CA1202892A - Tumor imaging with radiolabeled monoclonal antibodies - Google Patents
Tumor imaging with radiolabeled monoclonal antibodiesInfo
- Publication number
- CA1202892A CA1202892A CA000414208A CA414208A CA1202892A CA 1202892 A CA1202892 A CA 1202892A CA 000414208 A CA000414208 A CA 000414208A CA 414208 A CA414208 A CA 414208A CA 1202892 A CA1202892 A CA 1202892A
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- Prior art keywords
- antibody
- chelating agent
- radionuclide
- group
- monoclonal antibody
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
- A61K51/1048—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell determinant being a carcino embryonic antigen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
- A61K51/1066—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
ABSTRACT
Described herein are monoclonal antibodies to tumor associated antigens to which are bound a metallic radionuclide, either directly or by means of a chelating agent such as di-ethylenetriaminepentaacetic acid (DTPA) conjugated to the antibody. When parenterally administered to a subject, the labeled monoclonal antibody localizes in the tumor and can be detected by photoscanning to detect emitted radiation.
Described herein are monoclonal antibodies to tumor associated antigens to which are bound a metallic radionuclide, either directly or by means of a chelating agent such as di-ethylenetriaminepentaacetic acid (DTPA) conjugated to the antibody. When parenterally administered to a subject, the labeled monoclonal antibody localizes in the tumor and can be detected by photoscanning to detect emitted radiation.
Description
1Z()2-~9~
This invention relates to the detection of tumors.
In another aspect it relates to monoclonal antibodies labeled with radionuclides.
A reliable techni~ue for the site-specific detection of tumors has long been sought~ Methods have been described for detecting, for example, carcinoembryonic antigen ~CEA) associated with human carcinoma circulating in the blood stream.
See United States 3,663,684 and United States 3,697,638. These methods obviously cannot be used to determine the tumor location.
More recently, it has been proposed to use antibodies labeled with radioactive isotopes of iodine to detect an antigenic substance associated with a tumor. Thus, in United States 3J927,193 a process using antibodies to CEA labeled with 125I and 131I is described. According to that patent, in experiments conducted using male Syrian hamsters in which human signet-ring cell carcinoma was introduced, injection of labeled goat anti-CEA
followed by e~in~tion of the animals' organs demonstrated that radioisotope localized in the tumor. Therefore, it was proposed that the location of a tumor in a human could be determined by
This invention relates to the detection of tumors.
In another aspect it relates to monoclonal antibodies labeled with radionuclides.
A reliable techni~ue for the site-specific detection of tumors has long been sought~ Methods have been described for detecting, for example, carcinoembryonic antigen ~CEA) associated with human carcinoma circulating in the blood stream.
See United States 3,663,684 and United States 3,697,638. These methods obviously cannot be used to determine the tumor location.
More recently, it has been proposed to use antibodies labeled with radioactive isotopes of iodine to detect an antigenic substance associated with a tumor. Thus, in United States 3J927,193 a process using antibodies to CEA labeled with 125I and 131I is described. According to that patent, in experiments conducted using male Syrian hamsters in which human signet-ring cell carcinoma was introduced, injection of labeled goat anti-CEA
followed by e~in~tion of the animals' organs demonstrated that radioisotope localized in the tumor. Therefore, it was proposed that the location of a tumor in a human could be determined by
2~ _ vivo administration of a parenteral solution of the antibody followed by photoscan~;n~ the host.
~;Z0~2~92 The actual use of radioiodinated antibodies has not met the expectations the early work generated. For example, Goldenberg et al, N. Eng. J. Med., 298 1384-1388 (1978) re-ported some success in 1978 in scanning humans with radioio-dinated anti-CEA. However, because of residual background radioactivity the limited success reported was dependent on the use of subtraction techniques. Mach et al, N. Eng. J.
Med., 303, 5-lO (1980) re~orted even less success and deter-mined that only 0.1% of the dose administered localized in the tumor. However, in selected cases, imaging the tumor was still possible.
It is well known that the affinity purification of heterologous antisera to obtain the antibodies used in prior art tumor imaging processes usually results in the loss of high affinity antibody and that the antibodies obtained, being a mix of specific antibodies for different determinants on the antigen, comprise largely antibodies of low affinity and anti-bodies which give non-specific reaction. Monoclonal atibod-ies, on the other hand, can be selected to exhibit high affinity to selected sites on the antigen and low non-specific binding~ However, surprisingly we have found that monoclonal antibodies to tumor antigens labeled with radioisotopes of iodine according to well-known procedures still show poor localization at the tumor site notwithstandinq the fact that in vitro immunoreact:ivity can be demonstrated. This may, and likely does, result from the loss of radioactive iodine by the labeled antibody. Accordingly, there still remains a need for a reliable technique for detecting, n vivo, the precise loca-tion of a tumor.
~:Z0289:~
Accordingly, one aspect of the invention provides a monoclonal antibody to a tumor associated antigen to which a metallic radionuclide is bound directly or through a chelating agent conjugated with the antibody.
Thus, accorcling to the present invention, a monoclonal antibody or antibody fragment to a tumor antigen is labeled either directly with a metallic radionuclide or with a chelate bound radionuclide, for example, DTPA bound lllIn, by conjugat-ing a chelating agent to the antibody followed by forming a complex between the chelating agent and radionuclide. The resulting labeled antibody, or fragment thereof, when injected into a tumor bearing host, localizes rapidly and with high specificity in the tumor itself. In some cases, localization in distant metastases may occur as well to indicate spread of the tumor. The location of the tumor can be detected by photoscanning techniques. Mixtures of labeled monoclonal antibodies can be used as well.
Another aspect of the present invention provides a process for the preparation of a monoclonal antibody to a tumor associated antigen to which a metallic radionuclide is bound directly or through a chelating agent conjugated with the antibody, which process comprises:
a) reacting the metallic radionuclide with the antibody to cause the radionuclide to bind directly to the antibody; or b) reacting the antibody with a chelating agent to conjugate the chelating agent to the antibody, and reacting the conjugate of antibody and chelating agent with the radio-nuclide to bind the radionuclide to the chelating agent.
A still further aspect of the invention provides a process for the preparation of a composition comprising a solution for parenteral administration of a monoclonal antibody ~j;" ~!
to a tumor associated antigen mentioned above, which process comprises admixing the monoclonal antibody with an agent suitable for parenteral administration of the antibody.
In the accompanying drawings:
Figure 1 is a group of graphs illustrating the distribution in tissue of lllIn and 125I compared with the distribution of 67Ga as a function of time after injection into nude mice infected with human colon cancer of anti-CEA
labeled with lllIn and 125I and 67G it Figure 2 is a group of graphs illustrating the amount of lllIn and 125I and 67Ga excreted by nude mi~e with increasing - 3a -~t time after injection of lllIn and 125I labeled anti-CEA and Ga citrate;
Figure 3 is a group of graphs illustrating the tissue distribution of lllIn and 1~5I in nude mice ~8 hours after in-jection of the In and 5I labeled anti-CEA compared to In citrate and 125I labeled human albumin; and Figure 4 is a group of graphs illustrating the tumor/
tissue ratios of lllIn and 125I compared with 67Ga, after in-jection of 1 In and 1 5I labeled anti-CEA and 67Ga citrate into nude mice, as a function of time.
As pointed out, according to the present invention, monoclonal antibodies or fragments thereof to tumor associated antigens are labeled directly with metallic radio-nuclides or with chelate bound radionuclides. The monoclonal antibodies useful in the invention are products of the hybridoma technology which is now well-known. Reference may be made, for example, to the original work of Milstein and Kohler rep~rted in Nature, 256, 495-497 (197S). Basically, the process involves injecting a mouse or other suitable animal with an immunogen, 2Q in this case a tumor associated antigen such as CEA. The immunized mouse is subsequently sacrificed and cells taken from its spleen are fused with myeloma cells to yield hybrid cells referred to as "hybridomas" which can be reproduced in vitro.
The population of cells producing antibodies is selectively cultured and screened to isolate individual clones, each of which secretes a single antibody specie specific to a particular region ("determinant") on the surface of the antigen molecule.
The individual clones can further be screened to deter-mine those producing antibodies of the highest affinity for the antigen and which exhibit low non-specific binding. The anti-body selected for radiolabeling (normally isolated from the ascites) is caused to react with the metallic radionuclide or is conjugated with a suitable chelating agent which is subse-quently used to bind the radionuclide. The chelating agent may be bound to the antibody using a wide variety of procedures.
In general, since the antibody is a polypeptide, a chelating agent having a reactive group of the kind known to be useful for introducing a moiety to a proteinaceous substrate can be used. Among suitable reactive groups can be mentioned the following:
,, ~ N
Ch NCS C~-- C - N
Ch \ ~ Ch - S02Cl Q
jO ,~
Ch \S / Ch - C - O - ~
o F~ F
Ch C o- < ~ F Ch N S02 CH CH2 O~ ~ F
Ch - N~ Ch - N2 (from Ch - NH2) O O
Ch - NH -C - CH2~r Ch- C - O -C- CH2C~(CH3)2 O O
wherein Ch is the residue of the chelating agent.
Suitable chelating agents include a wide variety of polydentate chelating agents whose ligands complex with metallic radionuclides. Among such agents may be mentioned the poly-aminocarboxylic acids of the formula:
HO2C (CH2)Y /(CH2)Y CO2H\ (CH2)Y C2H
N- C~I (CH2)z N - fH - (CH2)z N
H02C (CH2 ) y x (CH2 ) y C02H
(1) wherein x is O or an integer (x = 0 or x = 1 being preferred) y = 1 or 2 (Y = 1 being preferred) and z = an integer of from 1 to 7 (z = 1 or 7 being preferred) and wherein R is hydrogen or a group through which the chelating agent is conjugated with the antibody or a group which affects the binding constant of the chelating agent for the radionuclide. ~nong useful R
groups can be mentioned, in particular, the group - ~ -A
wherein A is the group through which conjugation is accomplished. For example, A may be -N2~ which is obtained by diazotization of the group -NH2, which itself may be obtained by reduction of a nitro (-NO2) group which, in its turn, can be obtained by nitration of the phenyl nucleus.
The group A may also be 0 , obtained by acetylation -NH-C-CH L
of the group -NH2 by known procedures, in which L is a leaving group displaced during conjugation such as a halogen, e.g., Cl, Br or I (Br being preferred).
In other instances R may be -CH3 or -CH2C6H4OCH2CO2H.
~;~0;2~2 In the case of the polyaminocarboxylic acids, conjuga-tion may be accomplished through the carboxylic acid group it-self by formation, for example, of an acid anhydride intermed-iate as taught by Krejcarek and Tucker, ~iochemical and Bio-physical Research Communications, 77, 581 (1977).
Of the polyaminocarboxylic acids, the polyaminoacetic acids are presently preferred. Specific polyaminocarboxylic acids which may be used include ethylenediaminotetraacetic acid (EDTA) and derivatives thereof such as diethylenetriaminopenta-acetic acid (DTPA), p-bromoacetamidophenyl (ethylenedinitrilo-tetraacetic) acid and l-(p-aminophenyl)-ethylenediaminotetra-acetic acid, the last of which is converted to a diazonium salt to permit conjugation. Of these chelating agents, DTPA is pre-sently preferred.
Among the other suitable chelating agents are polyamino (alkylene phosphoric) acids, particularly those of the formula:
.. O O
HO-P - (CH2) bH \ / 2 N- CH - (CH2~z N CH (CH2 ~ N
HO-P (CH2)y (CH2 ~ P-OH
OH OH
(2) wherein x, y, z and R are as previously defined. Preferably, x is 0 or 1/ y = 1, and z = 1. Specific polyamino ~methylene phosphoric) acids (y = 1) include ethylenediaminotetra (methy-lene phosphoric) acid, hexamethylenediaminetetra (methylene lZOX~8~Z
phosphoric) acid and diethylenetriaminepenta (methylene phos-phoric) acid for use in the invention.
Further chelating agents are the polyamines of the formula:
H2N (CH2~CH2 ~CH2 (CH2 )~NH~\ C~-(CH2~ Z NH2 N--CH--(CH2 zt N--IH ~CH2~
H2N (CH2 ~ CH2 CH ~ CH2)Z NH2
~;Z0~2~92 The actual use of radioiodinated antibodies has not met the expectations the early work generated. For example, Goldenberg et al, N. Eng. J. Med., 298 1384-1388 (1978) re-ported some success in 1978 in scanning humans with radioio-dinated anti-CEA. However, because of residual background radioactivity the limited success reported was dependent on the use of subtraction techniques. Mach et al, N. Eng. J.
Med., 303, 5-lO (1980) re~orted even less success and deter-mined that only 0.1% of the dose administered localized in the tumor. However, in selected cases, imaging the tumor was still possible.
It is well known that the affinity purification of heterologous antisera to obtain the antibodies used in prior art tumor imaging processes usually results in the loss of high affinity antibody and that the antibodies obtained, being a mix of specific antibodies for different determinants on the antigen, comprise largely antibodies of low affinity and anti-bodies which give non-specific reaction. Monoclonal atibod-ies, on the other hand, can be selected to exhibit high affinity to selected sites on the antigen and low non-specific binding~ However, surprisingly we have found that monoclonal antibodies to tumor antigens labeled with radioisotopes of iodine according to well-known procedures still show poor localization at the tumor site notwithstandinq the fact that in vitro immunoreact:ivity can be demonstrated. This may, and likely does, result from the loss of radioactive iodine by the labeled antibody. Accordingly, there still remains a need for a reliable technique for detecting, n vivo, the precise loca-tion of a tumor.
~:Z0289:~
Accordingly, one aspect of the invention provides a monoclonal antibody to a tumor associated antigen to which a metallic radionuclide is bound directly or through a chelating agent conjugated with the antibody.
Thus, accorcling to the present invention, a monoclonal antibody or antibody fragment to a tumor antigen is labeled either directly with a metallic radionuclide or with a chelate bound radionuclide, for example, DTPA bound lllIn, by conjugat-ing a chelating agent to the antibody followed by forming a complex between the chelating agent and radionuclide. The resulting labeled antibody, or fragment thereof, when injected into a tumor bearing host, localizes rapidly and with high specificity in the tumor itself. In some cases, localization in distant metastases may occur as well to indicate spread of the tumor. The location of the tumor can be detected by photoscanning techniques. Mixtures of labeled monoclonal antibodies can be used as well.
Another aspect of the present invention provides a process for the preparation of a monoclonal antibody to a tumor associated antigen to which a metallic radionuclide is bound directly or through a chelating agent conjugated with the antibody, which process comprises:
a) reacting the metallic radionuclide with the antibody to cause the radionuclide to bind directly to the antibody; or b) reacting the antibody with a chelating agent to conjugate the chelating agent to the antibody, and reacting the conjugate of antibody and chelating agent with the radio-nuclide to bind the radionuclide to the chelating agent.
A still further aspect of the invention provides a process for the preparation of a composition comprising a solution for parenteral administration of a monoclonal antibody ~j;" ~!
to a tumor associated antigen mentioned above, which process comprises admixing the monoclonal antibody with an agent suitable for parenteral administration of the antibody.
In the accompanying drawings:
Figure 1 is a group of graphs illustrating the distribution in tissue of lllIn and 125I compared with the distribution of 67Ga as a function of time after injection into nude mice infected with human colon cancer of anti-CEA
labeled with lllIn and 125I and 67G it Figure 2 is a group of graphs illustrating the amount of lllIn and 125I and 67Ga excreted by nude mi~e with increasing - 3a -~t time after injection of lllIn and 125I labeled anti-CEA and Ga citrate;
Figure 3 is a group of graphs illustrating the tissue distribution of lllIn and 1~5I in nude mice ~8 hours after in-jection of the In and 5I labeled anti-CEA compared to In citrate and 125I labeled human albumin; and Figure 4 is a group of graphs illustrating the tumor/
tissue ratios of lllIn and 125I compared with 67Ga, after in-jection of 1 In and 1 5I labeled anti-CEA and 67Ga citrate into nude mice, as a function of time.
As pointed out, according to the present invention, monoclonal antibodies or fragments thereof to tumor associated antigens are labeled directly with metallic radio-nuclides or with chelate bound radionuclides. The monoclonal antibodies useful in the invention are products of the hybridoma technology which is now well-known. Reference may be made, for example, to the original work of Milstein and Kohler rep~rted in Nature, 256, 495-497 (197S). Basically, the process involves injecting a mouse or other suitable animal with an immunogen, 2Q in this case a tumor associated antigen such as CEA. The immunized mouse is subsequently sacrificed and cells taken from its spleen are fused with myeloma cells to yield hybrid cells referred to as "hybridomas" which can be reproduced in vitro.
The population of cells producing antibodies is selectively cultured and screened to isolate individual clones, each of which secretes a single antibody specie specific to a particular region ("determinant") on the surface of the antigen molecule.
The individual clones can further be screened to deter-mine those producing antibodies of the highest affinity for the antigen and which exhibit low non-specific binding. The anti-body selected for radiolabeling (normally isolated from the ascites) is caused to react with the metallic radionuclide or is conjugated with a suitable chelating agent which is subse-quently used to bind the radionuclide. The chelating agent may be bound to the antibody using a wide variety of procedures.
In general, since the antibody is a polypeptide, a chelating agent having a reactive group of the kind known to be useful for introducing a moiety to a proteinaceous substrate can be used. Among suitable reactive groups can be mentioned the following:
,, ~ N
Ch NCS C~-- C - N
Ch \ ~ Ch - S02Cl Q
jO ,~
Ch \S / Ch - C - O - ~
o F~ F
Ch C o- < ~ F Ch N S02 CH CH2 O~ ~ F
Ch - N~ Ch - N2 (from Ch - NH2) O O
Ch - NH -C - CH2~r Ch- C - O -C- CH2C~(CH3)2 O O
wherein Ch is the residue of the chelating agent.
Suitable chelating agents include a wide variety of polydentate chelating agents whose ligands complex with metallic radionuclides. Among such agents may be mentioned the poly-aminocarboxylic acids of the formula:
HO2C (CH2)Y /(CH2)Y CO2H\ (CH2)Y C2H
N- C~I (CH2)z N - fH - (CH2)z N
H02C (CH2 ) y x (CH2 ) y C02H
(1) wherein x is O or an integer (x = 0 or x = 1 being preferred) y = 1 or 2 (Y = 1 being preferred) and z = an integer of from 1 to 7 (z = 1 or 7 being preferred) and wherein R is hydrogen or a group through which the chelating agent is conjugated with the antibody or a group which affects the binding constant of the chelating agent for the radionuclide. ~nong useful R
groups can be mentioned, in particular, the group - ~ -A
wherein A is the group through which conjugation is accomplished. For example, A may be -N2~ which is obtained by diazotization of the group -NH2, which itself may be obtained by reduction of a nitro (-NO2) group which, in its turn, can be obtained by nitration of the phenyl nucleus.
The group A may also be 0 , obtained by acetylation -NH-C-CH L
of the group -NH2 by known procedures, in which L is a leaving group displaced during conjugation such as a halogen, e.g., Cl, Br or I (Br being preferred).
In other instances R may be -CH3 or -CH2C6H4OCH2CO2H.
~;~0;2~2 In the case of the polyaminocarboxylic acids, conjuga-tion may be accomplished through the carboxylic acid group it-self by formation, for example, of an acid anhydride intermed-iate as taught by Krejcarek and Tucker, ~iochemical and Bio-physical Research Communications, 77, 581 (1977).
Of the polyaminocarboxylic acids, the polyaminoacetic acids are presently preferred. Specific polyaminocarboxylic acids which may be used include ethylenediaminotetraacetic acid (EDTA) and derivatives thereof such as diethylenetriaminopenta-acetic acid (DTPA), p-bromoacetamidophenyl (ethylenedinitrilo-tetraacetic) acid and l-(p-aminophenyl)-ethylenediaminotetra-acetic acid, the last of which is converted to a diazonium salt to permit conjugation. Of these chelating agents, DTPA is pre-sently preferred.
Among the other suitable chelating agents are polyamino (alkylene phosphoric) acids, particularly those of the formula:
.. O O
HO-P - (CH2) bH \ / 2 N- CH - (CH2~z N CH (CH2 ~ N
HO-P (CH2)y (CH2 ~ P-OH
OH OH
(2) wherein x, y, z and R are as previously defined. Preferably, x is 0 or 1/ y = 1, and z = 1. Specific polyamino ~methylene phosphoric) acids (y = 1) include ethylenediaminotetra (methy-lene phosphoric) acid, hexamethylenediaminetetra (methylene lZOX~8~Z
phosphoric) acid and diethylenetriaminepenta (methylene phos-phoric) acid for use in the invention.
Further chelating agents are the polyamines of the formula:
H2N (CH2~CH2 ~CH2 (CH2 )~NH~\ C~-(CH2~ Z NH2 N--CH--(CH2 zt N--IH ~CH2~
H2N (CH2 ~ CH2 CH ~ CH2)Z NH2
(3) wherein X, Z and R are as previously defined.
Another class of chelating agents are those of the formula:
OH HO
--CH2 ~ CH2 N--CH--CH --N
o ~ 1 2 ~ o ~ / R
OH OH
Another class of chelating agents are those of the formula:
OH HO
--CH2 ~ CH2 N--CH--CH --N
o ~ 1 2 ~ o ~ / R
OH OH
(4) wherein R is as previously defined, except that when R is not a group which permits conjugation, at least one of the phenol groups is a group having the formula ~ wherein A is as pre~viously defined. OH
The porphyrins represent another particularly useful class of chelating agents. Those useful in the invention in-clude synthetic and naturally occurring porphyrins including ~, ~2~)Z8~Z
those porphyrins of the fo~mula:
R~ Rl R\ ~ C/ R
N
N N ~ 1 R
C ~ C
/ \ / R
R ~
Rl Rl
The porphyrins represent another particularly useful class of chelating agents. Those useful in the invention in-clude synthetic and naturally occurring porphyrins including ~, ~2~)Z8~Z
those porphyrins of the fo~mula:
R~ Rl R\ ~ C/ R
N
N N ~ 1 R
C ~ C
/ \ / R
R ~
Rl Rl
(5) wherein R is as previously defined and Rl is hydrogen or a sub-stituent occuring as a part of a naturally occurring porphyrin molecule or a group introduced during synthesis, for example, to allow conjugation or to affect the binding constant of the porphyrin. Preferred porphyrins are tetraphenylporphyrin (R =
phenyl, Rl = H) or tetrapyridylporphyrin (R = 4-pyridyl, Rl =
H). One or more of the phenyl or pyridyl groups is substituted with -NH2 or O , where L is as previously defined, or other groups to permit conjugation with the antibody. Sub-stitution will normally be at the para position for a phenyl group and the 2- position or 4- position of a pyridyl group.
Other chelating agents which may be used in the invention are the crown ethers and their cryptand analogues of _ g ~2~Z~Z
the general formulas:
R R R R
R~ ~ R ~ \H ~ ~R
R O O R N/~ ` ~N R
\~ ~
R R R R
phenyl, Rl = H) or tetrapyridylporphyrin (R = 4-pyridyl, Rl =
H). One or more of the phenyl or pyridyl groups is substituted with -NH2 or O , where L is as previously defined, or other groups to permit conjugation with the antibody. Sub-stitution will normally be at the para position for a phenyl group and the 2- position or 4- position of a pyridyl group.
Other chelating agents which may be used in the invention are the crown ethers and their cryptand analogues of _ g ~2~Z~Z
the general formulas:
R R R R
R~ ~ R ~ \H ~ ~R
R O O R N/~ ` ~N R
\~ ~
R R R R
(6) (7) wherein R is as previously defined. Alternatively, the group which is used to permit conjugation is incorporated directly into the crown ether or cryptand structure as illustrated by the following formula:
wherein A is as previously defined.
~20~a~
Derivatives of desferrioxamine B can also be employed as the chelating agent. These have the formula:
O OH O O OH
ll l ll ll l R-CH2-C-N - (CH2)5 NH-C - (CH ~ C-N
OH O O
.. ..
H2N----tCH2 ~ N-C--~CH~ ~ C-NH-- (CH2)5 (g) wherein R is as previously defined.
Derivatives of enterobactin are also useful ch~lating agents. Preferred are those of the formula:
OH OH
_~
/ \
OH OHfH2-O -C- CH- NH -C ~ - A
~ C NH - f H
O=C
O
. I
O CH2 - CH C=O
I
NH
~_= O
H
~ H
(9) wherein A is as previously defined.
rl ~02~
Carbocylic analogues of enterobactin are also suit-able. Among those can be mentioned compounds of the formula:
OH OH
C _HN - CH 2 l H2 fH2 H
C=O
¢~aH
(10 ) wherein A is as previously defined.
Other carbocyclic systems useful as chelating agents include those of the formula:
OH OH
H ~ ~O O o ~
~ C - O - CHCH2 - O - C- ~ A
ll H2 0=~
~ OH
(11) OH
120~
and the corresponding anilides wherein A is as previously de-fined.
Those skilled in the art will appreciate that the ring shown for the enterobactin and carbocyclic analogues may be further substituted by substituents which do not materially impair their capability of binding metallic radionuclides and that such substituents may be introduced for the purpose of providing sites for conjugation to the antibody.
Yet another group of useful chelating agents are the siloxanes of the formula:
O ~ OH CHR CHR CHR O ~ I
(CH2)3 i- o~ O - i (CH
CH3 (CH2)3 CH3 OH
(12) wherein R is as previously defined, except that when R is not a group which permits conjugation, at least one of the catechol groups is a group having the formula ~ A wherein A is as previously defined.
Those skilled in the art will appreciate that the foregoing list of chelating agents is not exhaustive but, rather, illustrative of suitable chelating agents.
The direct labeling of antibodies with metallic radio-nuclides may be carried out using known methods for introducinga metallic ion onto an antibody by its attachment to a site on the antibody itself. In some cases, the metallic radionuclide ~;~0~892 ~ay be incorporated by exposing the antibody, usually in solu-tion to the radionuclide in its appropriate oxidation state.
For example, a presently preferred technique involves the direct metallation of an antibody with l03Ru+3 by contacting the anti-body with a suitable ruthenium salt, for example, ruthenium tri-chloride, in a buffered solution. Among the buffering agents which may be used are potassium acetate, sodium bicarbonate and trihydroxyethylamine (Tris).
Another suitable technique involves the direct metal-lation of the antibody with chromium. Chromium radionuclidescan be introduced by admixing a chromate salt of the radionuc-lide, for example, sodium or potassium chromate, with the anti-body in solution.
In other circumstances, the antibody may be first chemically modified to accept the radionuclide and then reacted with it. For example, disulfide groups within the antibody may be reduced followe(~ by the formation of a -S-M or -S-M-S group wherein M is the radionuclide. For example, disulfide groups can be reduced using dithiothreitol, to first form a thiol group which can be caused to react with appropriate metal ions, for example, those of mercury, silver, zinc, lead or cadmium, in their oxide or salt form.
Xadionuclides which may be used in the invention are those which form stable complexes with polydentate chelating agents or, under suitable conditions, combine directly with the antibody. The radionuclides are selected to have a half life and other radiation characteristics which permit safe disposal and safe introduction into humans. A large number of radionuclides have been investigated for use in humans which would be useful in the present invention. These may be grouped l2a2~z as follows:
The porphyrin (formula 5) t crown ether (formula 6) and cryptand (formula 7) chelating agents are most useful for binding radionuclides having a +2 oxidation state. For exam-195m 57 i 57C 105Ag 67CU and 52Mn form stable complexes in their +2 oxidation state.
The chelating agents of formulas 1-5 and 9-12 form stable complexes with radionuclides having a +3 oxidation state. Among radionuclides which form stable complexes in the +3 oxidation state are 52Fe, lllIn 113mI 99m 68 67 Yb, Co, 7Tm, 166Tm, 146Gd 157Dy 95mNb 103R 97 99Rh, 101mRh and 201Tl. The chelating agents having catechol groups, formulas 9-12, are particularly suited for use with radionuclides that are highly mobile in vivo, particularly radionuclides of iron such as 52Fe.
Radionuclides which can directly bond to antibodies include the fOllowing 203Hg 197Hg 105Ag 203pb 97R 103 99Rh llmRh and 48Cr.
The most suitable radionuclides are gamma emitters with an energy between 100 and 400 keV having half lives in the range from about 5 hrs. to 5 days.
For reasons which are not clearly understood at this time, certain individual monoclonal antibodies to tumor anti-gens resist direct metallation. Thus, for incorporation of the radioisotope by this route it is necessary to carefully select specific antibodies. Therefore, we presently prefer to bind a radionuclide to an antibody using the more flexible approach of first conjugating a chelating agent to the antibody and then forming a complex with the radionuclide. In that regard, we particularly prefer to use In (T ~ = 2.8 days) as the ~, ;
~o~g~
radlonuclide in conjunction with DTPA as a chelating agent.
The process of the present invention can be employed for the detection of tumors using monoclonal antibodies to tumor associated antigens in general. In addition to CEA, melanoma associated antigens, ~-feto-protein, ferritin, human choriogonadotropin, zinc gylcinate marker, and prostatic acid phosphatase (PAP) can, for example, be employed as immunogens to stimulate the production of the desired monoclonal antibodies as described above. In actual use, the labeled antibody in a suitable parenteral solution is injected into the patient.
After sufficient time has passed, the patient is photoscanned to detect any site of localized radiation indicative of a tumor and/or one of its metastases.
The following experiments conducted with monoclonal antibody to CEA labeled with 125I and 111In demonstrate the advantage of using monoclonal antibodies labeled with chelate bound radionuclides for tumor imaging according to the present invention compared to processes relying upon iodine labeled antibodies.
1. Preparation of Radiolabeled Anti-CEA
Monoclonal anti-CEA, available from Hybritech, Inc., La Jolla, Ca., was labeled with 125I by using the method of Bolten and Hunter, Biochem. J., 133, 529-39 (1973). The product was passed through a Sephadex G-25 column and the final material contained less than 0.5% free iodide. Radiolabeled monoclonal antibody binding was greater than 80% with horse anti-mouse anti-body bound to sepharose and greater than 75% with CEA bound to sepharose.
~202~Si~
The same monoclonal anti-CEA was labeled with ~ n using conjugated DTPA as the chelating agent according to the procedure of Krejcarek and Tucker, Biochemical and Biophysical Research Communications, _, 581 (1977). The reaction product was purified by passage through a Sephadex G-75 column. Anti-gen binding was comparable to the exhibited by the 125I
labeled antibody. The process of labeling did not affect the affinity constant, Ka, for the antibody relative to unchelated antibody as determined by titration with CEA.
The in vitro stabilities of the labeled anti-CEA
antibodies were determined by incubating a portion of the material in a parenteral solution comprising sterile normal saline, U.S.P., at 37C for 72 hours and determining free radionuclide chromatographically. The antibody solutions were spotted on Baker alumina lB strips and developed in 50% aqueous acetone. The protein bound radionuclide remains at the origin while free radionuclide migrates. After the incubation period, the radioiodinated material showed less than 3% activity present as free iodide. Storage at 4C reduced this by 50%.
Under the same conditions the lllIn anti-CEA showed comparable stability.
2. Distribution Studies In Nude Mice Tissue distribution studies of the radiolabeled mono-clonal mouse anti-CEA were performed in nude mice bearing 0.5-1.0 gm human colon tumors. The mice ranged in weight from 17-28 gm (mean of about 23 gm). The tumors were initiated by subcutaneous injection of a mince containing CEA-producing human colon adenocarcinoma cells. Distribution studies were undertaken three weeks later. Two experiments were performed.
~o;~æ
In the first, 19 animals were divided into four groups of 4-6 and were injected intravenously (IV) with 0.1 ml of a sterile normal saline U.S.P. solution containing 0.5 microcurie of 125I
anti-CEA antibody (about 0.3 microgram antibody) and, as a control, carrier-free 67Ga citrate, a known non-specific human colon tumor imaging agent. Injections were made without anesthesia using a 100 microliter Hamilton syringe to insure accuracy. Animals in which the dose was partially infiltrated into the tail were rejected from the study. Following injection, the mice were placed in sterile metabolic cages having elevated wire mesh flooring for collection of urine and feces. Beneath the flooring, sterile 4x4 gauze was packed in such a manner that it could not be gnawed by the animals. The mice, maintained in a clean room at 40F with sterile food and water ad libitum, were sacrificed in groups at 4, 24, 48 and 72 hours afer the injection of tracer.
The mice were anesthetized with ether and blood samples were obtained by direct cardiac puncture. The mice were then sacrificed by cervical dislocation. During subsequent dissection, the tumor, liver, kidney, muscle, heart, lung, femur and spleen were taken, rinsed twice in water, once in 10% formalin, blotted dry and wet weighed on an analytical balance. The intact stomach and intestines were also removed and counted. Blood, muscle and bone were assumed to represent
wherein A is as previously defined.
~20~a~
Derivatives of desferrioxamine B can also be employed as the chelating agent. These have the formula:
O OH O O OH
ll l ll ll l R-CH2-C-N - (CH2)5 NH-C - (CH ~ C-N
OH O O
.. ..
H2N----tCH2 ~ N-C--~CH~ ~ C-NH-- (CH2)5 (g) wherein R is as previously defined.
Derivatives of enterobactin are also useful ch~lating agents. Preferred are those of the formula:
OH OH
_~
/ \
OH OHfH2-O -C- CH- NH -C ~ - A
~ C NH - f H
O=C
O
. I
O CH2 - CH C=O
I
NH
~_= O
H
~ H
(9) wherein A is as previously defined.
rl ~02~
Carbocylic analogues of enterobactin are also suit-able. Among those can be mentioned compounds of the formula:
OH OH
C _HN - CH 2 l H2 fH2 H
C=O
¢~aH
(10 ) wherein A is as previously defined.
Other carbocyclic systems useful as chelating agents include those of the formula:
OH OH
H ~ ~O O o ~
~ C - O - CHCH2 - O - C- ~ A
ll H2 0=~
~ OH
(11) OH
120~
and the corresponding anilides wherein A is as previously de-fined.
Those skilled in the art will appreciate that the ring shown for the enterobactin and carbocyclic analogues may be further substituted by substituents which do not materially impair their capability of binding metallic radionuclides and that such substituents may be introduced for the purpose of providing sites for conjugation to the antibody.
Yet another group of useful chelating agents are the siloxanes of the formula:
O ~ OH CHR CHR CHR O ~ I
(CH2)3 i- o~ O - i (CH
CH3 (CH2)3 CH3 OH
(12) wherein R is as previously defined, except that when R is not a group which permits conjugation, at least one of the catechol groups is a group having the formula ~ A wherein A is as previously defined.
Those skilled in the art will appreciate that the foregoing list of chelating agents is not exhaustive but, rather, illustrative of suitable chelating agents.
The direct labeling of antibodies with metallic radio-nuclides may be carried out using known methods for introducinga metallic ion onto an antibody by its attachment to a site on the antibody itself. In some cases, the metallic radionuclide ~;~0~892 ~ay be incorporated by exposing the antibody, usually in solu-tion to the radionuclide in its appropriate oxidation state.
For example, a presently preferred technique involves the direct metallation of an antibody with l03Ru+3 by contacting the anti-body with a suitable ruthenium salt, for example, ruthenium tri-chloride, in a buffered solution. Among the buffering agents which may be used are potassium acetate, sodium bicarbonate and trihydroxyethylamine (Tris).
Another suitable technique involves the direct metal-lation of the antibody with chromium. Chromium radionuclidescan be introduced by admixing a chromate salt of the radionuc-lide, for example, sodium or potassium chromate, with the anti-body in solution.
In other circumstances, the antibody may be first chemically modified to accept the radionuclide and then reacted with it. For example, disulfide groups within the antibody may be reduced followe(~ by the formation of a -S-M or -S-M-S group wherein M is the radionuclide. For example, disulfide groups can be reduced using dithiothreitol, to first form a thiol group which can be caused to react with appropriate metal ions, for example, those of mercury, silver, zinc, lead or cadmium, in their oxide or salt form.
Xadionuclides which may be used in the invention are those which form stable complexes with polydentate chelating agents or, under suitable conditions, combine directly with the antibody. The radionuclides are selected to have a half life and other radiation characteristics which permit safe disposal and safe introduction into humans. A large number of radionuclides have been investigated for use in humans which would be useful in the present invention. These may be grouped l2a2~z as follows:
The porphyrin (formula 5) t crown ether (formula 6) and cryptand (formula 7) chelating agents are most useful for binding radionuclides having a +2 oxidation state. For exam-195m 57 i 57C 105Ag 67CU and 52Mn form stable complexes in their +2 oxidation state.
The chelating agents of formulas 1-5 and 9-12 form stable complexes with radionuclides having a +3 oxidation state. Among radionuclides which form stable complexes in the +3 oxidation state are 52Fe, lllIn 113mI 99m 68 67 Yb, Co, 7Tm, 166Tm, 146Gd 157Dy 95mNb 103R 97 99Rh, 101mRh and 201Tl. The chelating agents having catechol groups, formulas 9-12, are particularly suited for use with radionuclides that are highly mobile in vivo, particularly radionuclides of iron such as 52Fe.
Radionuclides which can directly bond to antibodies include the fOllowing 203Hg 197Hg 105Ag 203pb 97R 103 99Rh llmRh and 48Cr.
The most suitable radionuclides are gamma emitters with an energy between 100 and 400 keV having half lives in the range from about 5 hrs. to 5 days.
For reasons which are not clearly understood at this time, certain individual monoclonal antibodies to tumor anti-gens resist direct metallation. Thus, for incorporation of the radioisotope by this route it is necessary to carefully select specific antibodies. Therefore, we presently prefer to bind a radionuclide to an antibody using the more flexible approach of first conjugating a chelating agent to the antibody and then forming a complex with the radionuclide. In that regard, we particularly prefer to use In (T ~ = 2.8 days) as the ~, ;
~o~g~
radlonuclide in conjunction with DTPA as a chelating agent.
The process of the present invention can be employed for the detection of tumors using monoclonal antibodies to tumor associated antigens in general. In addition to CEA, melanoma associated antigens, ~-feto-protein, ferritin, human choriogonadotropin, zinc gylcinate marker, and prostatic acid phosphatase (PAP) can, for example, be employed as immunogens to stimulate the production of the desired monoclonal antibodies as described above. In actual use, the labeled antibody in a suitable parenteral solution is injected into the patient.
After sufficient time has passed, the patient is photoscanned to detect any site of localized radiation indicative of a tumor and/or one of its metastases.
The following experiments conducted with monoclonal antibody to CEA labeled with 125I and 111In demonstrate the advantage of using monoclonal antibodies labeled with chelate bound radionuclides for tumor imaging according to the present invention compared to processes relying upon iodine labeled antibodies.
1. Preparation of Radiolabeled Anti-CEA
Monoclonal anti-CEA, available from Hybritech, Inc., La Jolla, Ca., was labeled with 125I by using the method of Bolten and Hunter, Biochem. J., 133, 529-39 (1973). The product was passed through a Sephadex G-25 column and the final material contained less than 0.5% free iodide. Radiolabeled monoclonal antibody binding was greater than 80% with horse anti-mouse anti-body bound to sepharose and greater than 75% with CEA bound to sepharose.
~202~Si~
The same monoclonal anti-CEA was labeled with ~ n using conjugated DTPA as the chelating agent according to the procedure of Krejcarek and Tucker, Biochemical and Biophysical Research Communications, _, 581 (1977). The reaction product was purified by passage through a Sephadex G-75 column. Anti-gen binding was comparable to the exhibited by the 125I
labeled antibody. The process of labeling did not affect the affinity constant, Ka, for the antibody relative to unchelated antibody as determined by titration with CEA.
The in vitro stabilities of the labeled anti-CEA
antibodies were determined by incubating a portion of the material in a parenteral solution comprising sterile normal saline, U.S.P., at 37C for 72 hours and determining free radionuclide chromatographically. The antibody solutions were spotted on Baker alumina lB strips and developed in 50% aqueous acetone. The protein bound radionuclide remains at the origin while free radionuclide migrates. After the incubation period, the radioiodinated material showed less than 3% activity present as free iodide. Storage at 4C reduced this by 50%.
Under the same conditions the lllIn anti-CEA showed comparable stability.
2. Distribution Studies In Nude Mice Tissue distribution studies of the radiolabeled mono-clonal mouse anti-CEA were performed in nude mice bearing 0.5-1.0 gm human colon tumors. The mice ranged in weight from 17-28 gm (mean of about 23 gm). The tumors were initiated by subcutaneous injection of a mince containing CEA-producing human colon adenocarcinoma cells. Distribution studies were undertaken three weeks later. Two experiments were performed.
~o;~æ
In the first, 19 animals were divided into four groups of 4-6 and were injected intravenously (IV) with 0.1 ml of a sterile normal saline U.S.P. solution containing 0.5 microcurie of 125I
anti-CEA antibody (about 0.3 microgram antibody) and, as a control, carrier-free 67Ga citrate, a known non-specific human colon tumor imaging agent. Injections were made without anesthesia using a 100 microliter Hamilton syringe to insure accuracy. Animals in which the dose was partially infiltrated into the tail were rejected from the study. Following injection, the mice were placed in sterile metabolic cages having elevated wire mesh flooring for collection of urine and feces. Beneath the flooring, sterile 4x4 gauze was packed in such a manner that it could not be gnawed by the animals. The mice, maintained in a clean room at 40F with sterile food and water ad libitum, were sacrificed in groups at 4, 24, 48 and 72 hours afer the injection of tracer.
The mice were anesthetized with ether and blood samples were obtained by direct cardiac puncture. The mice were then sacrificed by cervical dislocation. During subsequent dissection, the tumor, liver, kidney, muscle, heart, lung, femur and spleen were taken, rinsed twice in water, once in 10% formalin, blotted dry and wet weighed on an analytical balance. The intact stomach and intestines were also removed and counted. Blood, muscle and bone were assumed to represent
7%, 40% and 10% of the total body weight, respectively, for total organ uptake calculations. The other organs were weighed in their entirety. Radioactivity counting was performed in an auto Gamma well counter with windows set over the 27-35 keV
photopeak of 125I and the 93 keV photopeak of 57Ga. The counter was programmed to reject at 10 minutes or one million counts to ~202~
achieve statistical significance for those samples with lowest activity levels. Corrections for background and reciprocal contribution of iostopic activity between window settings was made by using prepared sources and solving simultaneous equations. The activity in the samples was then compared to standards of the injected material, with the results expressed in terms of percent injected dose per gram and percent injected dose per organ. Tumor to tissue ratios were calculated from the percent dose/gm data. Activity in the gut, urine and feces was calculated as percent of the total injected dose.
In the second study, 37 nude mice bearing tumors grown from the same human colon cancer were divided into five groups of 7-8 animals. One group served as a control and was simultaneously injected IV with 1 microcurie of 125I human serum albumin (0.01 mg HSA) and 3 microcuries carrier-free lllIn citrate (0.01 mg citrate) and sacrificed 48 hours after injec-tion. In the rPm~in;ng four groups, the animals received simultaneous injections of 3 microcuries of lllIn labeled anti~
CEA (1.5 microgram antibody) and 5 microcuries carrier-free 67Ga citrate. The groups were sacrificed at 4, 24, 48 and 72 hours, respectively. Tissue dissection, sample preparation for radio~
assay, counting techniques and calculations were performed as described above. In the case of lllIn, the spectrometer was set to count the 247 keV photopeak using a 40 keV window (210-250 keV) while Ga was counted with a 30 keV window overlapping the 93 keV photopeak. These settings resulted in less than 10%
reciprocal contributions of radioactivity.
As shown in Fig. 1, the concentrations of In anti-CEA localizing in the tumor increased steadily throughout the time scale of the experiment. Twenty-three percent of the ;
~20~9~
injected dose was accumulated by 72 hours and the results sug-gest that this level would have been even higher had later samp-ling been attempted. The blood level, by contrast, decreased throughout the time period. The liver showed a rapid uptake at four hours, followed by a period of little change, then an increase to 72 hours. The other tissues showed little variation over the time period studied.
The level of 67Ga in the tumor remained fairly con-stant over the course of the experiment, being about 1/4 that of lllIn anti-CEA at 72 hours. The blood level of 67Ga dropped steadily, while the liver concentration showed a pattern some-what similar to the lllIndium labeled antibody. Muscle concen-tration of 67Ga dropped until 48 hours then leveled, while the bone concentration remained the same throughout.
The quantity of iodide in the tumor peaked at four hours, being initially somewhat greater than the quantities of lllIndium, then decreased irregularly throughout the remainder of the experiment, being only 1/10 that of lllIn at 72 hours.
At thht time twice as much 67Ga appeared in the tumor as 125I.
The count rate of 125I in all of the tissues decreased rapidly from their maxima at four hours, and in some cases, reached background levels by 48 hours.
The excretion data (Fig. 2) show a steady increase in urinary lllIn between 24 and 72 hours, reaching a maximum of 14%
of the injected dose. The feces concentration of lllIn remained fairly constant at 24 and 48 hours, then increased to 17% at 72 hours, while the amount in the intestine remained relatively con-stant over the entire period, never exceeding 5% of the dose.
The 67Ga excretion pattern was somewhat similar to that of lllIn yet showed more tracer in the intestine and feces lZOZ~92 then was observed for lllIn and less in the urine.
Nearly 60% of the 125I radioactivity was eliminated in the urine during the flrst 24 hours, and 75% by 48 hours.
Another 5% of the 125I was lost in the feces. Thusl after 72 hours about 80% of the 125I initially injected had been excreted by the animal. Chromatography of the 125I in the urine showed most of it to be free iodide with a second species probably representing free 125I complexed to something other than a protein.
Fig. 3 presents the data for lllIn labeled anti-CEA
at 48 hours and compares it to lllIndium citrate and 1 5IHSA.
The lllIn citrate was used as a control for any Indium that might have dissociated from the antibody, while the 125IHSA was used as a non-specific protein control. The llIn anti-CEA
demonstrated a much different distribution pattern from the lllIn citrate, concentrating more effectively in the tumor by a factor of 10. Uptakes of the two tracers by the other tissues were also dissimilar. The tumor concentrations of 12 IHSA was 20% that of 111In anti-CEA, but still 50~ greater than that of 125I anti-CEA. The 125IHSA distribution in the non-tumor tissue is also much different from that of the lllIn anti-CEA product.
It is especially significant that 125IHSA, long considered a stable iodinated protein, showed nearly 60% excretion (43% into the urine) by 48 hours" remarkably consistent with the above finding that I anti-CEA was also found in urine. The urinary fraction probably represents free iodide.
The tumor/tissue ratios (Fig. 4) show lllIn anti-CEA
to be superior to 67Ga with respect to all tissues but blood, being about the same as 7Ga in that case. The I anti-CEA
ratios, while apparently preferable to those exhibited by lllIn ~.~0:28a~2 and 67Ga in some tissues, are misleading since they arise from low 125I levels in the normal tissues rather than from local-ization in the tumor.
In view of the foregoing experiments, 1 I anti-CEA
is clearly unsuitable for a tumor localizinq agent. The rapid and extensive dehalogenation decreases tumor tracer concentra-tion and raises the blood background prohibiting its use in imaging without sophisticated subtraction techniques. Indeed, at 72 hours the tumor contains twice the quantity of the non-specific 67Ga on a per gram basis as 12 I. Furthermore, thetumor/blood ratios are better for 67Ga than for the iodinated antibody. When one considers that 67Ga is considered inadequate for imaging adenocarcinoma of the colon, the difficulties encountered by Mach et al, N. Eng. J. Med., 303, 5-10 (1980~, are easily explained. These data usinq 125I unequivocally demonstrate that, in the absence of subtraction techniques, if 131I were used, very large doses of the 131I labeled material would have to be administered to achieve the necessary tumor concentrations for effective imaging. This would result in the patient receiving a high radiation dose due to the 8 day physical half life and 608 keV Beta component of 131I.
By contrast, the large percentage of the injected dose of lllIn anti-CEA acquired by the tumor, and the more favorable physical characteristic of lllIn permit the injection of lower quantities of radiopharmaceutical, while improving the image and minimizing the radiation dose to the patient.
The tumor-to-background ratios formed by lllIn anti-CEA are well within the range required for imaging purposes because of the large amount of radionuclide localization in the tumor. Further-more, the data suggest that the tumor acquires radiolabel thathad once been incorporated into, or adhered to, other tissues.
1~0~
Individual mice were photoscanned at 4, 24, 48 and 72 hours after injection of the l11In labeled anti-CEA using a Phogam H P Anger gamma camera. By 48 hours, the tumor was well defined without the need to resort to subtraction techniques.
At 72 hours, tumor definition was even further enhanced.
The in vivo stability and, therefore, suitability ror use in tumor imaging of a directly labeled monoclonal antibody is demonstrated by the following experiments using a monoclonal anti-melanoma labeled with 103Ru as compared to the same antibody labeled with 12 I.
1. Preparation of Ru Labeled Anti-Melanoma An aqueous solution of 220 ~1 of 1 3RuC13 (available from New England Nuclear, Inc.) was added to 100 ~1 of a satu-rated potassium acetate solution. The pH was adjusted to 5.3 with 70 ~1 of 10 M NaOH. This solution of radionuclide was added to 500 ~1 of a 0.5 ~g/~l solution of mouse anti-melanoma antibody and the mixture incubated for 1 hour at room tempera-ture. The pH was adjusted to 7.2 by addition o 20 ~1 of 10 M
NaOH and the preparation stored overnight at room temperature.
The unbound metal was separated from antibody bound metal by application of the reaction mixture to a Sephadex G-50 column and elution with phosphate buffered saline pH 7.2. Protein containing fractions were collected and pooled for ln vivo studies in mice.
2. Distribution Studies in Normal Mice Tissue distribution studies were done in normal BALB-C mice using the 103Ru labeled anti-melanoma prepared according to the oregoing procedure and 125I labeled anti-~202a~2 melanoma prepared in the same manner as described above for labeling anti-CEA.
The mice were segregated into two groups which were injected intravenously, respectively, with 125I labeled and 03Ru labeled antibody. The mice were sacrificed at 4, 24, 48, 72 and 96 hours, dissected and organ radioactivity determined as described above for the studies with monoclonal anti-CEA.
The tissue distribution of 125I and 103RU is shown in the table below.
TISSUE DISTRIBUTION OF I-125 AND Ru-103 FOLLO~ING INJECTION OF LABELED MELANOMA
ANTIBODY COMPARED AT VARIOUS TIMES IN NORMAL BALB-C MICE *
4 Hours 24 Hours 48 Hours 72 Hours 96 Hours % Dose/Organ % Dose/Organ % Dose/Organ % Dose/Organ % Dose/Organ I-125 Ru-103 I-125 Ru-103 I-125 Ru-103 I-125 Ru-103 I-125 Ru-103 BLOOD X 4604 28.3 1107 12.5 13.0 6.97 1108 5.54 1302 3.30 +s.d. 4.17 3.32 10.8 1.74 6.66 .550 6.88 o678 .929 .244 LIVER X 12.6 4801 5022 4209 5.27 47.2 4.54 41.2 3.86 39.3 Ts.à. 1.87 6.63 o864 4084 .904 6.42 1.03 4.14 O395 4.34 SPLEEN X 1.27 3.38 .520 2.64 .340 1.95 .284 1094 .252 1098 +s.d. .181 .467 .053 .498 .049 .248 .025 .311 .022 .263 KIDNEY X 1.94 7.11 o784 5015 o641 4021 o542 3.68 .460 3.21 +s.d. .135 .284 oO91 .352 .073 o284 o063 .203 .034 o188 HEART X .792 o629 O403 .327 .340 o301 o263 .236 o220 .207 Ts.d. .161 oll9 O075 ,049 .036 o017 o060 o051 .~37 .015 MUSCLE X 1502 7.61 1205 8.64 11.6 8,17 9.56 6.98 8.05 6.66 +s.d. 2.56 1.02 2.34 1.55 2048 1.88 1.00 o906 .641 ~482 FEMUR X o360 .346 o189 o253 .163 o241 .150 o252 .106 .200 +s.d. .040 o027 .046 .045 O035 .041 .019 .030 .014 o015 LUNG X 2O58 2.01 1.13 o817 o878 .612 1.17 .585 .855 .475 +s.d. .736 .464 .271 o133 .131 .079 .281 .082 .159 .075 INTESTINE X 23.8 7.72 5.61 6011 6.04 5051 5.22 4O70 3.94 4001 +s.d. 4.24 .646 3016 o817 .704 .460 .501 .523 .129 .296 ~IMAL WT. X 17.96 + 3.88 1603 + 1.68 14.8 + 1.79 14095 + 1.80 15.95 + 1.21 TABLE (cont d) 4 Hours ~ Hours 4` Hours 72 Hours ~6 Hours % Dose/Organ% se/Organ% D--se/Organ% ~ose/Organ% ~ose/Organ I-125 Ru-103I-_ 5 Ru-103 I-1 5 Ru-103 I-_25 Ru-103 I-_25 Ru-103 RANGE ~g)12.1 - 22.21406 - 19.8 11.6 - 17.1 13.1 - 18.Z 14.0 - 17.5 =Data represents mean ~X) of n animals + one standard deviation (sOdO) ~Blood estimated to be 7% muscle 40% of animal weight P~ ~
J
~2o2a~æ
The data in the table demonstrate that the 125I
labeled anti-melanoma rapidly dehalogenat~d, just as occurred with 125I labeled anti-CEA, whereas the 103Ru labeled anti-melanoma was stable. These observations are confirmed by the fact that the level of 125I in the blood is very high 4 hours after injection and drops to low, but constant levels from 24-96 hours. By contrast, the level of 103Ru is relatively rapidly cleared from the blood and concentrates in the liver as would be expected in a non-tumor infected host. Spleen and kidney levels for the mice injected with 1O3RU labeled anti-melanoma are also consistent with ln vivo stability.
It is well known that CEA and some other tumor as;o-ciated antigens are secreted or shed by the tumor. In such cases, the incidence of background radiation arising because the antigen circulating in the blood or other tissue combines with labeled antibody can be reduced by initially administering un-labeled antibody to combine with circulating antigen. This would also tend to reduce the high liver uptake of labeled anti-body. After a suitable period, labeled antibody is added and the subject photoscanned to determine the site of localized radia-tion.
While the foregoing discussion has stressed the use of a single monoclonal antibody labeled with a radionuclide, it is within the scope of the invention to use two or even more labeled monoclonal antibodies for different antigenic determin-ants. The different antibodies are selected to be non-inter-fering with the binding to the antigen of another antibody.
By using plural, non-interfering labeled antibodies, the tumor image can be enhanced since the antigen will have a higher capacity for labeled antibody. The use of plural antibodies is particularly useful for imaging of a tumor whose associated anti-i,~
~L20Z89Z
gen is present in a low concentration.
The foregoing description of the invention relates to presently preferred embodiments and it is contemplated that other variations will be apparent to those skilled in the art.
Accordingly, the scope of the present invention is intended to be limited only by the scope of the appended claims.
photopeak of 125I and the 93 keV photopeak of 57Ga. The counter was programmed to reject at 10 minutes or one million counts to ~202~
achieve statistical significance for those samples with lowest activity levels. Corrections for background and reciprocal contribution of iostopic activity between window settings was made by using prepared sources and solving simultaneous equations. The activity in the samples was then compared to standards of the injected material, with the results expressed in terms of percent injected dose per gram and percent injected dose per organ. Tumor to tissue ratios were calculated from the percent dose/gm data. Activity in the gut, urine and feces was calculated as percent of the total injected dose.
In the second study, 37 nude mice bearing tumors grown from the same human colon cancer were divided into five groups of 7-8 animals. One group served as a control and was simultaneously injected IV with 1 microcurie of 125I human serum albumin (0.01 mg HSA) and 3 microcuries carrier-free lllIn citrate (0.01 mg citrate) and sacrificed 48 hours after injec-tion. In the rPm~in;ng four groups, the animals received simultaneous injections of 3 microcuries of lllIn labeled anti~
CEA (1.5 microgram antibody) and 5 microcuries carrier-free 67Ga citrate. The groups were sacrificed at 4, 24, 48 and 72 hours, respectively. Tissue dissection, sample preparation for radio~
assay, counting techniques and calculations were performed as described above. In the case of lllIn, the spectrometer was set to count the 247 keV photopeak using a 40 keV window (210-250 keV) while Ga was counted with a 30 keV window overlapping the 93 keV photopeak. These settings resulted in less than 10%
reciprocal contributions of radioactivity.
As shown in Fig. 1, the concentrations of In anti-CEA localizing in the tumor increased steadily throughout the time scale of the experiment. Twenty-three percent of the ;
~20~9~
injected dose was accumulated by 72 hours and the results sug-gest that this level would have been even higher had later samp-ling been attempted. The blood level, by contrast, decreased throughout the time period. The liver showed a rapid uptake at four hours, followed by a period of little change, then an increase to 72 hours. The other tissues showed little variation over the time period studied.
The level of 67Ga in the tumor remained fairly con-stant over the course of the experiment, being about 1/4 that of lllIn anti-CEA at 72 hours. The blood level of 67Ga dropped steadily, while the liver concentration showed a pattern some-what similar to the lllIndium labeled antibody. Muscle concen-tration of 67Ga dropped until 48 hours then leveled, while the bone concentration remained the same throughout.
The quantity of iodide in the tumor peaked at four hours, being initially somewhat greater than the quantities of lllIndium, then decreased irregularly throughout the remainder of the experiment, being only 1/10 that of lllIn at 72 hours.
At thht time twice as much 67Ga appeared in the tumor as 125I.
The count rate of 125I in all of the tissues decreased rapidly from their maxima at four hours, and in some cases, reached background levels by 48 hours.
The excretion data (Fig. 2) show a steady increase in urinary lllIn between 24 and 72 hours, reaching a maximum of 14%
of the injected dose. The feces concentration of lllIn remained fairly constant at 24 and 48 hours, then increased to 17% at 72 hours, while the amount in the intestine remained relatively con-stant over the entire period, never exceeding 5% of the dose.
The 67Ga excretion pattern was somewhat similar to that of lllIn yet showed more tracer in the intestine and feces lZOZ~92 then was observed for lllIn and less in the urine.
Nearly 60% of the 125I radioactivity was eliminated in the urine during the flrst 24 hours, and 75% by 48 hours.
Another 5% of the 125I was lost in the feces. Thusl after 72 hours about 80% of the 125I initially injected had been excreted by the animal. Chromatography of the 125I in the urine showed most of it to be free iodide with a second species probably representing free 125I complexed to something other than a protein.
Fig. 3 presents the data for lllIn labeled anti-CEA
at 48 hours and compares it to lllIndium citrate and 1 5IHSA.
The lllIn citrate was used as a control for any Indium that might have dissociated from the antibody, while the 125IHSA was used as a non-specific protein control. The llIn anti-CEA
demonstrated a much different distribution pattern from the lllIn citrate, concentrating more effectively in the tumor by a factor of 10. Uptakes of the two tracers by the other tissues were also dissimilar. The tumor concentrations of 12 IHSA was 20% that of 111In anti-CEA, but still 50~ greater than that of 125I anti-CEA. The 125IHSA distribution in the non-tumor tissue is also much different from that of the lllIn anti-CEA product.
It is especially significant that 125IHSA, long considered a stable iodinated protein, showed nearly 60% excretion (43% into the urine) by 48 hours" remarkably consistent with the above finding that I anti-CEA was also found in urine. The urinary fraction probably represents free iodide.
The tumor/tissue ratios (Fig. 4) show lllIn anti-CEA
to be superior to 67Ga with respect to all tissues but blood, being about the same as 7Ga in that case. The I anti-CEA
ratios, while apparently preferable to those exhibited by lllIn ~.~0:28a~2 and 67Ga in some tissues, are misleading since they arise from low 125I levels in the normal tissues rather than from local-ization in the tumor.
In view of the foregoing experiments, 1 I anti-CEA
is clearly unsuitable for a tumor localizinq agent. The rapid and extensive dehalogenation decreases tumor tracer concentra-tion and raises the blood background prohibiting its use in imaging without sophisticated subtraction techniques. Indeed, at 72 hours the tumor contains twice the quantity of the non-specific 67Ga on a per gram basis as 12 I. Furthermore, thetumor/blood ratios are better for 67Ga than for the iodinated antibody. When one considers that 67Ga is considered inadequate for imaging adenocarcinoma of the colon, the difficulties encountered by Mach et al, N. Eng. J. Med., 303, 5-10 (1980~, are easily explained. These data usinq 125I unequivocally demonstrate that, in the absence of subtraction techniques, if 131I were used, very large doses of the 131I labeled material would have to be administered to achieve the necessary tumor concentrations for effective imaging. This would result in the patient receiving a high radiation dose due to the 8 day physical half life and 608 keV Beta component of 131I.
By contrast, the large percentage of the injected dose of lllIn anti-CEA acquired by the tumor, and the more favorable physical characteristic of lllIn permit the injection of lower quantities of radiopharmaceutical, while improving the image and minimizing the radiation dose to the patient.
The tumor-to-background ratios formed by lllIn anti-CEA are well within the range required for imaging purposes because of the large amount of radionuclide localization in the tumor. Further-more, the data suggest that the tumor acquires radiolabel thathad once been incorporated into, or adhered to, other tissues.
1~0~
Individual mice were photoscanned at 4, 24, 48 and 72 hours after injection of the l11In labeled anti-CEA using a Phogam H P Anger gamma camera. By 48 hours, the tumor was well defined without the need to resort to subtraction techniques.
At 72 hours, tumor definition was even further enhanced.
The in vivo stability and, therefore, suitability ror use in tumor imaging of a directly labeled monoclonal antibody is demonstrated by the following experiments using a monoclonal anti-melanoma labeled with 103Ru as compared to the same antibody labeled with 12 I.
1. Preparation of Ru Labeled Anti-Melanoma An aqueous solution of 220 ~1 of 1 3RuC13 (available from New England Nuclear, Inc.) was added to 100 ~1 of a satu-rated potassium acetate solution. The pH was adjusted to 5.3 with 70 ~1 of 10 M NaOH. This solution of radionuclide was added to 500 ~1 of a 0.5 ~g/~l solution of mouse anti-melanoma antibody and the mixture incubated for 1 hour at room tempera-ture. The pH was adjusted to 7.2 by addition o 20 ~1 of 10 M
NaOH and the preparation stored overnight at room temperature.
The unbound metal was separated from antibody bound metal by application of the reaction mixture to a Sephadex G-50 column and elution with phosphate buffered saline pH 7.2. Protein containing fractions were collected and pooled for ln vivo studies in mice.
2. Distribution Studies in Normal Mice Tissue distribution studies were done in normal BALB-C mice using the 103Ru labeled anti-melanoma prepared according to the oregoing procedure and 125I labeled anti-~202a~2 melanoma prepared in the same manner as described above for labeling anti-CEA.
The mice were segregated into two groups which were injected intravenously, respectively, with 125I labeled and 03Ru labeled antibody. The mice were sacrificed at 4, 24, 48, 72 and 96 hours, dissected and organ radioactivity determined as described above for the studies with monoclonal anti-CEA.
The tissue distribution of 125I and 103RU is shown in the table below.
TISSUE DISTRIBUTION OF I-125 AND Ru-103 FOLLO~ING INJECTION OF LABELED MELANOMA
ANTIBODY COMPARED AT VARIOUS TIMES IN NORMAL BALB-C MICE *
4 Hours 24 Hours 48 Hours 72 Hours 96 Hours % Dose/Organ % Dose/Organ % Dose/Organ % Dose/Organ % Dose/Organ I-125 Ru-103 I-125 Ru-103 I-125 Ru-103 I-125 Ru-103 I-125 Ru-103 BLOOD X 4604 28.3 1107 12.5 13.0 6.97 1108 5.54 1302 3.30 +s.d. 4.17 3.32 10.8 1.74 6.66 .550 6.88 o678 .929 .244 LIVER X 12.6 4801 5022 4209 5.27 47.2 4.54 41.2 3.86 39.3 Ts.à. 1.87 6.63 o864 4084 .904 6.42 1.03 4.14 O395 4.34 SPLEEN X 1.27 3.38 .520 2.64 .340 1.95 .284 1094 .252 1098 +s.d. .181 .467 .053 .498 .049 .248 .025 .311 .022 .263 KIDNEY X 1.94 7.11 o784 5015 o641 4021 o542 3.68 .460 3.21 +s.d. .135 .284 oO91 .352 .073 o284 o063 .203 .034 o188 HEART X .792 o629 O403 .327 .340 o301 o263 .236 o220 .207 Ts.d. .161 oll9 O075 ,049 .036 o017 o060 o051 .~37 .015 MUSCLE X 1502 7.61 1205 8.64 11.6 8,17 9.56 6.98 8.05 6.66 +s.d. 2.56 1.02 2.34 1.55 2048 1.88 1.00 o906 .641 ~482 FEMUR X o360 .346 o189 o253 .163 o241 .150 o252 .106 .200 +s.d. .040 o027 .046 .045 O035 .041 .019 .030 .014 o015 LUNG X 2O58 2.01 1.13 o817 o878 .612 1.17 .585 .855 .475 +s.d. .736 .464 .271 o133 .131 .079 .281 .082 .159 .075 INTESTINE X 23.8 7.72 5.61 6011 6.04 5051 5.22 4O70 3.94 4001 +s.d. 4.24 .646 3016 o817 .704 .460 .501 .523 .129 .296 ~IMAL WT. X 17.96 + 3.88 1603 + 1.68 14.8 + 1.79 14095 + 1.80 15.95 + 1.21 TABLE (cont d) 4 Hours ~ Hours 4` Hours 72 Hours ~6 Hours % Dose/Organ% se/Organ% D--se/Organ% ~ose/Organ% ~ose/Organ I-125 Ru-103I-_ 5 Ru-103 I-1 5 Ru-103 I-_25 Ru-103 I-_25 Ru-103 RANGE ~g)12.1 - 22.21406 - 19.8 11.6 - 17.1 13.1 - 18.Z 14.0 - 17.5 =Data represents mean ~X) of n animals + one standard deviation (sOdO) ~Blood estimated to be 7% muscle 40% of animal weight P~ ~
J
~2o2a~æ
The data in the table demonstrate that the 125I
labeled anti-melanoma rapidly dehalogenat~d, just as occurred with 125I labeled anti-CEA, whereas the 103Ru labeled anti-melanoma was stable. These observations are confirmed by the fact that the level of 125I in the blood is very high 4 hours after injection and drops to low, but constant levels from 24-96 hours. By contrast, the level of 103Ru is relatively rapidly cleared from the blood and concentrates in the liver as would be expected in a non-tumor infected host. Spleen and kidney levels for the mice injected with 1O3RU labeled anti-melanoma are also consistent with ln vivo stability.
It is well known that CEA and some other tumor as;o-ciated antigens are secreted or shed by the tumor. In such cases, the incidence of background radiation arising because the antigen circulating in the blood or other tissue combines with labeled antibody can be reduced by initially administering un-labeled antibody to combine with circulating antigen. This would also tend to reduce the high liver uptake of labeled anti-body. After a suitable period, labeled antibody is added and the subject photoscanned to determine the site of localized radia-tion.
While the foregoing discussion has stressed the use of a single monoclonal antibody labeled with a radionuclide, it is within the scope of the invention to use two or even more labeled monoclonal antibodies for different antigenic determin-ants. The different antibodies are selected to be non-inter-fering with the binding to the antigen of another antibody.
By using plural, non-interfering labeled antibodies, the tumor image can be enhanced since the antigen will have a higher capacity for labeled antibody. The use of plural antibodies is particularly useful for imaging of a tumor whose associated anti-i,~
~L20Z89Z
gen is present in a low concentration.
The foregoing description of the invention relates to presently preferred embodiments and it is contemplated that other variations will be apparent to those skilled in the art.
Accordingly, the scope of the present invention is intended to be limited only by the scope of the appended claims.
Claims (30)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A monoclonal antibody to a tumor associated antigen to which a metallic radionuclide is bound directly or through a chelating agent conjugated with the antibody.
2. A monoclonal antibody according to claim 1 wherein the chelating agent is selected from polyaminocarboxylic acids, polyamino (alkylene phosphoric) acids, polyamines, porphyrins, crown ethers, cryptands, desferrioxamine B and derivatives thereof, and enterobactins and carbocyclic analogues thereof.
3. A monoclonal antibody according to claim 2 wherein the chelating agent is a polyaminocarboxylic acid chelating agent of the formula wherein x is 0 or an integer, y is 1 or 2, z is an integer from 1-7 and R is hydrogen or a group through which the chelating agent is conjugated with the antibody or a group which affects the binding constant of the chelating agent for the radionuclide.
4. A monoclonal antibody according to claim 2 wherein the polyamino (alkylene phosphoric) acid is of the formula wherein X is 0 or an integer, y is 1 or 2, z is an integer from 1-7 and R is hydrogen or a group through which the chelating agent is conjugated with the antibody or a group which affects the binding constant of the chelating agent for the radionuclide.
5. A monoclonal antibody according to claim 3 or 4 wherein R is selected from hydrogen, methyl, -CH2C6H4OCH2CO2H, and -C6H4-A wherein A is a diazonium salt or precursor thereof, or wherein L is a group displaced during conjuga-tion.
6. A monoclonal antibody according to claim 3 or 4 where-in X is 0 and R is
7. A monoclonal antibody according to claim 3 wherein the chelating agent is a polyaminocarboxylic acid and X is 1 and R is
8. A monoclonal antibody according to claim 2 wherein the chelating agent is selected from the group consisting of ethylenediaminotetraacetic acid, p-bromoacetamidodiphenyl (ethylenedinitrilotetraacetic acid) 1-(p-aminophenyl)-ethylenediaminotetraacetic acid and diethylenetriaminopenta-acetic acid.
9. A monoclonal antibody according to claim 1, 2 or 3 wherein the radionuclide is a gamma-emitter having a half life of from 5 hours to 5 days and whose gamma emissions have an energy of from 100-400 keV.
10. A monoclonal antibody according to claim 4, 7 or 8 wherein the radionuclide is a gamma-emitter having a half life of from 5 hours to 5 days and whose gamma emissions have an energy of from 100-400 keV.
11. A monoclonal antibody according to claim 1, 2 or 3 wherein the radionuclide is selected from 195mPt, 57Ni, 57Co, 105Ag, 67Cu, 52Mn, 52Fe, 111In, 113mIn, 99mTc, 67Ga, 68Ga, 169Yb, 166Tm, 167Tm, 146Gd, 157Dy, 95mNb, 103Ru, 97Ru, 99Ru, 101mRh, and 201T1, 203Hg, 197Hg, 105Hg, 203Pb, 99Rh and 48Cr.
12. A monoclonal antibody according to claim 4, 7 or 8 wherein the radionuclide is selected from 195mPt, 57Ni, 57Co, 105Ag, 67Cu, 52Mn, 52Fe, 111In, 113mIn, 99mTc, 67Ga, 68Ga, 169Yb, 166Tm, 167Tm, 146Gd, 157Dy, 95mNb, 103Ru, 97Ru, 99Ru, 101mRh, and 201Tl, 203Hg, 197Hg, 105Hg, 203Pb, 99Rh and 48Cr.
13. A monoclonal antibody according to claim 1, 2 or 3 wherein the tumor associated antigen is one of the group, carcinoembryonic antigen, .alpha.-feto-protein, ferritin, human choriogonadotropin, zinc glycinate marker, prostatic acid phosphatase and melanoma associated antigens.
14. A monoclonal antibody according to claim 4, 7 or 8 wherein the tumor associated antigen is one of the group, carcinoembryonic antigen, .alpha.-feto-protein, ferritin, human choriogonadotropin, zinc glycinate marker, prostatic acid phosphatase and melanoma associated antigens.
15. A composition comprising a solution for parenteral administration of a monoclonal antibody to a tumor associated antigen according to claim 1.
16. A composition comprising a solution for parenteral administration of a monoclonal antibody to a tumor associated antigen according to claim 2, 3 or 4.
17. A composition comprising a solution for parenteral administration of a monoclonal antibody to a tumor associated antigen according to claim 7 or 8.
18. A composition according to claim 15 comprising a mixture of two or more of said monoclonal antibodies.
19. A process for the preparation of a monoclonal anti-body to a tumor associated antigen to which a metallic radio-nuclide is bound directly or through a chelating agent con-jugated with the antibody, which process comprises:
a) reacting the metallic radionuclide with the antibody to cause the radionuclide to bind directly to the antibody; or b) reacting the antibody with a chelating agent to con-jugate the chelating agent to the antibody, and reacting the conjugate of antibody and chelating agent with the radionuclide to bind the radionuclide to the chelating agent.
a) reacting the metallic radionuclide with the antibody to cause the radionuclide to bind directly to the antibody; or b) reacting the antibody with a chelating agent to con-jugate the chelating agent to the antibody, and reacting the conjugate of antibody and chelating agent with the radionuclide to bind the radionuclide to the chelating agent.
20. A process according to claim 19, wherein the chelat-ing agent is selected from polyaminocarboxylic acids, polyamino (alkylene phosphoric) acids, polyamines, porphyrins, crown ethers, cryptands, desferrioxamine B and derivatives thereof, and enterobactins and carbocyclic analogues thereof.
21. A process according to claim 20 wherein the chelat-ing agent is a polyaminocarboxylic acid chelating agent of the formula wherein x is 0 or an integer, y is 1 or 2, z is an integer from 1-7 and R is hydrogen or a group through which the chel-ating agent is conjugated with the antibody or a group which affects the binding constant of the chelating agent for the radionuclide.
22. A process according to claim 20 wherein the poly-amino (alkylene phosphoric) acid is of the formula wherein X is 0 or an integer, y is 1 or 2, z is an integer from 1-7 and R is hydrogen or a group through which the chel-ating agent is conjugated with the antibody or a group which affects the binding constant of the chelating agent for the radionuclide.
23. A process according to claim 21 or 22 wherein R is selected from hydrogen, methyl, -CH2C6H4OCH2CO2H, and -C6H4-A
wherein A is a diazonium salt or precursor thereof, or wherein L is a group displaced during conjugation.
wherein A is a diazonium salt or precursor thereof, or wherein L is a group displaced during conjugation.
24. A process according to claim 21 or 22 wherein X is 0 and R is
25. A process according to claim 21 wherein the chelat-ing agent is a polyaminocarboxylic acid and X is 1 and R is
26. A process according to claim 20 wherein the chelat-ing agent is selected from the group consisting of ethylene-diaminotetraacetic acid, p-bromoacetamidodiphenyl (ethylene-dinitrilotetraacetic acid) 1-(p-aminophenyl)-ethylenedi-aminotetraacetic acid and diethylenetriaminopentaacetic acid.
27. A process according to claim 19, 20 or 21 wherein the radionuclide is a gamma-emitter having a half life of from 5 hours to 5 days and whose gamma emissions have an energy of from 100-400 keV.
28. A process according to claim 19, 20 or 21 wherein the radionuclide is selected from 195mPt, 57Ni, 57Co, 105Ag, 67Cu, 52Mn, 52Fe, 111In, 113mIn, 99mTc, 67Ga, 68Ga, 169Yb, 166Tm, 167Tm, 146Gd, 157Dy, 95mNb, 103Ru, 97Ru, 99Ru, 101mRh, and 201T1, 203Hg, 197Hg, 105Hg, 203Pb, 99Rh and 48Cr.
29. A process according to claim 19, 20 or 21 wherein the tumor associated antigen is one of the group, carcino-embryonic antigen, .alpha.-feto-protein, ferritin, human chorio-gonadotropin, zinc glycinate marker, prostatic acid phos-phatase and melanoma associated antigens.
30. A process for the preparation of a composition com-prising a solution for parenteral administration of a mono-clonal antibody to a tumor associated antigen according to claim 1, which process comprises admixing the monoclonal anti-body with an agent suitable for parenteral administration of the antibody.
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| US315,286 | 1981-10-27 |
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| CA000414208A Expired CA1202892A (en) | 1981-10-27 | 1982-10-26 | Tumor imaging with radiolabeled monoclonal antibodies |
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| AT (1) | AT392004B (en) |
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| US5688488A (en) * | 1989-04-03 | 1997-11-18 | Purdue Research Foundation | Composition and method for tumor imaging |
| US5792444A (en) * | 1989-05-09 | 1998-08-11 | The General Hospital Corporation | Labeled chemotactic peptides to image focal sites of infection or inflammation |
| US5807535A (en) * | 1992-07-31 | 1998-09-15 | Australian Nuclear Science & Technology Organisation | Metal complexes of hydroxyaryl containing aminocarboxylic acid chelating agents |
| US5550160A (en) * | 1992-07-31 | 1996-08-27 | Australian Nuclear Science & Technology Organization | Metal complexes of hydroxyaryl containing aminocarboxylic acid chelating agents |
| US7521531B2 (en) | 1996-08-28 | 2009-04-21 | Immunomedics, Inc. | Methods for the purification of stable radioiodine conjugates |
| US6663866B1 (en) | 1996-08-28 | 2003-12-16 | Immunomedics, Inc. | Stable radioiodine conjugates and methods for their synthesis |
| US6558669B1 (en) | 1996-08-28 | 2003-05-06 | Immunomedics, Inc. | Stable radioiodine conjugates and methods for their synthesis |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4323546A (en) * | 1978-05-22 | 1982-04-06 | Nuc Med Inc. | Method and composition for cancer detection in humans |
| US4272503A (en) * | 1978-05-25 | 1981-06-09 | New England Nuclear Corporation | Reductant composition for technetium-99m and method for making technetium-99m labelled ligands |
| US4311688A (en) * | 1979-10-29 | 1982-01-19 | Serono Laboratories Inc. | Composition and method for cancer detection in humans |
| DE3173342D1 (en) * | 1980-03-03 | 1986-02-13 | Milton David Goldenberg | Agent for tumor localization and therapy with labeled antibodies and antibody fragments |
-
1982
- 1982-10-25 NO NO823530A patent/NO169947C/en unknown
- 1982-10-25 NL NL8204108A patent/NL8204108A/en not_active Application Discontinuation
- 1982-10-25 DE DE19823239410 patent/DE3239410A1/en active Granted
- 1982-10-25 ES ES516797A patent/ES516797A0/en active Granted
- 1982-10-25 AT AT0391682A patent/AT392004B/en not_active IP Right Cessation
- 1982-10-25 FI FI823633A patent/FI82379C/en not_active IP Right Cessation
- 1982-10-25 GB GB08230356A patent/GB2109407B/en not_active Expired
- 1982-10-25 FR FR8217800A patent/FR2515046B1/en not_active Expired
- 1982-10-26 IL IL67068A patent/IL67068A0/en not_active IP Right Cessation
- 1982-10-26 JP JP57188921A patent/JPS58135820A/en active Granted
- 1982-10-26 LU LU84441A patent/LU84441A1/en unknown
- 1982-10-26 ZA ZA827806A patent/ZA827806B/en unknown
- 1982-10-26 DK DK474182A patent/DK164682C/en active
- 1982-10-26 IT IT23932/82A patent/IT1153857B/en active
- 1982-10-26 CH CH6239/82A patent/CH653040A5/en not_active IP Right Cessation
- 1982-10-26 SE SE8206073A patent/SE8206073L/en not_active Application Discontinuation
- 1982-10-26 CA CA000414208A patent/CA1202892A/en not_active Expired
- 1982-10-27 BE BE0/209345A patent/BE894829A/en not_active IP Right Cessation
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1984
- 1984-01-31 ES ES529310A patent/ES8505482A1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| NO823530L (en) | 1983-04-28 |
| ZA827806B (en) | 1983-08-31 |
| LU84441A1 (en) | 1983-06-13 |
| ES529310A0 (en) | 1985-05-16 |
| JPS58135820A (en) | 1983-08-12 |
| SE8206073L (en) | 1983-04-28 |
| IT8223932A0 (en) | 1982-10-26 |
| DK474182A (en) | 1983-04-28 |
| DK164682B (en) | 1992-08-03 |
| ES8505482A1 (en) | 1985-05-16 |
| IT1153857B (en) | 1987-01-21 |
| NL8204108A (en) | 1983-05-16 |
| FI823633L (en) | 1983-04-28 |
| DE3239410C2 (en) | 1991-12-19 |
| FI82379B (en) | 1990-11-30 |
| BE894829A (en) | 1983-02-14 |
| DK164682C (en) | 1992-12-21 |
| JPH0564130B2 (en) | 1993-09-14 |
| ES8404857A1 (en) | 1984-05-16 |
| FR2515046A1 (en) | 1983-04-29 |
| DE3239410A1 (en) | 1983-05-19 |
| FI823633A0 (en) | 1982-10-25 |
| FR2515046B1 (en) | 1985-11-29 |
| ES516797A0 (en) | 1984-05-16 |
| CH653040A5 (en) | 1985-12-13 |
| ATA391682A (en) | 1990-07-15 |
| NO169947B (en) | 1992-05-18 |
| IL67068A0 (en) | 1983-02-23 |
| NO169947C (en) | 1992-08-26 |
| GB2109407A (en) | 1983-06-02 |
| AT392004B (en) | 1991-01-10 |
| SE8206073D0 (en) | 1982-10-26 |
| FI82379C (en) | 1991-03-11 |
| GB2109407B (en) | 1985-12-18 |
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