L, 10 .mu.L, and 20
.mu.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%.
[0097] 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).
[0098] 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.
[0099] 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.
[0100] 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 .beta.t.sub.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 .mu.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.
[0101] FIG. 23. Twenty BALB/c mice were each injected with 1.1
.mu.Ci of radiolabeled conjugate (100 .mu.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.
[0102] FIG. 24. Twenty BALB/c mice were each injected intravenously
with approximately 1.0 .mu.Ci (in 100 .mu.l) of radiolabeled
conjugate formulated in 1.times. 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.
[0103] FIG. 25. Athymic mice bearing Ramos B-cell tumors were
injected intravenously with 24 .mu.Ci of .sup.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.
[0104] FIG. 26. Binding assay for determination of immunoreactivity
of "mix-&-shoot" .sup.90Y-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 .sup.90Y-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 .sup.90Y radioactivity over bound radioactivity (AT/B) was
plotted. Linear extrapolation allowed calculation of the
y-intercept (1.139). The reciprocal of the y-intercept.times.100
yielded an immunoreactivity value of 87.9% at infinite antigen
excess. No binding was observed with CD20-negative cells (HSB).
[0105] FIG. 27. Autoradiograms obtained from SDS-PAGE analysis of
.sup.90Y-labeled 2B8-MX-DTPA incubated at 4.degree. 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 .mu.L (lanes 1,2), 10 .mu.L (lanes
5,6). The gels were exposed to x-ray film for approximately 15 min
at ambient temperature and photographed.
[0106] FIG. 28. Densitometric scan of time zero autoradiogram
obtained from SDS-PAGE analysis of .sup.90Y-labeled 2B8-MX-DTPA
incubated at 4.degree. 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 .mu.L, 10 .mu.L, and 20 .mu.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%.
[0107] FIG. 29. Densitometric scan of 48 h autoradiogram obtained
from SDS-PAGE analysis of .sup.90Y-labeled 2B8-MX-DTPA incubated at
4.degree. 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 .mu.L, 10 .mu.L, and 20 .mu.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%.
[0108] FIG. 30. Autoradiograms obtained from SDS-PAGE analysis of
"mix-&-shoot" .sup.90Y-labeled 2B8-MX-DTPA incubated at
37.degree. 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 .mu.L (lanes 1, 2), 10
.mu.L (lanes 3, 4), and 20 .mu.L (lanes 5, 6). The gels were
exposed to x-ray film for approximately 15 min at ambient
temperature and photographed.
[0109] FIG. 31. Densitometric scan of time zero autoradiogram
obtained from SDS-PAGE analysis of "mix-&-shoot"
.sup.90Y-labeled 2B8-MX-DTPA incubated at 37.degree. 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 .mu.L, 10
.mu.L, and 20 .mu.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%.
[0110] FIG. 32. Densitometric scan of 72 h autoradiogram obtained
from SDS-PAGE analysis of "mix-&-shoot" .sup.90Y-labeled
2B8-MX-DTPA incubated at 37.degree. 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 .mu.L, 10 .mu.L, and 20
.mu.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%.
[0111] FIG. 33. Twenty BALB/c mice were each injected intravenously
with 5 .mu.Ci .sup.90Y-labeled 2B8-MX-DTPA formulated in 1.times.
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.
[0112] 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)'.sub.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.
[0113] 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.times.10.sup.-10 M and
2.5.times.10.sup.-10 M, respectively. An irrelevant isotype
antibody (S004), was included for comparison.
[0114] 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. For binding assessment, 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.
[0115] FIG. 37. A) SB cells were washed and resuspended to
90.times.10.sup.6 cells/mL with dilution buffer (1.times. 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.times.100.
Immunoreactivity and correlation coefficient (R) values were 80.6%
and 0.981, respectively.
[0116] FIG. 38. A) SB cells were washed and resuspended to
90.times.10.sup.6 cells/mL with dilution buffer (1.times. 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.
DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
[0117] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described. For
purposes of the present invention, the following terms are defined
below.
[0118] low metal--refers to reagents treated to reduce metal
contamination to a level which does not impact
radioincorporation
[0119] 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.
[0120] % 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.
[0121] % binding--refers to the amount of antibody from a sample
which binds to the target antigen, with or without specificity.
[0122] % 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 .sup.111In which can effect diagnostic
imaging of tumors and antigen positive cells.
[0123] therapeutic antibody--refers to an antibody conjugated to a
alpha or beta emitting radiolabel (such as .sup.90Y) which can
effect cell killing when bound to the targeted antigen.
DESCRIPTION OF THE INVENTION
[0124] Pre-clinical Development of Murine Monoclonal Anti-CD20
Antibody 2B8, Conjugated 2B8, .sup.111In and .sup.90Y-Labeled
2B8
[0125] I. Materials and Methods for Development of Murine
Monoclonal Anti-CD20 Antibody 2B8, Conjugate 2B8-MX-DTPA,
.sup.111In-Labeled 2B8-MX-DTPA and HPLC-Purified
.sup.90Y-MX-DTPA
[0126] A. Materials.
[0127] 1. Cells.
[0128] 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.
[0129] 2. Antibodies.
[0130] The anti-CD20 antibodies Bi and Leu 16 were purchased from
Coulter Immunology and Becton/Dickinson, respectively. The
.sup.125I-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.
[0131] 3. Reagents.
[0132] 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 .sup.90Y 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.
[0133] 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.
[0134] B. Methods.
[0135] 1. Production and Screening of 2B8 Hybridoma Supernatants by
RIA.
[0136] 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.
[0137] Two animals at a time were sacrificed by cervical
dislocation and their spleens removed for fusion with the murine
myeloma SP2/0. Animals were chosen based on the ability of
post-immune sera to effectively inhibit the binding of radiolabeled
Coulter B1 anti-CD20 antibody to human SB cells. Three days prior
to each fusion the selected animals were given one last intravenous
(tail vein) injection of 20 million SB cells in PBS. Upon sacrifice
the spleens were removed under aseptic conditions and the
splenocytes fused with SP2/0 cells at a ratio of 5:1
(splenocytes:SP2/0). Fused cells were washed in tissue culture
media and distributed into 96 well plates containing HAT selection
media. Hybridomas were screened by inhibition radioimmunoassay
using Coulter B1 antibody after 10-14 days.
[0138] Screening of hybrids secreting anti-CD20 antibody was
accomplished using established radioimmunoassay methods. Briefly,
Coulter B1 anti-CD20 antibody was purified by Protein A affinity
chromatography. Fifty micrograms of purified antibody was coupled
to .sup.125I by brief oxidation in the presence of lodobeads
(Pierce Chemical Co.), following the manufacturer's procedure. The
radiolabeled antibody was desalted on amberlite resin and stored in
dilution buffer (PBS, pH 7.4, containing 0.2% gelatin, 0.02% sodium
azide, and 1.0% BSA). Ten nanograms of radiolabeled antibody was
placed in each well of a previously blocked filter assay plate
(blocking buffer: dilution buffer containing 10% FBS) along with 50
.mu.L of hybridoma supernatant from test wells and 100,000 SB cells
suspended in 50 .mu.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 .mu.g to 2 ng.
[0139] 2. Flow Cytometry Studies.
[0140] a. Cell Lines
[0141] Preliminary flow cytometry studies were performed with
supernatants from 2B8 hybridoma cultures. One hundred microliters
of hybridoma supernatant was incubated with SB cells for one hour
at ambient temperature; a secondary antibody (goat F(ab')2
anti-mouse IgG; Cappel), used at a 1/400 dilution, was added
subsequently and the incubation continued for 1 hour in the dark.
The cells were washed for 5 times. Controls included cells only (no
primary or secondary antibody) from which autofluorescence was
determined, cells with secondary antibody only to determine
non-specific binding and commercially available fluorescein
isothiocyanate-conjugated B1 (B1-FITC) for a CD20 population
control.
[0142] For some experiments, 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
.mu.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.
[0143] 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 .mu.L added per well into 96 well U-bottom plates. Primary
antibody (50 .mu.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 .mu.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. in a Sorvall centrifuge and the
supernatants removed by gently "flicking" the plates. Secondary
antibody, if needed, was added and incubated for an additional 30
min to 1 h at ambient temperature in the dark; wells were then
washed as above. Finally, 200 .mu.L of fixing buffer (0.15 M sodium
chloride containing 1% paraformaldehyde, pH 7.4) was added to each
sample and the treated cells transferred to 12.times.75 mm tubes
for analysis.
[0144] b. Whole Blood From Cynomolgus Monkeys.
[0145] After removal of plasma, 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. Fluorescently-labeled monoclonal antibodies were
added as follows:
1 Tube #1: Murine anti-CD2-FITC (AMAC), 2.5 .mu.g/mL, 5 .mu.g; Tube
#2: Goat anti-Human IGM-FITC (Fisher) 2.5 .mu.g/mL, 5 .mu.g;
Tube#3: Goat anti-mouse IgG-RPE (Fisher) 2.5 .mu.g/mL, 5 .mu.g;
Tube #4: Goat anti-Human IgM-FITC + Goat anti-mouse IgG-RPE
(absorbed), 2.5 .mu.g/mL, 5 .mu.g; Tube #5: anti-human CD20-FITC
(anti-Leu 16, Becton Dickinson), 5 .mu.g; Tube #6: Cells only
(auto-fluorescence).
[0146] Labeled antibodies and cells were centrifuged for 2 min at
1500 rpm to mix cells and antibodies and all 6 samples were then
placed on ice and incubated for 30 min. Subsequently the tubes were
removed from the ice and lysing buffer (prewarmed to 37.degree. C.)
was added to a volume of 12 mL. The samples were then incubated for
15 min at room temperature, centrifuged for 5 min at 40.degree. C.
at 1500 rpm, and the supernatants removed. Cell pellets were then
washed twice in labeling buffer (PBS containing 1% BSA and 0. 05%
sodium azide).
[0147] Subsequently the cells were fixed by the addition of 0.5 mL
of fixation buffer (0.15 M sodium chloride, pH 7.4, containing 1%
paraformaldehyde) per tube and analyzed on a Becton Dickinson
FACScan instrument using autocompensation and precalibration with
Calibrite beads. Green fluorescence from fluorescein was measured
in FL1 mode and red fluorescence from phycoeretherin was measured
in FL2 mode. Data were expressed in log form. Viable lymphocyte
populations were initially identified by forward vs. right angle
light scatter in a dot plot bitmap. The total lymphocyte population
was then isolated by gating out all other events. Subsequent
fluorescence measurements reflected only those specific events
which occurred within the gated area.
[0148] For high-dose pharmacology/toxicology studies the 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.
[0149] After injection of 2B8 into the monkeys, when the CD20
antigen was saturated with 2B8, the percentage of B cells in the
total population was approximated using goat anti-human IgM-FITC,
anti-mouse IgG-RPE and the double staining population containing
these two markers. The double staining population was used for
quantitation until all of the 2B8 was cleared from the peripheral
blood of the animals. The percentage of T cells in the total
lymphocyte population was estimated using anti-CD2-FITC. Data were
averaged from three, 10,000 event measurements made with each
sample. Cells from each of the designated blood samples were
evaluated subsequently, enumerating in each case the T- and B-cell
subpopulations within the total lymphocyte population. The T:B
ratios were also examined. Depletion of B-cells was calculated as
the percent of reduction of B-cells relative to original B-cell
levels for each individual monkey.
[0150] 3. Radioiodination and Immunoprecipitation of CD20.
[0151] One hundred million SB cells were divided into two equal
parts after surface iodination with 1251 and lodobeads (Pierce
Chemical Co.). The cells were washed repeatedly by centrifugation
until radioactivity levels in the supernatant returned to
background. One hundred micrograms of 2B8 or B1 (Coulter
Immunology) antibody were added to either of the two samples of
labeled B cells. The antibodies and SB cells were incubated
overnight and then washed three times by centrifugation until all
of the unbound antibody was removed. The cell pellets containing
bound 2B8 and B1 were then lysed and extracted by addition of 1%
NP-40 detergent in 0.1 M Tris-HC1, pH 8.0, followed by incubation
at room temperature for 1 h. 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 .mu.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.degree. C.; subsequently the film
was developed and analyzed.
[0152] 4. Scatchard Analysis of 2B8 Binding.
[0153] Purified 2B8 was evaluated for apparent affinity by
Scatchard analysis. Radiolabeled 2B8 was prepared by reaction with
.sup.125I 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
.sup.1251-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.
[0154] 5. Preparation of 2B8-MX-DTPA
[0155] a. Source of MX-DTPA
[0156] For some pre-clinical studies, carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid
(MX-DTPA) was provided as a dry solid by Dr. Otto Gansow at the
National Institute of Health and stored desiccated at 4.degree. C.
protected from light. Stock solutions of the chelate were prepared
in Milli-Q water and the concentration determined by assessing the
radioactivity and using the specific activity of the compound.
Stock solutions were generally 2-5 mM and were stored at
-70.degree. C. in polypropylene tubes. For other studies, MX-DTPA
was obtained from Coulter Immunology as the disodium salt in water
and stored at -70.degree. C.
[0157] b. Maintenance of Metal-Free Conditions
[0158] In addition to using metal-free reagents, all manipulations
of reagents were performed so as to minimize the possibility of
metal contamination. When possible, polypropylene plastic
containers such as flasks, beakers and graduated cylinders were
used. These were washed with Alconox and exhaustively rinsed with
Milli-Q water before use. In addition, metal-free pipette tips
(BioRad) were used for accurately manipulating small volumes. For
manipulating larger volumes of reagents, sterile, plastic
serological pipettes (available in 1 to 25 mL sizes) were used.
Reactions were conveniently performed in screw-top, polypropylene
microfuge tubes (Sardstedt Industries; 1.5 .mu.L capacity) or
polypropylene conical tubes (Costar; 15 mL and 50 .mu.L). When
dialysis tubing was manipulated, disposable surgical gloves,
previously rinsed with Milli-Q water, were worn.
[0159] c. Preparation of Antibody
[0160] 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.
[0161] 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. In some studies,
buffer exchange was effected using repetitive ultrafiltration with
Centricon 30 (Amicon) spin filters (30,OOOD MWCO). In general,
50-200 .mu.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.degree. C. in a Sorval SS-34 rotor at 6,000 rpm for 45 min.
Retentate volume was approximately 50-100 .mu.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.degree. C. until used for
conjugation. For some studies, 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.
[0162] d. Conjugation Protocol
[0163] 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
.mu.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.degree. C. in a polypropylene
tube until radiolabeled.
[0164] e. Determination of Chelate Incorporation
[0165] 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 .sup.90Y of known concentration and
specific activity.
[0166] Briefly, a stock solution of yttrium chloride of known
concentration was prepared in metal-free 0.05 N HC1 to which
carrier-free .sup.90Y (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.
[0167] After a 5 min incubation the entire volume was purified by
high-performance size-exclusion chromatography as described below.
The eluted protein-containing fractions were combined, the protein
concentration determined, and an aliquot assayed for radioactivity.
The chelate incorporation was calculated using the specific
activity of the .sup.90Y chloride preparation and the protein
concentration.
[0168] f. Immunoreactivity of 2B8-MX-DTPA
[0169] The immunoreactivity of conjugated 2B8 was assessed using
whole-cell ELISA. Mid-log phase SB cells were harvested from
culture by centrifugation and washed two times with 1.times. HBSS.
Cells were diluted to 1-2.times.10.sup.6 cells/mL in HBSS and
aliquoted into 96-well polystyrene microliter plates at
50,000-100,000 cells/well. The plates were dried under vacuum for 2
h at 40-45.degree. C. to fix the cells to the plastic. The plates
were stored dry at -20.degree. C. until used. For assay, the plates
were warmed to ambient temperature immediately before use, then
blocked with 1.times. PBS, pH 7.2-7.4 containing 1% BSA (2 h).
Samples for assay were diluted in 1.times. 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.times. PBS. Secondary antibody (goat
anti-mouse IgGl-specific HRP conjugate) (50 .mu.L) was added to
wells (1:1500 dilution in 1.times. PBS/1% BSA) and incubated 1 h at
ambient temperature. Plates were washed four times with 1.times.
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.
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.
[0170] g. In Vitro Stability of Native 2B8 and 2B8-MX-DTPA
[0171] For this 12-week assessment of antibody and conjugate
stability, aliquots of 2B8 antibody and 2B8-MX-DTPA were formulated
in either normal saline or normal saline containing 10 mM
glycine-HCl, pH 6.8. Duplicate sets of samples were incubated at
both 4.degree. and 30.degree. C. and samples assayed weekly using
the following methods: SDS-PAGE (both reducing and nonreducing),
immunoreactivity by whole-cell enzyme immunoassay using either SB
(antigen-positive) or HSB (antigen-negative) cells as capture, and
isoelectric focusing gel electrophoresis (pH range, 3-10). In
addition, the radiolabeling efficiency of the conjugate was
assessed at weeks 4, 8, and 12 by radiolabeling the conjugate with
.sup.90Y and analyzing the product by SDS-PAGE and autoradiographic
analysis. Finally, in a separate study, aliquots of 2B8-MX-DTPA
incubated at 4.degree. and 30.degree. C. for 10 weeks were
radiolabeled with .sup.111In and evaluated in a biodistribution
study in BALB/c mice as described below.
[0172] h. Immunohistology Studies.
[0173] Immunohistology studies with both the native and conjugated
(2B8-MX-DTPA) antibodies were performed by IMPATH Laboratories
using sections of human tissues fixed with acetone. The antibody
was purified from hollow-fiber bioreactor supernatants by
chromatography on protein A and Q Sepharose. Clinical-grade
conjugate was prepared using MX-DTPA from Coulter Immunology
according to the protocol described above.
[0174] i. In Vitro Immunoreactivity of Radiolabeled
2B8-MX-DTPA.
[0175] For some experiments, the whole-cell ELISA protocol used for
unlabeled 2B8-MX-DTPA was used. In later experiments,
immunoreactivity of .sup.111In and .sup.90Y-labeled conjugates
(each prepared at IDEC Pharmaceuticals or, alternatively, at MPI
Pharmacy Services, Inc.) was determined using a modified version of
the whole-cell binding assay described by Lindmo (3). Briefly,
increasing concentrations of mid-log phase, antigen-positive SB
cells or antigen-negative HSB cells [20-30.times.10.sup.6 cells/mL
in dilution buffer (PBS, pH 7.4 containing 1% BSA, 0.1% gelatin,
and 0.02% sodium azide)] were added to duplicate sets of tubes. 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.
[0176] j. In Vitro Stability of Radiolabeled 2B8-MX-DTPA in Human
Serum.
[0177] The in vitro stability of .sup.111In- and .sup.90Y-labeled
2B8-MX-DTPA was assessed by incubation in human serum at 37.degree.
C. for 96 hours. The conjugated antibody was prepared and
radiolabeled with .sup.111In ("mix-and-shoot" protocol) or .sup.90Y
as described above. The specific activities of the .sup.111In and
.sup.90Y-labeled conjugates were 2.5 and 14.6 mCi/mg, respectively;
the radiolabeled conjugates were suspended in buffer containing 75
mg/mL human serum albumin (HSA) and 1 mM DTPA (yttrium-labeled
conjugate) or buffer containing 50 mg/mL HSA (indium-labeled
conjugate). The 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.degree. 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.
[0178] k. In Vitro Stabilitv of Clinically-Formulated
.sup.111In-2B8-MX-DTPA.
[0179] The 2B8-MX-DTPA conjugate was radiolabeled with .sup.111In
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. The specific
activity of the formulated radiolabeled conjugate was 2.2 mCi/mg.
The formulated conjugate was subsequently incubated at 4.degree. 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.
[0180] 1. In Vitro Stability of Clinically-Formulated
.sup.90Y-2B8-MX-DTPA.
[0181] The 2B8-MX-DTPA conjugate was radiolabeled with .sup.90Y and
purified by size-exclusion chromatography on HPLC using 1.times.
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.degree. 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.
[0182] 2. Animal Studies.
[0183] a. Primate High Dose Pharmacology/Toxicology Study Using
2B8.
[0184] 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).
[0185] Prior to initiation of the study, blood was obtained from
all 10 animals and used to determine reagent backgrounds and
initial B cell populations. All subsequent blood samples were drawn
prior to each antibody injection. Groups III and IV were sacrificed
at day 13 for complete necropsy and histopathology.
[0186] Animals in groups I, II, and V were bled on days 0, 1, 3, 7,
13, 21, 37 and 52; approximately 5 mL whole blood was drawn in
heparinized tubes. Whole blood was kept at 4.degree. C. and
analyzed within 24 hours. Blood from each animal was centrifuged at
2000 rpm for 5 min. and the supernatant plasma was removed for
assay of serum 2B8 levels by RIA (see RIA procedure for specific
assay methods). The pelleted material containing PBLs and RBCs was
resuspended in FCS for FACS analysis.
[0187] b. Pharmacokinetic Studies with 2B8 and 2B8-MX-DTPA.
[0188] 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 .mu.g per ml in 10 mM borate
buffer, pH 9.6, and 50 .mu.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.degree. C., or for 2 h at ambient
temperature. Each plate was blocked for 30 min. at ambient
temperature with 150 .mu.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 .mu.g antibody
per mL serum.
[0189] In addition, the .beta.t.sub.1/2 values of 2B8 and
2B8-MX-DTPA were determined in BALB/c mice. Unconjugated 2B8 stored
at -70.degree. C. in 1.times. 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.degree. C. with the
native antibody until used.
[0190] Six-to-eight week old mice were injected with 100 .mu.L of
purified 2B8 antibody at a concentration of 250 .mu.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
.beta.t.sub.1/2 values.
[0191] c. Pharmacology/Toxicology Study of [891-Y-2B8-MX-DTPA in
Cynomolgus Monkeys.
[0192] Yttrium-[89]-bearing 2B8-MX-DTPA was prepared using the
protocol described for insertion of .sup.90Y, except that HPLC
purification was not used. The non-radioactive, metal-bearing
conjugate was formulated in 1.times. 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). The animals 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.
[0193] d. Biodistribution of Radiolabeled 2B8-MX-DTPA
[0194] In a preliminary study .sup.111In-labeled 2B8-MX-DTPA was
evaluated for tissue biodistribution in six-to-eight week old
BALB/c mice. The radiolabeled conjugate was prepared using
clinical-grade 2B8-MX-DTPA following the "mix and shoot" protocol
described above. The specific activity of the conjugate was 2.3
mCi/mg and the conjugate was formulated in PBS, pH 7.4 containing
50 mg/mL HSA. Mice were injected intravenously with 100 .mu.L of
.sup.111In-labeled 2B8-MX-DTPA (approximately 21 .mu.Ci) and groups
of three mice were sacrificed by cervical dislocation at 0, 24, 48,
and 72 hours. After sacrifice, the tail, heart, lungs, liver,
kidney, spleen, muscle, and femur were removed, washed, weighed; a
sample of blood was also removed for analysis. Radioactivity
associated with each specimen was determined by gamma counting and
the percent injected dose per gram tissue subsequently determined.
No attempt was made to discount the activity contribution
represented by the blood associated with individual organs.
[0195] In a separate protocol, aliquots of 2B8-MX-DTPA incubated at
40 and 30.degree. C. for 10 weeks were radiolabeled with .sup.111In
to a specific activity of 2.1 mCi/mg for both preparations. These
conjugates were then used in biodistribution studies in mice as
described above.
[0196] For dosimetry determinations, 2B8-MX-DTPA was radiolabeled
with .sup.111In to a specific activity of 2.3 mCi/mg and
approximately 1.1 .mu.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.
[0197] Using a similar approach, 2B8-MX-DTPA was also radiolabeled
with .sup.90Y and its biological distribution evaluated in BALB/c
mice over a 72-hour time period. Following purification by HPLC
size exclusion chromatography, four groups of five mice each were
injected intravenously with approximately 1 .mu.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.
[0198] e. Tumor Localization of .sup.111In-Labeled 2B8-MX-DTPA.
[0199] The localization of radiolabeled 2B8-MX-DTPA was determined
in athymic mice bearing Ramos B-cell tumors. Six-to-eight week old
athymic mice were injected subcutaneously (left-rear flank) with
0.1 mL of RPMI-1640 containing 1.2.times.10.sup.7 Ramos tumor cells
which had been previously adapted for growth in athymic mice.
Tumors arose within two weeks and ranged in weight from 0.07 to 1.1
grams. Mice were injected intravenously with 100 .mu.L of
.sup.111In-labeled 2B8-MX-DTPA (16.7 .mu.Ci) and groups of three
mice were sacrificed by cervical dislocation at 0, 24, 48, and 72
hours. After sacrifice the tail, heart, lungs, liver, kidney,
spleen, muscle, femur, and tumor were removed, washed, weighed; a
sample of blood was also removed for analysis. Radioactivity
associated with each specimen was determined by gamma counting and
the percent injected dose per gram tissue determined.
[0200] 3. Dosimetry Calculations
[0201] Using the biodistribution data obtained using BALB/c mice
injected with either the .sup.111In or .sup.90Y-labeled 2B8-MX-DTPA
(Tables 1-4 and 5-8), estimates of the radiation dose absorbed from
a 1.0 mCi dose administered to a 70 Kg patient were calculated
using the approach formalized by Medical Internal Radiation Dose
(MIRD) Committee of the Society of Nuclear Medicine. The biological
half-lives of the radiolabeled conjugates were determined from the
injected dose per organ values determined from the biodistribution
data for each radioimmunoconjugate. For some tissues, e.g. blood,
it was assumed that the biological decay of the radioconjugate
followed a two-compartrnent model with an exponential decay from
these compartments. For other tissues, e.g. the liver, whose
activity levels remained roughly constant throughout the 72-hour
biodistribution study, it was assumed that the biological half-life
was very long and assigned a value of 1000 hours.
2TABLE 1 Distribution of Activity 1.0 Hour Following I.V. Injection
of .sup.111In-2B8-MX-DTPA Into Normal BALB/c Mice Mean Values .+-.
SD Organ Weight % ID/ % ID per Sample Gram Gram Organ Blood 1.47
.+-. 0.17 40.3 .+-. 5.32 58.4 .+-. 3.1 Heart 0.087 .+-. 0.01 5.88
.+-. 0.76 0.51 .+-. 0.05 Lung (2) 0.149 .+-. 0.01 14.2 .+-. 1.4
2.10 .+-. 0.17 Kidney (1) 0.127 .+-. 0.02 9.82 .+-. 0.86 1.22 .+-.
0.12 Liver 1.06 .+-. 0.20 10.32 .+-. 1.58 10.76 .+-. 1.93 Spleen
0.090 .+-. 0.01 6.94 .+-. 1.17 0.61 .+-. 0.03 Muscle 8.39 .+-. 0.98
0.70 .+-. 0.25 5.67 .+-. 1.35 Bone 3.15 .+-. 0.35 2.97 .+-. 0.71
9.10 .+-. 1.09 Skin 3.15 .+-. 0.35 0.96 .+-. 0.29 3.0 .+-. 1.12 GI
Tract 2.58 .+-. 0.31 6.10 .+-. 2.00 7.80 .+-. 1.80 Urine -- Feces
-- TOTAL 99.04 .+-. 4.8 No. Mice = 5 Mean Weight = 20.97 .+-. 2.46
grams
[0202]
3TABLE 2 Distribution of Activity 24 Hours Following I.V. Injection
of .sup.111In-2B8-MX-DTPA Into Normal BALB/c Mice Mean Values .+-.
SD Organ Weight % ID/ % ID per Sample Gram Gram Organ Blood 1.47
.+-. 0.07 21.97 .+-. 1.87 32.22 .+-. 1.35 Heart 0.128 .+-. 0.03
4.02 .+-. 0.23 0.38 .+-. 0.01 Lung (2) 0.152 .+-. 0.02 7.90 .+-.
1.61 1.20 .+-. 0.18 Kidney (1) 0.128 .+-. 0.01 5.94 .+-. 0.40 0.76
.+-. 0.04 Liver 1.11 .+-. 0.10 10.08 .+-. 1.83 11.20 .+-. 2.23
Spleen 0.082 .+-. 0.01 5.04 .+-. 0.75 0.40 .+-. 0.02 Muscle 8.41
.+-. 0.38 1.24 .+-. 0.05 10.44 .+-. 0.76 Bone 3.15 .+-. 0.14 2.02
.+-. 0.33 6.31 .+-. 0.81 Skin 3.15 .+-. 0.14 3.75 .+-. 0.39 11.77
.+-. 1.09 GI Tract 2.91 .+-. 0.27 4.50 .+-. 0.52 6.65 .+-. 0.56
Urine 0.98 Feces 2.54 TOTAL 87.10 .+-. 1.68 No. Mice = 5 Mean
Weight = 21.03 .+-. 0.94 grams
[0203]
4TABLE 3 Distribution of Activity 48 Hours Following I.V. Injection
of .sup.111In-2B8-MX-DTPA Into Normal BALB/c Mice Mean Values .+-.
SD Organ Weight % ID/ % ID per Sample Gram Gram Organ Blood 1.45
.+-. 0.13 22.41 .+-. 3.95 31.90 .+-. 2.89 Heart 0.090 .+-. 0.01
4.05 .+-. 0.94 0.36 .+-. 0.06 Lung (2) 0.155 .+-. 0.02 8.45 .+-.
0.53 1.31 .+-. 0.19 Kidney (1) 0.125 .+-. 0.01 6.16 .+-. 1.15 0.76
.+-. 0.07 Liver 1.040 .+-. 0.11 9.41 .+-. 2.33 9.84 .+-. 3.18
Spleen 0.082 .+-. 0.01 5.32 .+-. 0.71 0.48 .+-. 0.11 Muscle 8.26
.+-. 0.77 1.42 .+-. 0.58 11.62 .+-. 4.67 Bone 3.10 .+-. 0.29 2.08
.+-. 0.16 6.41 .+-. 0.44 Skin 3.10 .+-. 0.29 3.43 .+-. 0.59 10.54
.+-. 1.69 GI Tract 2.96 .+-. 0.20 5.05 .+-. 0.63 7.46 .+-. 0.60
Urine 1.46 Feces 6.41 TOTAL 88.49 .+-. 6.87 No. Mice = 5 Mean
Weight = 20.65 .+-. 1.93 grams
[0204]
5TABLE 4 Distribution of Activity 72 Hours Following I.V. Injection
of .sup.111In-2B8-MX-DTPA Into Normal BALB/c Mice Mean Values .+-.
SD Organ Weight % ID/ % ID per Sample Gram Gram Organ Blood 1.52
.+-. 0.06 18.97 .+-. 1.31 28.51 .+-. 2.03 Heart 0.094 .+-. 0.01
3.71 .+-. 0.31 0.35 .+-. 0.04 Lung (2) 0.161 .+-. 0.01 7.60 .+-.
0.30 1.18 .+-. 0.09 Kidney (1) 0.135 .+-. 0.01 5.55 .+-. 0.53 0.76
.+-. 0.09 Liver 1.11 .+-. 0.11 9.90 .+-. 1.77 11.00 .+-. 2.03
Spleen 0.095 .+-. 0.01 5.12 .+-. 0.75 0.48 .+-. 0.04 Muscle 8.58
.+-. 0.34 1.04 .+-. 0.09 8.95 .+-. 0.68 Bone 3.22 .+-. 0.12 1.73
.+-. 0.34 6.04 .+-. 0.51 Skin 3.22 .+-. 0.12 3.16 .+-. 0.60 10.19
.+-. 2.03 GI Tract 2.79 .+-. 0.19 4.53 .+-. 0.83 6.37 .+-. 1.38
Urine 2.49 Feces 11.50 TOTAL 87.80 .+-. 4.79 No. Mice = 5 Mean
Weight = 21.46 .+-. 0.84 grams
[0205]
6TABLE 5 Distribution of Activity 1.0 Hour Following I.V. Injection
of .sup.90Y-2B8-MX-DTPA Into Normal BALB/c Mice Mean Values .+-. SD
Organ Weight % ID/ % ID per Sample Gram Gram Organ Blood 1.27 .+-.
0.06 39.23 .+-. 2.45 49.77 .+-. 1.72 Heart 0.086 .+-. 0.01 5.80
.+-. 0.84 0.50 .+-. 0.09 Lung (2) 0.137 .+-. 0.01 12.11 .+-. 1.08
1.66 .+-. 0.17 Kidney (1) 0.120 .+-. 0.01 10.23 .+-. 1.30 1.15 .+-.
0.12 Liver 0.921 .+-. 0.05 12.12 .+-. 1.72 11.17 .+-. 1.66 Spleen
0.080 .+-. 0.01 9.27 .+-. 0.46 0.74 .+-. 0.07 Muscle 7.27 .+-. 0.32
0.78 .+-. 0.13 5.72 .+-. 1.05 Bone 2.73 .+-. 0.12 4.35 .+-. 0.39
11.89 .+-. 1.47 Skin 2.73 .+-. 0.12 2.12 .+-. 0.78 5.82 .+-. 2.24
GI Tract 2.22 .+-. 0.06 3.52 .+-. 1.12 4.22 .+-. 0.84 Urine -- --
-- Feces -- -- -- TOTAL 94.85 .+-. 3.47 No. Mice = 5 Mean Weight =
18.17 grams .+-. 0.81 grams
[0206]
7TABLE 6 Distribution of Activity at 24 Hours Following I.V.
Injection of .sup.90Y-2B8-MX-DTA Into Normal BALB/c Mice Mean
Values .+-. SD Organ Weight % ID/ % ID per Sample Gram Gram Organ
Blood 1.517 .+-. 0.090 8.35 .+-. 2.547 12.83 .+-. 4.60 Heart 0.092
.+-. 0.005 2.63 .+-. 0.142 0.240 .+-. 0.006 Lung 0.141 .+-. 0.005
4.56 .+-. 0.393 0.644 .+-. 0.047 Kidney 0.138 .+-. 0.007 5.63 .+-.
0.222 0.779 .+-. 0.040 Liver 0.438 .+-. 0.098 5.22 .+-. 0.335 2.259
.+-. 0.399 Spleen 0.081 .+-. 0.003 4.23 .+-. 0.180 0.345 .+-. 0.011
Muscle 8.668 .+-. 0.514 0.976 .+-. 0.164 8.55 .+-. 1.945 Bone 3.246
.+-. 0.186 1.326 .+-. 0.102 4.289 .+-. 0.154 No. Mice = 3 Mean
Weight = 21.671 .+-. 1.11 gram
[0207]
8TABLE 7 Distribution of Activity at 48 Hours Following I.V.
Injection of .sup.90Y-2B8-MX-DTPA Into Normal BALB/c Mice Mean
Values .+-. SD Organ Weight % ID/ % ID per Sample Gram Gram Organ
Blood 1.33 .+-. 0.06 17.34 .+-. 2.0 23.03 .+-. 1.95 Heart 0.088
.+-. 0.01 3.56 .+-. 0.31 0.31 .+-. 0.04 Lung (2) 0.139 .+-. 0.01
7.54 .+-. 0.88 1.05 .+-. 0.15 Kidney (1) 0.122 .+-. 0.01 6.53 .+-.
0.42 0.79 .+-. 0.01 Liver 0.968 .+-. 0.04 9.05 .+-. 1.70 8.92 .+-.
1.57 Spleen 0.087 .+-. 0.01 6.52 .+-. 1.13 0.57 .+-. 0.07 Muscle
7.26 .+-. 0.36 1.05 .+-. 0.18 8.01 .+-. 1.17 Bone 2.86 .+-. 0.14
3.34 .+-. 0.42 9.53 .+-. 1.08 Skin 2.86 .+-. 0.14 4.13 .+-. 0.76
11.75 .+-. 1.82 GiTract 2.84 .+-. 0.19 2.74 .+-. 0.34 3.80 .+-.
0.30 Urine -- -- 4.29 Feces -- -- 7.67 TOTAL 79.72 .+-. 3.23 No.
Mice = 5 Mean Weight = 19.07 .+-. 0.91 grams
[0208]
9TABLE 8 Distribution of Activity at 72 Hours Following I.V.
Injection of .sup.90Y-2B8-MX-DTPA Into Normal BALB/c Mice Mean
Values .+-. SD Organ Weight % ID/ % ID per Sample Gram Gram Organ
Blood 1.35 .+-. 0.02 15.40 .+-. 1.63 20.71 .+-. 2.13 Heart 0.088
.+-. 0.01 3.12 .+-. 0.24 0.28 .+-. 0.01 Lung (2) 0.142 .+-. 0.01
8.23 .+-. 1.05 1.17 .+-. 0.20 Kidney (1) 0.123 .+-. 0.01 6.45 .+-.
0.57 0.79 .+-. 0.07 Liver 0.02 .+-. 0.06 8.39 .+-. 1.04 8.58 .+-.
1.31 Spleen 0.103 .+-. 0.01 5.90 .+-. 1.19 0.59 .+-. 0.08 Muscle
7.68 .+-. 0.11 1.01 .+-. 0.15 7.73 .+-. 1.05 Bone 2.88 .+-. 0.05
3.20 .+-. 0.25 9.20 .+-. 0.61 Skin 2.88 .+-. 0.05 3.97 .+-. 0.49
11.42 .+-. 1.36 GI Tract 2.86 .+-. 0.18 2.90 .+-. 0.65 4.06 .+-.
0.93 Urine -- -- 3.00 Feces -- -- 11.08 TOTAL 78.62 .+-. 2.63 No.
Mice = 5 Mean Weight = 19.21 .+-. 0.27 grams
[0209] In a similar manner the other biological half-life values
were assigned or calculated using the standard equation for
calculating the t.sub.1/2 for an exponential decay. Once these
values had been determined, the variables for T.sub.ue, T.sub.e1,
T.sub.e2, A.sup.1, A.sub.2, and A, listed in Tables 9 and 10, were
determined for each radiolabeled conjugate using the equations
provided at the top of these tables (output variables). These
values, as well as those shown in the subsequent tables, were
calculated using a program written in the Symphony spreadsheet
(Lotus Development Corp.) by Mr. Phillip Hagan, MS, Nuclear
Medicine Service, VA Medical Center, La Jolla, Calif. 92161.
10TABLE 9 INPUT VARIABLES OUTPUT VARIABLES A0 = Administered dose
Tue = Effective uptake half-time Tp = Physical half-life of
radionuclide Te1 = Effective disappearance half-time of first
component Tu = Biologic uptake half-time Te2 = Effective
disappearance half-time of second component Tb1 = Biological
disappearance half-time of first component A1 = Cumulated activity
of first component Tb2 = Biological disappearance half-time of
second component A2 = Cumulated activity of second component f1 =
Fraction of A0 with biological half-time of Tb1 A = Total cumulated
activity f2 = Fraction of A0 with biological half-time of Tb2 Tue =
Tu * Tp/(Tu + Tp) A1 - 1.44*f1*A0*Te1*(Tue/Tu) S = Mean Dose/Unit
Cumulated Activity Te1 = Tb1 * Tp/(Tb1 + Tp) A2 - 1.44*f2*A0*Te2
Te2 = Tb2 * Tp/(Tb2 + Tp) A - A1 + A2 Example: A1 FOR LIVER - 1.44
* 11.000% * 1000 * 63.2 * 1.00 = 10007.5 microcuries cumulated
activity TABLE OF INPUT AND OUTPUT VALUES USED TO EVALUATE
CUMULATED ACTIVITY (A) Tu Tb1 Tb2 Tue Te1 Te2 A1 A2 A (hr) f1 f2
(hr) (hr) (hr) (hr) (hr) (uCi-hr) (uCi-hr) (uCi-hr) ADRENALS
2.78E-04 0.000% 0.00% 1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 BLAD
CONTENTS 2.78E-04 1.000% 0.00% 4 0 2.78E-04 3.8 0.0 54.4 0.0 54
STOMACH CONTENTS 2.78E-04 6.650% 0.00% 1.5 0 2.78E-04 1.5 0.0 140.5
0.0 141 SM. INT. CONTENTS 2.78E-04 6.650% 0.00% 3.5 0 2.78E-04 3.3
0.0 318.6 0.0 319 ULI_CONTENTS 2.78E-04 6.650% 0.00% 4.5 0 2.78E-04
4.2 0.0 404.0 0.0 404 LLI_CONTENTS 2.78E-04 6.650% 0.00% 4.2 0
2.78E-04 4.0 0.0 378.6 0.0 379 KIDNEYS 2.78E-04 1.220% 0.00% 35 0
2.78E-04 23.0 0.0 404.8 0.0 405 LIVER 2.78E-04 11.000% 0.00% 1000 0
2.78E-04 63.2 0.0 10007.5 0.0 10008 LUNGS 2.78E-04 2.100% 1.20% 30
1000 2.78E-04 20.8 63.2 627.9 1091.7 1720 OTH TISS (TOTAL) 2.78E-04
0.000% MUSCLE 2.78E-04 10.400% 0.00% 1000 0 2.78E-04 63.2 0.0
9461.7 0.0 9462 ADIPOSE 2.78E-04 0.000% 0.00% 1000 0 2.78E-04 63.2
0.0 0.0 0.0 0 BLOOD 2.78E-04 58.400% 32.22% 15 1000 2.78E-04 12.3
63.2 10319.2 29313.1 39632 BRAIN 2.78E-04 0.000% 0.00% 1000 0
2.78E-04 63.2 0.0 0.0 0.0 0 HEART 2.78E-04 0.510% 0.38% 57 1000
2.78E-04 30.9 63.2 226.9 345.7 573 PVARIES 2.78E-04 0.000% 0.00%
1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 PANCREAS 2.78E-04 0.000% 0.00%
1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 SKELETON (TOTAL) 2.78E-04 0.000%
CORTICAL BONE 2.78E-04 0.000% 0.00% 1000 0 2.78E-04 63.2 0.0 0.0
0.0 0 TRABECULAR BONE 2.78E-04 9.100% 6.30% 45 1000 2.78E-04 27.0
63.2 3536.8 5731.6 9268 NARROW (RED) 2.78E-04 0.000% 0.00% 1000 0
2.78E-04 63.2 0.0 0.0 0.0 0 MARROW (YELLOW) 2.78E-04 0.000% 0.00%
1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 CARTILAGE 2.78E-04 0.000% 0.00%
1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 OTHER CONSTIT. 2.78E-04 0.000%
0.00% 1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 SKIN 2.78E-04 11.770%
0.00% 1000 0 2.78E-04 63.2 0.0 10708.1 0.0 10708 SPLEEN 2.78E-04
0.610% 0.40% 39 1000 2.78E-04 24.7 63.2 217.1 363.9 581 TESTES
2.78E-04 0.000% 0.00% 1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 THYROID
2.78E-04 0.000% 0.00% 1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 TOTAL BODY
2.78E-04 0.000% 0.00% 1000
[0210]
11TABLE 10 INPUT VARIABLES OUTPUT VARIABLES A0 = Administered dose
Tue = Effective uptake half-time Tp = Physical half-life of
radionuclide Te1 = Effective disappearance half-time of first
component Tu = Biologic uptake half-time Te2 = Effective
disappearance half-time of second component Tb1 = Biological
disappearance half-time of first component A1 = Cumulated activity
of first component Tb2 = Biological disappearance half-time of
second component A2 = Cumulated activity of second component f1 =
Fraction of A0 with biological half-time of Tb1 A = Total cumulated
activity f2 = Fraction of A0 with biological half-time of Tb2 Tue =
Tu * Tp/(Tu + Tp) A1 - 1.44*f1*A0*Te1*(Tue/Tu) S = Mean Dose/Unit
Cumulated Activity Te1 = Tb1 * Tp/(Tb1 + Tp) A2 - 1.44*f2*A0*Te2
Te2 = Tb2 * Tp/(Tb2 + Tp) A - A1 + A2 Example: A1 FOR LIVER - 1.44
* 9.000% * 1000 * 60.2 * 1.00 = 7795.5 microcuries cumulated
activity TABLE OF INPUT AND OUTPUT VALUES USED TO EVALUATE
CUMULATED ACTIVITY (A) Tu Tb1 Tb2 Tue Te1 Te2 A1 A2 A (hr) f1 f2
(hr) (hr) (hr) (hr) (hr) (uCi-hr) (uCi-hr) (uCi-hr) ADRENALS
2.78E-04 0.000% 0.00% 1000 0 2.78E-04 63.2 0.0 0.0 0.0 0 BLAD
CONTENTS 2.78E-04 1.000% 0.00% 4 0 2.78E-04 3.8 0.0 54.2 0.0 54
STOMACH CONTENTS 2.78E-04 4.220% 0.00% 1.5 0 2.78E-04 1.5 0.0 89.1
0.0 89 SM. INT. CONTENTS 2.78E-04 4.220% 0.00% 3.5 0 2.78E-04 3.3
0.0 201.7 0.0 202 ULI_CONTENTS 2.78E-04 4.220% 0.00% 4.5 0 2.78E-04
4.2 0.0 255.5 0.0 255 LLI_CONTENTS 2.78E-04 4.220% 0.00% 4.2 0
2.78E-04 3.9 0.0 239.5 0.0 240 KIDNEY 2.78E-04 1.150% 0.87% 70 1000
2.78E-04 33.4 60.2 553.6 753.6 1307 LIVER 2.78E-04 9.000% 0.00%
1000 0 2.78E-04 60.2 0.0 7795.5 0.0 7795 LUNGS 2.78E-04 1.200%
0.00% 1000 0 2.78E-04 60.2 0.0 1039.4 0.0 1039 OTH TISS (TOTAL)
2.78E-04 0.000% MUSCLE 2.78E-04 8.720% 0.00% 1000 0 2.78E-04 60.2
0.0 7552.9 0.0 7553 ADIPOSE 2.78E-04 0.000% 0.00% 1000 0 2.78E-04
60.2 0.0 0.0 0.0 0 BLOOD 2.78E-04 49.770% 25.90% 13 1000 2.78E-04
10.8 60.2 7743.9 22433.7 30178 BRAIN 2.78E-04 0.000% 0.00% 1000 0
2.78E-04 60.2 0.0 0.0 0.0 0 HEART 2.78E-04 0.500% 0.36% 51 1000
2.78E-04 28.4 60.2 204.4 311.8 516 OVARIES 2.78E-04 0.000% 0.00%
1000 0 2.78E-04 60.2 0.0 0.0 0.0 0 PANCREAS 2.78E-04 0.000% 0.00%
1000 0 2.78E-04 60.2 0.0 0.0 0.0 0 SKELETON (TOTAL) 2.78E-04 0.000%
CORTICAL BONE 2.78E-04 0.000% 0.00% 1000 0 2.78E-04 60.2 0.0 0.0
0.0 0 TRABECULAR BONE 2.78E-04 11.890% 9.28% 67 1000 2.78E-04 32.7
60.2 5604.4 8038.0 13642 MARROW (RED) 2.78E-04 0.000% 0.00% 1000 0
2.78E-04 60.2 0.0 0.0 0.0 0 MARROW (YELLOW) 2.78E-04 0.000% 0.00%
1000 0 2.78E-04 60.2 0.0 0.0 0.0 0 CARTILAGE 2.78E-04 0.000% 0.00%
1000 0 2.78E-04 60.2 0.0 0.0 0.0 0 OTHER CONSTIT. 2.78E-04 0.000%
0.00% 1000 0 2.78E-04 60.2 0.0 0.0 0.0 0 SKIN 2.78E-04 15.600%
0.00% 1000 0 2.78E-04 60.2 0.0 13512.1 0.0 13512 SPLEEN 2.78E-04
0.740% 0.56% 60 1000 2.78E-04 31.0 60.2 330.0 485.1 815 TESTES
2.78E-04 0.000% 0.00% 1000 0 2.78E-04 60.2 0.0 0.0 0.0 0 THYROID
2.78E-04 0.000% 0.00% 1000 0 2.78E-04 60.2 0.0 0.0 0.0 0 TOTAL BODY
2.78E-04 0.000% 0.00% 1000 0 2.78E-04 60.2 0.0 0.0 0.0 0
[0211] Using the Total Cumulated Activity (A) values from Tables 9
and 10, and the S values provided from MIRD Pamphlet Number 11
(Tables 11 and 12, and 13 and 14), the radiation absorbed dose
estimates were determined for each of the radiolabeled conjugates
for the listed tissues (Tables 15, 16, 17 and 18). In determining
the summary radiation dose estimates for the indium-labeled
conjugate provided in Table 19, the self-dose of a given organ was
summed with the absorbed dose produced by activity in adjacent
organs or tissues. However, in calculating the radiation dose
estimate values attributed to the yttrium-labeled conjugate (Table
20), certain of the values are absent for the listed tissues (e.g.
adrenals). This is due to the shorter path length of the released
13 particle, relative to the path-length of the emitted g particle,
hence providing a negligible activity contribution from adjacent
tissues, and to the absence of primary biodistribution data for
these tissues.
12TABLE 11 S. ABSORBED DOSE PER UNIT CUMULATED ACTIVITY,
(RAD/UCI-H) INDIUM-[111] HALF-LIFE 67.44 HOURS SOURCE ORGANS
Intestinal Tract Other Target Bladder Stomach Si Uli Lli Tissue
Organs Adrenals Contents Contents Contents Contents Contents
Kidneys Liver Lungs (Muscle) ADRENALS 7.4E-03 5.7E-07 7.3E-06
4.4E-06 2.8E-06 1.3E-06 3.4E-05 1.5E-05 7.6E-06 4.8E-06 BLADDER
WALL 3.6E-07 4.5E-04 7.5E-07 8.0E-06 6.4E-06 2.0E-05 9.3E-07
5.2E-07 1.5E-07 5.5E-06 BONE 5.2E-06 2.3E-06 2.3E-06 3.2E-06
2.9E-06 4.2E-06 3.7E-06 2.9E-06 3.8E-06 3.2E-06 GI (STOM WALL)
8.8E-06 8.5E-07 3.4E-04 1.1E-05 1.2E-05 5.4E-06 1.0E-05 5.8E-06
5.7E-06 4.3E-06 GI (SI) 2.5E-06 8.6E-06 7.9E-06 2.1E-04 5.4E-05
3.0E-05 8.6E-06 5.0E-06 6.1E-07 4.8E-06 GI (ULI WALL) 2.8E-06
6.9E-06 1.1E-05 8.3E-05 3.3E-04 1.4E-05 8.6E-06 7.5E-06 7.4E-07
5.0E-06 GI (LLI WALL) 7.1E-07 2.2E-05 3.8E-06 2.4E-05 9.5E-06
4.7E-04 2.5E-06 7.3E-07 3.0E-07 5.2E-06 KIDNEYS 3.7E-05 8.5E-07
1.1E-05 9.2E-06 8.3E-06 2.8E-06 5.2E-04 1.2E-05 2.7E-06 4.4E-06
LIVER 1.5E-05 6.3E-07 5.9E-06 5.6E-06 7.8E-06 8.4E-07 1.2E-05
1.3E-04 7.7E-06 3.4E-06 LUNGS 7.6E-06 8.2E-08 5.2E-06 7.5E-07
8.3E-07 2.6E-07 2.5E-06 7.8E-06 1.4E-04 4.2E-06 MARROW (RED)
9.4E-06 5.3E-06 4.0E-06 1.1E-05 9.1E-06 1.3E-05 9.6E-06 4.1E-06
4.8E-06 5.3E-06 OTH TISS (MUSC) 4.8E-06 5.5E-06 4.3E-06 4.8E-06
4.5E-06 5.2E-06 4.4E-06 3.4E-06 4.4E-06 7.5E-06 OVARIES 1.8E-06
2.3E-05 1.3E-06 3.3E-05 3.7E-05 6.4E-05 3.6E-06 1.4E-06 3.6E-07
6.3E-06 PANCREAS 2.6E-05 8.6E-07 5.7E-05 6.1E-06 7.1E-06 2.1E-06
2.0E-05 1.2E-05 7.7E-06 5.7E-06 SKIN 1.8E-06 1.7E-06 1.4E-06
1.4E-06 1.4E-06 1.6E-06 1.8E-06 1.6E-06 1.8E-06 2.5E-06 SPLEEN
2.0E-05 7.6E-07 3.1E-05 4.6E-06 4.2E-06 2.4E-06 2.8E-05 2.8E-06
7.1E-06 4.6E-06 TESTES 1.4E-07 1.4E-05 1.8E-07 I.0E-06 9.8E-07
5.9E-06 3.4E-07 2.5E-07 3.9E-08 3.6E-06 THYROID 4.7E-07 1.2E-08
3.5E-07 6.9E-08 7.5E-08 2.7E-08 2.0E-07 6.2E-07 2.6E-06 4.3E-06
UTERUS (NONGRVD) 5.8E-06 4.9E-05 2.4E-06 2.9E-05 1.5E-05 2.1E-05
3.1E-06 1.2E-06 2.8E-07 7.4E-06 TOTAL BODY 6.6E-06 6.2E-06 6.1E-06
7.3E-06 6.8E-06 6.9E-06 6.6E-06 6.6E-06 5.9E-06 5.6E-06 REFERENCE -
MIRD PAMPHLET NO. 11, PAGE 164
[0212]
13TABLE 12 S. ABSORBED DOSE PER UNIT CUMULATED ACTIVITY,
(RAD/UCI-H) INDIUM-[111] HALF-LIFE 67.44 HOURS SOURCE ORGANS Target
Skeleton Organs Ovaries Pancreas R Marrow Cort Bone TRA Bone Skin
Spleen Testes Thyroid Total Body ADRENALS 1.1E-06 2.6E-05 7.9E-06
3.9E-06 3.9E-06 2.4E-06 2.0E-05 1.4E-07 4.7E-07 7.0E-06 BLADDER
WALL 2.1E-05 4.7E-07 2.4E-06 1.5E-06 1.5E-06 1.6E-06 4.7E-07
1.5E-05 1.2E-08 6.9E-06 BONE 3-8E-06 3.6E-06 1.2E-05 3.0E-05
2.6E-05 2.9E-06 2.9E-06 2.4E-06 2.6E-06 6.9E-06 GI (STOM WALL)
2.4E-06 5.9E-05 3.2E-06 1.7E-06 1.1E-06 1.7E-06 3.0E-05 1.5E-07
1.5E-07 7.1E-06 GI (SI) 3.8E-05 5.5E-06 7.9E-06 2.3E-06 2.3E-06
1.5E-06 4.2E-06 1.2E-06 4.2E-08 7.5E-06 GI (ULI WALL) 3.7E-05
6.6E-06 6.4E-06 2.2E-06 2.2E-06 1.4E-06 3.8E-06 1.1E-06 3.3E-08
7.0E-06 GI (LLI WALL) 4.8E-05 1.7E-06 9.0E-06 3.2E-06 3.2E-06
1.5E-06 1.9E-06 8.3E-06 2.2E-08 6.7E-06 KIDNEYS 2.9E-06 1.9E-05
6.8E-06 2.7E-06 2.7E-06 2.0E-06 2.8E-05 1.7E-07 1.2E-07 6.6E-06
LIVER 1.7E-06 1.3E-05 2.9E-06 2.0E-06 2.0E-06 1.7E-06 3.0E-06
1.2E-07 3.5E-07 6.5E-06 LUNGS 2.2E-07 7.6E-06 3.7E-06 3.0E-06
3.0E-06 1.9E-06 6.9E-06 3.4E-08 2.9E-06 5.9E-06 MARROW (RED)
1.3E-05 6.8E-06 7.5E-05 1.3E-05 2.6E-05 2.7E-06 4.4E-06 1.9E-06
2.9E-06 7.7E-06 OTH TISS (MUSC) 6.3E-06 5.7E-06 3.8E-06 3.2E-06
3.2E-06 2.5E-06 4.6E-06 .3.6E-06 4.3E-06 5.6E-06 OVARIES 1.0E-02
1.0E-06 7.7E-06 2.2E-06 2.2E-06 1.4E-06 1.7E-06 0.0E+00 2.5E-08
7.0E-06 PANCREAS 1.5E-06 1.6E-03 4.9E-06 3.IE-06 3.1E-06 1.7E-06
6.IE-05 2.IE-07 3.0E-07 7.8E-06 SKIN 1.4E-06 1.3E-06 2.0E-06
2.3E-06 2.3E-06 3.7E-05 1.5E-06 4.9E-06 2.5E-06 3.7E-06 SPLEEN
1.6E-06 6.2E-05 2.7E-06 2.0E-06 2.0E-06 1.7E-06 9.1E-04 8.9E-08
3.5E-07 6.8E-06 TESTES 0.0E+00 2.1E-07 1.0E-06 1.9E-06 1.9E-06
3.4E-06 2.0E-07 3.6E-03 3.3E-09 4.9E-06 THYROID 2.5E-08 4.5E-07
2.2E-06 2.8E-06 2.8E-06 2.4E-06 3.4E-07 3.3E-09 5.8E-03 5.2E-06
UTERUS (NONGRVO) 6.5E-05 1.9E-06 6.7E-06 1.8E-06 1.8E-06 1.2E-06
1.2E-06 0.0E+00 2.4E-08 7.8E-06 TOTAL BODY 7.7E-06 7.5E-06 6.4E-06
5.9E-06 5.9E-06 3.8E-06 6.6E-06 5.6E-06 5.3E-06 5.8E-06 REFERENCE -
MIRD PAMPHLET NO. 11. PAGE 165
[0213]
14TABLE 13 S. ABSORBED DOSE PER UNIT CUMULATED ACTIVITY,
(RAD/UCI-H) YTTRIUM-[90] HALF-LIFE 64 HOURS SOURCE ORGANS
Intestinal Tract Target Bladder Stomach SI ULI LLI Other Tissue
Organs Adrenals Contents Contents Contents Contents Contents
Kidneys Liver Lungs (Muscle) ADRENALS 1.4E-01 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 BLADDER WALL 0.0 5.0E-03 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 BONE 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 GI (STOM WALL)
0.0 0.0 4.0E-03 0.0 0.0 0.0 0.0 0.0 0.0 0.0 GI (SI) 0.0 0.0 0.0
2.5E-03 0.0 0.0 0.0 0.0 0.0 0.0 GI (ULI WALL) 0.0 0.0 0.0 0.0
4.5E-03 0.0 0.0 0.0 0.0 0.0 GI (LLI WALL) 0.0 0.0 0.0 0.0 0.0
7.4E-03 0.0 0.0 0.0 0.0 KIDNEYS 0.0 0.0 0.0 0.0 0.0 0.0 6.4E-03 0.0
0.0 0.0 LIVER 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.1E-03 0.0 0.0 LUNGS 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0E-03 0.0 MARROW (RED) 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 OTH TISS (MUSC) 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 7.1E-05 OVARIES 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
PANCREAS 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 SKIN 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 SPLEEN 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 TESTES 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 THYROID 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 UTERUS (NONGRVD) 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 TOTAL BODY 2.8E-05 3.2E-06 8.5E-05
2.3E-05 1.4E-05 1.7E-05 2.8E-05 2.8E-05 2.8E-05 2.8E-05 REFERENCE -
MIRD PAMPHLET NO. 11, PAGE 144
[0214]
15TABLE 14 S. ABSORBED DOSE PER UNIT CUMULATED ACTIVITY,
(RAD/UCI-H) YTTRIUM-[90] HALF-LIFE 64 HOURS SOURCE ORGANS Target
Skeleton Organs Ovaries Pancreas R Marrow Cort Bone TRA Bone Skin
Spleen Testes Thyroid Total Body ADRENALS 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 2.8E-05 BLADDER WALL 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 2.8E-05 BONE 0.0 0.0 1.1E-04 4.0E-04 2.3E-04 0.0 0.0 0.0 0.0
2.8E-05 GI (STOM WALL) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05
GI (SI) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05 GI (ULI WALL)
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05 GI (LLI WALL) 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05 KIDNEYS 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 2.8E-05 LIVER 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
2.8E-05 LUNGS 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05 MARROW
(RED) 0.0 0.0 8.6E-04 3.3E-05 5.7E-04 0.0 0.0 0.0 0.0 2.8E-05 OTH
TISS (MUSC) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05 OVARIES
1.8E-01 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05 PANCREAS 0.0
2.0E-02 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05 SKIN 0.0 0.0 0.0 0.0
0.0 7.6E-04 0.0 0.0 0.0 2.8E-05 SPLEEN 0.0 0.0 0.0 0.0 0.0 0.0
1.1E-02 0.0 0.0 2.8E-05 TESTES 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.7E-02
0.0 2.8E-05 THYROID 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 9.9E-02 2.8E-05
UTERUS (NONGRVO) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8E-05 TOTAL
BODY 2.8E-05 2.8E-05 2.8E-05 2.8E-05 2.8E-05 2.8E-05 2.8E-05
2.8E-05 2.8E-05 2.8E-05 REFERENCE - MIRD PAMPHLET NO. 11. PAGE
145
[0215]
16TABLE 15 RADIATION ABSORBED DOSE (RAD = A * S) INDIUM-[111]
HALF-LIFE 67.44 HOURS SOURCE ORGANS Intestinal Tract Target Bladder
Stomach SI ULI LLI Other Organs Adrenals Contents Contents Contents
Contents Contents Kidneys Liver Lungs Tissue ADRENALS 0.0E+00
3.1E-05 1.0E-03 1.4E-03 1.1E-03 4.9E-04 1.4E-02 1.5E-01 1.3E-02
2.4E-01 BLADDER WALL 0.0E+00 2.4E-02 1.1E-04 2.5E-03 2.6E-03
7.6E-03 3.8E-04 5.2E-03 2.6E-04 2.7E-01 GI (STOM WALL) 0.0E+00
4.6E-05 4.8E-02 3.5E-03 2.2E-02 2.0E-03 4.0E-03 5.8E-02 9.8E-03
2.1E-01 GI (SI) 0.0E+00 4.7E-04 1.1E-03 6.7E-03 2.2E-02 1.1E-02
3.5E-03 5.0E-02 1.0E-03 2.4E-01 GI (ULI WALL) 0.0E+00 3.8E-04
1.5E-03 2.6E-02 1.3E-01 5.3E-03 3.5E-03 7.5E-02 1.3E-03 2.5E-01 GI
(LLI WALL) 0.0E+00 1.2E-03 5.3E-04 7.6E-03 3.8E-03 1.8E-01 1.0E-03
7.3E-03 5.2E-04 2.6E-01 KIDNEYS 0.0E+00 4.6E-05 1.5E-03 2.9E-03
3.4E-03 1.1E-03 2.1E-01 1.2E-01 4.6E-03 2.2E-01 LIVER 0.0E+00
3.4E-05 8.3E-04 1.8E-03 3.2E-03 3.2E-04 4.9E-03 1.3E+00 1.3E-02
1.7E-01 LUNGS 0.0E+00 4.5E-06 7.3E-04 2.4E-04 3.4E-04 9.8E-05
1.0E-03 7.8E-02 2.4E-01 2.1E-01 OTHER TISSUES MUSCLE 0.0E+00
3.0E-04 6.0E-04 1.5E-03 1.8E-03 2.0E-03 1.8E-03 3.4E-02 7.6E-03
3.7E-01 ADIPOSE 0.0E+00 3.0E-04 6.0E-04 1.5E-03 1.8E-03 2.0E-03
1.8E-03 3.4E-02 7.6E-03 3.7E-01 BLOOD 0.0E+00 3.0E-04 6.0E-04
1.5E-03 1.8E-03 2.0E-03 1.8E-03 3.4E-02 7.6E-03 3.7E-01 BRAIN
0.0E+00 3.0E-04 6.0E-04 1.5E-03 1.8E-03 2.0E-03 1.8E-03 3.4E-02
7.6E-03 3.7E-01 HEART 0.0E+00 4.1E-05 4.4E-03 1.5E-03 1.7E-03
9.1E-04 1.1E-02 2.8E-02 1.2E-02 3.7E-01 OVARIES 0.0E+00 1.3E-03
1.8E-04 1.1E-02 1.5E-02 2.4E-02 1.5E-03 1.4E-02 6.2E-04 2.8E-01
PANCREAS 0.0E+00 4.7E-05 8.0E-03 1.9E-03 2.9E-03 8.0E-04 8.1E-03
1.2E-01 1.3E-02 1.2E-01 SKELETON CORTICAL BONE 0.0E+00 1.3E-04
3.2E-04 1.0E-03 1.2E-03 1.6E-03 1.5E-03 2.9E-02 6.5E-03 1.6E-01
TRABECULAR BONE 0.0E+00 1.3E-04 3.2E-04 1.0E-03 1.2E-03 1.6E-03
1.5E-03 2.9E-02 6.5E-03 1.6E-01 MARROW (RED) 0.0E+00 2.9E-04
5.6E-04 3.5E-03 3.7E-03 4.6E-03 3.9E-03 4.1E-02 8.3E-03 2.6E-01
MARROW (YELLOW) 0.0E+00 2.9E-04 5.6E-04 3.5E-03 3.7E-03 4.9E-03
3.9E-03 4.1E-02 8.3E-03 2.6E-01 CARTILAGE 0.0E+00 1.3E-04 3.2E-04
1.0E-03 1.2E-03 1.6E-03 1.5E-03 2.9E-02 6.5E-03 1.6E-01 OTHER
CONSTIT. 0.0E+00 1.3E-04 3.2E-04 1.0E-03 1.2E-03 1.6E-03 1.5E-03
2.9E-02 6.5E-03 1.6E-01 SKIN 0.0E+00 9.2E-05 2.0E-04 4.5E-04
5.7E-04 6.1E-04 7.3E-04 1.6E-02 3.1E-03 1.2E-01 SPLEEN 0.0E+00
4.1E-05 4.4E-03 1.5E-03 1.7E-03 9.1E-04 1.1E-02 2.8E-02 1.2E-02
2.3E-01 TESTES 0.0E+00 7.6E-04 2.5E-05 3.2E-04 4.0E-04 2.2E-03
1.4E-04 2.5E-03 6.7E-05 1.8E-01 THYROID 0.0E+00 6.SE-07 4.9E-05
2.2E-05 3.0E-05 1.0E-05 8.1E-05 6.2E-03 4.5E-03 2.1E-01 UTERUS
(NONGRVD) 0.0E+00 3.4E-04 8.6E-04 2.3E-03 2.7E-03 2.6E-03 2.7E-03
6.6E.02 1.0E-02 3.7E-01 TOTAL BODY 0.0E+00 2.7E-03 3.4E.04 9.2E-03
6.1E-03 8.0E.03 1.3E-03 1.2E-02 4.8E-04 2.8E-01
[0216]
17TABLE 16 RADIATION ABSORBED DOSE (RAD = A * S) INDIUM-[111]
HALF-LIFE 67.44 HOURS SOURCE ORGANS Target Skeleton Organs Ovaries
Pancreas R Marrow Cort Bone TRA Bone Skin Spleen Testes Thyroid
Total Body ADRENALS 0.0E+00 0.0E+00 0.0E+00 0.0E+00 3.6E-02 2.6E-02
1.2E-02 0.0E+00 0.0E+00 0.0E+00 BLADDER WALL 0.0E+00 0.0E+00
0.0E+00 0.0E+00 1.4E-02 1.7E-02 2.7E-04 0.0E+00 0.0E+00 0.0E+00 GI
(STOM WALL) 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1.6E-02 1.8E-02 1.7E-02
0.0E+00 0.0E+00 0.0E+00 GI (SI) 0.0E+00 0.0E+00 0.0E+00 0.0E+00
2.1E-02 1.6E-02 2.4E-03 0.0E+00 0.0E+00 0.0E+00 GI (ULI WALL)
0.0E+00 0.0E+00 0.0E+00 0.0E+00 2.0E-02 1.5E-02 2.2E-03 0.0E+00
0.0E+00 0.0E+00 GI (LLI WALL) 0.0E+00 0.0E+00 0.0E+00 0.0E+00
3.0E-02 1.6E-02 1.1E-03 0.0E+00 0.0E+00 0.0E+00 KIDNEYS 0.0E+00
0.0E+00 0.0E+00 0.0E+00 2.5E-02 2.1E-02 1.6E-02 0.0E+00 0.0E+00
0.0E+00 LIVER 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1.9E-02 1.8E-02
1.7E-03 0.0E+00 0.0E+00 0.0E+00 LUNGS 0.0E+00 0.0E+00 0.0E+00
0.0E+00 2.8E-02 2.0E-02 4.0E-03 0.0E+00 0.0E+00 0.0E+00 OTHER
TISSUES MUSCLE 0.0E+00 0.0E+00 0.0E+00 0.0E+00 3.0E-02 2.7E-02
2.7E-03 0.0E+00 0.0E+00 0.0E+00 ADIPOSE 0.0E+00 0.0E+00 0.0E+00
0.0E+00 3.0E-02 2.7E-02 2.7E-03 0.0E+00 0.0E+00 0.0E+00 BLOOD
0.0E+00 0.0E+00 0.0E+00 0.0E+00 3.0E-02 2.7E-02 2.7E-03 0.0E+00
0.0E+00 0.0E+00 BRAIN 0.0E+00 0.0E+00 0.0E+00 0.0E+00 3.0E-02
2.7E-02 2.7E-03 0.0E+00 0.0E+00 0.0E+00 HEART 0.0E+00 0.0E+00
0.0E+00 0.0E+00 3.0E-02 2.7E-03 2.7E-03 0.0E+00 0.0E+00 0.0E+00
OVARIES 0.0E+00 0.0E+00 0.0E+00 0.0E+00 2.0E-02 1.5E-02 9.9E-04
0.0E+00 0.0E+00 0.0E+00 PANCREAS 0.0E+00 0.0E+00 0.0E+00 0.0E+00
2.9E-02 1.8E-02 3.5E-02 0.0E+00 0.0E+00 0.0E+00 SKELETON CORTICAL
BONE 0.0E+00 0.0E+00 0.0E+00 0.0E+00 2.4E-01 3.1E-02 1.7E-03
0.0E+00 0.0E+00 0.0E+00 TRABECULAR 0.0E+00 0.0E+00 0.0E+00 0.0E+00
2.4E-01 3.1E-02 1.7E-03 0.0E+00 0.0E+00 0.0E+00 BONE MARROW (RED)
0.0E+00 0.0E+00 0.0E+00 0.0E+00 2.4E-01 2.9E-02 2.6E-03 0.0E+00
0.0E+00 0.0E+00 MARROW 0.0E+00 0.0E+00 0.0E+00 0.0E+00 2.4E-01
2.9E-02 2.6E-03 0.0E+00 0.0E+00 0.0E+00 (YELLOW) CARTILAGE 0.0E+00
0.0E+00 0.0E+00 0.0E+00 2.4E-01 3.1E-02 1.7E-03 0.0E+00 0.0E+00
0.0E+00 OTHER CONSTIT. 0.0E+00 0.0E+00 0.0E+00 0.0E+00 2.4E-01
3.1E-02 1.7E-03 0.0E+00 0.0E+00 0.0E+00 SKIN 0.0E+00 0.0E+00
0.0E+00 0.0E+00 2.1E-02 4.0E-01 8.7E-04 0.0E+00 0.0E+00 0.0E+00
SPLEEN 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1.9E-02 1.8E-02 5.3E-01
0.0E+00 0.0E+00 0.0E+00 TESTES 0.0E+00 0.0E+00 0.0E+00 0.0E+00
1.8E-02 3.6E-02 1.2E-04 0.0E+00 0.0E+00 0.0E+00 THYROID 0.0E+00
0.0E+00 0.0E+00 0.0E+00 2.6E-02 2.6E-02 2.0E-04 0.0E+00 0.0E+00
0.0E+00 UTERUS (NONGRVO) 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1.7E-02
0.0E+00 7.0E-04 0.0E+00 0.0E+00 0.0E+00 TOTAL BODY 0.0E+00 0.0E+00
0.0E+00 0.0E+00 5.5E-02 4.1E-02 3.8E-03 0.0E+00 0.0E+00 0.0E+00
[0217]
18TABLE 17 RADIATION ABSORBED DOSE (RAD = A *S) YTTRIUM-[90]
HALF-LIFE 64 HOURS SOURCE ORGANS Intestinal Tract Target Bladder
Stomach SI ULI LLI Other Organs Adrenals Contents Contents Contents
Contents Contents Kidneys Liver Lungs Tissue ADRENALS 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 BLADDER WALL 0.0E+00 2.7E-01 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 GI (STOM WALL) 0.0E+00
0.0E+00 3.6E-01 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 GI (SI) 0.0E+00 0.0E+00 0.0E+00 5.0E-01 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 GI (ULI WALL) 0.0E+00 0.0E+00
0.0E+00 0.0E+00 1.1E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 GI
(LLI WALL) 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 KIDNEYS 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 8.4E+00 0.0E+00 0.0E+00 0.0E+00 LIVER 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E-00 0.0E+00 0.0E+00 8.6E+00 0.0E+00
0.0E+00 LUNGS 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 2.1E+00 0.0E+00 OTHER TISSUES MUSCLE 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
2.7E+00 ADIPOSE 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 2.7E+00 BLOOD 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 2.7E+00 BRAIN
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 2.7E+00 HEART 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 2.7E+00 OVARIES 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
PANCREAS 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 SKELETON CORTICAL BONE 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
TRABECULAR BONE 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 MARROW (RED) 0.0E+00 0.0E-00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
MARROW (YELLOW) 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 CARTILAGE 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 OTHER
CONSTIT. 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 SKIN 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 SPLEEN 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 TESTES 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 THYROID 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 UTERUS
(NONGVO) 0.0E+00 1.7E-04 7.6E-03 4.6E-03 3.6E-03 4.1E-03 3.7E-02
2.2E-01 2.9E-02 0.0E+00 TOTAL BODY 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1.1E+00
[0218]
19TABLE 18 RADIATION ABSORBED DOSE (RAD = A * S) INDIUM-[111]
HALF-LIFE 64.00 HOURS SOURCE ORGANS Target Skeleton Organs Ovaries
Pancreas R Marrow Cort Bone TRA Bone Skin Spleen Testes Thyroid
Total Body ADRENALS 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 BLADDER WALL 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 GI
(STOM WALL) 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 GI (SI) 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 GI (ULI WALL)
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 GI (LLI WALL) 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 KIDNEYS 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 LIVER 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 LUNGS 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 OTHER
TISSUES MUSCLE 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 ADIPOSE 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 BLOOD
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 BRAIN 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 HEART 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
OVARIES 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 PANCREAS 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 SKELETON CORTICAL
BONE 0.0E+00 0.0E+00 0.0E+00 0.0E+00 3.1E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 TRABECULAR BONE 0.0E+00 0.0E+00 0.0E+00
0.0E+00 3.1E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 MARROW
(RED) 0.0E+00 0.0E+00 0.0E+00 0.0E+00 7.8E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 MARROW (YELLOW) 0.0E+00 0.0E+00 0.0E+00
0.0E+00 7.8E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 CARTILAGE
0.0E+00 0.0E+00 0.0E+00 0.0E+00 3.1E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 OTHER CONSTIT. 0.0E+00 0.0E+00 0.0E+00 0.0E+00
3.1E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 SKIN 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 SPLEEN 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
9.0E+00 0.0E+00 0.0E+00 0.0E+00 TESTES 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 THYROID
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 UTERUS (NONGRVO) 0.0E+00 0.0E+00 0.0E+00 0.0E+00
0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 0.0E+00 TOTAL BODY 0.0E+00
0.0E+00 0.0E+00 0.0E+00 3.8E-01 3.8E+01 2.3E-02 0.0E+00 0.0E+00
0.0E+00
[0219]
20TABLE 19 Radiation Dosimetry Estimates Resulting from the
Administration of Indium-[111] Labeled 2B8-MX Uniformly Distributed
in Standard Man(70 Kg) and Based on Animal Distribution Data Over
72 Hours after Injection 1000 AMOUNT OF ACTIVITY =
MICROCIJRIES/PATIENT DOSE RADS RADS ADRENALS 0.493 OVARIES 0.387
BLADDER WALL 0.348 PANCREAS 0.362 STOMACH WALL 0.412 SKELETON SMALL
INTESTINE 0.434 CORTICAL BONE 0.474 UL INTEST. WALL 0.533
TRABECULAR BONE 0.474 LL INTEST. WALL 0.505 MARROW (RED) 0.602
KIDNEYS 0.625 MARROW (YELLOW) 0.602 LIVER 1.533 CARTILAGE 0.474
LUNGS 0.582 OTHER CONSTIT. 0.474 OTHER TISSUES SKIN 0.564 MUSCLE
SPLEEN 0.854 ADIPOSE TESTES 0.239 BLOOD THYROID 0.276 BRAIN UTERUS
(NONGRVD) 0.473 HEART TOTAL BODY 0.417 Ref: A Schema for
Absorbed-dose Calculation for Biologically Distributed
Radionuclides, MIRD J. of Nucl. Med./Suppl. #1, 2/68
[0220] Calculations Performed Using a Spreadsheet Template in
Symphony (Lotus Development Corporation) and Created by
[0221] Phillip L. Hagan, MS
[0222] Nuclear Medicine Service
[0223] VA Hospital
[0224] San Diego, Calif. 92161
21TABLE 20 Radiation Dosimetry Estimates Resulting from the
Administration of Yttrium-[90] Labeled 2B8-MX Uniformly Distributed
in Standard Man(70 Kg) and Based on Animal Distribution Data Over
72 Hours After Injection 1000 AMOUNT OF ACTIVITY =
MICROCURIES/PATIENT DOSE RADS RADS ADRENALS 0.000 OVARIES 0.000
BLADDER WALL 0.271 PANCREAS 0.000 STOMACH WALL 0.356 SKELETON SMALL
INTESTINE 0.504 CORTICAL BONE 3.138 UL INTEST. WALL 1.150
TRABECULAR BONE 3.138 LL INTEST. WALL 1.772 MARROW (RED) 7.776
KIDNEYS 8.366 MARROW (YELLOW) 7.776 LIVER 8.575 CARTILAGE 3.138
LUNGS 2.079 OTHER CONSTIT. 3.138 OTHER TISSUES SKIN 10.269 MUSCLE
2.716 SPLEEN 8.965 ADIPOSE 2.716 TESTES 0.000 BLOOD 2.716 THYROID
0.000 BRAIN 2.716 UTERUS (NONGRYD) 0.304 HEART 2.176 TOTAL BODY
1.854 Ref: A Schema for Absorbed-dose Calculation for Biologically
Distributed Radionuclides, MIRD J. of Nucl. Med./Suppl. #1,
2/68
[0225] Calculations Performed Using a Spreadsheet Template in
Symphony (Lotus Development Corporation) and Created by
[0226] Phillip L. Hagan, MS
[0227] Nuclear Medicine Service
[0228] VA Hospital
[0229] San Diego, Calif. 92161
[0230] II. Results
[0231] A. In Vitro Studies With 2B8 and 2B8-MX-DTPA
[0232] 1. Production and Characterization of the Anti-CD20 Antibody
2B8
[0233] A total of nine fusions resulted in three hybridomas
producing antibodies which effectively competed with radiolabeled
Coulter B1 antibody. In each case, the hybridoma was expanded into
a 24 well plate. The first two antibodies isolated from fusions 3
and 4, were isotyped and both identified as IgM. The third
antibody, produced in fusion 5 and designated 2B8, was determined
to be an IgGl kappa isotype and was selected for continuation
studies. Clone 2B8.H11 was expanded and placed in long term storage
in liquid nitrogen. Clone 2B8.H11 was subcloned to produce clone
2B8.H11.G3 and again to produce clone 2B8.H11.G3.G9. This clone was
expanded for further study and the antibody was purified by protein
A affinity chromatography.
[0234] Competition assays using unlabeled 2B8, B1 and Leu 16 and
radiolabeled Coulter B1 demonstrated that 2B8 was able to inhibit
B1 binding to CD20 more effectively than equal concentrations of
either B1 or Leu 16 (FIG. 1). Similar results were obtained (data
not shown) in a competition study using FITC-conjugated 2B8, native
B1 and the irrelevant antibodies UPC-10 and S-003 (IgG 2a and 1
isotypes, respectively).
[0235] Direct binding to cellular CD20 antigen by 2B8 and B1
antibodies was compared by FACS analysis using CD20-positive SB
cells and CD20-negative HSB cells. The results shown in FIG. 2
indicate that for comparable amounts of antibody, more 2B8 than B1
was bound to the SB cells. No significant binding to SB cells was
observed with the irrelevant antibodies. Only background
fluorescence was observed with any reagent used with HSB cells.
These results confirm the specificity of interaction of 2B8 with
the CD20 antigen and suggest that 2B8 may have higher affinity for
the cell-surface antigen than B1.
[0236] To determine the apparent affinity of 2B8, purified antibody
was radiolabeled with .sup.125I and increasing concentrations of
the labeled antibody were incubated with antigen-positive SB cells;
cell-associated radioactivity was determined following a 1 hour
incubation period (FIG. 3). The results suggest that the 2B8
antibody binds to the CD20 antigen with an apparent affinity
constant of 4.3.times.10.sup.-9M.
[0237] Flow cytometry studies with human normal peripheral blood
lymphocytes indicated that 2B8 was specific for B-cells and did not
react with other types of lymphocytes (e.g. T-cells, monocytes,
macrophages). FITC-labeled 2B8 was compared to B1-FITC and Leu
16-FITC using the same population of human lymphocytes. The results
shown in Table 21 indicate that 2B8 reacted with approximately 14
percent of the peripheral blood lymphocytes versus approximately 12
percent for Leu 16 and 11 percent for B1. The lymphocyte population
based on another B lymphocyte marker (CD-19) was between 11 and 14
percent. Finally, when human peripheral blood lymphocytes were
incubated with 2B8 and either B1 or Leu 16 and then counterstained
with the CD19 marker (Becton/Dickinson) the double staining
population of B lymphocytes was 9 percent with 2B8, and 10 percent
with either B1 or Leu 16. These results confirm the similarity of
these reagents.
22TABLE 21 Comparison of Binding of 2B8 to Human Peripheral Blood
Lymphocytes with other B- and T-Lymphocyte Specific Reagents
Antibody Marker Percent of CD45 Gated Lymphocytes A. Single
Staining: None (autofluorescence) 0 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
[0238] Immunoprecipitation of radiolabeled cellular CD20 antigen by
either 2B8 or Bi resulted in the precipitation of indistinguishable
doublet protein species with molecular weights of approximately 33
and 35 KD (data not shown).
[0239] 2. Production and Characterization of 2B8-.MX-DTPA
[0240] The 2B8-MX-DTPA conjugate was produced by reacting the
antibody with a 4:1 molar excess of
isothiocyanatobenzyl-3-methyldiethylene-triami- nepentaacetic 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 .mu.g/mL, respectively. These results were
obtained using .sup.125I-labeled B1 antibody in a whole-cell
radioimmunoassay performed using SB cells. Similar results were
obtained using 2B8 or 2B8-MX-DTPA as inhibitors of
.sup.125I-labeled 2B8 binding to SB cells; both 2B8 and its MX-DTPA
conjugate inhibited .sup.125I-2B8 binding to SB cells at
concentrations of approximately 3-4 .mu.g/mL (data not shown).
[0241] To assess the in vitro stability of the native 2B8 antibody
and the 2B8-.MX-DTPA conjugate, samples in normal saline or saline
containing 10 mM glycine-HCl, pH 6.8, were incubated at 40 and
30.degree. C. for 12 weeks and aliquots were assayed weekly using
the following assays: immunoreactivity by whole-cell enzyme
immunoassay, SDS-PAGE under reducing and non-reducing conditions,
and isoelectric focusing gel electrophoresis. While
immunoreactivity assays detected no loss of antigen recognition by
antibody samples incubated at either temperature (FIG. 5), the
isoelectric focusing range for the antibody (pH 7.30-8.40 at week
zero), which was stable at 4.degree. C., did exhibit a decrease of
0.2 pH unit at 30.degree. C. after week six (Table 22). This result
may be equivocal, however, as it is at the limit of experimental
error for the assay.
23TABLE 22 2B8/2B8-MX-DTPA pI SUMMARY 2B8 2B8 2B8 2B8 2B8-MX 2B8-MX
2B8-MX 2B8-MX WEEK 4 SAL 30 SAL 4 GLY 30 GLY 4 SAL 30 SAL 4 GLY 30
GLY 0 7.46-8.37 7.46-8.37 6.30-8.21 6.30-8.21 1 7.39-8.24 7.42-8.27
7.46-8.31 7.46-8.24 6.39-8.26 6.39-8.26 6.32-8.24 6.25-8.24 2
7.38-8.27 7.45-8.34 7.45-8.40 7.45-8.34 6.02-8.40 6.02-8.34
6.02-8.40 5.95-8.27 3 7.47-8.35 7.33-8.35 7.40-8.29 7.33-8.29
6.0-8.29 6.0-8.29 6.0-8.22 6.0-8.15 4 7.38-8.24 7.38-8.24 7.38-8.35
7.38-8.28 5.99-8.28 5.99-8.35 5.99-8.35 5.99-8.35 5 7.29-8.25
7.29-8.25 7.37-8.32 7.37-8.32 5.90-8.32 5.90-8.27 5.90-8.32
5.90-8.27 6 7.24-8.12 7.20-8.27 7.27-8.27 7.20-8.12 5.85-8.27
5.85-8.27 5.85-8.27 5.85-7.95 7 7.39-8.32 7.17-8.32 7.35-8.25
7.17-8.47 6.02-8.25 5.95-8.32 5.95-8.32 8 7.33-8.29 7.26-8.36
7.40-8.36 5.86-8.36 5.86-8.36 5.86-8.36 5.86-8.21 9 7.49-8.53
7.26-8.45 7.41-8.45 7.34-8.30 5.93-8.45 5.93-8.45 5.93-8.45
5.93-8.23 10 7.26-8.27 7.19-8.27 7.26-8.27 7.19-8.27 5.95-8.35
5.95-8.35 5.88-8.35 5.95-8.13 11 7.40-8.27 7.18-8.27 7.40-8.35
7.18-8.13 5.93-8.35 5.93-8.27 5.93-8.27 5.93-8.13 12 7.26-8.18
7.04-8.18 7.26-8.18 7.19-8.11 5.90-8.26 5.90-8.18 5.90-8.26
5.90-8.18 Samples of native 2B8 and 2B8-MX-DTPA were formulated in
different buffers and incubated at either 40 or 30.degree. C. for
12 weeks. During this period various assays, including isoelectric
point determinations, were performed. The values shown above show
the isoelectric point range for the native and conjugated antibody
incubated at each temperature, in each of the formulations, and for
each of the twelve weeks # during the stability study. The headings
represent: 2B8 4 SAL, 2B8 incubated at 4.degree. C. in saline; 2B8
30 SAL, 2B8 incubated at 30.degree. C. in saline; 2B8 4 GLY, 2B8
incubated at 4.degree. C. in normal saline containing 10 mM
glycine; 2B8 30 GLY, 2B8 incubated at 30.degree. C. in normal
saline containing 10 mM glycine; 2B8-MX 4 SAL; 2B8-MX-DTPA
(conjugate) incubated at 4.degree. C. in saline; 2B8-MX 30 SAL, #
conjugate incubated at 30.degree. C. in saline; 2B8-MX 4' GLY,
conjugate incubated at 4.degree. C. in normal saline containing 10
mM glycine; and, 2B8-MX 30 GLY, conjugate incubated at 30.degree.
C. in normal saline containing 10 mM glycine.
[0242] Finally, using non-reducing SDS-PAGE, the 30.degree. C.
antibody samples exhibited high molecular weight aggregates after
week 1 (Table 23). Densitometric analyses of the gels indicated
that the aggregates represented between 8 and 17% of the samples
(Table 23). However, when these samples were analyzed by reducing
SDS-PAGE, no evidence of the high molecular weight species was
found, suggesting the formation of covalent antibody aggregates at
30.degree. C. Again, no loss of immunoreactivity was observed.
24TABLE 23 In Vitro Stability of 2B8 Percentage Sample High MW
Monomer Low MW A. Desensitometric Scans of Non-Reducing SDS Gels
Reference 0 100.00 0 12 wk/4.degree. C./saline 0 95.42 4.58 12
wk/4.degree. C./glycine 0 100.00 0 12 wk/30.degree. C./saline 7.63
83.34 9.03 12 wk/30.degree. C./glycine 16.70 72.11 11.18 B.
Desensitometric Scans of Reducing SDS Gels Reference 0 100.00 0 12
wk/30.degree. C./saline 0 100.00 0 12 wk/30.degree. C./glycine 0
10.00 0
[0243] During the course of this stability study, samples of
2B8-MX-DTPA, incubated at both 4.degree. and 30.degree. C. were
also tested for radiometal incorporation using 90Y. Samples assayed
at weeks 4, 8, and 12 incorporated >90% of the .sup.90Y,
regardless of the incubation temperature.
[0244] Finally, in a separate study, aliquots of 2B8-MX-DTPA
incubated at 40 and 30.degree. C. for 10 weeks were radiolabeled
with .sup.125In and their tissue biodistribution assessed in BALB/c
mice. Conjugate from both incubation temperatures produced similar
biodistributions (data not shown). Moreover, the results obtained
were similar to biodistribution results obtained in BALB/c mice
using .sup.125I-labeled conjugate stored at 4.degree. C. (see
below).
[0245] The radiolabeling protocols for both .sup.111In and .sup.90Y
were found to be reproducible. Typically, radioincorporations of
>95% for .sup.111In and >90% for .sup.90Y were obtained.
Specific activities for .sup.111I- and .sup.90Y-labeled conjugates
were routinely in the range of 2-3 and 10-15 mCi/mg antibody,
respectively. In initial development of the .sup.111I- and .sup.90Y
radiolabeling protocols, 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.
[0246] The immunoreactivity of .sup.111In and .sup.90Y-labeled
preparations of 2B8-MX-DTPA were analyzed by the method of Lindmo
(3). The .sup.111In labeled 2B8-MX-DTPA was found to be 100%
immunoreactive (FIG. 6), and the .sup.90Y-labeled conjugate was
determined to be 60% immunoreactive (data not shown).
[0247] 3. Characterization of .sup.111I- and .sup.90Y-Labeled
2B8-MX-DTPA
[0248] Preliminary experiments with the .sup.90Y-labeled conjugate
demonstrated that significant antibody degradation and loss of
immunoreactivity occurred at specific activities >10 mCi/mg
antibody. Therefore, a formulation was developed to minimize the
effects of radiolysis. While a number of low molecular weight
free-radical scavengers were evaluated and found to be effective,
high concentrations of human serum albumin (HSA) were the most
effective in preserving antibody integrity and immunoreactivity
(FIGS. 7-9).
[0249] The .sup.90Y-labeled antibody was formulated in 1.times.
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 .sup.90Y 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.degree. C. Analysis using
instant thin layer chromatography showed that the loss of .sup.90Y
was less than 2% during the 48 h incubation (Table 24). The
immunoreactivity was also relatively constant at 60% (Table
24).
25TABLE 24 Stability of Clinically-Formulated .sup.90Y-2B8-MX-DTPA
Percent Conjugate- Percent Time (Hours at 4.degree. C.) Associated
Radioactivity Immunoreactivity 0 97.2 62 24 96.2 60 48 96.2 60
Radiolabeled conjugate (14.6 mCi/mg specific activity) was
formulated in PBS, pH 7.4, containing 75 mg/mL human serum albumin
and 1 MM DTPA and aliquots incubated at 4.degree. C. Conjugate
stability was analyzed at the times shown by SDS-PAGE and
autoradiography, instant thin-layer chromatography and by
whole-cell binding assay. The results show that approximately 96%
of the radiometal remained associated with the conjugate after 48
hours at 4.degree. C., and that #immunoreactivity remained constant
at approximately 60%.
[0250] Formulation studies were also performed with the
.sup.111In-labeled conjugate; the specific activity was 2.2 mCi/mg.
The radiolabeled antibody was evaluated in 1.times. PBS, pH 7.4
containing 50 mg/mL HSA. 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 .sup.125In (Table
25); moreover, immunoreactivity was maintained at approximately
100% (Table 25).
26TABLE 25 Stability of Clinically-Formulated
.sup.111In-2B8-MX-DTPA Percent Conjugate- Percent Time (Hours at
4.degree. C.) Associated Radioactivity Immunoreactivity 0 94.0 105
24 96.5 104 48 96.0 100 The radiolabeled conjugate (2.2 mCi/mg
specific activity) was formulated in PBS, pH 7.4, containing 50
mg/mL human serum albumin and aliquots incubated at 4.degree. C.
Conjugate stability was analyzed by SDS-PAGE and autoradiography,
by instant thin-layer chromatography, and by whole-cell binding
assay. The results show that approximately 96% of the radiolabel
was retained with the conjugate after 48 hours at 4.degree. C., and
that antibody immunoreactivity remained constant # at approximately
100%.
[0251] When a clinically-formulated preparation of 2B8-MX-DTPA,
radiolabeled with 90Y to a specific activity 14.6 mCi/mg, was
incubated for 96 hours at 37.degree. C. in human serum and analyzed
by non-reducing SDS-PAGE and autoradiography, less than 4% of the
radioisotope was lost during the course of the incubation period.
Densitometric scans of the autoradiograms at time zero and 96 h
indicated no significant degradation of the radiolabeled conjugate
(FIGS. 13-15). These results were corroborated by analytical
thin-layer chromatographic analyses of the time zero and 96-hour
samples (Table 26). Taken together these results suggest that the
yttrium-labeled conjugate is stable under the conditions used in
this study. Similar results were obtained with the .sup.111In
labeled 2B8-MX-DTPA conjugate (FIGS. 16-18).
27TABLE 26 Analytical Thin-Layer Chromatographic Analysis of
.sup.90Y-2B8-MX-DTPA Conjugate Incubated in Human Serum for 96
Hours at 37.degree. C. Percent Conjugate- Time (Hours at 37.degree.
C.) Associated Radioactivity 0 95.1 24 95.2 48 93.2 72 92.0 96 91.4
Human serum samples containing .sup.90Y-2B8-MX-DTPA (specific
activity 14.6 mCi/mg) were analyzed at the times shown by spotting
1 .mu.l of a 1:20 dilution of samples on instant thin-layer
chromatography strips; samples were analyzed in triplicate.
Chromatography strips were developed by ascending chromatography in
10% ammonium acetate in methanol:water (1:1; v/v), dried, and cut
in half crosswise. The radioactivity associated with the bottom and
top halves of # each strip was then determined and the percent
conjugate-associated radioactivity calculated. (Free radiometal
migrates with the solvent front while protein-associated
radioactivity remains at the origin.) The means of each
determination of conjugate-associated radioactivity are shown.
[0252] B. Animal Studies.
[0253] 1. High-Dose Pharmacology/Toxicology Studies with 2B8 and
2B8-MX-DTPA
[0254] In a GLP study performed at White Sands Research Center
(Study Number 920111), cynomolgus monkeys were given intravenous
injections of various doses of 2B8. Blood samples were taken before
each new injection and the blood was processed for flow cytometric
evaluation of the lymphocyte populations (Table 27).
28TABLE 27 Primate B cell Populations Determined by Flow Cytometry,
Following Infusion of Anti-CD20 Murine Monoclonal Antibody 2B8
Animal # Dose Day B cells.sup.a,b % Depletion Group I 452 saline 0
20.1 0 1 18.3 9 7 21.6 0 13 14.6 27 38 15.5 23 52 18.6 7 424 saline
0 12.4 0 1 11.6 6 7 11.2 10 13 8.4 32 38 7.7 38 52 13.1 0 Group II
540 0.6 mg/kg 0 16.1 0 1 7.1 54 7 6.0 63 13 5.7 65 38 10.8 33 52
14.4 11 804 0.6 mg/kg 0 17.6 0 1 8.3 53 7 6.1 65 13 6.6 62 38 5.1
71 52 5.2 68 Group III 701 2.5 mg/kg 0 21.6 0 1 10.7 50 7 3.0 86 13
10.7 50 754 2.5 mg/kg 0 19.9 0 1 11.2 44 7 10.5 47 13 9.0 55 Group
IV 782 10 mg/kg 0 15.9 0 1 3.0 81 7 3.5 78 13 6.5 59 164 10 mg/kg 0
17.7 0 1 8.4 47 7 7.9 50 13 7.7 42 Group V 705 10 mg/kg 0 17.2 0 1
5.2 70 7 1.3 69 13 8.2 52 38 17.1 1 52 13.3 22 716 10 mg/kg 0 34.7
0 1 18.6 46 7 8.1 77 13 3.5 90 38 6.9 80 52 9.2 61 .sup.aPercent of
total lymphocytes. .sup.bB cell population quantitated by double
staining marker reagents anti mouse IGG-RPE + anti human IG-FITC
(anti mouse IgG RPE detects 2B8 blocked CD20 and anti human IgG
FITC detects monkey B cell surface Ig) Animals in groups I through
IV were injected every 48 hours for a total of seven injections;
animals in group V were injected once on day 0. The animals in
Groups III and IV were sacrificed on day 14.
[0255] No significant pharmacotoxic effects related to the
administration of the anti-CD20 antibody 2B8 were noted in any
clinical parameter evaluated during or following the study.
Similarly, no abnormalities were noted during analysis of the
various histopathology specimens obtained from animals in groups
III and IV.
[0256] 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.
[0257] When the blood from the treated animals was analyzed for
levels of circulating T- and B-cells, animals in groups II through
V exhibited >50% loss of circulating B-cells through day 13
(FIG. 19); administration of the antibody had no effect on T-cell
levels (data not shown). All groups receiving 2B8 showed saturation
of B-cells and excess antibody in the plasma (not shown). The
animals in group V, which received a single 10.0 mg/Kg dose of 2B8
also exhibited reduction in circulating B-cell levels equivalent to
that observed in animals in the other groups.
[0258] The animals in groups I, II, and V were examined through day
52 (FIG. 20). The levels of B-cells returned to >70% of normal
by day 38, except for one animal in Group II (PRO804) and one
animal in Group V (PRO716). The levels of circulating B-cells in
these animals remained at approximately 40% of normal levels after
52 days.
[0259] In addition to this study, the pharmacotoxic effects of
.sup.89Y-2B8-MX-DTPA were assessed in cynomolgus monkeys in a GLP
study performed at White Sands Research Center (Study No. 920611).
Clinical-grade conjugate was loaded with non-radioactive .sup.89Y.
The yttrium-bearing conjugate was formulated in PBS pH 6.8,
containing 75 mg/mL human serum albumin and 1 mM DTPA (clinical
formulation) and administered intravenously as described in the
Methods Section.
[0260] As shown by the results in FIG. 21, the .sup.89Y-labeled
2B8-MX-DTPA had little, if any, effect on circulating B-cells in
these animals, regardless of the dose administered. In addition,
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.
[0261] 2. Pharmacokinetic Studies with 2B8 and 2B8-MX-DTPA
[0262] As described above, the animals in group V of the GLP study
received a single dose of 10.0 mg/Kg of 2B8. Linear regression
analysis of the data suggest that the native antibody was cleared
from the circulation of these monkeys with a 13 t1/2 value of
approximately 4.5 days. In a similar study using BALB/c mice, the
.beta. t.sub.1/2 values for native and conjugated 2B8 were
determined by linear regression analysis (not shown) to be 8.75
days (FIG. 22). These results suggest that conjugation of 2B8 had
no effect on its clearance from BALB/c mice.
[0263] 3. Biodistribution and Tumor Localization Studies with
Radiolabeled 2B8-MX-DTPA
[0264] Building on the preliminary biodistribution experiment
described above (Section 2d), conjugated 2B8 was radiolabeled with
.sup.111In to a specific activity of 2.3 mCi/mg and roughly 1.1
.mu.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.
The level of radioactivity in the blood dropped from 40.3% of the
injected dose per gram at 1 hour to 18.9% at 72 hours (Tables 1-4;
FIG. 23). Values for the heart, kidney, muscle and spleen remained
in the range of 0.7-9.8% throughout the experiment. Levels of
radioactivity found in the lungs decreased from 14.2% at 1 hour to
7.6% at 72 hours; similarly the respective liver injected dose per
gram values were 10.3% and 9.9%. These data were used in
determining radiation absorbed dose estimates
.sup.111In-2B8-MX-DTPA (Table 19).
[0265] The biodistribution of .sup.90Y-labeled conjugate, having a
specific activity of 12.2 mCi/mg antibody, was evaluated in BALB/c
mice. Radioincorporations of >90% were obtained and the
radiolabeled antibody was purified by HPLC. Tissue deposition of
radioactivity was evaluated in the major organs, and the skin,
muscle, bone, and urine and feces over 72 hours and expressed as
percent injected dose/g tissue. The results shown in Tables 5-8 and
FIG. 24 demonstrate that while the levels of radioactivity
associated with the blood dropped from approximately 39.2% injected
dose per gram at 1 hour to roughly 15.4% after 72 hours; the levels
of radioactivity associated with tail, heart, kidney, muscle and
spleen remained fairly constant at 10.2% or less throughout the
course of the experiment. Importantly, the radioactivity associated
with the bone ranged from 4.4% of the injected dose per gram bone
at 1 hour to 3.2% at 72 hours. Taken together, these results
suggest that little free yttrium was associated with the conjugate
and that little free radiometal was released during the course of
the study. These data were used in determining radiation absorbed
dose estimates for .sup.90Y-2B8-MX-DTPA (Table 20).
[0266] For tumor localization studies, 2B8-MX-DTPA was prepared and
radiolabeled with .sup.111In to a specific activity of 2.7 mCi/mg.
One hundred microliters of labeled conjugate (approximately 24
.mu.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. At time points of 0, 24, 48, and 72 hours following
injection, 50 .mu.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.
After processing and weighing the tissues, the radioactivity
associated with each tissue specimen was determined using a gamma
counter and the values expressed as percent injected dose per
gram.
[0267] The results (FIG. 25) demonstrate that the tumor
concentrations of the .sup.111In-2B8-MX-DTPA increased steadily
throughout the course of the experiment. Thirteen percent of the
injected dose was accumulated in the tumor after 72 hours. The
blood levels, by contrast, dropped during the experiment from over
30% at time zero to 13% at 72 hours. All other tissues (except
muscle) contained between 1.3 and 6.0% of the injected dose per
gram tissue by the end of the experiment; muscle tissue contained
approximately 13% of the injected dose per gram.
[0268] C. Dosimetry
[0269] The summary dosimetry data derived from biodistribution
studies in normal BALB/c mice and presented in Tables 19 and 20,
for the indium- and yttrium-labeled conjugates, respectively, are
in agreement with data presented in the literature when compared
per millicurie of injected dose (5) and suggest that both the
yttrium- and indium-labeled conjugates of 2B8 may be safely
evaluated for clinical efficacy in lymphoma patients.
[0270] D. Toxicology.
[0271] 1. 2B8: Single Dose General Safety Test.
[0272] 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.
[0273] 2. 2B8 and 2B8-MX-DTPA: Immunohistology Studies with Human
Tissues.
[0274] The 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.
[0275] 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 propria (Peyer's Patches) of the
large intestines. Finally, 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.
[0276] All simple epithelial cells, as well as the stratified
epithelia and squamous epithelia of different organs, were found to
be unreactive. Similarly, no reactivity was seen with
neuroectodermal cells, including those in the brain, spinal cord
and peripheral nerves. Mesenchymal elements, such as skeletal and
smooth muscle cells, fibroblasts, endothelial cells, and
polymorphonuclear inflammatory cells were also found to be
negative.
[0277] 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.
[0278] III. Discussion
[0279] 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. Cytofluorographic
analysis of the specificity of the 2B8 antibody for peripheral
blood lymphocytes demonstrates that the antibody reacts
specifically with B-cells and has no demonstrated reactivity with
T-cells or other types of lymphocytes. Finally, preliminary
stability data suggest that the antibody is stable at 30.degree. C.
for 12 weeks with no loss of immunoreactivity.
[0280] When the 2B8 antibody was conjugated to methylbenzyl
diethylenetriamine-pentaacetic acid (MX-DTPA), virtually no
reduction in immunoreactivity, relative to the native antibody, was
observed. Further, radiolabeling the conjugate with either
.sup.111In or .sup.90Y produced labeled conjugates with
immunoreactivities of 100% and 60%, respectively. Stability studies
of .sup.111In or .sup.90Y-labeled conjugates incubated in human
serum for 96 hours at 37.degree. C. indicated negligible loss of
the radiometal during the course of the study, suggesting that the
conjugates will be stable when used clinically.
[0281] Tumor localization studies in athymic mice using an
indium-labeled preparation of 2B8-MX-DTPA demonstrated that
increasing amounts of the conjugate bound to the tumor cells during
the course of the experiment without unusual accumulations in other
tissues. Moreover, dosimetry estimates derived from the
biodistribution. Moreover, dosimetry estimates derived from the
biodistribution studies are in agreement with data published in the
literature. Finally, human tissue cross-reactivity studies with the
native and conjugated antibodies indicated that both antibodies
recognize an antigen with highly restricted tissue distribution,
reacting only with a subset of cells in lymphoid tissues, including
those of hematopoietic origin. Taken together, these results
suggest that conjugation did not alter the tissue specificity of
the antibody, and that the radiolabeled conjugates are stable in
vivo and recognize the CD20 antigen present on the surface of
tumors produced experimentally in athymic mice.
[0282] When 2B8 was used in a high-dose pharmacology/toxicology
study, the antibody produced no significant pharmacotoxic effects
in any parameter evaluated, either during or following the study.
Similarly, no abnormalities were noted during analysis of the
various histopathology specimens examined by light microscopy.
Surprisingly, all doses of the antibody used produced marked
depletion of circulating B-cells. Circulating B-cell levels did,
however, return to roughly normal levels once administration of the
antibody ceased. In the single-dose group of monkeys (Group V) the
native antibody was cleared from the circulation with an apparent
.beta.t.sub.1/2 value of approximately 4.5 days. Predictably, when
this pharmacokinetic study was performed in BALB/c mice the 2B8
antibody was cleared with a .beta.t.sub.1/2 value of 8.75 days.
Thus, taken together, these data suggest that the native antibody
may also provide some clinical effect when administered as an
adjunct to the radiolabeled conjugates.
[0283] Overall our data indicate that the high affinity 2B8
antibody and its MX-DTPA conjugate exhibit a restricted pattern of
human tissue reactivity. Moreover, in primates, the native antibody
is non-toxic and produces transient clearance of B-cells; however,
once the antibody is cleared from the circulation the B-cell levels
return reasonably rapidly. Additionally, the indium- and
yttrium-labeled 2B8-MX-DTPA conjugates appeared stable in vitro,
exhibiting no loss of radiometal during prolonged incubation in
human serum. Finally, radiation dose estimates derived from the
biodistribution of .sup.90Y- or .sup.111In-labeled 2B8-MX-DTPA in
BALB/c mice are in agreement, per millicurie of injected dose, with
dose estimates derived from human clinical studies using conjugated
anti-shared idiotype antibodies radiolabeled with these
isotopes.
[0284] IV. Summary of Pre-clinical Development of "Mix-&-shoot"
Radiolabeling Protocol for Preparation of .sup.90Y-2b8-MX-DTPA
[0285] A. Introduction
[0286] A .sup.90Y-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. Unfortunately, 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. Additionally, 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.
[0287] Accordingly, a revised radiolabeling procedure was
developed, referred to as the "mix-and-shoot" method, which
obviates the need for HPLC purification while maintaining a high
radioincorporation and improved retention of immunoreactivity. In
vitro stability studies as well as biodistribution studies in mice
showed that radiolabeled antibody prepared using the
"mix-and-shoot" method is comparable to material produced using the
current HPLC process. The results of these pre-clinical studies
indicate that this new "mix-&-shoot" protocol can be used to
prepare .sup.90Y-labeled 2B8-MX-DTPA suitable for use in clinical
trials.
[0288] B. Materials and Methods
Materials
[0289] 1. Cells
[0290] 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.
[0291] 2. Antibodies
[0292] 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).
[0293] 3. Additional Reagents
[0294] 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.
[0295] Methods
[0296] 1. Preparation of 2B8-D4X-DTPA
[0297] Clinical-grade MX-DTPA was obtained from Coulter Immunology
as the disodium salt in water and stored at -70.degree. 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.degree. C.
[0298] 2. Maintenance of Metal-free Conditions
[0299] All manipulations of reagents were performed to minimize the
possibility of metal contamination. Polypropylene or polystyrene
plastic containers such as flasks, beakers and graduated cylinders
were used. These were washed with Alconox and exhaustively rinsed
with Milli-Q water or Water for Irrigation (WFIr) before use.
Metal-free pipette tips (BioRad) were used for accurately
manipulating small volumes. Larger volumes of reagents were
manipulated using sterile, plastic serological pipettes. Reactions
were conveniently performed in 1.8 mL screw-top microfuge tubes
made from polypropylene.
[0300] 3. Determination of Radioincorporation Radioincorporation
was determined using instant thin-layer chromatography (ITLC) in
triplicate according to SOP SP-13-008. In general, the protocol was
as follows: radiolabeled conjugate was diluted 1:20 in 1.times. PBS
containing 1 mM DTPA or 5 mM EDTA, then 1.mu.-L spotted 1.5 cm from
one end of a 1.times.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.
[0301] 4. "Mix and-Shoot" Protocol for Yttrium-[90]-Labeled
2B8-MX-DTPA
[0302] Antibodies were radiolabeled with carrier-free .sup.90Y
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.times. 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.times. PBS with 75 mg/mL human
serum albumin (HSA) and 1 mM diethylenetriaminepentaacetic acid
(DTPA) and gently mixed. The radiolabeled antibody was stored at
2-8.degree. C.
[0303] Specific activities were determined by measuring the
radioactivity of an appropriate aliquot of the radiolabeled
conjugate. This value was corrected for the counter efficiency,
related to the protein concentration of the conjugate, determined
by absorbance at 280 nm and expressed as mCi/mg proteins.
[0304] 5. In Vitro Immunoreactivity of Yttrium-[90]-2B8-MX-DTPA
Immunoreactivity of .sup.90Y-labeled conjugate was determined using
SOP #SP13-009 based on a modified version of the whole-cell binding
assay described by Lindmo. Increasing concentrations of log phase,
CD20-positive SB cells or CD20-negative HSB cells were added to
duplicate sets of 1.5 mL polypropylene tubes; final volume of
cells, 0.40 mL. The radiolabeled conjugate was diluted to a final
antibody concentration of 1-2.5 ng/nL and 0.35 mL was added to each
tube. Following a 90 min incubation, the cells were pelleted by
centrifugation and the supernatants collected. 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.
[0305] 6. In Vitro Stability of Clinically-Formulated
Yttrium-F90]-2B8-MX-DTPA
[0306] The 2B8-MX-DTPA conjugate was radiolabeled with .sup.90Y 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.degree. 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.
[0307] 7. In Vitro Stability of Yttrium-[90]-2B8-NTX-DTPA in Human
Serum
[0308] The stability of .sup.90Y-labeled 2B8-MX-DTPA was assessed
by incubation in human serum at 37.degree. 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.degree. C. At selected
times, samples were removed and analyzed by non-reducing SDS-PAGE
and autoradiography.
[0309] 8. Biodistribution of Yttrium-[90]-2B8-MX-DTPA
[0310] 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 .mu.Ci of
.sup.90Y-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.
[0311] 9. Dosimetry
[0312] Biodistribution data obtained using mice injected with
.sup.90Y-labeled 2B8-MX-DTPA were used to calculate estimates of
the radiation doses absorbed from a 1.0 mCi dose administered to a
70 Kg patient. Estimates were made according to methods adopted by
the Medical Internal Radiation Dose (MIRD) Committee of the Society
of Nuclear Medicine. These calculations were performed Mr. Phillip
Hagan, Nuclear Medicine Service, VA Medical Center, La Jolla,
Calif. 92161. 10. Validation of Protocol for Preparation of
Clinical Doses of Yttrium-[90]-2B8-MX-DTPA
[0313] (Reference R&D report titled "Validation of
"Mix-and-Shoot" Radiolabeling Protocol for the Preparation of
Clinical Doses of .sup.90Y-2B8-MX-DTPA; author, P. Chinn; dated
Apr. 22, 1994).
[0314] C. Results
[0315] 1. Preparation of Yttrium-[90]-Labeled 2B8-MX-DTPA Using
"Mix-&-Shoot" Protocol
[0316] Preliminary experiments evaluating the kinetics of the
radiolabeling reaction with 2B8-MX-DTPA and 90Y showed that at pH
3.6-4.0, 95% of the radioisotope was incorporated for a reaction
time of 5 to 10 min. The reproducibility of this radioincorporation
(95.7%.+-.1.7%) was subsequently confirmed in a validation study
for the scale-up protocol (Reference R&D report titled
"Validation of "Mix-and-Shoot" Radiolabeling Protocol for the
Preparation of Clinical Doses of .sup.90Y-2B8-MX-DTPA; author, P.
Chinn; dated Apr. 22, 1994). The preparation of .sup.90Y-labeled
2B8-MX-DTPA using this "mix-&-shoot" protocol gave a product
comparable to that produced with the, HPLC method (see BB-IND
4850/4851). The radiolabeling protocol was found to be reproducible
with specific activities typically ranging from 10 to 15 mCi/mg
antibody.
[0317] The immunoreactivity of the .sup.90Y-labeled 2B8-MX-DTPA
prepared using this protocol was typically greater than 70%,
compared with the 55-60% observed for the validations runs for the
HPLC protocol (FIG. 26). This difference is probably due to the
reduced effects of radiolysis because of the reduced incubation
time with the "mix-and-shoot" protocol. This result was typical,
and, as discussed below, was representative of the validation runs
for the scale-up protocol for preparing clinical doses of the
radiolabeled conjugate.
[0318] 2. In vitro Stability of .sup.90Y-Labeled 2B8-MX-DTPA
[0319] Preliminary experiments with unprotected .sup.90Y-labeled
antibody conjugate prepared using the HPLC process demonstrated
that radiolysis caused significant antibody degradation and loss of
immunoreactivity. Therefore, a formulation buffer was developed to
minimize the effects of radiolysis. Human serum albumin (HSA) was
shown to be effective in minimizing. antibody degradation due to
radiolysis. An evaluation was made with the radiolabeled conjugate
prepared with the "mix-&-shoot" method to confirm the efficacy
of the formulation in minimizing radiolysis. The .sup.90Y-labeled
antibody, radiolabeled to a specific activity of 14.5 mCi/mg
antibody, was formulated in 1.times. PBS, pH 7.4 containing 75
mg/mL HSA and 1 mM DTPA. Degradation of the conjugate 2B8-MX-DTPA
was evaluated at 0, 24, and 48 hours using SDS-PAGE and
autoradiography. FIGS. 2, 3, and 4 show that the radiolabeled
conjugate exhibited no significant degradation over a period of 48
h when incubated at 4.degree. C. Instant thin-layer chromatography
analysis showed no loss of .sup.90Y 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).
29TABLE 28 Stability of "Mix-&-Shoot" .sup.90Y-2B8-MX-DTPA in
PBS Containing Human Serum Albumin and DTPA Percent of Conjugate-
Associated Radioactivity Time (h) ITLC SDS/PAGE 0 92.9 96.0 24 95.5
95.4 48 91.3 94.6
[0320]
30TABLE 29 Immunoreactivity of "Mix-&-Shoot"
.sup.90Y-2B8-MX-DTPA in PBS Containing Human Serum Albumin and DTPA
Percent Time (Hours at 4.degree. C.) Immunoreactivity 0 87.9 24
88.5 48 90.4
[0321] A clinically-formulated .sup.90Y-labeled 2B8-MX-DTPA at a
specific activity 15.7 mCi/mg was incubated for 72 hours at
37.degree. C. in human serum. Samples analyzed by non-reducing
SDS-PAGE and autoradiography (FIG. 30) showed no loss of
radioisotope during the course of the incubation period (Table 30).
Densitometric scans of the autoradiograms at time zero and 72 h
indicated no significant degradation of the radiolabeled conjugate
(FIGS. 31 and 32). These results were corroborated by thin-layer
chromatographic analyses (Table 30). It should be noted that the
radioincorporation for the antibody used in this study was lower
than that obtained in the validation studies of the labeling
protocol. This lower radioincorporation was due to the reduced
quality of the batch of .sup.90Y chloride used for this particular
preparation of radiolabeled antibody. The lower radioincorporation
did not alter the conclusion that the yttrium-labeled conjugate
prepared with the "mix-and-shoot" method is stable under these
incubation conditions.
31TABLE 30 Stability of .sup.90Y-2B8-MX-DTPA Conjugate Incubated in
Human Serum Percent Conjugate- Associated Radioactivity Time (hours
at 37.degree. C.) ITLC SDS-PAGE/Autoradiography 0 85.7 88.8 24 76.4
90.0 72 87.6 88.7 Human serum samples containing
.sup.90Y-2B8-MX-DTPA (specific activity 15.7 mCi/mg) were analyzed
for non-protein bound .sup.90Y at the times shown using instant
thin-layer chromatography strips and SDS-PAGE/autoradiography.
[0322] Human serum samples containing [.sup.90] Y-2B8-MX-DTPA
(specific activity 15.7 mCi/mg) were analyzed for non-protein bound
.sup.90Y at the times shown using instant thin-layer chromatography
strips and SDS-PAGE/autoradiography.
[0323] 3. Biodistribution Studies with Yttrium-[90] 2B8-MX-DTPA
[0324] The biodistribution of the .sup.90Y-labeled conjugate, with
a specific activity of 11.5 mCi/mg antibody and a
radioincorporation of >95%, was evaluated in BALB/c mice.
Deposition of radioactivity in tissues was evaluated for major
organs, skin, muscle, bone, urine and feces over 72 hours and
expressed as percent injected dose per g tissue and as percent
injected dose per organ. The results shown in Tables 31-34 and FIG.
33 show that the levels of radioactivity associated with the blood
decreased from approximately 43% injected dose per gram (%ID/g) at
1 hour to approximately 16% after 72 hours; at 24 h and later, the
levels of radioactivity associated with heart, kidney, and spleen
remained fairly constant at 4-8%. For lung and liver, radioactivity
decreased from 10-12% at 1 h to 8%-10% at 72 h. For the skin,
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.
32TABLE 31 Distribution of Activity 1.0 Hour Following I.V.
Injection of .sup.90Y-2B8-MX-DTPA Into Normal BALB/c Mice Mean
Values .+-. SD Organ Weight % ID/ % ID per Sample Gram Gram Organ
Blood 1.37 .+-. 0.053 42.74 .+-. 0.78 58.52 .+-. 1.74 Heart 0.101
.+-. 0.01 8.03 .+-. 3.33 0.82 .+-. 0.37 Lung (2) 0.126 .+-. 0.01
12.44 .+-. 0.94 1.56 .+-. 0.05 Kidney (1) 0.129 .+-. 0.01 7.81 .+-.
1.24 0.997 .+-. 0.10 Liver 0.899 .+-. 0.07 10.08 .+-. 1.28 9.01
.+-. 0.52 Spleen 0.077 .+-. 0.004 10.74 .+-. 0.96 0.823 .+-. 0.04
Muscle 7.83 .+-. 0.28 0.44 .+-. 0.08 3.43 .+-. 0.51 Bone 2.94 .+-.
0.11 3.44 .+-. 0.57 10.11 .+-. 1.80 Skin 2.94 .+-. 0.11 1.46 .+-.
0.58 4.24 .+-. 1.57 GI Tract 2.33 .+-. 0.08 1.02 .+-. 0.19 2.36
.+-. 0.35 Urine -- -- -- Feces -- -- -- TOTAL 94.66 .+-. 3.47 No.
Mice = 3 Mean Weight = 19.58 grams .+-. 0.71 grams
[0325]
33TABLE 32 Distribution of Activity at 24 Hours Following I.V.
Injection of .sup.90Y-2B8-MX-DTA Into Normal BALB/c Mice Mean
Values .+-. SD Organ Weight % ID/ % ID per Sample Gram Gram Organ
Blood 1.55 .+-. 0.12 19.77 .+-. 2.42 30.77 .+-. 6.04 Heart 0.105
.+-. 0.01 4.44 .+-. 0.55 0.47 .+-. 0.08 Lung (2) 0.127 .+-. 0.02
8.78 .+-. 1.61 1.11 .+-. 0.21 Kidney (1) 0.139 .+-. 0.01 5.02 .+-.
0.52 0.69 .+-. 0.05 Liver 0.966 .+-. 0.09 8.62 .+-. 2.73 8.20 .+-.
1.97 Spleen 0.083 .+-. 0.01 6.75 .+-. 1.27 0.55 .+-. 0.064 Muscle
8.83 .+-. 0.69 0.692 .+-. 0.01 6.12 .+-. 0.52 Bone 3.31 .+-. 0.26
2.24 .+-. 0.31 7.47 .+-. 1.53 Skin 3.31 .+-. 0.26 3.33 .+-. 0.76
10.88 .+-. 1.76 GI Tract 2.89 .+-. 0.43 0.73 .+-. 0.09 1.02 .+-.
0.05 Urine 2.31 Feces 1.23 Total: 73.52 .+-. 6.18% No. Mice = 3
Mean Weight = 22.09 .+-. 1.73 gram
[0326]
34TABLE 33 Distribution of Activity at 48 Hours Following I.V.
Injection of .sup.90Y-2B8-MX-DTPA Into Normal BALB/c Mice Mean
Values .+-. SD Organ Weight % ID/ % ID per Sample Gram Gram Organ
Blood 1.50 .+-. 0.14 14.97 .+-. 5.77 22.53 .+-. 8.48 Heart 0.104
.+-. 0.01 3.99 .+-. 1.43 0.415 .+-. 0.16 Lung (2) 0.122 .+-. 0.02
8.41 .+-. 1.57 1.04 .+-. 0.31 Kidney (1) 0.124 .+-. 0.01 3.99 .+-.
1.62 0.49 .+-. 0.19 Liver 0.966 .+-. 0.13 6.12 .+-. 3.21 5.69 .+-.
2.25 Spleen 0.079 .+-. 0.01 6.05 .+-. 2.38 0.46 .+-. 0.16 Muscle
8.59 .+-. 0.82 0.54 .+-. 0.19 4.67 .+-. 1.67 Bone 3.22 .+-. 0.31
2.07 .+-. 0.84 6.65 .+-. 2.56 Skin 3.22 .+-. 0.31 2.30 .+-. 0.70
7.34 .+-. 1.95 GI Tract 2.63 .+-. 0.40 0.652 .+-. 0.30 1.67 .+-.
0.64 Urine -- -- 2.83 Feces -- -- 2.06 TOTAL 57.28 .+-. 17.60 No.
Mice = 3 Mean Weight = 21.48 .+-. 2.05 grams
[0327]
35TABLE 34 Distribution of Activity at 72 Hours Following I.V.
Injection of .sup.90Y-2B8-MX-DTPA Into Normal BALB/c Mice Mean
Values .+-. SD Organ Weight % ID/ % ID per Sample Gram Gram Organ
Blood 1.45 .+-. 0.07 15.87 .+-. 4.81 23.14 .+-. 7.26 Heart 0.093
.+-. 0.01 4.16 .+-. 1.27 0.392 .+-. 0.13 Lung (2) 0.123 .+-. 0.02
10.67 .+-. 3.79 1.30 .+-. 0.45 Kidney (1) 0.123 .+-. 0.01 4.79 .+-.
1.03 0.596 .+-. 0.16 Liver 0.876 .+-. 0.07 7.26 .+-. 1.79 6.39 .+-.
1.76 Spleen 0.081 .+-. 0.01 7.37 .+-. 2.34 0.584 .+-. 0.16 Muscle
8.30 .+-. 0.39 0.67 .+-. 0.13 5.58 .+-. 1.22 Bone 3.11 .+-. 0.15
2.58 .+-. 0.51 8.05 .+-. 1.76 Skin 3.11 .+-. 0.15 3.09 .+-. 0.82
9.66 .+-. 2.68 GI Tract 2.59 .+-. 0.20 0.79 .+-. 0.18 2.05 .+-.
0.53 Urine -- -- 3.56 Feces -- -- 2.82 TOTAL 65.47 .+-. 14.0 No.
Mice = 3 Mean Weight = 20.76 .+-. 0.97 grams
[0328] 4. Dosimetry
[0329] The radiation absorbed doses for a "standard" 70 Kg human
calculated for the .sup.90Y-labeled conjugate using the mouse
biodistribution data (%ID/organ values in Tables 31-34) are
presented in Table 35. These results are comparable to results
obtained previously using .sup.90Y-labeled 2B8-MX-DTPA prepared
using the HPLC radiolabeling method.
36TABLE 35 Radiation Dosimetry Estimates Resulting from the
Administration of Yttrium-[90] Labeled 2B8-MX Uniformly Distributed
in Standard Man(70 Kg) and Based on Animal Distribution Data Over
72 Hours After Injection 1000 AMOUNT OF ACTIVITY =
MICROCURIES/PATIENT DOSE RADS RADS ADRENALS 0.534 OVARIES 0.534
BLADDER WALL 0.534 PANCREAS 0.534 STOMACH WALL 0.534 SKELETON SMALL
INTESTINE 1.158 CORTICAL BONE 1.466 UL INTEST. WALL 1.657
TRABECULAR BONE 1.466 LL INTEST. WALL 2.380 MARROW (RED) 4.452
KIDNEYS 7.015 MARROW (YELLOW) 2.096 LIVER 7.149 CARTILAGE 1.466
LUNGS 2.157 OTHER CONSTIT. 1.466 OTHER TISSUES SKIN 6.603 MUSCLE
2.646 SPLEEN 4.973 ADIPOSE 2.646 TESTES 0.534 BLOOD 2.646 THYROID
0.534 BRAIN 2.112 UTERUS (NONGRVD) 0.767 HEART 2.646 TOTAL BODY
1.755
[0330] 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
[0331] 5. Validation of Protocol for Preparation of Clinical Doses
of Yttrium-[90]-2B8-MX-DTPA
[0332] A total of ten validation lots were prepared at MPI Pharmacy
Services, Inc. The results of testing on each lot are summarized in
Table 36. The mean value for each test result was calculated and
standard deviations noted where appropriate. To evaluate the
variability of the process due to different labeling times, lot #1
through #8 were prepared using a 10 min labeling time; lot #9 and
#10 were prepared using a reaction time of 5 min. Based on the test
results for the ten validation lots, release specifications were
established. Release specifications are summarized in Table 37.
37TABLE 36 Assay Results for the Ten Validation Lots of
.sup.90Y-Labeled 2B8-MX-DTPA Prepared Using "Mix-&-Shoot" #1 #2
#3 #4 #5 #6 #7 #8 #9 #10 mean % imunoreactivity 72.8 93.3 71.7 70.2
60.6 68.2 79.5 72.4 88.2 68.5 74.5 .+-. 9.8 endotoxin (Eu/ml)
<0.12 <0.12 <0.12 <0.12 <0.12 <0.25 <0.25
<0.25 <0.12 <0.12 <0.162 .+-. 0.06 5 5 5 5 5 5 5 %
radioincorporation 97.5 97.0 93.5 96.0 94.7 94.9 95.9 96.5 97.5
93.5 95.7 .+-. 1.4 antibody conc. (mg/ml) 0.122 0.102 0.088 0.128
0.134 0.119 0.093 0.088 0.111 0.096 0.108 .+-. 0.017 radioactivity
(mCi/ml) 1.22 1.22 0.98 1.26 1.51 1.55 1.06 0.98 1.28 1.02 1.21
.+-. 0.21 specific act. (mCi/mg) 10.0 12.0 11.2 9.8 11.3 13.0 11.3
11.1 11.5 10.7 11.2 .+-. 0.9 vial radioactivity (mCi) 8.16 8.68
8.26 8.52 8.43 8.45 8.56 8.45 8.45 8.45 8.44 .+-. 0.15 protein
conc. (mg/ml) 76.2 76.1 73.6 73.1 72.4 76.1 76.4 74.3 71.2 74.8
74.4 .+-. 1.8 pH 6.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4 .+-.
0.0 sterility pass pass pass pass pass pass pass pass pass pass
[0333]
38TABLE 37 Release Specifications for .sup.90Y-Labeled 2B8-MX-DTPA
Prepared Using "Mix-&-Shoot" Protocol Test Specification Method
Immunoreactivity .gtoreq.60% RIA Endotoxin <5 EU/ml LAL
Radiolabel incorporation .gtoreq.90% ITLC Antibody conc.
0.075-0.150 mg/ml A.sub.280 Radioact. conc..sup.1 .gtoreq.6.0
mCi/ml dose calibration Specific activity.sup.1 .gtoreq.9.0 mCi/mg
antibody A.sub.280/dose calib. Total vial radioact..sup.1
.gtoreq.6.0 mCi dose calibration pH 6.0-8.0 pH paper total protein
conc. 65-85 mg/ml A.sub.280 sterility testing passes CFR 610.12
.sup.1(time zero calibration)
[0334] D. Discussion
[0335] The original radiolabeling protocol for preparing
.sup.90Y-labeled 2B8-MX-DTPA utilized a particularly laborious and
time-consuming HPLC purification step for removing non-protein
bound .sup.90Y from the preparation. In order to simplify this
process and make it more amenable to use at the clinical site,
efforts were directed at eliminating the 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. An additional benefit of this protocol was
increased retention of immunoreactivity (>70%), presumably due
to the shorter exposure time of the antibody to the high energy
radioisotope before the addition of human serum albumin which
provides protection against radiolysis. This retention of
immunoreactivity is superior to that previously observed using the
HPLC method.
[0336] Stability studies with .sup.90Y-labeled conjugate prepared
using the "mix-and-shoot" protocol incubated in formulation buffer
(1.times. PBS containing 75 mg/mL human serum albumin and 1 mM
DTPA) for up to 48 h at 4.degree. C. showed no loss of radioisotope
and complete retention of immunoreactivity. Stability studies
conducted with human serum for 72 hours at 37.degree. C. also
indicated minimal radioisotope loss. These stability results are
comparable to those previously seen with conjugate radiolabeled
using the HPLC protocol.
[0337] Biodistribution in BALB/c mice using .sup.90Y-labeled
conjugate prepared with the "mix-and-shoot" method indicated no
unusual tissue deposition. These results suggested that the
radiolabeled antibody was not altered significantly so as to
dramatically affect the in vivo characteristics of the antibody.
Also, these results are comparable to those obtained previously
with the radiolabeled conjugated prepared using the HPLC method of
radiolabeling (see BB-IND 4850/4851). Dosimetry estimates for a
"standard" 70 Kg human calculated from the biodistribution data for
mice are in agreement with those obtained with conjugate
radiolabeled using the HPLC procedure (see BB-IND 4850/4851).
Additionally, 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. For
six patients in the study, 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.
[0338] Before implementing the "mix-and-shoot" labeling protocol
for preparing clinical-grade .sup.90Y-2B8-MX-DTPA, it was necessary
to assess the reproducibility of the protocol. Therefore, ten
validation lots were prepared using different lots of .sup.90Y
chloride. For the ten lots prepared, the immunoreactivity values
obtained using the "mix-and-shoot" method were in the range of
60.6% to 93.3% with a mean of 74.5% and a median of 72.1%. This
retention of immunoreactivity is significantly better than the
approximately 60% previously obtained using the current HPLC method
(range of 54.9% to 65.1%; mean of 60.2%). The average
radioincorporation for the ten lots was 95.7% (range of 93.5% to
97.5%). This value is comparable to that previously seen with the
HPLC method (range of 91.7% to 93.7% and a mean of 93.1%). Also,
results for endotoxin, antibody concentration, radioactivity
concentration, specific activity, total vial radioactivity, total
protein concentration, pH, and sterility were comparable for the
ten lots. Together, these results confirmed the reproducibility of
the "mix-and-shoot" method. In addition, we evaluated the
variability of the process due to different labeling times by
performing reactions for 5 and 10 minutes. Since there were no
significant differences noted for the two reaction times, it was
decided that the shorter incubation time would be used in the final
protocol.
[0339] E. Summary
[0340] We have developed a labeling procedure, referred to as the
"mix-and-shoot" method, for the preparation of clinical doses of
.sup.90Y-labeled 2B8-MX-DTPA which obviates the need for the
currently used high performance liquid chromatographic (HPLC) step
for removal of non-protein bound radioisotope. 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.degree. C. for 48 hours based on
retention of radioisotope and immunoreactivity. Additionally, the
radiolabeled conjugate was stable when incubated in human serum at
37.degree. 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 .sup.90Y-labeled 2B8-MX-DTPA. The results of these studies
showed that .sup.90Y-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 .sup.90Y-labeled 2B8-MX-DTPA
suitable for use in clinical trials.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE 1.
Radioincorporation--Kits and Assays
[0341] I. Summary One objective of the present invention was to
devise radiolabeling kit protocols for preparation of .sup.111In
and .sup.90Y-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
.gtoreq.95% and .gtoreq.70%, respectively.
[0342] II. Introduction
[0343] A .sup.90Y-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, .sup.111In-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.
[0344] 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.times. 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.
[0345] This report summarizes the validation of this radiolabeling
kit which is simple and easy to use and which yields radiolabeled
antibodies with .gtoreq.95% radioincorporation and acceptable
retention of binding to antigen-positive cells. Release testing
specifications are recommended for the clinical products.
[0346] III. Materials and Methods for Radioincorporation
[0347] A. Reagents in Radiolabeling Kit
[0348] 1. 2B8-MX-DTPA, IDEC; Lot# 082395RM2
[0349] 2. 50 mM Sodium Acetate, low-metal, IDEC; Lot# 082395RM3
[0350] 3. Formulation Buffer (1.times. PBS, pH 7.4 containing 7.5%
(w/v) human serum albumin and 1 mM DTPA), IDEC, Lot# 082395RM1
[0351] 4. Reaction vial, 10 mL, IDEC
[0352] B. Materials and Equipment
[0353] 1. Biodex Tec-Control Radioincorporation Kit,
Cat.#151-770
[0354] 2. Gloves: powder-free
[0355] 3. Sterile polypropylene syringes
[0356] 4. Sterile syringe needles
[0357] 5. Small tubes with closure; 1.5 ml
[0358] C. Methods
[0359] 1. Preparation of Y2B8 and In2B8 Using Radiolabeling Kit Kit
reagents were prepared and filled into glass septum vials. Type I
borosilicate vials (2 or 10 mL) were rinsed with sterile water for
injection (WFI) and autoclaved before filling. Butyl rubber septa
were rinsed with sterile WFI and autoclaved before use. Reagents
were manually filled and crimped in a Class 100 room and tested for
pyrogenicity and sterility using USP methods.
[0360] a. Preparation of In2B8
[0361] Additional Reagents:
[0362] 1. Indium-[ 1 1]: chloride salt, carrier-free, in HCl.
[0363] Precautions:
[0364] 1. All steps are preferably performed using aseptic
technique.
[0365] 2. Radiolabeling kit components should be allowed to come to
room temperature before use.
[0366] 3. Final product should be administered to patient within 8
hours of completing step 9 below.
[0367] In2B8 Radiolabeling protocol
[0368] Procedure
[0369] 1. The volume of .sup.111InCl.sub.3 to add to the reaction
vial was calculated as follows:
[0370] a. Radioactivity concentration at the time of radiolabeling
in mCi/ml:
[0371] C.sub.0=Radioactivity concentration at time of calibration
(see manufacturer's Certificate of Analysis).
[0372] .DELTA.t=Change in time (positive number is post
calibration, negative number is pre calibration). 1 Radioactivity
Concentration at time of labeling = C 0 e 0.0103 ( t )
[0373] b. Volume of .sup.111InCl.sub.3 to add to the reaction vial:
2 5.5 mCi Radioactivity Concentration at time of labeling = Volume
to add to reaction vial
[0374] 2. The volume of 50 mM sodium acetate to be added to the
reaction vial was calculated as follows:
[0375] Volume of .sup.111InCl.sub.3 added (Step
1b).times.(1.2)=Volume of 50 mM sodium acetate to be added.
[0376] 3. The septa of the reaction vial and the 50 mM sodium
acetate vial were wiped with alcohol. Using a icc syringe, the
calculated volume of 50 mM sodium acetate (Step 2) was transferred
to the reaction vial.
[0377] 4. The septum of .sup.111InCl.sub.3 source was wiped with
alcohol. The vial was vented with a needle fitted with sterile 0.2
.mu.m filter. Using a lcc sterile syringe, the required volume
(Step 1b) of .sup.111InCl.sub.3 was transferred to the reaction
vial. The vial was mixed by inverting several times.
[0378] 5. The septum of the 2B8-MX-DTPA vial was wiped with
alcohol. Using a 1 cc syringe, 1.0 mL of 2B8-MX-DTPA was slowly
transferred to the reaction vial. The vial was mixed by inverting
several times.
[0379] 6. The reaction was allowed to proceed for 30 minutes.+-.5
minutes at ambient temperature.
[0380] 7. The total volume of reaction mixture was calculated by
adding together the volume of .sup.111InCl.sub.3 added (Step 4),
the volume of 50 mM sodium acetate added (Step 3) and the volume of
2B8-MX-DTPA added (Step 5).
[0381] 8. The volume of Formulation Buffer to add to the Reaction
Vial to obtain a final volume of 10 mL was calculated by
subtracting the total amount calculated in step 7 from 10.
[0382] 9. The Formulation Buffer vial was wiped with alcohol and
the vial was vented. Due to the viscosity of the Formulation
Buffer, the reaction vial was vented using a needle fitted with a
0.2 .mu.m syringe filter. Using a 10 cc sterile syringe fitted with
an appropriate gauge needle, the volume of Formulation Buffer
calculated in Step 8 was transferred to the reaction vial. The vent
needle was removed from the reaction vial and the vial was mixed by
inverting several times (Final Product). This vial was incubated at
least 5 minutes prior to doing the "Radioincorporation Assay". The
color of the solution was amber and the vial was full, confirming
that Formulation Buffer was added.
[0383] 10. Total radioactivity of the Final Product vial was
measured using the appropriate instrumentation set for .sup.111In
measurement.
[0384] 11. The Final Product was stored immediately at
2.degree.-8.degree. C. for "Binding Assay" and "Radioincorporation
Assay".
[0385] b. Preparation of Y2B8
[0386] Additional Reagents:
[0387] 1. Yttrium-[90]: chloride salt, carrier-free, in HCl.
[0388] Precautions:
[0389] 1. All steps should be performed using aseptic
technique.
[0390] 2. Radiolabeling kit components should be allowed to come to
room temperature before use.
[0391] 3. The product should be administered to the patient within
8 hours of completing step 8 below.
[0392] Y2B8 Radiolabeling Protocol
[0393] 1. The volume of .sup.90YCl.sub.3 to add to the reaction
vial was calculated as follows:
[0394] a. The radioactivity concentration at the time of
radiolabeling:
[0395] C.sub.0=Radioactivity concentration at time of calibration
(see manufacturer's Certificate of Analysis).
[0396] .DELTA.t =Change in time (positive number is post
calibration, negative number is pre calibration). 3 Radioactivity
Concentration at time of labeling = C 0 e 0.0108 ( t )
[0397] b. The volume of .sup.90YCl.sub.3 to add to the reaction
vial: 4 45 mCi Radioactivity Concentration time of labeling =
Volume added to reaction vial
[0398] 2. The volume of 50 mM sodium acetate to add to the reaction
vial was calculated as follows:
[0399] a. For .sup.90YCl.sub.3 in 0.040 M HCl (Amersham):
[0400] Volume .sup.90YCl.sub.3 (Step 1b).times.(0.8)=volume of
sodium acetate to add
[0401] b. For .sup.90YCl.sub.3 in 0.050 M HCl(Nordion):
[0402] Volume .sup.90YCl.sub.3 (Step 1b).times.(1.0)=volume of
sodium acetate to add
[0403] 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.
[0404] 4. The septum of the .sup.90YCl.sub.3 source vial was wiped
with alcohol. The vial with a needle fitted with sterile 0.2 .mu.m
filter. Using a lcc sterile syringe, was vented the required volume
(Step 1b) of .sup.90YCl.sub.3 was transferred to the reaction vial.
The vial was mixed by inverting several times.
[0405] 5. The septum of the 2B8-MX-DTPA vial was wiped with
alcohol. Using a 3 cc sterile syringe, 1.5 mL of 2B8-MX-DTPA was
transferred to the reaction vial. The vial was mixed by inverting
several times.
[0406] 6. The total volume of 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).
[0407] 7. The volume of Formulation Buffer to add to the Reaction
Vial to obtain a final volume of 10 mL was calculated by
subtracting the total reaction volume calculated in step 6 from
10.
[0408] 8. The Formulation Buffer vial was wiped with alcohol and
the vial was vented. Due to the viscosity of the Formulation
Buffer, the reaction vial using a needle fitted with a 0.20 .mu.m
syringe filter. Using a 10 cc sterile syringe fitted with an
appropriate gauge needle, the volume of Formulation Buffer
calculated in Step 7 was transferred to the reaction vial. The vent
needle was removed from the reaction vial and the vial was mixed by
inverting several times (Final Product). The vial was incubated at
least 5 minutes prior to doing the "Radioincorporation Assay". The
color of the solution was amber and the reaction vial was full
thereby confirming that Formulation Buffer was added.
[0409] 9. The total radioactivity of the Final Product vial was
measured using the appropriate instrumentation set for measurement
of .sup.90Y.
[0410] 10. The Final Product was immediately stored at
2.degree.-8.degree. C. until required for patient
administration.
[0411] 11. Immunoreactivity testing:
[0412] Using a 1 mL syringe, 0.1 mL was aseptically removed from
the reaction vial and transferred to a separate 1.5 mL screwcap
tube. The tube was immediately stored at 2.degree.-8.degree. C. for
"Binding Assay" and "Radioincorporation Assay".
[0413] Validation of the radiolabeling kit protocols was performed
at IDEC Pharmaceuticals (San Diego, Calif.), MD Anderson Health
Center (Houston, Tex.), Mayo Clinic (Rochester, MN), and City of
Hope (Duarte, Calif.). All kit components, including clinical-grade
2B8-MX-DTPA, were prepared by IDEC Pharmaceuticals under GMP
conditions (Good Manufacturing Conditions according to the Code of
Federal Regulations) and determined to be sterile and
pyrogen-free.
[0414] The radiolabeled antibodies were formulated with 1.times.
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.
[0415] Six validation lots each of In2B8 and Y2B8 were prepared by
five operators. These lots were designated as follows and performed
at the following facilities:
[0416] In2B8:
[0417] #1: IDEC Pharmaceuticals
[0418] #2: IDEC Pharmaceuticals
[0419] #3: IDEC Pharmaceuticals
[0420] #4: MD Anderson Health Center
[0421] #5: Mayo Clinic
[0422] #6: City of Hope
[0423] Y2B8:
[0424] #1: IDEC Pharmaceuticals
[0425] #2: IDEC Pharmaceuticals
[0426] #3: IDEC Pharmaceuticals
[0427] #4: MD Anderson Health Center
[0428] #5: Mayo Clinic
[0429] #6: City of Hope
[0430] 2. Preparation of Lyophilized SB and HSB Cells
[0431] 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.degree. C. and 5% CO.sub.2. Cells were typically
split 1:2 every other day and harvested at 0.5-2.5.times.10.sup.6
cells/mL and viability's >80%. Cell concentrations were
determined using a hemacytometer and viability determined by trypan
blue exclusion.
[0432] Cells were harvested at ambient temperature at a cell
density of 0.5-2.times.10.sup.6 cells/mL by centrifugation (1300
rpm in a Sorvall centrifuge) and washed twice with 1.times. HBSS.
Pelleted cells were resuspended to 50.times.10.sup.6 cells/mL in
1.times. HBSS containing 1% (w/v) bovine serum albumin (BSA) and
10% (w/v/) mannitol (lyophilization buffer), 0.5 mL dispensed into
1.5 mL polypropylene microfuge tubes with o-ring gaskets and stored
at -70.degree. C., and lyophilized overnight at 30-60 millitorr.
Tubes of lyophilized cells were stored desiccated at 2-8.degree. C.
and reconstituted in sterile water for assays; tubes of cells
lyophilized in microfuge tubes were stored with desiccant.
[0433] 3. Analytical Assays
[0434] The analytical methods used to test the validation lots of
In2B8 and Y2B8 are described below. The following assays were
performed for each validation lot:
[0435] 1. Binding Assay using lyophilized SB cells
[0436] 2. Y2B8/In2B8 Radioincorporation Assay using Biodex Kit
[0437] a. Binding Assays
[0438] 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".
[0439] i. In2B8 Binding Assay
[0440] Additional Reagents:
[0441] 1. Indium-[111]-2B8-MX-DTPA
[0442] 2. Lyophilized SB cells; three tubes containing
25.times.10.sup.6 cells/tube.
[0443] 3. Lyophilized HSB cells; three tubes containing
25.times.10.sup.6 cells/tube.
[0444] 4. Sterile water for irrigation or sterile water for
injection.
[0445] 5. Dilution buffer (1.times. PBS, pH 7.2-7.4 containing 1%
Bovine Serum Albumin (BSA), and 0.02% Sodium Azide 0.2 .mu.m
filtered and stored at room temperature.
[0446] 6. Glass or plastic test tubes for counting
radioactivity.
[0447] Procedure:
[0448] Assay set-up (Non-radioactive portion)
[0449] 1. Three tubes of lyophilized SB and HSB cells were
obtained.
[0450] 2. A volume of 0.50 mL of SWFI (sterile water for injection)
was added to each tube, and the tubes were vortexed until
homogenous suspensions were obtained.
[0451] 3. Four empty 1.5 mL microfuge tubes. To three of the tubes
0.50 mL of Dilution buffer was added, representing a control with
no cells.
[0452] 4. To the other 1.5 mL microfuge tube, 0.99 mL of Dilution
buffer was added; this tube was labeled 1:100.
[0453] 5. A 50 mL sterile polypropylene tube with cap was obtained
and 10 mL of Dilution buffer was added to the tube.
[0454] Assay Set-Up (Radioactive Portion)
[0455] 1. The radiolabeled antibody stored at 2.degree.-8.degree.
C. was obtained.
[0456] 2. A volume of 0.01 mL was withdrawn with a P20 and added to
the 1.5 mL microfuge tube containing 0.99 mL of Dilution buffer
(1:100 dilution). The tip was rinsed and the tube vortexed gently
to mix.
[0457] 3. A volume 0.20 mL was withdrawn with a P200 from the 1:100
dilution tube and added to the conical tube containing 10 mL of
Dilution buffer. The tube was mixed thoroughly.
[0458] Assay Protocol
[0459] 1. A volume of 0.50 mL of the diluted .sup.111In2B8-MX-DTPA
was added to all tubes.
[0460] 2. The caps were securely tightened on all tubes, and the
tubes mixed continuously for 60 minutes.
[0461] 3. After 60 minutes incubation at ambient temperature, all
tubes were centrifuged for 5 minutes at minimum of 2000 g.
[0462] 4. A volume of 0.75 mL of each supernatant was transferred
to tubes appropriate for the counting instrument.
[0463] 5. The radioactivity in tubes was counted using a gamma
counter, adjusting for background.
[0464] ii. Y2B8 Binding Assay
[0465] Additional Reagents
[0466] 1. .sup.90Y2B8-MX-DTPA
[0467] 2. Lyophilized SB cells
[0468] 3. Sterile water for irrigation or sterile water for
injection
[0469] 4. Dilution buffer (1.times. PBS, pH 7.2-7.4 containing 1%
Bovine Serum Albumin (BSA), and 0.02% Sodium Azide)
[0470] Procedure:
[0471] Radiolabeled Antibody Sample Prep
[0472] 1. The radiolabeled antibody stored at 2.degree.-8.degree.
C. was obtained.
[0473] 2. A volume of 10 .mu.L was withdrawn with a P20 and added
to a 1.5 mL microfuge tube containing 990 .mu.L of Dilution buffer
(1:100 dilution). The tip was rinsed and the tube was vortexed
slightly.
[0474] 3. A 50 mL sterile polypropylene tube with cap was obtained
and 10 mL of Dilution buffer to the tube, using a 10 mL serological
pipette.
[0475] 4. A volume of 35 .mu.L was withdrawn with a P200 from the
1:100 dilution tube and added to the conical tube containing 10 mL
of Dilution buffer. Mix thoroughly.
[0476] Lyophilized Cell Prep
[0477] 1. Three tubes of lyophilized SB Cells were obtained.
[0478] 2. A volume of 0.5 mL of SWFI was added to each tube, and
the tubes were vortexed until single cell suspensions were
obtained.
[0479] 3. Three empty 1.5 mL microfuge tubes were obtained; to
three of the tubes, 0.5 mL of Dilution buffer was added,
representing a control with no cells.
[0480] Assay Protocol
[0481] 1. A volume of 0.5 mL of the diluted .sup.90Y2B8-MX-DTPA was
added to each tube.
[0482] 2. The tubes were placed on end over mixer for 45 minutes,
after making sure caps are securely tightened.
[0483] 3. After 45 minutes incubation at ambient temperature, the
cells were pelleted by microcentrifugation for 5 minutes.
[0484] 4. A volume of 0.8 mL of the supernatant was transferred to
scintillation vials.
[0485] 5. Scintillation cocktail was added to each vial.
[0486] 6. The amount of radioactivity in each vial was determined
using a scintillation counter, adjusting for background.
[0487] b. Radioincorporation Assay
[0488] Percent radioincorporation was determined by instant
thin-layer chromatography (ITLC) using the Biodex Tec-Control
Radiochromatographic Kit according to the following protocol:
[0489] Additional Materials and Equipment:
[0490] 1. .sup.111In- or .sup.90Y-radiolabeled 2B8-MX-DTPA
[0491] 2. Tubes for counting radioactive TLC strips
[0492] 3. Scissors
[0493] 4. Sterile syringe, 1 cc
[0494] 5. Sterile needles, 26 G
[0495] 6. Gamma counter or scintillation counter
[0496] 7. Pipettor
[0497] Procedure:
[0498] 1. The entire Biodex Operation Manual should be read
first.
[0499] 2. Each radiolabeled sample in triplicate was tested
according to kit instructions; one strip per vial was
developed.
[0500] 3. To spot the radiolabeled sample on the chromatography
strip, a pipettor was used to spot 1 .mu.l on the origin line.
Alternatively, one small drop dispensed from a 26 G needle attached
to a sterile 1 cc syringe may be spotted.
[0501] 4. Each section was counted for activity using the
appropriate counter, i.e., gamma counter for .sup.111In and a
scintillation counter for .sup.90Y, adjusting for background.
[0502] 5. The Biodex instructions for calculating the percentage of
radiolabeled antibody were followed.
[0503] IV. Results
[0504] The results of testing on each validation lot of In2B8 or
Y2B8 are summarized in Tables 38 and 39.
39TABLE 38 Release Assay Results for Y2B8 Validation Lot Number %
Radioincorporation % Binding 1 99.5 78.6 2 99.3 87.0 3 99.4 85.9 4
99.2 81.8 5 99.2 79.6 6 96.3 80.8 Mean = 98.8 Mean = 82.3 Standard
Deviation = 1.24 Standard Deviation = 3.4 % CV = 1.25% CV =
4.2%
[0505]
40TABLE 39 Release Assay Results for In2B8 Validation Lots Lot
Number % Radioincorporation % Binding 1 99.4 86.2 2 98.7 86.8 3
99.3 85.8 4 98.3 86.7 5 99.0 82.1 6 99.3 83.0 Mean = 99.0 Mean =
85.2 Standard Deviation = 0.43 Standard Deviation = 2.06 % CV =
0.45% CV = 2.42%
[0506] V. Discussion and Conclusions
[0507] To simplify the current radiolabeling methods for In2B8 and
Y2B8, a four-component kit was 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.
[0508] Reproducibility and ruggedness of the radiolabeling
protocols for In2B8 and Y2B8 was evaluated by performing several
validation runs using different lots of each radioisotope. For the
six validation lots of In2B8 prepared, binding ranged from 82.1% to
86.8% with a mean of 85.1%; radioincorporation values for were
approximately 99% (range of 98.3% to 99.4%). For the six validation
lots of Y2B8 prepared, the percent binding obtained was in the
ranged from 78.6% to 87.0% with a mean of 82.3%. Radioincorporation
values for Y2B8 averaged 98.8% (range of 96.3% to 99.5%). Together,
these results confirm the reproducibility and ruggedness of the
radiolabeling kit methods for preparation of both In2B8 and Y2B8.
Based on these validation results, it is recommended that release
specifications for radioincorporation and binding be established at
.gtoreq.95% and .gtoreq.70%, respectively, for both In2B8 and Y2B8.
Additionally, because of the increased ease of use and reduced
potential for mistakes during preparation, it is recommended that
percent binding using lyophilized CD20-positive cells and
radioincorporation be used to release test In2B8 and Y2B8 at the
clinical sites.
[0509] To summarize, these results together indicate that In2B8 and
Y2B8 prepared using the radiolabeling kit are suitable for use in
the clinical setting. Additionally, for both radiolabeled
antibodies, release specifications are established reflecting the
results of several validation runs by the five different
operators.
EXAMPLE 2
Radioincorporation and Binding--Kits and Assays
[0510] I. Summary
[0511] 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.times.10.sup.-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.times.10.sup.-10 M. Optimal chelator
conjugation, as determined by measuring radioincorporation of
.sup.111In, was achieved after eight hours reaction. Radiolabeling
protocols for 2B8-MX-DTPA were optimized for .sup.90Y or .sup.111In
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.
[0512] II. Introduction
[0513] 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 .sup.90Y
and .sup.111In-labeled antibody products prepared using the
clinical radiolabeling kit protocols.
[0514] III. Materials and Methods
[0515] A. Reagents 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.degree. C. and 5% CO.sub.2. Cells were typically
split 1:2 every other day and harvested at 0.5-2.5.times.10.sup.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).
[0516] Pharmaceutical-grade .sup.111In chloride was purchased from
Amersham (U.K.) or Cyclotron Products Inc. (Coral Gables, Fla.).
Yttrium[90] chloride was obtained from Amersham (U.K.), Nordion
International (Kanatta, Canada), or Pacific Northwest National
Laboratory (Richland, Wash.). MX-DTPA prepared under GMP was
obtained from Hauser Chemical (Boulder, Colo.). Clinical-grade
calcium trisodium diethylenetriaminepentaacetic acid (DTPA) was
obtained from Heyl (Berlin, Germany). TAG-NHS was obtained from
IGEN Inc. (Rockville, Md.). Murine anti-CD19 beads were purchased
from Dynal Inc. (Lake Success, N.Y.). Goat anti-mouse FITC-labeled
F(ab').sub.2 was purchased from Jackson ImmunoResearch.
[0517] Reagents requiring removal of contaminating heavy metals
were batch treated with Chelex 100 (BioRad Industries) or with
Chelating Sepharose (Pharmacia) by passing solutions through a
column. Low-metal stock solutions were diluted with Sterile Water
for Irrigation (SWFIr). Solutions were stored in sterile plastic
containers.
[0518] Additional reagents are described below for specific
methods.
[0519] B. Materials and Equipment
[0520] 1. Origen Analyzer; IGEN Inc. Model #1100-1000; IDEC
#1492
[0521] 2. Top-Count scintillation counter; Packard, Model #A9912;
IDEC #1329
[0522] 3. Gamma counter; Isodata, Model # 20-10; IDEC #0628
[0523] 4. Tec-Control Radiochromatographic Kit, Biodex, Model
#151-770
[0524] 5. Lyophilizer; Virtis, Model Freezemobile 12; IDEC
#0458
[0525] Additional materials and equipment are described for
specific methods.
[0526] C. Methods
[0527] 1. Preparation of Lyophilized SB and HSB Cell
[0528] Cells were cultured as described above and harvested at
ambient temperature at a cell density of 0.5-2.times.10.sup.6
cells/mL by centrifugation (1300 rpm in a Sorvall centrifuge) and
washed twice with 1.times. HBSS. Pelleted cells were resuspended to
50.times.10.sup.6 cells/mL in 1.times. HBSS containing 1% (w/v)
bovine serum albumin (BSA) and 10% (w/v/) mannitol (lyophilization
buffer), 0.5 mL dispensed into 1.5 mL polypropylene microfuge tubes
with o-ring gaskets and stored at -70.degree. C., and lyophilized
overnight at 30-60 millitorr. Tubes of lyophilized cells were
stored desiccated at 2-8.degree. C. and reconstituted in sterile
water for assays; tubes of cells lyophilized in microfuge tubes
were stored with desiccant.
[0529] 2. FACS Binding Analysis
[0530] Direct binding of antibodies to human B-cells was determined
by flow cytometry. Increasing concentrations of antibody were
incubated in 1.times. PBS, pH 7.2, containing 1% (w/v) BSA (binding
buffer) with 5.times.10.sup.6 CD20-positive (SB) or CD20-negative
(HSB) cells for 30 min. on ice. Cells were washed by
centrifugation, resuspended in binding buffer, and incubated with
FITC-labeled goat anti-mouse F(ab').sub.2 for 30 min. on ice. After
incubation with the secondary reagent, cells were washed by
centrifugation and resuspended in 1.times. PBS containing 1.1%
(v/v) formaldehyde to fix cells. Mean fluorescence intensity was
determined using flow cytometry.
[0531] 3. Competitive Binding Assays
[0532] Immunoreactivity of 2B8 and 2B8-MX-DTPA was determined by
competitive binding to CD20-positive SB cells using the ORIGEN
electrochemiluminescent method (Leland and Powell). Log-phase SB
cells were harvested from culture and washed twice with 1.times.
HBSS. Cells were diluted in 1.times. PBS pH 7.2 containing 1% (w/v)
bovine serum albumin. In some experiments, lyophilized cells were
used after reconstitution with sterile water. Ruthenium-labeled
tracer antibody was prepared by incubating CHO-derived 2B8 (lot
#165) in 1.times. 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.times. 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.
[0533] Assays were performed in 12.times.75 mm polypropylene tubes.
Varying amounts of competing antibody (0.002-17 ug/mL) were
incubated in 1.times. 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:
y=((m1-m4)/(1+(m0/m3) m2))+m4;m1=;m2=;m3=;m4=
[0534] m0=independent variable
[0535] m1=zero signal response in relative ECL units
[0536] m2=curvature parameter
[0537] m3=EC50 in ug/mL
[0538] 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.
[0539] 4. Preparation of 2B8-MX-DTPA
[0540] The chelating agent,
1-isothiocyanatobenzyl-3-methyldiethylenetriam- inepentaacetic acid
(MX-DTPA) was provided as a dry powder (free-acid) and stored
desiccated at -20.degree. or -70.degree. 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.
[0541] 5. Preparation of In2B8 and Y2B8
[0542] 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
.sup.111In chloride was transferred to a metal-free microfuge tube
and adjusted to approximately pH 4.2 using a 1.2.times. 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.times. 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%.
[0543] 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.times. volume of low-metal 50 mM sodium acetate.
2B8-MX-DTPA at 2 mg/mL was added to the .sup.90Y acetate solution
and after incubation at ambient temperature for 5 min., the labeled
antibody was formulated to 0.3 mg/mL in 1.times. 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%.
[0544] The 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.
[0545] For radiolabeling kinetic studies evaluating the effect of
pH on radioincorporation and binding, the pH of the reaction
mixtures was adjusted. by adding varying amounts of low-metal 50 mM
sodium acetate (0.8 to 2.2.times. volume of radioisotope
solution).
[0546] 6. Determination of Radioincorporation for In2B8 and
Y2B8
[0547] 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 .mu.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.
[0548] 7. Determination of Immunoreactivity of In2B8 and Y2B8
Immunoreactivity was assessed using the method of Lindmo et al and
as described above in Example 1.
[0549] 8. Direct Binding Assay for In2B8 and Y2B8
[0550] The same protocols as described herein were used to
determine binding to CD20-positive SB cells for In2B8 and Y2B8,
respectively. In2B8 and Y2B8 were prepared and formulated as
described above. For 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.
[0551] Antigen-positive (SB) and antigen-negative (HSB) cells were
maintained in RPMI 1640 supplemented with 10% fetal calf serum at
37.degree. C. and 5% CO.sub.2. Cells (viability >90% as
determined by trypan blue exclusion) were harvested at ambient
temperature at a cell density of 0.5-2.times.10.sup.6 cells/mL by
centrifugation (1300 rpm in a Sorvall centrifuge) and washed twice
with 50 mL 1.times. HBSS. Pelleted cells were resuspended to
50.times.10.sup.6 cells/mL in prechilled 1.times. HBSS containing
1% (w/v) bovine serum albumin (BSA) and 10% (w/v/) mannitol
(lyophilization buffer). Cell suspensions were dispensed into 1.5
mL polypropylene microfuge tubes with o-ring gaskets at
50.times.10.sup.6 cells/mL (0.5 mL per tube) and lyophilized
overnight at 30 to 60 millitorr. Lyophilized cells were stored
desiccated at 2-8.degree. C. and reconstituted in sterile water for
assays. Lyophilized SB and HSB cells in 1.5 mL polypropylene tubes
were reconstituted to 50.times.10.sup.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.
[0552] 9. Radioactivity Measurement
[0553] 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 .sup.111In. The Isodata gamma counter was
also used for counting ITLC strips having .sup.90Y spotted on them.
The energy windows for detection of the bremstrulung radiation were
100-1000 KeV.
[0554] For the binding assays, 90Y samples were transferred to
24-well plates and MicroScint 40 cocktail and counted in a Packard
TopCount for 1 min using minimum and maximum energy settings.
Indium-[111] 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.
[0555] 10. Release Specifications for Clinical Doses of In2B8 and
Y2B8
[0556] 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.
[0557] IV. Results
[0558] A. Characterization of CHO-Derived 2B8
[0559] Using flow cytometric analysis, it was demonstrated that CHO
2B8 binds directly to CD20-positive SB cells without binding to
CD20-negative HSB cells (FIG. 34). No significant binding to SB or
HSB cells was noted for art irrelevant isotype (.gamma.1.kappa.)
antibody (S004).
[0560] 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.times.10.sup.-10 M;
the 2B8 antibody derived from hollow-fiber bioreactors gave an
affinity value of 2.5 10.sup.-10 M. Non-specific binding was
negligible as demonstrated by the lack of competition with the
irrelevant isotype antibody, S004.
[0561] B. Characterization of CHO-Derived 2B8-MX-DTPA
[0562] 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 .sup.111In.
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. 36), affinity constants for the
unconjugated and conjugated antibodies ranged from
2.3.times.10.sup.-10 M to 5.9.times.10.sup.-10 M (Table 40).
Radioincorporation was >95% for conjugation times of 8 to 24 h
(Table 30).
41TABLE 40 Effect of Conjugation Reaction Time on
Radioincorporation and Immunoreactivity of CHO 2B8-MX-DTPA
Incubation Time (h) Radioincorporation (%) Affinity (M) 0 ND 2.3
.times. 10.sup.-10 2 83.5 ND 4 90.5 ND 8 96.1 5.9 .times.
10.sup.-10 17 97.3 5.9 .times. 10.sup.-10 24 98.8 4.4 .times.
10.sup.-10
[0563] C. Characterization of In2B8 and Y2B8 Prepared From
CHO-Derived 2B8-MX-DTPA
[0564] 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 .sup.111In 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. For comparison, the conjugate prepared
from hybridoma-produced antibody(2B8-49) was incubated with
.sup.111In acetate for 30 min. at pH 4.2 (conditions previously
established for this antibody).
[0565] Kinetics studies were performed to determine labeling
conditions providing maximal retention of binding to CD20-positive
cells and high radioincorporation (Tables 41 and 42). Conjugated
antibody (CHO-derived 2B8-MX-DTPA) was incubated at ambient
temperature with .sup.111In acetate at pH 4.2 for the times
indicated (Table 42).
42TABLE 41 In2B8 Radiolabeling Kinetics: Effect of pH on
Radioincorporation and Binding to CD20-Positive Cells Reaction pH
Radioincorporation (%) Binding (%) 3.0 97.7 85.3 3.7 98.5 83.9 4.0
98.6 84.1 4.3 98.0 84.0 4.6 98.9 83.4 Control (2B8-49) 99.3
86.5
[0566]
43TABLE 42 In2B8 Radiolabeling Kinetics: Effect of Incubation Time
on Radioincorporation and Binding to CD20-Positive Cells Incubation
Time (min) Radioincorporation (%) Binding (%) pH 2.9: 15 97.2 85.3
30 99.1 85.2 45 97.2 84.8 pH 4.6: 15 99.0 87.2 30 97.2 86.8 45 99.4
86.3 Control (2B8-49) 99.4 87.8
[0567] Results demonstrated that for the range of pH 3.0 to 4.6 and
an incubation time of 30 min, >97% radioincorporation of the
radioisotope was attained while maintaining binding at
approximately 84%. Radioincorporation and binding values were
invariant for incubation times of 15 to 45 min for reactions at pH
2.9 to 4.6 (Table 42). Results were comparable to those obtained
using the 2B8-49 antibody (Tables 41 and 42).
[0568] Yttrium-[90]-labeled antibody was prepared by incubating
conjugated antibody (CHO-derived 2B8-MX-DTPA) with .sup.90Y 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).
[0569] Similar kinetic studies were performed to evaluate the
preparation of the .sup.90Y-labeled antibody (Y2B8). For
radiolabeling reactions in the range of pH 3.9 to 4.7 at an
incubation time of 5 min, radioincorporation was >96% with
>80% retention of binding to CD20-positive cells (Table 43).
Similar results were obtained for incubation times of 3, 5, and 10
min for the range of pH 2.9 to 4.6 (Table 44). Then, conjugated
antibody (CHO-derived 2B8-MX-DTPA) was incubated at ambient
temperature with .sup.90Y acetate at pH 4.2 for the times indicated
(Table 44). Results were comparable to those obtained using the
2B8-49 antibody (Tables 43 and 44).
44TABLE 43 Y2B8 Radiolabeling Kinetics: Effect of pH on
Radioincorporation and Binding to CD20-Positive Cells Reaction pH
Radioincorporation (%) Binding (%) 3.9 98.4 80.7 4.2 97.8 81.0 4.4
96.1 80.0 4.6 97.0 80.2 4.7 97.4 80.6 Control (2B8-49) 99.3
82.6
[0570]
45TABLE 44 Y2B8 Radiolabeling Kinetics: Effect of Incubation Time
on Radioincorporation and Binding to CD20-Positive Cells Incubation
Time (min) Radioincorporation (%) Binding (%) pH 3.9: 3 97.0 82.0 5
98.9 82.1 10 99.2 82.3 pH 4.7: 3 97.2 82.5 5 96.7 81.8 10 97.6 81.5
Control (2B8-49) 99.2 84.2
[0571] 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).
[0572] D. Release Specifications for CHO-Derived In2B8 and Y2B8
[0573] Two lots of clinical-grade In2B8/Y2B8 radiolabeling kits
were used to prepare six lots each of In2B8 and Y2B8. In2B8 and
Y2B8 were prepared using small-scale versions of the radiolabeling
protocols currently used in the clinical trials. Each lot of
radiolabeled 2B8-MX-DTPA was tested for radioincorporation and
binding to CD20-positive (SB) and CD20-negative (HSB) human cells.
These results are summarized in Tables 45 and 46. For the six lots
of In2B8 prepared, radioincorporation ranged from 98.9% to 99.3%
with a mean of 99. 1%. 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%. For the six lots of Y2B8 prepared, 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%.
46TABLE 45 Release Assay Results for CHO-Derived In2B8 Prepared
Using the Radiolabeling Kit Protocol Radio- Binding (%) Run #
incorporation (%) SB Cells HSB Cells #1 (Lot #0219) 99.1 81.9 2.8
#2 (Lot #0219) 99.3 83.2 2.8 #3 (Lot #0219) 99.2 83.6 3.7 #4 (Lot
#0220) 99.0 83.8 2.6 #5 (Lot #0220) 98.9 84.1 2.6 #6 (Lot #0220)
98.9 85.1 3.3 Mean = 99.1% Mean = 83.6% Mean = 2.9% SD = 0.2% SD =
1.1% SD = 0.4%
[0574]
47TABLE 46 Release Assay Results for CHO-Derived Y2B8 Prepared
Using the Radiolabeling Kit Protocol Radio- Binding (%) Run #
incorporation (%) SB Cells HSB Cells #1 (Lot #0219) 98.7 82.1 7.4
#2 (Lot #0219) 98..6 82.7 0.7 #3 (Lot #0219) 98.3 82.2 7.2 #4 (Lot
#0220) 97.4 81.8 1.7 #5 (Lot #0220) 97.6 81.4 2.2 #6 (Lot #0220)
98.4 81.4 1.1 Mean = 98.2% Mean = 81.9% Mean = 3.4% SD = 0.5% SD =
0.5% SD = 3.1%
[0575] V. Discussion and Conclusions
[0576] The anti-CD20 murine monoclonal antibody (2B8) cloned and
expressed in CHO cells (CHO-derived 2B8) 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.times.10.sup.-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.times.10.sup.-10 M.
[0577] 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 .sup.111In and was achieved after
eight hours incubation at ambient temperature. Radiolabeling
protocols for the 2B8-MX-DTPA conjugate were optimized for .sup.90Y
or .sup.111In with respect to pH and incubation time to insure
maximal radioincorporation and retention of immunoreactivity.
[0578] The results of several preparations of In2B8 and Y2B8
demonstrate the reproducibility of the radiolabeling protocol used
to prepare clinical doses. Based on these radiolabeling results, it
is suggested that release specifications for radioincorporation and
binding, using lyophilized CD20-positive cells, be established at
.gtoreq.95% and .gtoreq.70%, respectively. Taken together, these
results demonstrate the comparability of CHO-derived 2B8 and
hollow-fiber-derived 2B8-49, and indicate the suitability of the
CHO-derived 2B8-MX-DTPA for use in clinical trials.
[0579] Finally, 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. 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.degree. C. for 48
hours based on retention of radioisotope and immunoreactivity.
Additionally, the radiolabeled conjugate was stable when incubated
in human serum at 37.degree. C. for 72 hours. Biodistribution
studies in -BALB/c mice demonstrated no unusual tissue deposition,
and no significant accumulation in the bone. Estimates of radiation
absorbed doses to a "standard" 70 Kg human were comparable to those
obtained in an on-going clinical trial using .sup.90Y-labeled
2B8-MX-DTPA. The results of these studies showed that
.sup.90Y-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 .sup.90Y-labeled 2B8-MX-DTPA suitable for use in
clinical trials.
REFERENCES
[0580] Each of the following citations is herein incorporated by
reference:
[0581] 1. Adams, R. A., Flowers, A., and Davis, B. J. Direct
Implantation and Transplantation of Human Acute Lymphoblastic
Leukemia in Hamsters, SB-2. CancerResearch28: 1121-1125, 1968.
[0582] 2. Adams, R. A. Formal Discussion: The Role of
Transplantation in the Experimental Investigation of Human Leukemia
and Lymphoma. Cancer Res. 27(1): 2479-2482, 1967.
[0583] 3. Lindmo, T., Boven, E., Cuttitta, F., Fedoroko, J., and
Bunn, P. A., J. Immunol. Methods, 72: 77-1984.
[0584] 4. Kozak, R. W., Raubitschek, A., Mirzadeh, S., Brechbiel,
M. W., Junghaus, R., Gansow, O. A., and Waldmann, T. A. Cancer Res.
(1989) 49:2639-2644.
[0585] 5. Parker, B. A., Halpern, S. E., Miller, R. A., Hupf, H.,
Shawler, D. L., Collins, H. A., Amox, D., White, C. A. and Royston,
I. N. Eng. J. Med., submitted.
[0586] 6. Leland, J. K. and Powell, M. J. J. (1990) Electrochem.
Soc. 137, 3127.
[0587] 7. Muller, R. J. Immunological Methods (1980) 34, 345.
[0588] 8. Mirzadeh, S., Brechbiel, M. W., Atcher, R. W. and Gansow,
O. A. (1990) Bioconjugate Chemistry 1(1), 59.
[0589] 9. Brechbiel, M. W., Gansow, O. A., Atcher, R. W., Sclom,
J., Esteban, J., Simpson, D. E. and Colcher, D. (1986) 25,
2772.
* * * * *