CA2077670C - Method for enhancing antibody transport through capillary barriers - Google Patents
Method for enhancing antibody transport through capillary barriers Download PDFInfo
- Publication number
- CA2077670C CA2077670C CA002077670A CA2077670A CA2077670C CA 2077670 C CA2077670 C CA 2077670C CA 002077670 A CA002077670 A CA 002077670A CA 2077670 A CA2077670 A CA 2077670A CA 2077670 C CA2077670 C CA 2077670C
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- cationized
- organ
- isoelectric point
- central nervous
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
- A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S424/00—Drug, bio-affecting and body treating compositions
- Y10S424/806—Drug, bio-affecting and body treating compositions involving IgM
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/863—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving IgM
Abstract
A method for increasing the transcytosis of an antibody across the microvascular barrier and into the interstitial fluid of organs is disclosed. The method consists of cationizing the antibody with a cationizing agent to increase the isoelectric point of the antibody by between about 1 to about 7 to produce a cationized antibody having an isoelectric point which is less than about 11.5. The increased rate of transport across the microvascular barrier of organs makes such canonized antibodies useful for both therapeutic and diagnostic purposes.
Description
,;~~W~ 91/13631 ~ ~ ~ ~ ~ ~ '~ PCT/1JS91/01533 r~r~ i METHOD FOR ENFIAIdCIIdG AI~TTIIBOIDY TRANSPQRT THROUGH
clIPILLARY B~rRRTHRS
BAOKGROUND OF THE INVENTION
1. Field of the Invention The present invention relates generally to the use of antibodies for treatmewt and diagnosis of diseases, most notably tumors and cancerous lesions. More particularly it relates to the modification and use of cationized antibodies for transport through capillary barriers into the interstitial fluid of organs.
clIPILLARY B~rRRTHRS
BAOKGROUND OF THE INVENTION
1. Field of the Invention The present invention relates generally to the use of antibodies for treatmewt and diagnosis of diseases, most notably tumors and cancerous lesions. More particularly it relates to the modification and use of cationized antibodies for transport through capillary barriers into the interstitial fluid of organs.
2. Description of Related Art Antibodies in general, and especially monoclonal antibodies, are widely used in diagnostic tests as a means for detecting the presence of specific antigens and in the treatment of diseases associated with a specific antigen. More particularly, antibodies have been used as targeting vehicles .for radioisotopes, magnetic resonance imaging agents, toxins and cytotoxic drugs, especially in the diagnosis and treatment of cancer, tumors, and certain infectious diseases.
Enzyme linked immunoassay and radioimmunoassay are common diagnostic techniques which utilize antibodies as targeting vehicles and detect antigens in vitro.
Antigens may also be detected in vivo by administering radiolabelled or paramagnetic labelled antibodies to a living subject followed by the external detection of the radiolabelled antibody sequestered by a particular organ bearing the respective antigen.
One of the limitations in using antibodies as targeting vehicles in either the in vivo 'treatment or diagnosis of cancer and infectious diseases has been the inability to obtain effective concentrations of the targeting antibody at the target site. The low antibody dose at the site is largely due to poor antibody uptake by the tumor or infected site. The poor uptake is due to the microvascular or endothelial barrier which is present in most organs. This endothelial barrier has WO 91/13631 ~ ~ ~:~ ~'~ ~ . PCT/1JS91/~133 ..':
pores which are too small to allow for rapid organ uptake of circulating antibodies. Also the small size of the aqueous pores in the walls of the vessels which perfuse organs greatly restricts antibody transport from the vessels into the organ.' Transport across the endothelial barrier is a particular problem for large plasma proteins, such as antibodies that have molecular weights in excess of 150,000 Daltons. These antibodies are excluded or cross the microvascular barrier only very slowly. Hot only does the size of these large antibodies restrict their transport across the endothelial barrier, but, their electrical charges also present transport problems.
More specifically, the molecules on the surface of capillaries are anionically charged and, therefore, present an electrical barrier to the neutral or slightly negatively charged antibodies.
Another limitation to an optimum concentration of targeting antibody at the target organ site is the '20 higher permeability of the liver and spleen vascular barrier. The liver and spleen do not exclude the transport of large molecules to the same degree as other organs. Consequently, these two organs will preferentially remove administered antibodies from the blood leaving only a small concentration for therapeutic or diagnostic delivery to other organs.
Since most of the radioisotopes or complexes used in targeting systems are somewhat toxic and dose limiting, merely increasing the dose of the antibody with the expectation that more will become available to the organ of interest is not a practical solution.
r Strategies have been developed to administer .
effective amounts of antibodies by an invasive regional route to the location of the tumor or diseased area.
This avoids a high concentration of a potentially toxic agent in the blood. For systemic administration, however, it is necessary to use methods which control or enhance the blood clearance of the targeted antibodies. Such techniques aid in avoiding toxic blood levels of radioisotopes or other therapeutic agents, but still require large doses of the antibody because of their restricted transport across capillary barriers.
Accordingly there presently is a need to provide an improved method for the diagnosis and treatment of cancer and infectious diseases which are responsive to antibodies used as target vehicles. Further, there is a need to provide improved methods for delivering effective amounts of antibodies to organ tissue without sustaining toxic amounts of the antibody target vehicle in the blood. There is also a need to provide improved means for transporting antibodies across the microvascular barrier of organs and into the interstitial pores of organs.
SUMMARY OF THE INVENTION
It is one object of the invention to provide chemically modified yet active antibodies for delivery to organ tissue in effective amounts for therapeutic or diagnostic applications. It is another objective to effectively deliver the chemically modified antibodies without maintaining toxic levels of the antibody target vehicle in the blood. Accordingly, the present invention provides a method for increasing the transytosis rate of an antibody across the microvascular barrier and into the interstitial fluid of organs. The invention is based upon the discovery that cationized antibodies have increased rates of delivery across organ vascular beds when compared with the transcytosis of antibodies which are not cationized.
The present invention also provides a system for increasing transcytosis of an antibody across microvascular 3a barrier and into interstitial fluid of mammalian non-central nervous system tissues and organs, comprising: a) an antibody which has been cationized by treatment with a sufficient amount of a can onization agent to increase the isoelectric point of the antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than 11.5; b) a suitable pharmaceutically acceptable diluent or carrier; and c) a means for delivering the cationized antibody in admixture with the diluent or carrier, to a site where the can onized antibody is transcytosed across the microvascular barrier and into the interstitial fluid of said organs.
The present invention also provides for the use of a cationized antibody for increasing transcytosis of said antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
The present invention also provides for the use of a cationized antibody for the manufacture of a medicament for the treatment of conditions where transcytosis of said antibody is desired across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
The present invention also provides a pharmaceutical composition comprising an antibody which has been cationized, in admixture with a suitable pharmaceutically acceptable diluent or carrier, for increasing transcytosis of said antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
The present invention also provides for a commercial package containing as an active ingredient a cationized antibody, together with instructions for its use for increasing transcytosis 3b of the antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
The effectiveness of antibodies for both diagnostic and therapeutic purposes is increased by cationizing the antibodies to provide can onized antibodies having elevated isoelectric points (pI). These antibodies WO 91/13631 ~ ~ ~ r~~ ~ ~ ~ . PCf/US91/01533 carry a net positive charge and have been found to cross microvascular barriers at rates which are much higher than the transcytosis rates for negatively charged or neutral antibodies which typically have isoelectric points in the range of 5 to 7. Isoelectric points for the cationized antibodies will vary depending upan the particular organ ar organs to which the antibody is targeted. Generally, however, it is desireable to raise the isoelectric point of the antibody by from about 2 to about 6 points. The resulting modified antibody preferably has an isoelectric point in the range of from about 8 to about 11.
The cationized antibodies in accordance with the present invention are prepared by treating a given mono clonal or polyclonal antibody with a cationizat:ion agent such as hexamethylenediamine. The amine cationization agent replaces surface carboxyl groups on the antibody with a more basic group, such as a primary amine group in the case of hexamethylenediamine and related amine compounds. The amount of cationization agent and reaction conditions are controlled so that the resulting cationized antibody has the desired isoelectric point of between from about 8 to about 11.
It is known that antibodies retain nearly 90% of their antigen binding properties following catonization.
Thus, the chemical process of cationization does not destroy the innate biologic properties of the antibody.
If preferred, however, the immunoreactive sites may be blocked prior to the cationizing process by reacting the antibody with an excess of the appropriate antigen.
These blocked immunoreactive sites are unreactive during the subsequent cationization steps. The antigens are .
then decoupled from the cationized antibodies after the cationization step to thereby reactivate the blocked immunoreactive sites.
The cationization and utilization of antibodies in accordance with the present invention is useful whenever ;~'~ 91/13631 ~ ~ ~ 4 ~ ~ ~~ PCT/US91/01533 ::;
,.:~;
it is necessary to introduce an antibody into the interstitial fluid of an organ. Both therapeutic and diagnostic uses for antibodies is contemplated.
Diagnostic uses include targeting a cationized antibody 5 carrying a radionuclide or a paramagneta.c label to a specific organ containing the antigen for that antibody.
Once the antibody and antigen are complexed, subsequent diagnostic techniques for the radionuclide or the paramagnetic label may be used to detect the antigen.
Therapeutic uses include targeting drugs to specific organs containing cancerous or diseased tissue. Such therapeutic utility contemplates using cationized antibodies which are antibodies for the antigen of interest as the carrying vehicle for the drug, The above discussed and many other features and attendant advantages of the present invention will become apparent as the invention becomes better understood by reference to the following detailed description.
BRIEF DESCRTPTION OF THE DRAWINGS
Fig. 1 is a plot of serum radioactivity (DPM/mL/%
injected) of [3H]-native albumin or ['H]-cationized IgG
versus time after a single intravenous injection of the isotope in the anesthetized rat.
Fig. 2 is a plot of the volume of distribution (vp) of [3H]-cationized IgG for liver, kidney, lung, and myocardium versus the time after single intravenous injection of the isotope in anesthetized rats.
Fig. 3 is a plat of serum [lzsl]-bovine serum albumin radioactivity and [~H]-cationized IgG radioactivity over a 60 minute period after a single intravenous injection of isotope in the anesthetized cynomologous monkey.
DETAILED DESCRIPTION OF THE INVENTION
Publications and other references will be referred to in this detailed description. For convenience, the reference materials are numerically referenced and grouped in the bibliography which is located at the end of the detailed description.
The present invention involves the transport of antibodies through the microvascular barrier or organs. The invention has wide application to any antibody which is useful as a targeting vehicle in diagnosing or treating cancers, tumors, or diseased tissue. Antibodies in general do not readily cross capillary barriers and enter the interstitial fluid area of organs. To the degree that antibodies do cross capillary barriers their movement is very slow. Thus, when antibodies are administered for the purpose of treating or diagnosing diseased tissue associated with specific organs, the antibody dose at the infected site is too low.
The vascular beds of most organs have a net negative charge. These charged sites are attributed to the presence of negatively charged molecules on the surface of capillary walls.
It is believed that these negatively charged surfaces also provide an added electrical barrier to the neutral or slightly negative charge associated with antibodies.
In addition, the size of a molecule is important in determining the ability of that molecule to cross capillary walls.
Since antibodies have relatively high molecular weights their capillary permeation rate is much slower than that for similar molecules with a smaller size. In the case of IgG the molecular 6a weight is in the region of 150,000 Daltons. For IgM it is on the order of 1,000,000 Daltons. Antibodies having lower molecular weights are transported at higher rates, but WO 91/13631 ~ ~ ~ ~ ~ ~ ~~ PCT/US91/01533 ~ :., c.;,:.:.
..
these are still well below the desired rates for therapeutic and diagnostic applications. In accordance with the present invention the transport rate of all antibodies is increased. 7For very large antibodies, e.g. IgM, the present invention provides a method for their therapeutic and diagnostic utility which has not been available.
This invention is based upon 'the discovery that the uptake or transport of antibodies across 'the microvascular barrier of organs can be increased by cationizing the antibodies to form cationized antibodies having an isoelectric point of between about 8 and about 11. Antibodies are proteins which have both positive and negative charges with the net charge depending upon the pH of the antibody solution. The pH at which the positive and negative charges are equal is called the "isoelectric point" (pI).
Antibodies with a relatively high pI (> ~ 7.5) have a net positive charge at normal physiological pH's 'of '20 about 7.4. The higher the pI, the greater the positive charge. Conversely, antibodies with pI less than neutral have a net negative charge at normal physiological pH's.
Techniques far measuring the pI of a given antibody or protein are well known and generally involve isoelectric focusing according to conventional electrophoresis procedure. As previously mentioned, most antibodies have an isoelectric paint of between about 5 to 7.
The slightly acidic to neutral isoelectric points characteristic of most antibodies is attributed to the carboxy functionalities on the antibody. The present invention involves reacting a diamine with the carboxy groups of the antibody. One amine group of the diamine reacts with a carboxy group of the antibody to form an amide bond. The second amine functionality associated with the diamine cationization reagent provides the antibody with a basic group which raises the isoelectric point. A sufficient amount of the cationizing diamine W~ 91/13631 ~ ~ ~ "'~ ~ ~ ~ ' . p~,T/US91/01533 ~, .,:
is utilized to form a cationized antibody with the desired isoelectric point.
Cationization of the antibody can be carried out according to any of the known procedures for reacting carboxy groups on proteins to provide functionalities which give the protein high isoelectric points.
Preferred cationization agents are diamine compounds such as hexamethylenediamine. Hexamethylenediamine is the most preferred cationization agent because it is widely available and the techniques for its use in cationizing proteins are well known. The amount of r cationizing agent arid the conditions for reaction with the antibody can be varied so long as the final cationized antibody has an isoelectric point within the desired range.
In accordance with the present invention, the higher the isoelectric point of the antibody the greater the degree of uptake by organ tissues. Thus, in general, higher isoelectric points are preferred.
~20 However, antibodies with isoelectric points in excess of about 11.5 are known to form aggregates. In addition to being non-therapeutic and non-useful for diagnostic purposes, the aggregates will cause toxic responses when administered. Accordingly, when choosing the appropriate isoelectric point, consideration must be given to the possibility of antibody aggregate formation at high diamine substitutions or high isoelectric point.
Another consideration in choosing the isoelectric point for the cationized antibody is the specific organ to be targeted. The microvessels which perfuse the organ contain surface anionic charges with each organ having a characteristic anionic charge density. It is believed that the positively charged cationized antibodies permeate the electrical barrier caused by the .
net positive charge on the microvessel surface. For monoclonal antibodies that are directed against organs perfused by vessels with a paucity of anionic charges, WO 91/13631 ~ ' ~ ~ ~ ~ PCT/US9l/01533 :,;.
v_~::~-.,>
it is necessary to markedly increase 'the cationization of these antibodies relative to antibodies that are targeted toward organs perfused by capillaries with a high degree of anionic charges on the surface of the microvessels.
The above mentioned characteristics of cationized antibodies and organ vascular beds are the factors which are considered in accordance with the present invention when establishing the degree of cationization of an antibody that is necessary to enhance its organ uptake.
The first factor is the isoelectric point of the antibody. If the antibody happens to be neutral or even slightly positively charged, the degree of cationization that is necessary may not be as high as that necessary in the case of a monoclonal antibody with a net negative charge. The second factor is the degree t.o which the targeted organ is perfused by microvessels and the anionic charge density. By varying the resulting isoelectric point of the cationized antibody, an organ specific compound can be prepared. The third factor to consider is the isoelectric point at which the cationized antibody forms an antibody aggregate. Since aggregate formation is undesirable, the isoelectric point must be less than that at which the aggregates form. The pI of the antibody may be raised between 1 to 7 points in accordance with the present invention provided that aggregates are not formed. For antibodies having a neutral pI, cationization will be limited to raising the pI only 1 to 4 points. The increase in pI
for neutral antibodies directed to organs having relatively high anionic charge such as kidney or lung will be less 'than for organs such as intestines, which have lower anionic charges. For example, when targeting the kidney, the pI increase for a neutral antibody will be in the range of 1 to 3. In contrast, when targeting the intestines, the cationization should be increased to ., provide a pI which is 2 to 4 points higher than that of the neutral antibody.
For acidic antibodies, the pI should be increased from 5 to 7 points. Again, the specific preferred V
increase will depend upon the organ being targeted. The amount of increase in pI can be easily determined experimentally for each organ and each awtibody.
The particular antibodies which can be used are virtually unlimited, provided that they have some use in connection with diagnosing or treating cancer, tumors, or diseased tissue. Monoclonal antibodies are preferred because of their increased diagnostic or therapeutic potential. Monoclonal antibodies which are organ specific for specific antigens are of particular importance. The invention has application to antibodies with molecular weights greater than 20,000 Daltons.
Typical antibodies which can be cationized for organ transcytosis include antibodies to carcinoembryonic antigen (CEA) which can be useful for imaging or treatment of colon cancer (1) or a monoclonal antibodies to T-lymphocyte receptors which are useful in the imaging or detection of cancers of lymphoid tissue such as lymphoma (2).
Additionally, monoclonal antibodies to a surface antigen on melanoma cells may be useful in the treatment ar imaging of malignant melanoma, a skin cancer (3).
Any of a number of antibodies to surface antigens specific for lung cancer are suitable for use in the treatment and diagnosis of lung cancer (4). Monoclonal antibodies to surface antigens peculiar to human prostrate tissue may be useful in the imaging or treatment of prostate cancer (5). Further, monoclonal antibodies to surface proteins or antigens on human breast cancer, kidney cancer, esophageal cancer, and .
pancreatic cancer are particularly suitable for chemical modification and use in the treatment or diagnosis of cancer (6), (7), (8), (9).
WO 91/13631 PC."T/US91/01533 Since monoclonal antibodies and other large proteins have difficulty in traversing the vascular barrier in colon, skin, lymph tissue, lung, prostate, breast tissue, kidney, esophagus, and pancreas, the cationization of any of these specific monoclonal antibodies in accordance with the present invention allows for marked increase in the uptake of these organ-specific monoclonal antibodies by their respective organs.
Antibodies to any of the above mentioned antigens may be tagged with a specific tracer for diagnostic purposes or a specific drug for therapeutic purposes and cationized to an isoelectric point which is selected for the specific antibody and the specific organ. The cationization agent is preferentially hexamethylenediamine and the isoelectric point is generally from about 8 to about 11. The amount that the isoelectric point for an antibody must be raised can be determined experimentally by first establishing the point at which aggregates form and then reducing the pI
depending upon the particular organ being targeted.
The resulting tagged or drug carrying cationized antibody may be utilized as a specific organ targeted vehicle. Accordingly, it can be administered intravenously to the patient using a suitable pharmaceutically acceptable carrier solution. The tagged cationized antibody will cross the microvascular bed of the specific organ in sufficient quantities to effectively treat the cancer or detect the antigen of interest. Additionally, because of the enhanced uptake by the specific organ, dangerously high levels of the tagged antibody in the blood are avoided. When radionu-clides are utilized in conjunction with cationized anti-bodies there is a reduced background level due to the enhanced contrast between the target and background areas. Detection of radionuclide bound cationized Wt'3 91/13631 ~ ~~ ~ ~a ~ ~~ ~~ PtT/US91/01533 ',', antibody is accomplished by conventional radionualide scanning techniques.
Although hexamethylenediamine is the preferred compound for use in cationizing antibodies, other cationizing agents are possible. For example, ethylene diamine, N,N-dimethyl- 1,3-propanediamine, or polylysine may be used. Cationization is preferably catalyzed by carboxy activation using N-ethyl,N
(3-dimethyl-aminopropyl carbodiimide hydrochloride ~.0 (EDAC) using the method described by Hoare and Koshland.(10) It is known the cationizing antibodies does not significantly reduce its antigen binding properties. If desired however, the antibody may be pre-bound to the antigen of interest prior to cationization. This prebinding with the antigen effectively blocks the immunoreactive sites on the antibody and prevents them from reacting during the cationization process. After cationization is complete and the isoelectric point has '20 been raised to the desired level, the cationized antibody is treated to unbind the antigen from the antibody. The unbinding is accomplished according to well known procedures where the antibody-antigen complex is treated with an acid to break the antibody-antigen bond. The antibody is then recovered by column chromatography or other conventional separation and recovery techniques.
As an example of practice, bovine IgG was cationized and the pharmacokinetics of its uptake by several organs in both rat and monkey were tested.
Bovine serum albumin was used as a test control for comparison.
EXAMPLE
Clearance of [3HZ cationized IgG and l~~ ZsI] BSA in primate Bovine immunoglobulin G (IgG) having an initial isoelectric point of 5 - 7 was cationized to an isoelec-~~i'~<
.. !V0 91/13631 PC,'T/US91/01533 tric point >10.7 as determined by polyacrylamide gel isoelectric focusing (11). The cationized IgG was monomeric as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-F~AGE). Native bovine serum albumin (BSA) and cationized IgG were iodinated to a specific activity of 13 and 21 ~.Ci/microgram, respectively, with ['zsI]-iodine and chloramine T. (11 and 12) The radiolabeled protein was separated from unreacted iodine by Sephadex G25 gel filtration after passage over two 0.7 x 28 cm columns in series. Cationized IgG and native BSA were tritiated to a specific activity of 0.14 and 0.4 ~,Ci/microgram, respectively, with ['H]--sodium borohydride.
An 0.5 mL aliquot of physiologic buffer (10 mM
Hepes, pH =7.4, 141 mM NaCl, 4 mM KC1, 2.8 mM CaClz, 1mM
MgSO~, 1 mM Na HZP04, and 10 mM D-glucose) containing 5 ~tCi of [lzsZ]_cationized IgG plus 50 ~CCi of [3H]-BSA or 10 ~Ci of [3H]-cationized IgG was rapidly injected into a femoral vein of anesthetized rats. At 0.5, 5, 30,60,120, and 180 minutes after the injection, the animal was quickly laparotomized and 5mL of arterial blood was withdrawn from the descending aorta. An 0.5 mL aliquot was removed for liquid scintillation counting and trichloroacetic acid (TCA) precipitability measure-ments. The remaining blood was allowed to clot and the serum was separated and stored at -20 degrees C. far subsequent use in vitro studies.
The following organs were extirpated and weighed:
brain, heart, liver, spleen, testis, small intestine, skeletal muscle, fat, kidney, and lung. The tissues and blood samples were solubilized in soluene 350 and prepared for [lzsI], (3H] double isotope liquid scintillation spectrometry.
The blood [3H] and [lzsl] radioactivities were normalized to DPM/mL as a percent of injected dose and these data fitted to a biexponential function. The volume of distribution (VD) of the labeled protein in s WO 91/1353 ~ ~ '~ ~ ~ '~ y ~crius~nous~3 ~~:'~
W~~S' brain or other organs was determined from the ratio of DPM/Gm tissue divided by the integrated DPM/mL blood over the time period of the eacperiment. Only arterial blood which was trichloroacetic acid precipitated was counted f or [ 3H ] and [ ~ZSI ] .
Table 1 is a table of percent trichloroacetic acid precipitable serum [azsl] and [3H]-cationized immunoglobulin G (cIgG) measured at different time intervals after a single intravenous injection in rats.
The results indicate that substantially all the radiolabelled material is recovered.
WO~1/13631 ~~~'1 ~~~~
.t~,,,, P~'d'/US91 /01533 ,.
TA~3LE 1 Trichloroacetic Acid (TCA) Precipitability of Serum [l2sl]_ or ['13]-Canonized Immunoglobulin G (cIgG) 5 After a Single Intravenous Injection in Rats Time % TCA Precipitable 10 (min) [l2sl]-clgG [3H]cIgG
0.25 99.4 1-0.1 97.0 0.6 5 99.3 0.1 98.3 0.4 30 98.9 0.3 97.8 0.2 15 60 97.8 0.3 92.2 1.2 120 97.0 1.0 91.4 0.9 180 97.7 0.1 88.9 1.6 Mean : S.E. (n y 3).
The volume of distribution (VD) of [~H]-cationized IgG in kidney, lung, or myocardium rose linearly with the duration of the three hour period of observation following the single intravenous injection of isotope as shown in Fig. 2. Similarly, the organ VD values for [3H]-canonized IgG in brain, intestines, skeletal muscle, or fat increased linearly during the three hour observation period (data not shown). In contrast, the volume of distribution of [3H]-cationized IgG in liver (Fig. 2) or spleen (data not shown) reached a maximal value within five minutes after the intravenous injection and subsequent values actually declined from this maximal volume of distribution. The volume of distribution of [3H]-canonized TgG in testis peaked at 60 minutes, and this value remained constant between 60 and 180 minutes after injection. Table 2 provides the volume of distribution of [3H]-cationized IgG, [1251]-cationized IgG, and [3H]-native bovine serum I
WO 91/13631 ~ ~ ~ rl ~, ry ~ PCT/US91101533 16 , albumin (BS~1) for the ten organs measured at a single time point of 180 minutes after single intravenous injection. Table 2 illustrates the enhanced uptake of cationized immunoglobulin G as compared to native bovine serum albumin. The ratio of transport of [3H]-cationized IgG to ['H]-native bovine serum albumin ranged from 1.0 (testis) to 17.9 (spleen). However, these ratios refer only to the l80 minute time point and it is projected that in organs such as kidney, brain, lung, intestine, skeletel muscle, heart, or fat the ratio of eationized IgG to native serum protein will rise appreciably beyond the values shown in Table 2 at time points later than 180 minutes after administration.
..Yd'O 91/i3631 l fCT/US91/U1S33 ~. i ~.7 Tntegrated Volume of Distribution (VD) of ['H] _Native Bovine serum Albumin (BSA) , [1251]_~ationized Immunoglobulin G (clgG), and [3H]-cTgG 180 Minutes After a Single Intravenous Injection in Rats VD (~CLg 1) ['H]-cIgG VD
[ 3H ] -BSA [ lzsl ] -cIgG [ 3H ] --cIgG 3I-I ] -BSA VD
Spleen 196 30 951 79 3498 454 17.9 Liver 251 1 8 1005 35 3392 143 13.5 Kidney 272 8 605 35 3380 198 12.4 Brain 16 1 29 2 118 8 7.4 Lung 360 11 462 8 2611 264 7.2 Intes- 125 13 259 56 660 19 5.3 tine Muscle 42 1 64 3 202 13 4.8 Heart 193 4 227 5 525 92 2.7 Fat 60 15 76 19 139 19 2.3 Testis 129 13 232 18 128 18 1.0 Data are mean t S.E. (n = 3 rats).
In general, the organ VD values for [3H]-cationized IgG were several-fold above the organ VD values for [~zsI]-cationized IgG. Since the formation of [1251]_cationized IgG is a oxidative process while the tritiation of IgG is a reductive procedure, it is apparent that the axidized form ( [l2sl]-cationized IgG) binds serum factors that inhibit the uptake of [izsl]_cationized IgG. This conclusion is supported by evidence that serum factors may bind oxidative forms of [1251]_cationized BSA or [l2sl]_cationized human albumin.
(13) WO 91/13631 ~ ~ ~ ~ ~ r~ ~ PCf/US91/01533 Fig. 1 plots the serum .radioactivity (DPM/mL/%injected) of ['H]-native albumin or [3H]-cationized IgG versus time after a single intravenous injection of the isotope. only the TCA
precipitable counts indicated in Table 1 were plotted in the decay curves. The [3H]-albumin data were fit to a monoexponential function while the [3H]-cationized IgG
data were fit to a biexponential function. Following initial rapid clearance from blood, the rate of egress of cationized IgG is relatively slow.
The initial rapid rate of cationized IgG clearance appears to be due to rapid uptake of the IgG by liver and spleen. However, these organs have a limited number of binding sites for the cationized IgG that the clearance by liver and spleen reaches a maximum value within 5 minutes after administratian. Owing to this rapid saturation, subsequent clearance of cationized IgG
from blood is relatively slow, and this maintenance of a relatively constant blood concentration throughout the '20 experimental period allows for the progressive uptake of the cationized IgG by other organs. Were it not for the limited number of binding sites for cationized IgG in liver and spleen, the rate of clearance of this protein from blood might be extremely rapid and it would be difficult to maintain a relatively constant blood level of the antibody for availability to other organs. This characteristic of cationized antibodies allows them to be present at the targeted organ in sufficient quantity for effective diagnostic or therapeutic purposes.
Clearance of f'H1-cationized IaG and fl2sl~-BSA following a single intravenous infection in a t~rimate An 0.5 mL aliquot of the same physiologic buffer as example 1 containing 500 microCi of [3H]-cationized IgG
and 50 microCi of [lzsl]-BSA was rapidly injected into a femoral vein of an adult, male anesthetized monkey. At ~n,,W~ 91/13631 PC'T/US91 /01533 r,,.,..:
Z9 ,_ different times up to 60 minutes, approximately 0.3 mh aliquot of blood were removed from the ipsilateral femoral artery. After 60 minutes, the monkey was sacrificed and the organs were removed. Samples were processed for double isotope liquid scintillation counting and TCA precipitability as described above.
Clearance and volume of distribution calculations were performed as described above.
Table 3 tabulates the integrated Vp of ('H]-cationized immunoglobulin G(clgG) and [12$I]-bovine serum albumin (BSA) 60 minutes after a single intravenous injection in the Cynomologous Monkey. In general, the monkey Vp values for native BSA at 60 minutes are comparable to Vp values in the rat.
Additionally, the uptake of cationized IgG by organs is substantially increased over BSA. Although the cationized IgG organ uptake in the primate was increased over that of the organ uptake of native bovine albumin, the enhanced uptake is relatively modest since the '20 primate experiment was restricted to organ measurements at a time period of only 60 minutes following the intravenous injection. dwing to the relatively slow second phase of clearance of the cationized IgG from the primate blood (see below), there is a linear increase in the volume of distribution of the can onized IgG by many organs in the primate, proportional to the duration following the intravenous injection of cationized antibody, similarly to that observed for the rat (Fig.
2).
WO 91/13b31 ~ ~~~ ~ ~ "~~ ,~ P(.T/US91/01533 TABLE
Integrated of Distribution Volume (VD) of [3H ]_Cati onized 5 ImmunoglobulinG (clgG) and [zxsl]_Bovine Albumin (BSA) 60 Serum Minutes Aftera Single Intravenous Injection Cynomologous Mon)tey in a 10 VD (~tLcJ') clgG VD
organ __ BSA cIgG BSA VD
Liver 350 -' 22 2537499 7.2 15 Spleen 387 8 2400216 6.2 Kidney 312 2 114323 3.7 Muscle 14 1 31 1- 2 2.2 White 15 -1 1 2.1 matter 7.2 0.6 Fat 19 * 4 31 -!- 4 1.6 .
20 Heart 128 4 177 10 1.4 Lung 439 11 590 17 1.3 Gray matter 22 1 1.2 Intestine100 7 118 12 1.2 Testis 164 4 154 3 0.94 Cheroid 279 0.93 Plex_ us tiara are mean : S.E. (n = 3 samples from one monkey,L
Fig. 3 illustrates the decay in serum [1251]_native BSA and [3H]-cationized IgG radioactivity following a single intravenous injection in a Macaca irus monkey.
As indicated in Fig. 3, the total DPMs injected'at zero time for the labelled BSA (0.254 DPM/ML/o injected) is about 14 fald lower than that for labelled BSA in the rat (3.5 DPM/ML/% injected, Fig. 1). Since the weight of the primate is approximately 14 fold greater than the weight of the rat it is likely that the difference is ~ ~_4 ~ d b) ~ 'i7 ~;1V0 91/13631 P~I"1US91101533 ''.':b:: ' '~Si~i ~'~
~i due to the larger primate blood volume. It is clear from the rat and primate experiments that the cationi-zation procedure in accordance with the present invention results in markedly increased rates of uptake of the IgG by organs after cationization of antibodies.
The data shown in Figs. 1 and 3 illustrate the highly favorable pharmacokinetics of ['H]-cationized IgG
clearance by organs. Owing to the rapid saturation of uptake sites in liver and spleen, there is a prolonged slow second phase of clearance of ['1-I]-cationized IgG
from blood. The maintenance of this prolonged slow phase of clearance from blood allows for progressive and linear increase of the cationized IgG by a number of different organs. The relatively long half-time of catianized IgG (e.g., 3.0 ~ 1.0 hours in rats or 2.9 ~
1.6 hours in a primatey indicates that the cationized IgG pharmaceutic need not be administered continuously, but could be administered on a once, twice, or three times a day basis.
~0 Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations and modifications may be made within the scope of the present invention. Accordingly the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.
WO91/13631 ~ 1 !~ PCT/US91/01533 ~
a~
R E F E R E ~T C E 8 1. Reynoso, G., Keane, M., and Reynoso, M.A. (.1985):
Monoclonal carcinoembryyonic antigen antibodies. ' In: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humans Press, Clifton, New Jersey, pp. 19°40. ' 2. Harden E.A., Palker, T.J., and Haynes, B.F. (1985):
Monoclonal antibodies. Probes for study of malignant T cells. Win: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humans Press, Clifton, New Jersey, pp. 121°145.
3. Reisfeld, R.A. (1985): Monoclonal antibodies as probes for the molecular structure and biological function of melanoma-associated antigens. In:
Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humans Press, Clifton, New Jersey, pp. 205-228.
Enzyme linked immunoassay and radioimmunoassay are common diagnostic techniques which utilize antibodies as targeting vehicles and detect antigens in vitro.
Antigens may also be detected in vivo by administering radiolabelled or paramagnetic labelled antibodies to a living subject followed by the external detection of the radiolabelled antibody sequestered by a particular organ bearing the respective antigen.
One of the limitations in using antibodies as targeting vehicles in either the in vivo 'treatment or diagnosis of cancer and infectious diseases has been the inability to obtain effective concentrations of the targeting antibody at the target site. The low antibody dose at the site is largely due to poor antibody uptake by the tumor or infected site. The poor uptake is due to the microvascular or endothelial barrier which is present in most organs. This endothelial barrier has WO 91/13631 ~ ~ ~:~ ~'~ ~ . PCT/1JS91/~133 ..':
pores which are too small to allow for rapid organ uptake of circulating antibodies. Also the small size of the aqueous pores in the walls of the vessels which perfuse organs greatly restricts antibody transport from the vessels into the organ.' Transport across the endothelial barrier is a particular problem for large plasma proteins, such as antibodies that have molecular weights in excess of 150,000 Daltons. These antibodies are excluded or cross the microvascular barrier only very slowly. Hot only does the size of these large antibodies restrict their transport across the endothelial barrier, but, their electrical charges also present transport problems.
More specifically, the molecules on the surface of capillaries are anionically charged and, therefore, present an electrical barrier to the neutral or slightly negatively charged antibodies.
Another limitation to an optimum concentration of targeting antibody at the target organ site is the '20 higher permeability of the liver and spleen vascular barrier. The liver and spleen do not exclude the transport of large molecules to the same degree as other organs. Consequently, these two organs will preferentially remove administered antibodies from the blood leaving only a small concentration for therapeutic or diagnostic delivery to other organs.
Since most of the radioisotopes or complexes used in targeting systems are somewhat toxic and dose limiting, merely increasing the dose of the antibody with the expectation that more will become available to the organ of interest is not a practical solution.
r Strategies have been developed to administer .
effective amounts of antibodies by an invasive regional route to the location of the tumor or diseased area.
This avoids a high concentration of a potentially toxic agent in the blood. For systemic administration, however, it is necessary to use methods which control or enhance the blood clearance of the targeted antibodies. Such techniques aid in avoiding toxic blood levels of radioisotopes or other therapeutic agents, but still require large doses of the antibody because of their restricted transport across capillary barriers.
Accordingly there presently is a need to provide an improved method for the diagnosis and treatment of cancer and infectious diseases which are responsive to antibodies used as target vehicles. Further, there is a need to provide improved methods for delivering effective amounts of antibodies to organ tissue without sustaining toxic amounts of the antibody target vehicle in the blood. There is also a need to provide improved means for transporting antibodies across the microvascular barrier of organs and into the interstitial pores of organs.
SUMMARY OF THE INVENTION
It is one object of the invention to provide chemically modified yet active antibodies for delivery to organ tissue in effective amounts for therapeutic or diagnostic applications. It is another objective to effectively deliver the chemically modified antibodies without maintaining toxic levels of the antibody target vehicle in the blood. Accordingly, the present invention provides a method for increasing the transytosis rate of an antibody across the microvascular barrier and into the interstitial fluid of organs. The invention is based upon the discovery that cationized antibodies have increased rates of delivery across organ vascular beds when compared with the transcytosis of antibodies which are not cationized.
The present invention also provides a system for increasing transcytosis of an antibody across microvascular 3a barrier and into interstitial fluid of mammalian non-central nervous system tissues and organs, comprising: a) an antibody which has been cationized by treatment with a sufficient amount of a can onization agent to increase the isoelectric point of the antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than 11.5; b) a suitable pharmaceutically acceptable diluent or carrier; and c) a means for delivering the cationized antibody in admixture with the diluent or carrier, to a site where the can onized antibody is transcytosed across the microvascular barrier and into the interstitial fluid of said organs.
The present invention also provides for the use of a cationized antibody for increasing transcytosis of said antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
The present invention also provides for the use of a cationized antibody for the manufacture of a medicament for the treatment of conditions where transcytosis of said antibody is desired across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
The present invention also provides a pharmaceutical composition comprising an antibody which has been cationized, in admixture with a suitable pharmaceutically acceptable diluent or carrier, for increasing transcytosis of said antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
The present invention also provides for a commercial package containing as an active ingredient a cationized antibody, together with instructions for its use for increasing transcytosis 3b of the antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
The effectiveness of antibodies for both diagnostic and therapeutic purposes is increased by cationizing the antibodies to provide can onized antibodies having elevated isoelectric points (pI). These antibodies WO 91/13631 ~ ~ ~ r~~ ~ ~ ~ . PCf/US91/01533 carry a net positive charge and have been found to cross microvascular barriers at rates which are much higher than the transcytosis rates for negatively charged or neutral antibodies which typically have isoelectric points in the range of 5 to 7. Isoelectric points for the cationized antibodies will vary depending upan the particular organ ar organs to which the antibody is targeted. Generally, however, it is desireable to raise the isoelectric point of the antibody by from about 2 to about 6 points. The resulting modified antibody preferably has an isoelectric point in the range of from about 8 to about 11.
The cationized antibodies in accordance with the present invention are prepared by treating a given mono clonal or polyclonal antibody with a cationizat:ion agent such as hexamethylenediamine. The amine cationization agent replaces surface carboxyl groups on the antibody with a more basic group, such as a primary amine group in the case of hexamethylenediamine and related amine compounds. The amount of cationization agent and reaction conditions are controlled so that the resulting cationized antibody has the desired isoelectric point of between from about 8 to about 11.
It is known that antibodies retain nearly 90% of their antigen binding properties following catonization.
Thus, the chemical process of cationization does not destroy the innate biologic properties of the antibody.
If preferred, however, the immunoreactive sites may be blocked prior to the cationizing process by reacting the antibody with an excess of the appropriate antigen.
These blocked immunoreactive sites are unreactive during the subsequent cationization steps. The antigens are .
then decoupled from the cationized antibodies after the cationization step to thereby reactivate the blocked immunoreactive sites.
The cationization and utilization of antibodies in accordance with the present invention is useful whenever ;~'~ 91/13631 ~ ~ ~ 4 ~ ~ ~~ PCT/US91/01533 ::;
,.:~;
it is necessary to introduce an antibody into the interstitial fluid of an organ. Both therapeutic and diagnostic uses for antibodies is contemplated.
Diagnostic uses include targeting a cationized antibody 5 carrying a radionuclide or a paramagneta.c label to a specific organ containing the antigen for that antibody.
Once the antibody and antigen are complexed, subsequent diagnostic techniques for the radionuclide or the paramagnetic label may be used to detect the antigen.
Therapeutic uses include targeting drugs to specific organs containing cancerous or diseased tissue. Such therapeutic utility contemplates using cationized antibodies which are antibodies for the antigen of interest as the carrying vehicle for the drug, The above discussed and many other features and attendant advantages of the present invention will become apparent as the invention becomes better understood by reference to the following detailed description.
BRIEF DESCRTPTION OF THE DRAWINGS
Fig. 1 is a plot of serum radioactivity (DPM/mL/%
injected) of [3H]-native albumin or ['H]-cationized IgG
versus time after a single intravenous injection of the isotope in the anesthetized rat.
Fig. 2 is a plot of the volume of distribution (vp) of [3H]-cationized IgG for liver, kidney, lung, and myocardium versus the time after single intravenous injection of the isotope in anesthetized rats.
Fig. 3 is a plat of serum [lzsl]-bovine serum albumin radioactivity and [~H]-cationized IgG radioactivity over a 60 minute period after a single intravenous injection of isotope in the anesthetized cynomologous monkey.
DETAILED DESCRIPTION OF THE INVENTION
Publications and other references will be referred to in this detailed description. For convenience, the reference materials are numerically referenced and grouped in the bibliography which is located at the end of the detailed description.
The present invention involves the transport of antibodies through the microvascular barrier or organs. The invention has wide application to any antibody which is useful as a targeting vehicle in diagnosing or treating cancers, tumors, or diseased tissue. Antibodies in general do not readily cross capillary barriers and enter the interstitial fluid area of organs. To the degree that antibodies do cross capillary barriers their movement is very slow. Thus, when antibodies are administered for the purpose of treating or diagnosing diseased tissue associated with specific organs, the antibody dose at the infected site is too low.
The vascular beds of most organs have a net negative charge. These charged sites are attributed to the presence of negatively charged molecules on the surface of capillary walls.
It is believed that these negatively charged surfaces also provide an added electrical barrier to the neutral or slightly negative charge associated with antibodies.
In addition, the size of a molecule is important in determining the ability of that molecule to cross capillary walls.
Since antibodies have relatively high molecular weights their capillary permeation rate is much slower than that for similar molecules with a smaller size. In the case of IgG the molecular 6a weight is in the region of 150,000 Daltons. For IgM it is on the order of 1,000,000 Daltons. Antibodies having lower molecular weights are transported at higher rates, but WO 91/13631 ~ ~ ~ ~ ~ ~ ~~ PCT/US91/01533 ~ :., c.;,:.:.
..
these are still well below the desired rates for therapeutic and diagnostic applications. In accordance with the present invention the transport rate of all antibodies is increased. 7For very large antibodies, e.g. IgM, the present invention provides a method for their therapeutic and diagnostic utility which has not been available.
This invention is based upon 'the discovery that the uptake or transport of antibodies across 'the microvascular barrier of organs can be increased by cationizing the antibodies to form cationized antibodies having an isoelectric point of between about 8 and about 11. Antibodies are proteins which have both positive and negative charges with the net charge depending upon the pH of the antibody solution. The pH at which the positive and negative charges are equal is called the "isoelectric point" (pI).
Antibodies with a relatively high pI (> ~ 7.5) have a net positive charge at normal physiological pH's 'of '20 about 7.4. The higher the pI, the greater the positive charge. Conversely, antibodies with pI less than neutral have a net negative charge at normal physiological pH's.
Techniques far measuring the pI of a given antibody or protein are well known and generally involve isoelectric focusing according to conventional electrophoresis procedure. As previously mentioned, most antibodies have an isoelectric paint of between about 5 to 7.
The slightly acidic to neutral isoelectric points characteristic of most antibodies is attributed to the carboxy functionalities on the antibody. The present invention involves reacting a diamine with the carboxy groups of the antibody. One amine group of the diamine reacts with a carboxy group of the antibody to form an amide bond. The second amine functionality associated with the diamine cationization reagent provides the antibody with a basic group which raises the isoelectric point. A sufficient amount of the cationizing diamine W~ 91/13631 ~ ~ ~ "'~ ~ ~ ~ ' . p~,T/US91/01533 ~, .,:
is utilized to form a cationized antibody with the desired isoelectric point.
Cationization of the antibody can be carried out according to any of the known procedures for reacting carboxy groups on proteins to provide functionalities which give the protein high isoelectric points.
Preferred cationization agents are diamine compounds such as hexamethylenediamine. Hexamethylenediamine is the most preferred cationization agent because it is widely available and the techniques for its use in cationizing proteins are well known. The amount of r cationizing agent arid the conditions for reaction with the antibody can be varied so long as the final cationized antibody has an isoelectric point within the desired range.
In accordance with the present invention, the higher the isoelectric point of the antibody the greater the degree of uptake by organ tissues. Thus, in general, higher isoelectric points are preferred.
~20 However, antibodies with isoelectric points in excess of about 11.5 are known to form aggregates. In addition to being non-therapeutic and non-useful for diagnostic purposes, the aggregates will cause toxic responses when administered. Accordingly, when choosing the appropriate isoelectric point, consideration must be given to the possibility of antibody aggregate formation at high diamine substitutions or high isoelectric point.
Another consideration in choosing the isoelectric point for the cationized antibody is the specific organ to be targeted. The microvessels which perfuse the organ contain surface anionic charges with each organ having a characteristic anionic charge density. It is believed that the positively charged cationized antibodies permeate the electrical barrier caused by the .
net positive charge on the microvessel surface. For monoclonal antibodies that are directed against organs perfused by vessels with a paucity of anionic charges, WO 91/13631 ~ ' ~ ~ ~ ~ PCT/US9l/01533 :,;.
v_~::~-.,>
it is necessary to markedly increase 'the cationization of these antibodies relative to antibodies that are targeted toward organs perfused by capillaries with a high degree of anionic charges on the surface of the microvessels.
The above mentioned characteristics of cationized antibodies and organ vascular beds are the factors which are considered in accordance with the present invention when establishing the degree of cationization of an antibody that is necessary to enhance its organ uptake.
The first factor is the isoelectric point of the antibody. If the antibody happens to be neutral or even slightly positively charged, the degree of cationization that is necessary may not be as high as that necessary in the case of a monoclonal antibody with a net negative charge. The second factor is the degree t.o which the targeted organ is perfused by microvessels and the anionic charge density. By varying the resulting isoelectric point of the cationized antibody, an organ specific compound can be prepared. The third factor to consider is the isoelectric point at which the cationized antibody forms an antibody aggregate. Since aggregate formation is undesirable, the isoelectric point must be less than that at which the aggregates form. The pI of the antibody may be raised between 1 to 7 points in accordance with the present invention provided that aggregates are not formed. For antibodies having a neutral pI, cationization will be limited to raising the pI only 1 to 4 points. The increase in pI
for neutral antibodies directed to organs having relatively high anionic charge such as kidney or lung will be less 'than for organs such as intestines, which have lower anionic charges. For example, when targeting the kidney, the pI increase for a neutral antibody will be in the range of 1 to 3. In contrast, when targeting the intestines, the cationization should be increased to ., provide a pI which is 2 to 4 points higher than that of the neutral antibody.
For acidic antibodies, the pI should be increased from 5 to 7 points. Again, the specific preferred V
increase will depend upon the organ being targeted. The amount of increase in pI can be easily determined experimentally for each organ and each awtibody.
The particular antibodies which can be used are virtually unlimited, provided that they have some use in connection with diagnosing or treating cancer, tumors, or diseased tissue. Monoclonal antibodies are preferred because of their increased diagnostic or therapeutic potential. Monoclonal antibodies which are organ specific for specific antigens are of particular importance. The invention has application to antibodies with molecular weights greater than 20,000 Daltons.
Typical antibodies which can be cationized for organ transcytosis include antibodies to carcinoembryonic antigen (CEA) which can be useful for imaging or treatment of colon cancer (1) or a monoclonal antibodies to T-lymphocyte receptors which are useful in the imaging or detection of cancers of lymphoid tissue such as lymphoma (2).
Additionally, monoclonal antibodies to a surface antigen on melanoma cells may be useful in the treatment ar imaging of malignant melanoma, a skin cancer (3).
Any of a number of antibodies to surface antigens specific for lung cancer are suitable for use in the treatment and diagnosis of lung cancer (4). Monoclonal antibodies to surface antigens peculiar to human prostrate tissue may be useful in the imaging or treatment of prostate cancer (5). Further, monoclonal antibodies to surface proteins or antigens on human breast cancer, kidney cancer, esophageal cancer, and .
pancreatic cancer are particularly suitable for chemical modification and use in the treatment or diagnosis of cancer (6), (7), (8), (9).
WO 91/13631 PC."T/US91/01533 Since monoclonal antibodies and other large proteins have difficulty in traversing the vascular barrier in colon, skin, lymph tissue, lung, prostate, breast tissue, kidney, esophagus, and pancreas, the cationization of any of these specific monoclonal antibodies in accordance with the present invention allows for marked increase in the uptake of these organ-specific monoclonal antibodies by their respective organs.
Antibodies to any of the above mentioned antigens may be tagged with a specific tracer for diagnostic purposes or a specific drug for therapeutic purposes and cationized to an isoelectric point which is selected for the specific antibody and the specific organ. The cationization agent is preferentially hexamethylenediamine and the isoelectric point is generally from about 8 to about 11. The amount that the isoelectric point for an antibody must be raised can be determined experimentally by first establishing the point at which aggregates form and then reducing the pI
depending upon the particular organ being targeted.
The resulting tagged or drug carrying cationized antibody may be utilized as a specific organ targeted vehicle. Accordingly, it can be administered intravenously to the patient using a suitable pharmaceutically acceptable carrier solution. The tagged cationized antibody will cross the microvascular bed of the specific organ in sufficient quantities to effectively treat the cancer or detect the antigen of interest. Additionally, because of the enhanced uptake by the specific organ, dangerously high levels of the tagged antibody in the blood are avoided. When radionu-clides are utilized in conjunction with cationized anti-bodies there is a reduced background level due to the enhanced contrast between the target and background areas. Detection of radionuclide bound cationized Wt'3 91/13631 ~ ~~ ~ ~a ~ ~~ ~~ PtT/US91/01533 ',', antibody is accomplished by conventional radionualide scanning techniques.
Although hexamethylenediamine is the preferred compound for use in cationizing antibodies, other cationizing agents are possible. For example, ethylene diamine, N,N-dimethyl- 1,3-propanediamine, or polylysine may be used. Cationization is preferably catalyzed by carboxy activation using N-ethyl,N
(3-dimethyl-aminopropyl carbodiimide hydrochloride ~.0 (EDAC) using the method described by Hoare and Koshland.(10) It is known the cationizing antibodies does not significantly reduce its antigen binding properties. If desired however, the antibody may be pre-bound to the antigen of interest prior to cationization. This prebinding with the antigen effectively blocks the immunoreactive sites on the antibody and prevents them from reacting during the cationization process. After cationization is complete and the isoelectric point has '20 been raised to the desired level, the cationized antibody is treated to unbind the antigen from the antibody. The unbinding is accomplished according to well known procedures where the antibody-antigen complex is treated with an acid to break the antibody-antigen bond. The antibody is then recovered by column chromatography or other conventional separation and recovery techniques.
As an example of practice, bovine IgG was cationized and the pharmacokinetics of its uptake by several organs in both rat and monkey were tested.
Bovine serum albumin was used as a test control for comparison.
EXAMPLE
Clearance of [3HZ cationized IgG and l~~ ZsI] BSA in primate Bovine immunoglobulin G (IgG) having an initial isoelectric point of 5 - 7 was cationized to an isoelec-~~i'~<
.. !V0 91/13631 PC,'T/US91/01533 tric point >10.7 as determined by polyacrylamide gel isoelectric focusing (11). The cationized IgG was monomeric as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-F~AGE). Native bovine serum albumin (BSA) and cationized IgG were iodinated to a specific activity of 13 and 21 ~.Ci/microgram, respectively, with ['zsI]-iodine and chloramine T. (11 and 12) The radiolabeled protein was separated from unreacted iodine by Sephadex G25 gel filtration after passage over two 0.7 x 28 cm columns in series. Cationized IgG and native BSA were tritiated to a specific activity of 0.14 and 0.4 ~,Ci/microgram, respectively, with ['H]--sodium borohydride.
An 0.5 mL aliquot of physiologic buffer (10 mM
Hepes, pH =7.4, 141 mM NaCl, 4 mM KC1, 2.8 mM CaClz, 1mM
MgSO~, 1 mM Na HZP04, and 10 mM D-glucose) containing 5 ~tCi of [lzsZ]_cationized IgG plus 50 ~CCi of [3H]-BSA or 10 ~Ci of [3H]-cationized IgG was rapidly injected into a femoral vein of anesthetized rats. At 0.5, 5, 30,60,120, and 180 minutes after the injection, the animal was quickly laparotomized and 5mL of arterial blood was withdrawn from the descending aorta. An 0.5 mL aliquot was removed for liquid scintillation counting and trichloroacetic acid (TCA) precipitability measure-ments. The remaining blood was allowed to clot and the serum was separated and stored at -20 degrees C. far subsequent use in vitro studies.
The following organs were extirpated and weighed:
brain, heart, liver, spleen, testis, small intestine, skeletal muscle, fat, kidney, and lung. The tissues and blood samples were solubilized in soluene 350 and prepared for [lzsI], (3H] double isotope liquid scintillation spectrometry.
The blood [3H] and [lzsl] radioactivities were normalized to DPM/mL as a percent of injected dose and these data fitted to a biexponential function. The volume of distribution (VD) of the labeled protein in s WO 91/1353 ~ ~ '~ ~ ~ '~ y ~crius~nous~3 ~~:'~
W~~S' brain or other organs was determined from the ratio of DPM/Gm tissue divided by the integrated DPM/mL blood over the time period of the eacperiment. Only arterial blood which was trichloroacetic acid precipitated was counted f or [ 3H ] and [ ~ZSI ] .
Table 1 is a table of percent trichloroacetic acid precipitable serum [azsl] and [3H]-cationized immunoglobulin G (cIgG) measured at different time intervals after a single intravenous injection in rats.
The results indicate that substantially all the radiolabelled material is recovered.
WO~1/13631 ~~~'1 ~~~~
.t~,,,, P~'d'/US91 /01533 ,.
TA~3LE 1 Trichloroacetic Acid (TCA) Precipitability of Serum [l2sl]_ or ['13]-Canonized Immunoglobulin G (cIgG) 5 After a Single Intravenous Injection in Rats Time % TCA Precipitable 10 (min) [l2sl]-clgG [3H]cIgG
0.25 99.4 1-0.1 97.0 0.6 5 99.3 0.1 98.3 0.4 30 98.9 0.3 97.8 0.2 15 60 97.8 0.3 92.2 1.2 120 97.0 1.0 91.4 0.9 180 97.7 0.1 88.9 1.6 Mean : S.E. (n y 3).
The volume of distribution (VD) of [~H]-cationized IgG in kidney, lung, or myocardium rose linearly with the duration of the three hour period of observation following the single intravenous injection of isotope as shown in Fig. 2. Similarly, the organ VD values for [3H]-canonized IgG in brain, intestines, skeletal muscle, or fat increased linearly during the three hour observation period (data not shown). In contrast, the volume of distribution of [3H]-cationized IgG in liver (Fig. 2) or spleen (data not shown) reached a maximal value within five minutes after the intravenous injection and subsequent values actually declined from this maximal volume of distribution. The volume of distribution of [3H]-canonized TgG in testis peaked at 60 minutes, and this value remained constant between 60 and 180 minutes after injection. Table 2 provides the volume of distribution of [3H]-cationized IgG, [1251]-cationized IgG, and [3H]-native bovine serum I
WO 91/13631 ~ ~ ~ rl ~, ry ~ PCT/US91101533 16 , albumin (BS~1) for the ten organs measured at a single time point of 180 minutes after single intravenous injection. Table 2 illustrates the enhanced uptake of cationized immunoglobulin G as compared to native bovine serum albumin. The ratio of transport of [3H]-cationized IgG to ['H]-native bovine serum albumin ranged from 1.0 (testis) to 17.9 (spleen). However, these ratios refer only to the l80 minute time point and it is projected that in organs such as kidney, brain, lung, intestine, skeletel muscle, heart, or fat the ratio of eationized IgG to native serum protein will rise appreciably beyond the values shown in Table 2 at time points later than 180 minutes after administration.
..Yd'O 91/i3631 l fCT/US91/U1S33 ~. i ~.7 Tntegrated Volume of Distribution (VD) of ['H] _Native Bovine serum Albumin (BSA) , [1251]_~ationized Immunoglobulin G (clgG), and [3H]-cTgG 180 Minutes After a Single Intravenous Injection in Rats VD (~CLg 1) ['H]-cIgG VD
[ 3H ] -BSA [ lzsl ] -cIgG [ 3H ] --cIgG 3I-I ] -BSA VD
Spleen 196 30 951 79 3498 454 17.9 Liver 251 1 8 1005 35 3392 143 13.5 Kidney 272 8 605 35 3380 198 12.4 Brain 16 1 29 2 118 8 7.4 Lung 360 11 462 8 2611 264 7.2 Intes- 125 13 259 56 660 19 5.3 tine Muscle 42 1 64 3 202 13 4.8 Heart 193 4 227 5 525 92 2.7 Fat 60 15 76 19 139 19 2.3 Testis 129 13 232 18 128 18 1.0 Data are mean t S.E. (n = 3 rats).
In general, the organ VD values for [3H]-cationized IgG were several-fold above the organ VD values for [~zsI]-cationized IgG. Since the formation of [1251]_cationized IgG is a oxidative process while the tritiation of IgG is a reductive procedure, it is apparent that the axidized form ( [l2sl]-cationized IgG) binds serum factors that inhibit the uptake of [izsl]_cationized IgG. This conclusion is supported by evidence that serum factors may bind oxidative forms of [1251]_cationized BSA or [l2sl]_cationized human albumin.
(13) WO 91/13631 ~ ~ ~ ~ ~ r~ ~ PCf/US91/01533 Fig. 1 plots the serum .radioactivity (DPM/mL/%injected) of ['H]-native albumin or [3H]-cationized IgG versus time after a single intravenous injection of the isotope. only the TCA
precipitable counts indicated in Table 1 were plotted in the decay curves. The [3H]-albumin data were fit to a monoexponential function while the [3H]-cationized IgG
data were fit to a biexponential function. Following initial rapid clearance from blood, the rate of egress of cationized IgG is relatively slow.
The initial rapid rate of cationized IgG clearance appears to be due to rapid uptake of the IgG by liver and spleen. However, these organs have a limited number of binding sites for the cationized IgG that the clearance by liver and spleen reaches a maximum value within 5 minutes after administratian. Owing to this rapid saturation, subsequent clearance of cationized IgG
from blood is relatively slow, and this maintenance of a relatively constant blood concentration throughout the '20 experimental period allows for the progressive uptake of the cationized IgG by other organs. Were it not for the limited number of binding sites for cationized IgG in liver and spleen, the rate of clearance of this protein from blood might be extremely rapid and it would be difficult to maintain a relatively constant blood level of the antibody for availability to other organs. This characteristic of cationized antibodies allows them to be present at the targeted organ in sufficient quantity for effective diagnostic or therapeutic purposes.
Clearance of f'H1-cationized IaG and fl2sl~-BSA following a single intravenous infection in a t~rimate An 0.5 mL aliquot of the same physiologic buffer as example 1 containing 500 microCi of [3H]-cationized IgG
and 50 microCi of [lzsl]-BSA was rapidly injected into a femoral vein of an adult, male anesthetized monkey. At ~n,,W~ 91/13631 PC'T/US91 /01533 r,,.,..:
Z9 ,_ different times up to 60 minutes, approximately 0.3 mh aliquot of blood were removed from the ipsilateral femoral artery. After 60 minutes, the monkey was sacrificed and the organs were removed. Samples were processed for double isotope liquid scintillation counting and TCA precipitability as described above.
Clearance and volume of distribution calculations were performed as described above.
Table 3 tabulates the integrated Vp of ('H]-cationized immunoglobulin G(clgG) and [12$I]-bovine serum albumin (BSA) 60 minutes after a single intravenous injection in the Cynomologous Monkey. In general, the monkey Vp values for native BSA at 60 minutes are comparable to Vp values in the rat.
Additionally, the uptake of cationized IgG by organs is substantially increased over BSA. Although the cationized IgG organ uptake in the primate was increased over that of the organ uptake of native bovine albumin, the enhanced uptake is relatively modest since the '20 primate experiment was restricted to organ measurements at a time period of only 60 minutes following the intravenous injection. dwing to the relatively slow second phase of clearance of the cationized IgG from the primate blood (see below), there is a linear increase in the volume of distribution of the can onized IgG by many organs in the primate, proportional to the duration following the intravenous injection of cationized antibody, similarly to that observed for the rat (Fig.
2).
WO 91/13b31 ~ ~~~ ~ ~ "~~ ,~ P(.T/US91/01533 TABLE
Integrated of Distribution Volume (VD) of [3H ]_Cati onized 5 ImmunoglobulinG (clgG) and [zxsl]_Bovine Albumin (BSA) 60 Serum Minutes Aftera Single Intravenous Injection Cynomologous Mon)tey in a 10 VD (~tLcJ') clgG VD
organ __ BSA cIgG BSA VD
Liver 350 -' 22 2537499 7.2 15 Spleen 387 8 2400216 6.2 Kidney 312 2 114323 3.7 Muscle 14 1 31 1- 2 2.2 White 15 -1 1 2.1 matter 7.2 0.6 Fat 19 * 4 31 -!- 4 1.6 .
20 Heart 128 4 177 10 1.4 Lung 439 11 590 17 1.3 Gray matter 22 1 1.2 Intestine100 7 118 12 1.2 Testis 164 4 154 3 0.94 Cheroid 279 0.93 Plex_ us tiara are mean : S.E. (n = 3 samples from one monkey,L
Fig. 3 illustrates the decay in serum [1251]_native BSA and [3H]-cationized IgG radioactivity following a single intravenous injection in a Macaca irus monkey.
As indicated in Fig. 3, the total DPMs injected'at zero time for the labelled BSA (0.254 DPM/ML/o injected) is about 14 fald lower than that for labelled BSA in the rat (3.5 DPM/ML/% injected, Fig. 1). Since the weight of the primate is approximately 14 fold greater than the weight of the rat it is likely that the difference is ~ ~_4 ~ d b) ~ 'i7 ~;1V0 91/13631 P~I"1US91101533 ''.':b:: ' '~Si~i ~'~
~i due to the larger primate blood volume. It is clear from the rat and primate experiments that the cationi-zation procedure in accordance with the present invention results in markedly increased rates of uptake of the IgG by organs after cationization of antibodies.
The data shown in Figs. 1 and 3 illustrate the highly favorable pharmacokinetics of ['H]-cationized IgG
clearance by organs. Owing to the rapid saturation of uptake sites in liver and spleen, there is a prolonged slow second phase of clearance of ['1-I]-cationized IgG
from blood. The maintenance of this prolonged slow phase of clearance from blood allows for progressive and linear increase of the cationized IgG by a number of different organs. The relatively long half-time of catianized IgG (e.g., 3.0 ~ 1.0 hours in rats or 2.9 ~
1.6 hours in a primatey indicates that the cationized IgG pharmaceutic need not be administered continuously, but could be administered on a once, twice, or three times a day basis.
~0 Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations and modifications may be made within the scope of the present invention. Accordingly the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.
WO91/13631 ~ 1 !~ PCT/US91/01533 ~
a~
R E F E R E ~T C E 8 1. Reynoso, G., Keane, M., and Reynoso, M.A. (.1985):
Monoclonal carcinoembryyonic antigen antibodies. ' In: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humans Press, Clifton, New Jersey, pp. 19°40. ' 2. Harden E.A., Palker, T.J., and Haynes, B.F. (1985):
Monoclonal antibodies. Probes for study of malignant T cells. Win: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humans Press, Clifton, New Jersey, pp. 121°145.
3. Reisfeld, R.A. (1985): Monoclonal antibodies as probes for the molecular structure and biological function of melanoma-associated antigens. In:
Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humans Press, Clifton, New Jersey, pp. 205-228.
4. Rittmann, L.S., Sobol, R.E., Astarita, R.W., and Martinis, J. (1985): Monoclonal antibodies to human small-cell lung cancer. In: Monoclonal Antibodies:
Diagnostic and Therapeutic Use in Tumor and Transplantation (Chatterjee, S.M., ed.). PSG
Publishing Company, Inc., Littleton, Massachusetts, pp. 73-83.
~3 0 Mulshine, J.L., Cuttitta, F., and Minna, J.D.
(1985): Lung cancer markers as detected by monoclonal anti bodies. _In: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humans Press, Clifton, New J~, pp. 229-246.
Diagnostic and Therapeutic Use in Tumor and Transplantation (Chatterjee, S.M., ed.). PSG
Publishing Company, Inc., Littleton, Massachusetts, pp. 73-83.
~3 0 Mulshine, J.L., Cuttitta, F., and Minna, J.D.
(1985): Lung cancer markers as detected by monoclonal anti bodies. _In: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humans Press, Clifton, New J~, pp. 229-246.
5. Chu, T.M. (1985): Monoclonal antibodies to human prostate cancer-related antigens. In: Monoclonal Antibodies to Cancer (Sell, S. and Reisfeld., R.A., eds.). Humans Press, Clifton, New Jersey, pp.
309-324.
Raynor, R.H., Mohanakumar, T., Monclure, C.W., and Hazra, T.A. (1985): Monoclonal antibodies to human prostate tissued. In: Monoclonal Antibodies (Chatter jee, S.M., ed.). PSG Publishing Company, Inc., Littleton, Massachusetts, pp.
155-161.
309-324.
Raynor, R.H., Mohanakumar, T., Monclure, C.W., and Hazra, T.A. (1985): Monoclonal antibodies to human prostate tissued. In: Monoclonal Antibodies (Chatter jee, S.M., ed.). PSG Publishing Company, Inc., Littleton, Massachusetts, pp.
155-161.
6. Schlom, J., Greiner, J., Hand, P.H., Colcher, D., ' Inghiram, G., Weeks, M., Pestka, S., Fisher, P.B., Noguchi, P., and Kufe, D. (1985): Human breast cancer markers defined by monoclonal antibodies.
In: Monoclonal Antibodies in Cancer (Sell, S. and iW~ 91/13631 ~ ~ ~ ~7 ~ ~ ~ PCT1US91/~D1533 .,.::._..
Reisfeld, R.A., eds.). Humana Press, Clifton, New Jersey, pp. 247-278.
In: Monoclonal Antibodies in Cancer (Sell, S. and iW~ 91/13631 ~ ~ ~ ~7 ~ ~ ~ PCT1US91/~D1533 .,.::._..
Reisfeld, R.A., eds.). Humana Press, Clifton, New Jersey, pp. 247-278.
7. Bander, N.I-I. and Cordon-Cardo, C. (1985) : Monoclonal antibodies to renal cancer markers. In: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humana Press, Clifton, New Jersey, pp.
325-338.
325-338.
8. Drew, S.I., Terasaki, P.I., Johnson, C., Chia, D., Wakisaka, A., Flardiwidjaja, S., Cicciarelli, J., Takasugi, M., Kaszubowski, P., Quinlan, T., Izuka, T., and Hirata, A. (1985): Phase I study of high-dose serotherapy with c~itotoxic monoclonal antibodies in patients with gastrointestinal malignancies. In: Monoclonal Antibodies (Chatterjee, S.N., ed.). PSG Publishing Company, Inc., Littleton, Massachusetts, pp. 127-136.
9. Hollingsworth, M.A. and Metzgar, R.S. (1985):
Antigens of normal and malignant human exocrine pancreatic cells. In: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humana Press, Clifton, New Jersey, pp. 279°308.
Antigens of normal and malignant human exocrine pancreatic cells. In: Monoclonal Antibodies in Cancer (Sell, S. and Reisfeld, R.A., eds.). Humana Press, Clifton, New Jersey, pp. 279°308.
10. Hoare, Koshland. A Method for the Quantitative Modification of Estimation of Carboxylic Acid Groups in Proteins. (1967) J.BioI.Chem. 342:2447°2453.
130 11. Triguero, D., Buciak, J.B., Yang, J., Pardridge, W.M. Blood Brain Barrier Transport of Cationized Immunoglobulin G. Proc. Natl. Acad. Sci., U.S.A.
86: 471--4765, 1989.
12. Pardridge, W.M., Eisenberg, J., and Cefalu, W.T.
Absence of albumin receptor on brain capillaries in vivo or in vitro. Am.J.Physio., 249: E264-E267, 1985.
13. Bergmann, P., Vizet, A., Sennesael, J., Beauwens, R., and Snauwaert, J. Bonding of cationized albumin to red cells. In: The Pathogenicity of Cationic Proteins (ed. P-.P. Lambert, P. Bergmann, and R.
Beauwens), Raven Press, New York, pp. 29-39, 1983.
130 11. Triguero, D., Buciak, J.B., Yang, J., Pardridge, W.M. Blood Brain Barrier Transport of Cationized Immunoglobulin G. Proc. Natl. Acad. Sci., U.S.A.
86: 471--4765, 1989.
12. Pardridge, W.M., Eisenberg, J., and Cefalu, W.T.
Absence of albumin receptor on brain capillaries in vivo or in vitro. Am.J.Physio., 249: E264-E267, 1985.
13. Bergmann, P., Vizet, A., Sennesael, J., Beauwens, R., and Snauwaert, J. Bonding of cationized albumin to red cells. In: The Pathogenicity of Cationic Proteins (ed. P-.P. Lambert, P. Bergmann, and R.
Beauwens), Raven Press, New York, pp. 29-39, 1983.
Claims (78)
1. A method for increasing the transcytosis of an antibody across the microvascular barrier and into the interstitial fluid of mammalian non-central nervous system tissues or organs, said method comprising the steps of: treating said antibody with a sufficient amount of a cationization agent to increase the isoelectric point of said antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than about pH 11.5; mixing said cationized antibody with a pharmaceutically acceptable carrier to provide a cationized antibody composition; and using said cationized antibody composition for increasing transcytosis of said cationized antibody across the microvascular barrier and into the interstitial fluid of said organs, compared to transcytosis of said antibody across said microvascular barrier.
2. A method for facilitating increase in transcytosis of an antibody across the microvascular barrier and into the interstitial fluid of mammalian non-central nervous system tissues or organs, said method comprising the steps of: treating said antibody with a sufficient amount of a cationization agent to increase the isoelectric point of said antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than about pH 11.5; mixing said cationized antibody with a pharmaceutically acceptable carrier to provide a canonized antibody composition; and wherein when said cationized antibody composition is administered to a mammal, transcytosis of said cationized antibody across the microvascular barrier and into the interstitial fluid of said organs is increased compared to transcytosis of said antibody across said barrier.
3. A method according to claim 1 or 2 wherein said antibody has a molecular weight greater than 20,000 Daltons.
4. A method according to claim 1 or 2 wherein said antibody is a monoclonal antibody.
5. A method according to claim 1 or 2 wherein said antibody is IgG.
6. A method according to claim 1 or 2 wherein said antibody is IgM.
7. A method according to claim 4 wherein said antibody is labeled with a detectable radionuclide.
8. A method according to claim 4 wherein said antibody is labeled with a detectable paramagnetic conjugate.
9. A method according to claim 4 wherein said antibody is labeled with a pharmaceutically active drug.
10. The method according to claim 4 wherein said monoclonal antibody is specific for an organ.
11. The method according to claim 4 wherein said non-central nervous system tissues or organs include one or more organ(s) selected from the group consisting of spleen, liver, kidney, lung, small intestine, heart, skeletal muscle, lymphoids, skin, prostate, pancreas, breast, esophagus, and fat.
12. The method according to claim 4 wherein said monoclonal antibody is selected from the group consisting of antibodies to carcinoembryonic antigen, T-lymphocyte receptors, melanoma antigens, lung cancer antigens, prostate cancer antigens, and human breast cancer antigens.
13. The method according to claim 4 wherein said cationization agent is an amine cationization agent.
14. The method according to claim 13 wherein said amine cationization agent is hexamethylenediamine.
15. A system for increasing transcytosis of an antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissues and organs, comprising:
a) an antibody which has been cationized by treatment with a sufficient amount of a cationization agent to increase the isoelectric point of the antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than 11.5;
b) a suitable pharmaceutically acceptable diluent or carrier; and c) means for delivering the cationized antibody in admixture with the diluent or carrier, to a site where the cationized antibody is transcytosed across the microvascular barrier and into the interstitial fluid of said organs.
a) an antibody which has been cationized by treatment with a sufficient amount of a cationization agent to increase the isoelectric point of the antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than 11.5;
b) a suitable pharmaceutically acceptable diluent or carrier; and c) means for delivering the cationized antibody in admixture with the diluent or carrier, to a site where the cationized antibody is transcytosed across the microvascular barrier and into the interstitial fluid of said organs.
16. A system according to claim 15 wherein said antibody has a molecular weight greater than 20,000 Daltons.
17. A system according to claim 15 wherein said antibody is a monoclonal antibody.
18. A system according to claim 15 wherein said antibody is IgG.
19. A system according to claim 15 wherein said antibody is IgM.
20. A system according to claim 17 wherein said antibody is labeled with a detectable radionuclide.
21. A system according to claim 17 wherein said antibody is labeled with a detectable paramagnetic conjugate.
22. A system according to claim 17 wherein said antibody is labeled with a pharmaceutically active drug.
23. The system according to claim 17 wherein said monoclonal antibody is specific for an organ.
24. The system according to claim 17 wherein said non-central nervous system tissues or organs include one or more organ(s) selected from the group consisting of spleen, liver, kidney, lung, small intestine, heart, skeletal muscle, lymphoids, skin, prostate, pancreas, breast, esophagus, and fat.
25. The system according to claim 17 wherein said monoclonal antibody is selected from the group consisting of antibodies to carcinoembryonic antigen, T-lymphocyte receptors, melanoma antigens, lung cancer antigens, prostate cancer antigens, and human breast cancer antigens.
26. The system according to claim 17 wherein said cationization agent is an amine cationization agent.
27. The system according to claim 26 wherein said amine cationization agent is hexamethylenediamine.
28. Use of a cationized antibody, wherein said cationized antibody has been cationized by treatment with a sufficient amount of a cationization agent to increase the isoelectric point of the antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than 11.5, for increasing transcytosis of said antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
29. Use of a cationized antibody, wherein said cationized antibody has been cationized by treatment with a sufficient amount of a cationization agent to increase the isoelectric point of the antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than 11.5, for the manufacture of a medicament for the treatment of conditions where transcytosis of said antibody is desired across microvascluar barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
30. A use according to claim 28 or 29 wherein said antibody has a molecular weight greater than 20,000 Daltons.
31. A use according to claim 28 or 29 wherein said antibody is a monoclonal antibody.
32. A use according to claim 28 or 29 wherein said antibody is IgG.
33. A use according to claim 28 or 29 wherein said antibody is IgM.
34. A use according to claim 31 wherein said antibody is labeled with a detectable radionuclide.
35. A use according to claim 31 wherein said antibody is labeled with a detectable paramagnetic conjugate.
36. A use according to claim 31 wherein said antibody is labeled with a pharmaceutically active drug.
37. The use according to claim 31 wherein said monoclonal antibody is specific for an organ.
38. The use according to claim 31 wherein said non-central nervous system tissues or organs include one or more organ(s) selected from the group consisting of spleen, liver, kidney, lung, small intestine, heart, skeletal muscle, lymphoids, skin, prostate, pancreas, breast, esophagus, and fat .
39. The use according to claim 31 wherein said monoclonal antibody is selected from the group consisting of antibodies to carcinoembryonic antigen, T-lymphocyte receptors, melanoma antigens, lung cancer antigens, prostate cancer antigens, and human breast cancer antigens.
40. The use according to claim 31 wherein said cationization agent is an amine cationization agent.
41. The use according to claim 40 wherein said amine cationization agent is hexamethylenediamine .
42. A pharmaceutical composition comprising cationized antibody, wherein said cationized antibody has been canonized by treatment with a sufficient amount of a cationization agent to increase the isoelectric point of the antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than 11.5, in admixture with a suitable pharmaceutically acceptable diluent or carrier, for increasing transcytosis of said antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
43. A composition according to claim 42 wherein said antibody has a molecular weight greater than 20,000 Daltons.
44. A composition according to claim 42 wherein said antibody is a monoclonal antibody.
45. A composition according to claim 42 wherein said antibody is IgG.
46. A composition according to claim 42 wherein said antibody is IgM.
47. A composition according to claim 44 wherein said antibody is labeled with a detectable radionuclide.
48. A composition according to claim 44 wherein said antibody is labeled with a detectable paramagnetic conjugate.
49. A composition according to claim 44 wherein said antibody is labeled with a pharmaceutically active drug.
50. The composition according to claim 44 wherein said monoclonal antibody is specific for an organ.
51. The composition according to claim 44 wherein said non-central nervous system tissues or organs include one or more organ(s) selected from the group consisting of spleen, liver, kidney, lung, small intestine, heart, skeletal muscle, lymphoids, skin, prostate, pancreas, breast, esophagus, and fat.
52. The composition according to claim 44 wherein said monoclonal antibody is selected from the group consisting of antibodies to carcinoembryonic antigen, T-lymphocyte receptors, melanoma antigens, lung cancer antigens, prostate cancer antigens, and human breast cancer antigens.
53. The composition according to claim 44 wherein said cationization agent is an amine cationization agent.
54. The composition according to claim 53 wherein said amine cationization agent is hexamethylenediamine.
55. A commercial package containing as an active ingredient a cationized antibody, wherein said cationized antibody has been cationized by treatment with a sufficient amount of a cationization agent to increase the isoelectric point of the antibody by between about 1 to about 7 pH units to produce a cationized antibody having an isoelectric point which is less than 11.5, together with instructions for its use for increasing transcytosis of the antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
56. A commercial package containing an antibody and cationization agent for forming as an active ingredient a cationized antibody, together with instructions for use of the cationized antibody, for increasing transcystosis of the antibody across microvascular barrier and into interstitial fluid of mammalian non-central nervous system tissue or organ.
57. A commercial package according to claim 55 or 56 wherein said antibody has a molecular weight greater than 20,000 Daltons.
58. A commercial package according to claim 55 or 56 wherein said antibody is a monoclonal antibody.
59. A commercial package according to claim 55 or 56 wherein said antibody is IgG.
60. A commercial package according to claim 55'or 56 wherein said antibody is IgM.
61. A commercial package according to claim 58 wherein said antibody is labeled with a detectable radionuclide.
62. A commercial package according to claim 58 wherein said antibody is labeled with a detectable paramagnetic conjugate.
63. A commercial package according to claim 58 wherein said antibody is labeled with a pharmaceutically active drug.
64. The commercial package according to claim 58 wherein said monoclonal antibody is specific for an organ.
65. The commercial package according to claim 58 wherein said non-central nervous system tissues or organs include one or more organ(s) selected from the group consisting of spleen, liver, kidney, lung, small intestine, heart, skeletal muscle, lymphoids, skin, prostate, pancreas, breast, esophagus, and fat.
66. The commercial package according to claim 58 wherein said monoclonal antibody is selected from the group consisting of antibodies to carcinoembryonic antigen, T-lymphocyte receptors, melanoma antigens, lung cancer antigens, prostate cancer antigens, and human breast cancer antigens.
67. The commercial package according to claim 58 wherein said cationization agent is an amine cationization agent.
68. The commercial package according to claim 67 wherein said amine cationization agent is hexamethylenediamine.
69. The method according to claim 11, wherein the mammalian non-central nervous system organ is kidney or lung, and the step of treating said antibody with cationization agent produces a cationized antibody having an isoelectric point which is between about 8 and 10 pH units.
70. The method according to claim 11, wherein the mammalian non-central nervous system organ is intestine, and the step of treating said antibody with cationization agent produces a cationized antibody having an isoelectric point which is between about 9 and 11 pH units.
71. The system according to claim 24, wherein said mammalian non-central nervous system organ is kidney or lung, and said cationized antibody has an isoelectric point which is between about 8 and 7n0 pH units.
72. The system according to claim 24, wherein said mammalian non-central nervous system organ is intestine, and said cationized antibody has an isoelectric point which is between about 9 and 11 pH units.
73. The use according to claim 38, wherein the mammalian non-central nervous system organ is kidney or lung, and said cationized antibody has an isoelectric point which is between about 8 and 10 pH units.
74. The use according to claim 38, wherein said mammalian non-central nervous system organ is intestine, and said cationized antibody has an isoelectric point which is between about 9 and 11 pH units.
75. The composition according to claim 51, wherein said mammalian non-central nervous system organ is kidney or lung, and said cationized antibody has an isoelectric point which is between about 8 and 10 pH units.
76. The composition according to claim 51, wherein said mammalian non-central nervous system organ is intestine, and said cationized antibody has an isoelectric point which is between about 9 and 11 pH units.
77. The commercial package according to claim 65, wherein said mammalian non-central nervous system organ is kidney or lung, and said cationized antibody has an isoelectric point which is between about 8 and 10 pH units.
78. The commercial package according to claim 65, wherein said mammalian non-central nervous system organ is intestine, and said cationized antibody has an isoelectric point which is between about 9 and 11 pH units.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US488,993 | 1990-03-06 | ||
| US07/488,993 US5130129A (en) | 1990-03-06 | 1990-03-06 | Method for enhancing antibody transport through capillary barriers |
| PCT/US1991/001533 WO1991013631A1 (en) | 1990-03-06 | 1991-03-06 | Method for enhancing antibody transport through capillary barriers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2077670A1 CA2077670A1 (en) | 1991-09-07 |
| CA2077670C true CA2077670C (en) | 2002-10-01 |
Family
ID=23941969
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002077670A Expired - Lifetime CA2077670C (en) | 1990-03-06 | 1991-03-06 | Method for enhancing antibody transport through capillary barriers |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US5130129A (en) |
| EP (1) | EP0518982A4 (en) |
| JP (1) | JP3210662B2 (en) |
| CA (1) | CA2077670C (en) |
| WO (1) | WO1991013631A1 (en) |
Families Citing this family (49)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6329508B1 (en) | 1989-09-07 | 2001-12-11 | Alkermes, Inc. | Transferrin receptor reactive chimeric antibodies |
| US5527527A (en) * | 1989-09-07 | 1996-06-18 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical agent conjugates |
| US5672683A (en) * | 1989-09-07 | 1997-09-30 | Alkermes, Inc. | Transferrin neuropharmaceutical agent fusion protein |
| US5977307A (en) * | 1989-09-07 | 1999-11-02 | Alkermes, Inc. | Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins |
| DE69222303T2 (en) | 1991-04-30 | 1998-01-22 | Eukarion Inc | CATIONIZED ANTIBODIES AGAINST INTRACELLULAR PROTEINS |
| US5607691A (en) * | 1992-06-12 | 1997-03-04 | Affymax Technologies N.V. | Compositions and methods for enhanced drug delivery |
| US6015555A (en) * | 1995-05-19 | 2000-01-18 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates |
| US5773292A (en) * | 1995-06-05 | 1998-06-30 | Cornell University | Antibodies binding portions, and probes recognizing an antigen of prostate epithelial cells but not antigens circulating in the blood |
| JP2000503004A (en) * | 1995-12-13 | 2000-03-14 | プレジデント アンド フェロウズ オブ ハーバード カレッジ | Use of toxin peptides and / or affinity handles to deliver compounds to cells |
| US20030096748A1 (en) * | 2001-06-04 | 2003-05-22 | The Regents Of The University Of Michigan | Methods and compositions for the treatment of diseases associated with signal transduction aberrations |
| US7074893B2 (en) * | 2002-06-03 | 2006-07-11 | Regents Of The University Of Michigan | Methods and compositions for the treatment of diseases associated with signal transduction aberrations |
| EP4001409A1 (en) | 2006-03-31 | 2022-05-25 | Chugai Seiyaku Kabushiki Kaisha | Methods for controlling blood pharmacokinetics of antibodies |
| WO2008100560A2 (en) * | 2007-02-14 | 2008-08-21 | University Of Southern California | Estrogen receptor modulators, associated pharmaceutical compositions and methods of use |
| CN101874042B9 (en) | 2007-09-26 | 2019-01-01 | 中外制药株式会社 | Method for changing isoelectric point of antibody by using amino acid substitution of CDR |
| US8549912B2 (en) | 2007-12-18 | 2013-10-08 | Teradyne, Inc. | Disk drive transport, clamping and testing |
| US7996174B2 (en) | 2007-12-18 | 2011-08-09 | Teradyne, Inc. | Disk drive testing |
| NZ602884A (en) | 2008-04-11 | 2014-08-29 | Chugai Pharmaceutical Co Ltd | Antigen-binding molecule capable of binding to two or more antigen molecules repeatedly |
| US7945424B2 (en) | 2008-04-17 | 2011-05-17 | Teradyne, Inc. | Disk drive emulator and method of use thereof |
| US20090262455A1 (en) | 2008-04-17 | 2009-10-22 | Teradyne, Inc. | Temperature Control Within Disk Drive Testing Systems |
| US7848106B2 (en) | 2008-04-17 | 2010-12-07 | Teradyne, Inc. | Temperature control within disk drive testing systems |
| US8117480B2 (en) | 2008-04-17 | 2012-02-14 | Teradyne, Inc. | Dependent temperature control within disk drive testing systems |
| US8305751B2 (en) | 2008-04-17 | 2012-11-06 | Teradyne, Inc. | Vibration isolation within disk drive testing systems |
| US8238099B2 (en) | 2008-04-17 | 2012-08-07 | Teradyne, Inc. | Enclosed operating area for disk drive testing systems |
| US8095234B2 (en) | 2008-04-17 | 2012-01-10 | Teradyne, Inc. | Transferring disk drives within disk drive testing systems |
| US8041449B2 (en) | 2008-04-17 | 2011-10-18 | Teradyne, Inc. | Bulk feeding disk drives to disk drive testing systems |
| US8160739B2 (en) | 2008-04-17 | 2012-04-17 | Teradyne, Inc. | Transferring storage devices within storage device testing systems |
| US8102173B2 (en) | 2008-04-17 | 2012-01-24 | Teradyne, Inc. | Thermal control system for test slot of test rack for disk drive testing system with thermoelectric device and a cooling conduit |
| US8086343B2 (en) | 2008-06-03 | 2011-12-27 | Teradyne, Inc. | Processing storage devices |
| US8547123B2 (en) | 2009-07-15 | 2013-10-01 | Teradyne, Inc. | Storage device testing system with a conductive heating assembly |
| US7920380B2 (en) | 2009-07-15 | 2011-04-05 | Teradyne, Inc. | Test slot cooling system for a storage device testing system |
| US8687356B2 (en) | 2010-02-02 | 2014-04-01 | Teradyne, Inc. | Storage device testing system cooling |
| US8116079B2 (en) | 2009-07-15 | 2012-02-14 | Teradyne, Inc. | Storage device testing system cooling |
| US7995349B2 (en) | 2009-07-15 | 2011-08-09 | Teradyne, Inc. | Storage device temperature sensing |
| US8466699B2 (en) | 2009-07-15 | 2013-06-18 | Teradyne, Inc. | Heating storage devices in a testing system |
| US8628239B2 (en) | 2009-07-15 | 2014-01-14 | Teradyne, Inc. | Storage device temperature sensing |
| KR102385507B1 (en) | 2010-11-30 | 2022-04-12 | 추가이 세이야쿠 가부시키가이샤 | Antigen-binding molecule capable of binding to plurality of antigen molecules repeatedly |
| US10441561B2 (en) | 2012-07-26 | 2019-10-15 | The William M. Yanbrough Foundation | Method for treating benign prostatic hyperplasia (BPH), prostatitis, and prostate cancer |
| RU2729831C2 (en) | 2012-08-24 | 2020-08-12 | Чугаи Сейяку Кабусики Кайся | Versions of fcγriib-specific fc-region |
| JP6774164B2 (en) | 2012-08-24 | 2020-10-21 | 中外製薬株式会社 | Mouse FcγRII specific Fc antibody |
| WO2014145159A2 (en) * | 2013-03-15 | 2014-09-18 | Permeon Biologics, Inc. | Charge-engineered antibodies or compositions of penetration-enhanced targeting proteins and methods of use |
| EP3783017A1 (en) | 2013-04-02 | 2021-02-24 | Chugai Seiyaku Kabushiki Kaisha | Fc region variant |
| SG11201700841QA (en) | 2014-12-19 | 2017-03-30 | Chugai Pharmaceutical Co Ltd | Anti-myostatin antibodies, polypeptides containing variant fc regions, and methods of use |
| CN114773469A (en) | 2015-02-05 | 2022-07-22 | 中外制药株式会社 | Antibodies comprising an ion concentration-dependent antigen-binding domain, FC region variants, IL-8-binding antibodies and uses thereof |
| US11359009B2 (en) | 2015-12-25 | 2022-06-14 | Chugai Seiyaku Kabushiki Kaisha | Anti-myostatin antibodies and methods of use |
| SG11201801024XA (en) | 2016-08-05 | 2018-05-30 | Chugai Pharmaceutical Co Ltd | Therapeutic or preventive compositions for il-8-related diseases |
| US11226390B2 (en) | 2017-08-28 | 2022-01-18 | Teradyne, Inc. | Calibration process for an automated test system |
| US10948534B2 (en) | 2017-08-28 | 2021-03-16 | Teradyne, Inc. | Automated test system employing robotics |
| US10983145B2 (en) | 2018-04-24 | 2021-04-20 | Teradyne, Inc. | System for testing devices inside of carriers |
| US10775408B2 (en) | 2018-08-20 | 2020-09-15 | Teradyne, Inc. | System for testing devices inside of carriers |
Family Cites Families (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4118379A (en) * | 1974-09-06 | 1978-10-03 | Behringwerke Aktiengesellschaft | Amidated immune globulins and process for preparing them |
| US4219335A (en) * | 1978-09-18 | 1980-08-26 | E. I. Du Pont De Nemours And Company | Immunochemical testing using tagged reagents |
| US4756907A (en) * | 1978-10-17 | 1988-07-12 | Stolle Research & Development Corp. | Active/passive immunization of the internal female reproductive organs |
| US4585651A (en) * | 1978-10-17 | 1986-04-29 | Stolle Research & Development Corporation | Active/passive immunization of the internal female reproductive organs |
| US4732763A (en) * | 1978-10-17 | 1988-03-22 | Stolle Research And Development Corporation | Active/passive immunization of the internal female reproductive organs |
| US4311688A (en) * | 1979-10-29 | 1982-01-19 | Serono Laboratories Inc. | Composition and method for cancer detection in humans |
| US4444744A (en) * | 1980-03-03 | 1984-04-24 | Goldenberg Milton David | Tumor localization and therapy with labeled antibodies to cell surface antigens |
| US4417967A (en) * | 1981-11-24 | 1983-11-29 | Georgetown University | Grooved gel |
| US4454106A (en) * | 1982-06-07 | 1984-06-12 | Gansow Otto A | Use of metal chelate conjugated monoclonal antibodies |
| US4741900A (en) * | 1982-11-16 | 1988-05-03 | Cytogen Corporation | Antibody-metal ion complexes |
| US4624846A (en) * | 1983-07-29 | 1986-11-25 | Immunomedics, Inc. | Method for enhancing target specificity of antibody localization and clearance of non-target diagnostic and therapeutic principles |
| US4649784A (en) * | 1985-01-31 | 1987-03-17 | Robert G. Fulks | Method and apparatus for sensing activity for a keyboard and the like |
| US4758421A (en) * | 1985-03-15 | 1988-07-19 | The Board Of Trustees Of The Leland Stanford Junior University | Bleomycin conjugates and method |
| US4735210A (en) * | 1985-07-05 | 1988-04-05 | Immunomedics, Inc. | Lymphographic and organ imaging method and kit |
| US4699784A (en) * | 1986-02-25 | 1987-10-13 | Center For Molecular Medicine & Immunology | Tumoricidal methotrexate-antibody conjugate |
| US4732974A (en) * | 1986-03-05 | 1988-03-22 | Mallinckrodt, Inc. | Metal ion labeling of carrier molecules |
| US4863713A (en) * | 1986-06-23 | 1989-09-05 | The Board Of Trustees Of Leland Stanford Jr. Univ. | Method and system for administering therapeutic and diagnostic agents |
| US5004697A (en) * | 1987-08-17 | 1991-04-02 | Univ. Of Ca | Cationized antibodies for delivery through the blood-brain barrier |
| US4859449A (en) * | 1987-09-14 | 1989-08-22 | Center For Molecular Medicine And Immunology | Modified antibodies for enhanced hepatocyte clearance |
| US5322678A (en) * | 1988-02-17 | 1994-06-21 | Neorx Corporation | Alteration of pharmacokinetics of proteins by charge modification |
-
1990
- 1990-03-06 US US07/488,993 patent/US5130129A/en not_active Expired - Lifetime
-
1991
- 1991-03-06 CA CA002077670A patent/CA2077670C/en not_active Expired - Lifetime
- 1991-03-06 WO PCT/US1991/001533 patent/WO1991013631A1/en not_active Application Discontinuation
- 1991-03-06 EP EP19910906119 patent/EP0518982A4/en not_active Ceased
- 1991-03-06 JP JP50617591A patent/JP3210662B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US5130129A (en) | 1992-07-14 |
| JPH05505606A (en) | 1993-08-19 |
| CA2077670A1 (en) | 1991-09-07 |
| JP3210662B2 (en) | 2001-09-17 |
| WO1991013631A1 (en) | 1991-09-19 |
| EP0518982A1 (en) | 1992-12-23 |
| EP0518982A4 (en) | 1993-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2077670C (en) | Method for enhancing antibody transport through capillary barriers | |
| EP0557270B1 (en) | Cationized antibodies for delivery through the blood-brain barrier | |
| US8258101B2 (en) | Peptide-based compounds | |
| US5990286A (en) | Antibodies with reduced net positive charge | |
| US4895714A (en) | Radioiodinated apotransferrin for use in tumor diagnosis, imaging, localization or treatment | |
| DE60111733T2 (en) | INTEGRIN BINDING PEPTIDE DERIVATIVES | |
| JPH06500563A (en) | Modified antibodies with controlled purification times | |
| US20090317327A1 (en) | Aqueous Dispersion of Superparamagnetic Single-Domain Particles, Production and Use Thereof in Diagnosis and Therapy | |
| AU2001292456A1 (en) | Peptide-based compounds | |
| JP2012131808A (en) | Antibody having reduced net positive charge | |
| DE69535094T2 (en) | PROTEIN MARKING WITH RADIOACTIVE PHOSPHORUS AND TARGETED RADIO THERAPY | |
| US5223242A (en) | Negatively charged specific affinity reagents | |
| EP0268707A2 (en) | Negatively charged specific affinity reagents | |
| US5380513A (en) | Methods for reducing non-target retention of immunoconjugates and metabolites thereof | |
| WO1997025069A9 (en) | Antibodies with reduced net positive charge | |
| Babich et al. | Targeted imaging of infection | |
| EP0090025A1 (en) | Specific mammary gland labelling | |
| KR20230124425A (en) | Nanoparticles for Molecular Imaging Diagnosis and Manufacturing Methods Thereof | |
| TIEFENAUER et al. | O Original Contribution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed | ||
| MKEC | Expiry (correction) |
Effective date: 20121202 |