CA1304293C - Immunoconjugates and methods for their use in tumor therapy - Google Patents
Immunoconjugates and methods for their use in tumor therapyInfo
- Publication number
- CA1304293C CA1304293C CA000541057A CA541057A CA1304293C CA 1304293 C CA1304293 C CA 1304293C CA 000541057 A CA000541057 A CA 000541057A CA 541057 A CA541057 A CA 541057A CA 1304293 C CA1304293 C CA 1304293C
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- immunoconjugate
- chemotherapeutic agent
- tumor
- spacer
- 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 - Fee Related
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
-
- 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
- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
-
- 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
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
Abstract
Abstract Novel pH-sensitive immunoconjugates which dissociate in low-pH tumor tissue, comprising a chemotherapeutic agent and an antibody reactive with a tumor-associated antigen are described. The chemotherapeutic agent is coupled to the antibody by a link which is unstable in low pH. The link may comprise a spacer consisting of a polyamino acid, Rep-resentative antibodies for use in these immunoconjugates include monoclonal antibodies which are not internalized by tumor cells.
149Z0531D7.9
149Z0531D7.9
Description
.~
~3~2~;3 'i~ld ~t ~h~ ~nv~ntion The present invention relates to the production of no~el immunoconjugates unstable at low p~, in particular to such immunoconjugates containing chemotherapeutic agents and ~o methods of using such immunoconjugates in chemotherapy.
nd of the rnvention :
Although various chemotherapeutic drugs have been found effective against certain tumors and even curative against some (Devita and ~ellman, eds., in Cancer, 5rincioles and Practice of Oncoloqy, Lippincott ~ Co. (1982)1, there is a great need for ~herapeutic agents ~hi-h kill cancer cells more efficiently and more selectivel~. An attractive approach ~owards meeting this need is to use antibodies to prepare anti~ody-drug complexes or "immunoconjugates" that direct or "target" anti-cancer agents to tumors. Antibodies are known in the art which recognize ar.tisens expressed on cancer cells, for example the antibody 96.5 which reac;s with the p97 antigen of human melanomas (3rown et al., J.
I~munol., 127 p. 539 (lsal) ) . Several immunoconjugates of this type have been shown to be selectively c-{totoxic to antigen-positive tumor cells ln vitro, ~o localize in tumors in vivo, and to have anti-tumor activi~y in mice ~hat is greater than that of he drug or antiDody alor.e (~owland e~
al-, L~~ D~ eL~ s~ 19, pp. 1 (1385)).
While the ability of such immunoconjuga~es to cure human tumors remains to be demonstrated, improvements in tumor targeting have been the focus of recent research efforts.
.
:
' `~9.
~3~2~;3 'i~ld ~t ~h~ ~nv~ntion The present invention relates to the production of no~el immunoconjugates unstable at low p~, in particular to such immunoconjugates containing chemotherapeutic agents and ~o methods of using such immunoconjugates in chemotherapy.
nd of the rnvention :
Although various chemotherapeutic drugs have been found effective against certain tumors and even curative against some (Devita and ~ellman, eds., in Cancer, 5rincioles and Practice of Oncoloqy, Lippincott ~ Co. (1982)1, there is a great need for ~herapeutic agents ~hi-h kill cancer cells more efficiently and more selectivel~. An attractive approach ~owards meeting this need is to use antibodies to prepare anti~ody-drug complexes or "immunoconjugates" that direct or "target" anti-cancer agents to tumors. Antibodies are known in the art which recognize ar.tisens expressed on cancer cells, for example the antibody 96.5 which reac;s with the p97 antigen of human melanomas (3rown et al., J.
I~munol., 127 p. 539 (lsal) ) . Several immunoconjugates of this type have been shown to be selectively c-{totoxic to antigen-positive tumor cells ln vitro, ~o localize in tumors in vivo, and to have anti-tumor activi~y in mice ~hat is greater than that of he drug or antiDody alor.e (~owland e~
al-, L~~ D~ eL~ s~ 19, pp. 1 (1385)).
While the ability of such immunoconjuga~es to cure human tumors remains to be demonstrated, improvements in tumor targeting have been the focus of recent research efforts.
.
:
' `~9.
-2- 1 ~ Q ~ 9 For a chemotherapeutiC agent to be able to exert an effect on tumors, it must be taken up by the tumor cells, since very few, if any, cancer drugs ~re otherwise cytotoxic. The immunoconjugates must, therefore, be directed to the cancer cells, for example by antibody recog-nition of tumor-associated antigens, and eithcr be taken up by the cancer cells ~with active drug being released inside the cells), or the active drug must bla released in the close vicinity of the cancer cells, and intl~rnalized in the same way as when the drug is used conventionally. The second alternative has several advantage~. First, while anti-cancer drugs can be taken up by most cells, the inter-nalization of immunoconjugates depends on both the antigenic target of the respective antibody and the cell in which the antigen is expressed. Antibodies to an~igens that undergo modulation, i.e., those antibodies that are internalized in the form of an antigen-antibody complex (Old et al., Proc.
Soc. xp._3iol. Med., 124, p. 63 (1967)), are the ones most easily used for drug targeting (Jansen et al., ~mmunol.
Rev., 62, p. 185 (1982)). Second, there is heterogeneity .n the expression by cells of most tumor antigens so that cells which do not express a given antigen, i.e., are antigen-negative, frequently occur within a tumor (Yeh et al., J.
Immunol., 126, p. 1312 ~1981); Albino et al., J. Exo. ,~ed., 154, p. 1764 (1981)). Although the difficulty of accumulat-ing effective levels of chemotherapeutic agents within a tumor as a result of tumor cell heterogeneity can be decreased by combining antibodies to different antigens expressed by the same tumor cells and forming immunoconjugates, it could be fureher minimized if a cherapeutic approach was ~eveloped in which the presence of some minim~l amount of cells possessing the given antigen within 2 tumor would be sufficient to allow locali7ation of effective amounts of immunoconjugates. Third, there are some tumor antigens, mucins, for example, which are present in larger amounts outside of th~ cells than at the cell mem-brane, (Rittenhouse et al~, Laboratorv Medicine, 16, p. 556 (1985)) suggesting the potential for targeting tumor regions.
The acidity ~pH) of tumor tissues appears to be lower ~han ~hat of normal tissues. Studie~; conducted more than half a century ago showed that m~lignant tumors metabolize carbohydrates mainly by anaerobic glycolysis, even under aerobic conditions (Warburg et al., ~iochem~ A., 152, p. 309 (1924)). The oxidation of glucose stops at the stage of glucose oxidation to pyruvic acid, followed by reduction to lactic acid (30xer and Devlin, Science, 134, p. lg95 (1961)). Most of this lactic acid is either removed or buffered by surrounding extracellular fluid, but some of it accumulates extracellularly. ~his results in a lower ?H
within the tumor than in normal tissues. Elevation of the blood-sugar by intravenous infusion of glucose should accel-erate anaerobic metabolism resulting in even more lactic acid in the tumor, and this should further increase the pH
difference between tumors and normal tissues.
Following Warburg's studies, there have been several reports of lower pH in tumors of both experimental animals, (Voegtlin et al., Nat'1. Inst. Hlth. 3ull., 164, p. l (1935); Xahler and Robertson, J Nat. Cancer Inst., 3, p. 495 (1943); and human patients, ~aeslund, Acta Soc. Med.
U~sal., 60, p. 150 (1955); Pampus, Acta ~eurochir., 11, p. 305 (1963)).
Meyer et al., in Cancer ~es., 8, p. 513 (19~8) reported that the pH of malignant human tumors is lower Ihan in nor-mal tissues. In twelve out of fQurteen cases, where both normal and neoplastic tissues ~rom the same pa~ients could be studied ln vivo, there was a difference in pH which aver-aged 0.~9 and ranged from 0.17 to 1.15.
Ashby, (Lancet, August 6, p. 312 (1966)), found that the mean pH of malignant tumors from nine patients was 6.~
(ranging between 6.6 and 6.9). Raising of the blood sugar by intraveneous infusion of dextrose further decreased the tumor pH to a mean of 6.5 (range 6.3 - 6.8).
Van Den seerg et al., Eur. J. Cc ~ , 18, p~ 457 (1982), showed that the pH of twenty-two human mammary carcinomas was 7.29 (~0.05, SEM), as compared to 7.63 (~0.03, SEM) in human subcutis, and observed similar differences in rat tumors. The differences between pH in tumors and normal tissues were highly statistically si~nifi-cant, although they were lower than those reported in the studies discussed above.
Thistlethwaite et al., Int. J. Radiation Oncoloqy 3iol.
~y~, 11, p. 1647 (1985), showed, likewise, that the pH of human tumors as measured by readings on fourteen tumors was below the physiological level with an average of 6.81'0.09 (SEM). They speculated that the reported therapeutic effec-tiveness of hyperthermia depends on the lower extracellular p~ of tumors as compared to normal tissues.
Trouet et al., U.S. Patent No. 4,376,765, describe drug compounds composed of a protein macromolecule ~carrier) linked via a peptide chain ("spacer arm") to an amino func-tion of a drug. The carrier facilitates endocytic take-up by target cells so that the spacer arm may be cleaved ~ithin the cell. Recently, attention has been directed to developing antibody drug conjugates ~hicn release a drug within a tumor cell once the conjugate has crossed the cell membrane and encountered acidic pH (3.5-5.5~ within the -5~
cell. U.5. Patent No. 4,569,789 by Blattler et al., describes chemical formation of conjugates using cross-linking structures which can link amino-group substances such as chemotherapeutic drugs to the sulfhydryl portion of a compound such as an antibody reactive with tumor cell sur-face antigens capable of crossing the tumor cell membrane.
One limitation of such a method of forming conjugates is that the antibody must contain a sulfhydryl group. This reduces the number of possible drug-antibody conjugates which may be formed using such procedures.
In spite of the published evidence that tumors have lower pH than normal tissues, and that acid-cleavable com-plexes may be formed between antibodies and drugs, this evi-dence has not yet resulted in the development of immunoconjugates which are composed of antibodies reactive with tumor associated antigens and chemotherapeutic agents, and which could be targeted to tumor tissues and are capable of selectively releasing the chemotherapeutic agents in the presence o the lower pH of cancer tissues for uptake by the tumor cells, but not at the pH of normal tissue.
Summary of the Invention In the present invention, pH sensitive immunoconjugates are provided for treating tumors in mammals by delivering a chemotherapeutic agent to tumor tissue. The immunocon-jugates comprise an antibody reactive with a tumor-associated antigen coupled to a chemotherapeutic agent by a link which renders the conjugate unstable at low pH. In particular, the immunoconjugates comprise a monoclonal anti-body which does not have to be internalized by tumor cells, and the chemotherapeutic agent is a compound such as an anthracycline compound effective in the treatment of tumors and possessing at least one free amino residue. A species of --b-- 3L3~ ~3 immunoconjugate showing particularly desirable ~ropertieS
for pH sensitivity in the range of pH of human tumor tissue, is that comprised of the L6 monoclonal antibody coupled by a poly-L-Lysine spacer to ~he drug Daunomycin.
Brief Descri~tion of the Drawinqs ; The present invention will be described in connection ~ with the accompanying drat~ings in which:
- : FIGURE 1 is a graph depicting gel chromatographs of the ; antibody-Daunomycin immunoconjugate reaction mixture FIGURE 2 is a photograph of an electrophoretic (5DS) gel of modified and unmodified antibody;
FIGURE 3 depicts the absorption spectra of the free and conjugated forms of Daunomycin;
FIGURE 4 illustrates the effects of changes in pH on the kinetics of release of Daunomycin conjugated to hu~nan : Ig~;
FIGURE 5 is a graph of the toxicity to melanoma cells of various doses of free Daunomycin as measured by 3[H¦
thymidine uptake by cells over time;
: `
FIGURE 6 is a graph of the toxicity to lung carcinoma cells of various doses of Daunomycin as measured by 3[H]
thymidine uptake by cells over time;
FIGURE 7 is a graph of the binding of the L6 antibody : to lung carcinoma cells, and cf the 96.5 antibody to melanoma cells at different pH;
FIGURE 8 is a graph of the competition binding assay of :~ the L6-Poly-L-Lysine-Daunomycin (L6-PLS~ADM) conjugate to :~ the fixed cell line 33~7 (M~85 designates the conjuga~e);
; ' : ` .
~ t33 FIGURE 9 is a graph of thymidine inhibition of the L6 - PLS -ADM con~ugate;
FrGuRE 10 is a graph of colony inhibition showing toxicity of the L6-PLS-ADM conjugate at pX 6 and pH 7 (M214 designates the conjugate);
FIGURE 11 is a graph showing the blood clearance of the L6-PLS-ADM conjugate in nude mice. Thi.s graph compares the blood clearance of the conjugate to nat:ive L6 antibody and to the non-specific antibody lF5;
FIGURE 12 is a graph of the in vivo tumor uptake of L6-PLS-ADM conjugate, L6 antibody alone and the non-specific IF5 antibody;
FIGURE 13 is a graph of the localization index (LoI~) as a function of time for the L6-P~S-ADM conjugate and the native L6 anti~ody;
FIGURE 14 is a graph of the 1n vlvo kidney uptake of the L6-PLS-ADM conjugate, L6 antibody alone and the I~5 non-specific antibody; and FIGURE 15 is a graph of the ln vivo liver uptake of the L6-PLS-ADM conjugate, L6 antibody alone and the IF5 non-specific antibody.
Accordingly, the present in~ention provides novel immunoconjugates composed of antibodies selectively reactive with tumor-associated antigens to target tumor tissues linked to chemotherapeutic agents. The immunoconjugates are unstable in low pH tumor tissues. The conjugates have a low toxicity at the pH of normal tissue, but when the conjugates localize in low pH tumor tissue as a result of recognition by the antibodies of ~he antigens associated with tumor ~L3~ 3 cells because of the chemical inst~bility of the conjugates, the chemotherapeutic agent is released and can be taken up by the tumor cells. Therefore, it is unnecessary for the entire conjugate to be internalized within the tumor cell, i.e., for the antibody to cross the cell membrane, for cell death to occur. In addition, those tumor cells which lack the target antigen can still be killed by the chemothera-p~utic agent, provided a sufficient number of cells within the tumor express the antigen recognized by the antibody of the immunoconjugate. In addition, the invention includes methods for using these pH-sensitive immunoconjugates in chemotherapy, by introducing the conjugates into a patient to localize in low pH tumor tissue, where the chemotherapeutic is released and allowed to diffuse into the tumor cells. Thus, the expres~ion of tumor-associated anti-gens in only a minimal number of the targeted tumor cells or tumor-associated tissue is required for tumor therapy, using the present invention. The examples set forth below demon-strate the ability of immunoconjugates prepared according to the invention, to localize in tumor tissue in an animal model.
To form the i~munoconjugates of this invention, suit-able antibodies must be selected or developed. The antibodies used for the conjugates are preferably monoclonal antibodies of either mouse or human origin, which are reactive with antigens that are expressed most strongly at the surface of tumor cells and/or in the close vicinity (i.e. outside the cell membrane~ of tumor cells. Monoclonal antibodies may be ?roduced using procedures such as those described by Kohler and Milstein in Nature, 256, p. 495, (1975). An example of one such monoclonal antibody, and the antibody preferred for use in this invention, is the L6 antibody (American Type Culture Collection "ATCC," No.
`-" ~ g ~iL3Q~.~9~
HB8677), an IgG2a mouse immunoglobulin which is specific for a gaglioside antigen and which reacts with most human carci-nomas. The ganglioside antigen (referred to as the "L6 antigen"), is expressed at the surface of cells of most human carcinomas, including non-small lung carcinomas, breast carcinomas, colon carcinomas and ovarian carcinomas~
The L6 antibody and the L6 antlgen are described in copending Canadian application Serial No. 497,251 filed 10 December, 1985 and assigned to the same assignee as the present invention.
.
The L6 antigen does not modulate in the presence of L6 antibody (i.e., the antigen antibody complex is not internalized), indicating that the L6 antibody remains at the cell surface and is not taken up by tumor cells.
Additional monoclonal antibodies of mouse, rat, human or other origin can be generated to the L6 antigen, or other tumor-associated antigens. Chimeric antibodies, obtained by splicing together genes for the variable region of the anti-body molecule (of mouse origin) and genes for the constant region (of human origin~ as are exemplified by the work of Morrison et al., Proc. Natl. Acad. Sci., 81, p. 6a51 (1984), and Takeda et al., Nature, 314, p. ~52 (1985), may also be used. The immunoconjugates can also be made by using polyclonal sera which are prepared in various species, including rabbits and monkeys. Various fragments which are, for example, obtained by proteolytic digestion of antibody molecules, and include Fab, (Fab')2, and Fc fragments can also be used. The present invention can equally well be carried out by using antibodies and fragments which are spe-cific for antigens other than the L6 antigen, as long as the A
10- ;~3~9 ~ r33 antibodies and fragments have a high affinity constant (103 M or better) and the antigen is either expressed in high levels at the tumor cell surface (at least 50,000 molecules per cell) or is present at relatively high levels in the immediate vicinity of the tumor cells~
Suitable chemotherapeutic agents for use in the present invention are those which have-a cytotoxic and/or grow~h inhi~itory effect on cancer cells. These include therapeutic agents of the type commonly used in the treat-ment of human cancer, including antineoplastic drugs such as the anthracycline compounds Daunomycin, Mitomycin C, Adriamycin, and antimetabolites such as the folic acid antagonist, for example, Methotrexate.
In the present invention, the i~munoconjugates must be unstable at lo~ p.~ to release the chemotherapeutic agent.
This may be ac-cmplished using several methods of chemical synthesis. In one approach, a pH-sensitive link such as aconitic anhydride, is attached to a chemotherapeutic agent and the carboxyl group (-COOH) of the agent is then coupled to the lysine group of the antibody. This approach is simi-lar to the chemistry described by Shen and ~yser, 3iochem.
3ioDhys. Res. Comm., 102, p. 10~8 (1981), . Stable immunoconjugates between toxins and anti~odies to certain lymphocyte populations for carry-ing the conjugate into the target cells, have been developed using such procedures; these immunotoxins have been found to be immuno-suppressive. Diener et al., Science, 231 p. 118 (1985).
The pH unstable immunoconjugates of the present inven-tion may also be formed using an aconitic anhydride link to couple the chemotherapeutic agent to the antibody. These reactions are depicted below. In Step I of such a .
11 ~
procedure, the labile gamma-carboxyl group of aconitic anhydride is reacted with a suitab].e chemotherapeutic agent, such as Daunomycin, containing at least one free amino group forming an intermediate compound (1). In the next step (II), this inte~nediate is reacted with an available antibody containing at least one lysine group, in the presence of carbodiimide reagent to form an immunoconjugate consisting of Daunomycin and antibody coupled by the link. This immunoconjugate ~2j will dissociate in low pH medium such as tumor tissue as #2 shown in Step III.
0 01~
I. ~ COR ~H-~ ~C/
CH30 0 Y ~ ~0 l ~ COOH
HO ~
Daunomycin ~ N-Cis Aconitic Anhydride (DM) ~
C~3 ~ ~ COR
CH3 OH O ~
~ ~ W (ADM) Hl 8 NH C -,CIH
~ C-COOH
COOH
~3~L2~3 - lla -IgG--(~)--[Lysine Group~ --Antibody I I 7M } Drug NH
~~C=O
LINK
~ \COOH ) (~) O=C--NH--IgG } Antibody I I I At Low pH
(~) ~ IgG-NH2 ~ DM + LINK
.
~' '' .
:
:: :
,, .
' .'~
-i2 ~3~
When the above chemistry is used to conjugate a monoclonal antibody such as L6 to a chemotherapeutic agent, for example, the anthracycline Daunomycin, relatively low yields of reaction may be obtained so that the amount of drug associated with antibody, which will be released, may be too low for optimum therapeutic effectiveness. In addi-tion, the reactivity of the antibody may be affected by a polymerization reaction induced by thle carbodilmide reagent used in the above reaction. Therefore, although the reac-tion may be used to form the immunoconjugates of this inven-tion, it is preferable to improve the above reaction, for example, by using activating reagents, or by the use of spacer molecules.
Thus, to improve the reactions, a succinated intermedi-ate of the anhydride modified chemotherapeutic agent and N-hydroxysuccinimide may be prepared using a carbodiimide reagent such as l-ethyl-3-(3-dimethylaminopropyl) carbo-diimide hydrochloride (EDC) to promote ~he activation of the carboxylic groups of the aconitic anhydride. This interme-diate is then reacted with the amino group of an available lysine of the antibody to form an immunoconjugate ~ith an amide bond. Such immunoconjugates are described, and the reactions shown, in Example II below.
Particularly useful immunoconjugates may be prepared which incorporate spacer molecuLes, preferably polyamino acids containing at least three amino acids such as poly-L-Lysine and poly-L-Glutamic acid and includins protein molecules, for e~ample, albumin. In a preferred conjugation process, the amino group of a lysine in a lysine-containing antibody is modiCied by thiolation, for example using S-acetylmercaptosuccinicanhydride (SACA) to provide free sulfhydryl groups (-SH). A spacer molecule, such as poly-L-Lysine is complexed ~ith the anhydride ~odified 2~`~3 chemotherapeutic agent prepared as described above, and ~he lysine group of the spacer molecule of the complex is then modified with a reagent such as maleiimide reagent for exam ple, sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate. The thiolated antibody is then conjugated ~ith the maleiimide-modified spacer molecule-chemotherapeutic agent complex to form an immunoconjugate capable of dissociation at low pH.
Alternatively, in a series of reactions mediated by a reagent such as N-succinimidyl 3-(2~pyridyldithio) propionate (SPDP), lysine groups in 2 spacer molecule such as albumin are attached to the carboxyl group of the anhydride-modified chPmotherapeutic agent (obtained as described above), and to the amino group of a lysine in the antibody. Immunoconjugates containing spacer molecules are set forth in Examples I'~ and V below.
Immunoconjugates having a spacer !ink may thus be pre-pared with several molecules of chemotherapeutic agent per antibody molecule (up to 50 molecules of agent per antibody molecule) which, in turn, enhances drug delivery to the tumor tissue, ~ithout significantly altering the reactivity of the antibody.
The level of conjugation using the above-described pro-cedures may be further improved by modifying the pH of the reactions so that the pH is in the range of from 6.5 to 8.5, or by increasing the temperature during the reactions in the range of from 4C to 37C. Additionally, the time of incu-bation may be modified to increase the amount of drug cou-pled to antibody from 3 up to 24 hours. Further, the ratio of chemotherapeutic agent introduced to the antibody in Step II of the reaction between antibody and the chemotherapeutic agent may be changed; final ratios of agent to antibody from lO to 50 are preferred.
Z~3 ~ or the above approach of making the low pH unstaole immunoconjugates usin~ an aconitic anhydride link, the chemotherapeutic agent should possess at least one free amino group. Since the amino group is believed to be neces-sary for biological activity, the spacer is preferably com-pletely hydrolyzed from the chemotherapeutic agen~ to free the amino group. Suitable chemotherapeutic agents which meet these requirements are the anthracycline compounds Daunomycin, Mitomycin C, Adriamycin, and methotrexate.
These compounds also contain a quinone structure and an acyl (-COR) moiety, both of which are believed to be important for tumor cell destruction. ~ pH stable conjugate can be made, as a control, by using another spacer, maleic anhydride, in place of the aconitic anhydride.
A second approach for linking a chemotherapeutic agent to an antibody ro form pH uns~able imm~noconjugates is based on chemical reac ons using cyanosen bromide, similar to those described by Axen et al,, in Nature, 214, p. 1302 (1967)~ Axen et al.
describes coupling proteins to polysaccharide resins such as Sephadex. To carry out these reactions, the chemo-therapeutic agen~, for example, Daunomycin, is activated using cyanogen bromide ~CNBR) at an alkaline pH (e.g., pH
11.0). The activated Daunomycin is then added to a solution of an appropriate antibody and a buffer solution, such as a sodium bicarbonate solution, to maintain an alkaline pH.
The resulting con,usate is purified, for example, by column chromatography. ~he immunoreac.ivity of the conjugated antibody is ~es,ed by procedures such as immunohistology using the PAP ~echnique (Garriques et a1., Inter. S. Cancer, 29 p. 511 (1982)), or by radioactive binding assays.
Immunoconjugates formed in this manner may then be tested in the pH range of tumor tissues, ?referably in the range of .~`' ~, .
3~4Z~
.
from pH 5.6 to pH 6.7. These reactions may be summarized as follows. 3riefly, the carbon of the cyanoqen bromide is reacted with hydroxyl ~-OH) groups of the chemotherapeutic agent to form a mixture of intermediates (l). The interme-diates ar-e reacted with the amino group of a lysine amino acid of the antibody to form a mixture of immunoconjugates (2), having an amino group of a lysine amino acid of the antibody coupled via a carboxyl link to hydroxyl groups of the chemotherapeutic agent.
These reactions are:
,OH ~0\
I DRUG~+ CNBr >DRUG C=NH + DRUG-0-C-N
OH ~0 ~ ~ O OH
~ IgG-NH-C-0-DRUG~OH
II W + IgG-NH2 ANTIBODY IgG-N=C~ ~DRUG
Since the i~munoconjugates (2) are not stable at !ow pH, the conjugate dissociates in low pH medium into free drug and antibody as shown below.
Acidic Medium ANTIBODY
III ~ > IgG-NH2+DRUG
The chemotherapeutic agent should contain at least two hydroxyl groups, and the antibody should contain at least one lysine amino acid for these reactions.
A third approach for forming acid-cleavable bonds uses diazotization, following a method described by Cuatrecasas for forming conjugates, J. Biol. Chem., 245, p. 3059 (1970), and consists of the fol-lowing steps. The chemotherapeutic agent, for example, Daunomycin, is activated using a reagent such as P-nitro benzyl chloride in solution. After incubation, the nitro group is reduced, using a stannous chloride solution. The ~3 product of this reduction is then diazotized by adding HCl while cooling on ice. Sodium nitrite is added to induce dia~otization. The activated Daunomycin is then conjugated with an available tyrosine amino acid of a suitable antibody using sodium bicarbonate to form a nitro-benzoyl link between the antibody and the drug. The pH is then adjusted, preferably to a pH of approximately ~Ø The conjugated antibody is then purified using column chromatography, and the immunoreactivity of the conjugated antibody is tested.
These immunoconjusates are tested for release of the Daunomycin at pH in the range of 5.6 to 6.7. These reac-tions are represented as follows:
I DRUG-OH ~ Cl-CH2- ~ No2 > DRUG- ~CH2 ~ No2 ~ Sn/HC' Reduc~ion, DRUG-0-CH2- ~ NH2 ~3~;~g~
_7--I I I (~) + NaN02/HCl , DRUG-O-CH2 ~; N+--N Cl Dlazotlzat1on Antibody IV ~ ~ IgG~ ~ ~IgG- ~ N= ~ 0-DRUG
ANTI~OD ~U OH OH
Tyrosine Since the immunoconjuyate is unstable at low pH, the drug will be released into the tumor tis~ue by dissocia-tion (Step V).
Low pH
V ~ ~ Ig5 ~ DRUG
The chemotherapeutic agent employed in the above reac tions (the third approach), should contain at least one hydroxyl (-OH) group, and preferably the antibody should contain at least one tyrosine amino acld in its structure.
The following examples are presented to illustrate the present invention and to assist one of ordinary skill in the art in making and using the same. The examples are not intended in any way to otherwise limit the scope of the dis-closure or the protection granted by Letters Pa~ent hereonO
XAMPLE I
pH of Tumor Tissue in Humans To investigate the in VLVO pH of several types of tumor tissues in humans, the following study was performed at the Virginia Mason Hospital in Seattle, Washington, in March of lg~6 .
Ten patients (8 females and 2 males; mean age 67.3 years) with different types of tumors were entered in an acute study during surgery. A flexible pH probe, diameter 1.2 mm (Microelectrode 20142, Microelectrodes, Inc., New Hampshire, U.S.A.) connected to a digital pH meter (Bechman Model 3500) was inserted into normal tissue and tumor tissue through a 14-gauge needle ~ith the patient's index finger connected to a reference electrode (NMI-401, Micro~
electrodes, Inc.). The probe was calibrated before and after the procedure for each patient by use of commercially available buffers, pH 7, (3eckman) and pH 2 (Ricca Chemi-cal , Arlington, TX). The probe was sterilized with Turgicos solution (Johnson & Johnson, Arlington, TX~. The pH values were recorded after stabilizataon, usually within S-10 minutes in normal tissue, and the same procedure was repeated in t~mor tissue. Two of the patients received 50 ml of a 50% glucose solution intraveneously (ni.v."). The glucose was given over a 30 min. period beginning one hour before surgery. The findings of this study are sumrnarized in Table 1, which demonstrates a consistent, highly signifi-cant difference (0.8 pH units) between various tumors and normal tissue.
9- ;~3~
Table 1 pH Measurements in ~.umors and Normal Tissues from Ten Patien~s A
5iven pH of pH of Difference i.v. nor~al tumor (A-a) Age Sex Diagnos1s ~lucose 1 e tissue 1) 76 F Cancer of the Yes 7.2 Subc. ;.9 1.3 colon with mats 2) 57 M Undif. mesenchymal Yes 7.4 5ubc. 6~6 0.8 tumor 3~ 80 F Rectal cancer No 6.9 Para- 6.4 0.5 rectal 4) 46 F Mammary cancer No 7.4 Subc. 6.7 0.7 5) 68 F Malignant melanoma No 6.9 Subc. 6.0 0.3 6) 48 M Lymphoma with No 7.4 Subc. 6.7 0.7 axillary me~s 7) 78 F Cancer of the No o.3 Subc. 6.0 0.9 cardia adenocarcinoma 8) 77 F Mammary cancer No 7.1 Subc. 6.5 0.6 mets 9~ 76 F Hypernephroma No 7.3 Subc. 6.6 0.7 10) 67 F Cancer of the No 7.3 Subc. 6.2 1.1 esophagus -Mean + SEM = 7.2 ~ 0.1 normal tissue.
Mean + SEM = 6.4 1 0.1 tumor tissue.
mets= metastasis Subc. = subcutaneous P value = 1.3 -06 -2~-EXA~PLE II
Daunomycin-Antibody Immunoconjugate Preparation of Anhydride-Modified Daun y~ DM~
12 mg of Daunomycin ("DM") (Signta Chemical Co., St.
Louis, MO) were dissolYed in ice-colcl water, and a solution of 3 ml dioxane containing 12 mg of c:is-aconitic anhydride was added drop-wise. The p~l was adjusted to 9.0 by the addition of 0.5 N NaOH. The mixture was stirred for fifteen minutes, after ~hich the pH was decreased to 7 by addinq 0.5 M HCI. The solution ("ADM solution") was ~irred for an additional hour. This derivative was designated "ADM".
The proportion of free (unmodified) to modified DM
("ADM") was estimated using thin-layer chromatography on a mixture of acetone:chloroform:acetic acid (17:3:1). The "~f" of free drug ~as approximately 0.1 and that of the spacer-DM (hereafter ADM) was approximately 0.5.
Spectroscopy showed that both DM and ADM had absorbance peaks at 475 nm and at 28~ nm. (Figure 3~.
Preparation of Antibody-Daunomycin Immunoconiuaate L6 antibody (ATCC No. H38677), was dissolved in phos-phate buffered saline (PBS), pH 7, and 0.6 ml of the ADM
solution prepared as described above was added drop-wise to 10 mg of the L6 antibody in 0.8 ml of PaS. Subsequently, 10 mg of (l-ethyl-3)3-dimethylamino-propyl) carbodiimide hydro chloride (EDC) was added, and the mixture was kept at ~C
and at a pH of ~.0 for 3 hours. The mixture was then loaded onto a Sephadex G-50 column (38 X 1.8 cm~, and l ml frac-tions were collected. The antibody-drug conjugate exhibiting the yellow color of the drug, was eluted in .
~, ~3~ 3~
.
fractions 16 and 17, and the free DM was eluted in fractions 35-42, as shown in Figure 1. The yield of the conjugation reaction was 7-10~, and a ratio of 3:1 DM molecules per antibody molecule was obtained.
Tests by i~munohistology, following the PAP procedures of Garrigues et al., supra, were performed to study the ability of the conjugate to bind to tumors expressing the L6 antigen. The tests showed that the immunoreactivity of the conjugate was pre-served, although it ~as weaker than that of the native anti-body. These tests were followed by cell binding assays using techniques desc~ibed by ~eaumier et al., J. Nuclear Med., 27 p.824 (1986). Approximately 80% of the original immunoreactivity was ~reserved. Gel-electrophersis (7% SDS) showed only one band of the conjugated protein. This band was identical ~o hat of unmodified TsG (MW, 150k), Figure 2, indicating that most of the conjugate remained in a monomeric s.ate and did not polymerize.
An absorption spectrum of the purified ?roduct showed a ne-~ 2eak at 370 nm. (Figure 3). This peak indicates that a - convaient bond ~as formed in the conjugation between the DM
and the L6 antibody.
Release of Daunomycin ~rom the I~munoconiuqate at Low ~ in a Cell-Free Mediu~
The purified immunoconjugate was mixed with citrate-phosphate buffers of .our different ?Hs: pH 4, 5, 6 or 7, after which the ."ixtures ~ere incubated a~ 37~C and 1 ml aliquots removed a~ cifferent time intervals. In order ~o separate DM which was released from the conjugate, conju-gates were fil~ered .hrough a Centricon-10 Fil~er (Amicon, Danvers, MA) which has a filtration cut-off at lO;OC0 ~ Trademark ,iJ ~
~9 J
~q?4~
daltons molecular weight, after which the absorbance of the supernatant was checked for presence of free DM (which absorbs at ~75 nm~. Figure 4 depicts data obtained with a conjugate prepared by coupling 3M to h~an IgG which serves as a readily available model for conjugation, rather than to the L6 antibody. Figure 4 shows that after 24 hours of incubation at pH 4 or 5, between 30-40% of the DM has been released from the conjugate. At pH 6 approximately 15% of the DM was released. No significant release was noticed at a neutral pH.
_ cin on Cultured Cell Lines The ability of DM to inhibit 3[H] thymidine uptake by cells from an explanated human lung carcinoma, 2981 (Oncogen, Seattle, wA), which can bind the L6 antibody, and by cells from melanoma M-2669 (Oncogen, Seattle, WA), which cannot, was measured. As shown in Figu.e 5, free DM ~as very effective even at a low dose, less than 0.5 ug/ml.
Cytotoxicity was observed after only 16 hours incubation with the drug, as illustrated in Figure 6.
3indinq of AntibodY to Tumor Cells The antibody used to form the immunoconjugate herein, L6, as well as another antibody 96.5, demonstrate the abil-ity to bind to eumor cells (lung carcinoma and melanoma) in the range of pH from 5 to 7. (Figure 7). Thus, antibody binding is not likely to be inhibited by the pH found in tumor eissue. (~able l).
23~
EXAMPLE III
Amide-Linked Daunomycin-Antibody Immunoconjugate Pre~aration of Succinated ADM
To maximize the amount of chemotherapeutic agent asso-ciated with the antibody of the i~munoconjugates of this invention, ADM solution was prepared as described above in ~ample II. To 4 ml of ADM solution, 10 mg of N-hydroxysuccinimide (Fluka, Basel, Switzerland) and 5 mg of EDC was added. This mi~ture was stirred at room temperature for 24 hours (pH 5) to make the succinated product (~ADM-SUC").
Coniuqation ~o Ant.bodv 1.0 ml of .~3~-SUC was added to 1 ~1 of L6 antibody (5 mg/ml in PBS buffer). The pH was adjus~ed to 8.5 with 1 M
NaO~. The mixt~ure ~as lncubated for 24 ~ours at ~C, then purified using a G-50 sephadex column. ~he in~unoconjugate was isolated as described in ~xample II, and c~ntains an amide link between the antibody and the Daunomycin. The reactions may be depicted as foLlows:
ADM - COOH + 1{0 - ~-O Cnrbodilmide, ADM - C - O
DI~UG O (~D~I-SUC) ~Anhydride- (,`I-I{ydroxysuccinimide) Daunomycin) O~
ADM - C - O ~ 1 + H ~
2~ ~ Ab ~ AD~'~i - C - NH - Ab (AD~_suc) (-' ntibody) *Trademark ., .. , . . , , , . ~ ~ ... . . .. . .
-24~
The conjugation yield was higher for the reactions in this example, and a DM to antibody ratio of 10:1 was obtained.
EXAMPLE _IV
Daunomycin-Antibody Immunoconjugate Using Albumin Spacer Modification of Ant _odY
- To l-ml of the antibody L6 (5 mg~m}) was added 63~1 of a solution of SPDP ~7 mg/5ml ethanol~, and the mixture was incubated for 30 minutes at room temperature to modify a lysine amino acid of the antibody. The SPDP-modified anti-body was then purified on a PD-10 (Pharmacia, Sweden) chromatography column, prewashed with a 0.1 M sodium acetate solution (pH 4.5). The eluted peak was then reduced with 0.24 ml of dithiothreitol (DTT) (O.SM) for 10 ~inutes.
Attachment of Albumin to Daunom~
.
1 ml of human serum albumin (HSA) was added to 0.65 ml of anhydride-modified Daunomycin (ADM) solution (prepared as described in Example II). 20 mg of ~DC were added to the mixture to form a DM-HSA complex and incubated for 20 hours at 4C. The comple~ed ~DM-HSA was then purified on a G-50 sephadex column. The molar ratio of ADM to HSA was 7:1.
Modification of ADM-HSA
.
The ADM-~SA solution was incubated with 21 ~1 of SPDP
solution (7 mg/5 ml of ethanol) for 30 minutes at room tem-perature to form SPDP modified (ADM-HS~) which was then purif ied on a PD-10 column.
:
: ' .
', ,' , ~4.3Q'~2~3 Coniuqation The reduced, SPDP modified L6 antibody and the S2DP
modified ADM-HSA were then mixed together to form an immunoconjugate of Daunomycin coupled to albumin by an albumin spacer. The ratio of DM to albumin was approxi-mately 7:1. The reactions were:
L6 + SPDP - > L6-SPDP DTT~ L6-SPDP-S~
ANTIBODY
ADM t HSA - > ADM - HSA ~ SPDP - > ADM - HSA - SPDP
ADM - HSA - SPDP + L6 - SPDP - SH- - -> ADM - HSA - L6 IMMUNOCONJU~ATF
EXAMPLE V
Daunomycin-Antibody Immunoconjugate Using Poly-~-Lysine Spacer Prepara~ion of Anhydride-Modified Daunomycin (ADM) Sixteen (16) mg of Daunomycin ("DM") (Sigma Chemical Co., St. Louis, MO) were dissolved in 1.5 ml of ice-cold water. 16 mg cis-aconitic anhydride was slowly added to the dissolved Daunomycin. The pH was adjusted to 9.0 by the addition of 0.5 N NaO~. The mixture was stirred for 15 min, and the pH was then decreased to 3 by adding HCl. The solu-tion was stirred in the cold (4C) for 15 min. The pellet wa~ then isolated by centrifugation for 15 min at 4C at 3000 rpm. The pellet was resuspended in 1 ml of PBS and the pH adjusted to approximately a. This derivative was desig-nated "ADM".
~93 Attachment of Poly-L-Lvsine to Dauno~ycin To lO mg of poly L-lysine (PLS) (Sigma Che~ical Co., St. Louis, MO), MW 53,000, 0.67 ml of ADM solution, pH 7, was added. Then 20 mg of EDC were added to the reaction mixture. The mixture was stirred for 20 hours at 4C, then the modified PLS (ADM-PLS) was purified on a G-50 sephadex column, as described in Example III. More than 70% of the ADM became associated with the eluted PLS.
Prevaration of DaunomYcin Poly-L-Lvsine-AntibodY
Immunocon~uqate Thiolation of_L6_Ant _ody (L6-SH) lO mg of L6 antibody were dissolved in l ml of PBS.
The pH was then adjusted to 6.5 with l N HCl. 40 ~l of S-acetylmercaptosuccinicanhydride solution ~SACA) (stock:
12.6 mg reagent in 0.1 ml dried dimethyl formamide~ was added to the antibody solution. The dimethyl formamide was freshly dried over molecular sieves (Aldrich Company, Milwaukee, WI). Thiolation was conducted for 30 min a~
25C. The following reagents were then added: 0.1 ml 0.1 M
Tris-HCl, pH 7, lO ~l 0.1 M EDTA pH 7, and 0.1 ml lM
hydroxlamine, pH 7. The mixture was incubated for 5 min at 30C and was then loaded on a G-25 Sephadex column (25 x 1.8 cm). The column was prewashed with phosphate buffer O.lM, pH 6, which contained 5 mM EDTA. The fractions of the modified antibody were collected, pooled and concentrated to a volume of 0.3 ml.
Maleiimide reaction of PLS-ADM
To 1 ml of PLS-ADM complex ~pH-7.2) 25 ~l of sulfosuccinimidyl-~-(N-maleimidomethyl) cyclohexane-1-carboxylate solution (maleiimide reagent, "ME") (17 mg -27- ~ ~Q~3 maleiimide reagent per 50 ~1 dried dimethylformamide) was added dropwise. The mixture was incubated for 30 min at 30C. It was then loaded on a Sephadex G-25 column (12 x 1.8 cm~ and eluted with PBS. Fractions containing modified (PLS-ADM-ME) were pooled and concentrated to a volume of 0.6 ml, Linkin~ of ADM-PLS tc, L6 antibodv and purlfication of the To 0.3 ml of modified L6 (L6-SH), 0.6 ml of modified PLS-ADM-ME were added dropwise. The pH was adjusted to 6.2.
Nitrogen was then purged into the mixture for 3 min. The mixture was incubated for one hour at 30C in a sealed tube.
2 mg of 2-ethyl maleiimide were then added to block excess -SH groups on the antibody. The reaction was continued for 20 min at 30C. The L6-PLS-ADM conjugate was purif;ed by precipitating ~ith saturated (55%) ammonium sulfate solution (30 min at 4C). The sample was then spun down at 90C0 xg for 10 min at 4C and the pellet which formed contained the conjugate. The pellet was resuspended in 0.5 ml of PBS
buffer. The pH was adjusted to 7.5. The molar ratio achieved by using this approach was between 18 and 25 mole-cules of Daunomycin for each antibody molecule. The purified conjugate was subsecuently tested for binding to tumor cells and for cytotoxicity.
The reactions were:
L6 + SACA > L6-SH
ANTIBODY
PLS + ADM > PLS-ADM > PLS-ADM-ME
(ME) PLS-ADM-ME + L6-SH > L6-PLS-ADM
IMMUNOCONJUGATE
;~3t~ '33 Bindinq of the L6-PLS-ADM immunoconiuqate to a carcinoma cell line The binding of the immunoconjugate L6-PLS-ADM prepared as described above to tumor cells was tested. The binding was done using a competition assay in which different amounts of both native and conjugated L6 antibody were incu-bated with the tumor (metastatic colon carcinoma) cell line 3347 (Oncogen, Seattle, WA) and displaced using 10 ng of a fluorescent derivative (fluorescein isothiocyanate (FITC)) of L6 (FITC-L6). The level of inhibition of binding was compared as a function of the amount of introduced ~cold"
antibody. The immunoconjugate and the native L6 antibody produced the same binding curve. A conjugate to a non-specific antibody (Ig2a), Pl.17, (ATCC No. TIB10) showed no binding. These data are shown in Figure 8.
Cytotoxicity of the L6-PLS-ADM immunoconjuqate The L6-PLS-ADM immunoconjugate was tested for its potential to inhibit growth of tumor cells. Two inhibition assays were used: 1) 3[H3Thymidine, and 2) colony assay.
soth assays correlated well. These results are summarized in Figures 9 and 10. The immunoconjugate was toxic even at pH 7-7.5, with an inhibition constant of 5 ~g/ml (based on drug concentration). The conjugate was less toxic, however, than free drugs: DM (at 0.2 ~g/ml) and ADM ( at 2 ~g/ml).
When the conjugate was exposed to low pH ~pH 6) the toxicity was 15-25% higher than in neutral pH and was similar to the free ADM toxicity. L6 antibody alone did not inhibit under these conditions.
13~ 2~?3 EXAMPLE VI
Localization of the L6-PLS-ADM Immunoconjugate in vivo The ability of ~6-PLS-ADM conjugate prepared as described in Example V to localize in tumors, compared to unconjugated L6 monoclonal antibody, was examined in nude mice bearing human tumor xenografts. Two randomized groups of 19 mice each were used. Each mouse bore 2 L6 antigen-positive bilateral subcutaneous human metastatic lung carci-noma tumors (H2981) (Oncogen, Seattle, WA) of appro~imately 7 x 7 mm at the start of the experiment. The viability of the tumors was deter~ined by observing enlargement of each tumor for two weeks following implantation.
125I was used to label the specific L6 antibody by the chloramine T method described by Beaumier et al, J. Nuc.
Med., 27, P. 824 (1986). Each mouse received approximately 5 ~Ci of L6 antibody (specific activity approximately 10 ~Ci/~g) along with either 50 ~9 L6 or 50 ~g unlabelled L6-PLS-A~M conjugate. In addition, each mouse also received a comparable 131I-labelled non-specific monoclonal antibody (IF5) (Oncogen, Seattle WA) of the same subclass (IgG2a) described by Clark et al, PNAS, 83 p. 4~94 ~1986), along with 50 ~9 of unlabelled IF5 antibody, coadministered i.v.
At selected time points, (6, 24, ~a, 72, and 120 hours), 4 animals from each group were anesthesized, exsanguinated through the orbital plexus and sacrificed.
Selected tissues, tumor, blood, liver, spleen and kidney were removed, weighed and counted in a gamma counter capable of differentiating between 125I and 131I.
Blood clearance was slower with the L6-PLS-ADM conju-gate compared to either L6 or IFS antibody, as seen in ~3~ 3~3 Figure ll. This was probably caused by the increased size of the L6-PLS-ADM conjugate.
Most significantly, the tumor uptake was similar for both the L6 antibody and the L6-PLS-ADM conjugate, both of which were much higher than the uptake of non-specific IF5 antibody as shown in Figure 12. The peak uptake was between 48 and 72 hrs. The localization index (L.I.) for the L6 antibody and the L~-PLS-ADM conjugate peaked at 72 hrs.!
with values of 3.3 and 20~ respectiveLy (Figure 13), Uptake by normal tissue, kidneys and liver was comparable for all the preparations, as illustrated in Figures 14 and 15.
The examples presented above demonstrate that pH sensi-tive immunoconjugates for joining antibodies reactive wi~h tumor associated antigens with chemotherapeutic agents may be formed according to the present invention and used to target tumor cells, without requiring ~hat the immunoconjugates cross the tumor cell membrane. As shown by these examples, the immunoconjugates will be unstable within the range of pH of human tumor tissue (p~ 4-6). While the examples demons~rate that up to 25 molecules of chemotherapeutic agent were bound per antibody molecule, higher ratios of drugs to antibody may be achieved using the procedures described herein, or by varying aspects of these procedures to maximize the association of chemotherapeutic agent with the antibody.
The immunoconjugates described herein have relatively little toxicity until they reach tumor tissue where, due to the low pH, the conjugates release Ihe active chemotherapeutic agent which can then diffuse into tumor cells.
.
~3~ 3~
-3i-From the pH values for tumor tissue obtained in the above study, set forth in Table 1, the efficacy of the immunoconjugates described in this invention may depend on the level of chemotherapeutic agent releasable in the range of pH 6.0 to pH 6.7. In addition, since only a small per-centage of an injected dose of immunoconjugate will reach the tumor vicinity (depending on the ability of the antibody to act as a carrier, i.e., to target the tumor site, which is a function of the antibody's affirlity for the tumor-associated antigen with which it reacts) it is necessary to obtain conjugates with as many molecules of chemotherapeutic agent bound to an antibody molecule as possible, preferably 10 to 50 molecules of agent per molecule of antibody.
In addition, during nonequilibrium pH conditions ln vivo in the tumor tissue region, portions of the agent may dissociate from the conjugate and be ~a;cen up by the tumor cells, so that higher amounts of chemotherapeutic agent will ultimately be released from the antibody leading to higher effective therapeutic levels of the drug at the tumor for a given dose of immunoconjugate. The ability of the immunoconjuqates of the present invention to take advantage of the low pH occurring in tumor tissues in chemotherapy may be enhanced by further lo~ering the pH of such tissue, for example by intravenous infusion of large doses of glucose.
Ashby, Lancet, Aug. 6, pp. 312-313 (1966).
The above esults demonstrate that the immunoconjugates of the present invention can localize in tumors in an animal model and may be useful for directing chemotherapeutic agents to tumors in humans for treatment.
The chemotherapeutic effectiveness of the immunoconjugates of the present invention may bP determined experimentally, for example, by administering a range of ~ -32- ~3~
doses of the immunoconjugateS into tumor-bearing animal models. Effectiveness of the immunoconjugates may then be assessed by determining the extent of destruction of tumor cells. In addition, where tumors consist of mixed popula-tions of antigen positive and antigen negative tumor ceLls, observations on the number of nonantigen bearing tumor cells (i.e., "antigen negative" cells) destroyed out of the total mixed population of tumor cells, can provide information on the number of an~.igen positive cells required to achieve chemotherapeutically effective levels of drug in tumor tis-sues using the immunoconjugates of the present invention.
In addition, the immunoconjugates may be radiolabeled using standard procedures for administration to humans, for exam-ple, to determine the dose per gram of immunoconjugate required to achieve a therapeutic effect.
While the present invention has been described in con-junction with preferred embodiments, one of the ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equiva-lents, and alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent thereon be limited only by the appended claims and equivalents thereof.
Soc. xp._3iol. Med., 124, p. 63 (1967)), are the ones most easily used for drug targeting (Jansen et al., ~mmunol.
Rev., 62, p. 185 (1982)). Second, there is heterogeneity .n the expression by cells of most tumor antigens so that cells which do not express a given antigen, i.e., are antigen-negative, frequently occur within a tumor (Yeh et al., J.
Immunol., 126, p. 1312 ~1981); Albino et al., J. Exo. ,~ed., 154, p. 1764 (1981)). Although the difficulty of accumulat-ing effective levels of chemotherapeutic agents within a tumor as a result of tumor cell heterogeneity can be decreased by combining antibodies to different antigens expressed by the same tumor cells and forming immunoconjugates, it could be fureher minimized if a cherapeutic approach was ~eveloped in which the presence of some minim~l amount of cells possessing the given antigen within 2 tumor would be sufficient to allow locali7ation of effective amounts of immunoconjugates. Third, there are some tumor antigens, mucins, for example, which are present in larger amounts outside of th~ cells than at the cell mem-brane, (Rittenhouse et al~, Laboratorv Medicine, 16, p. 556 (1985)) suggesting the potential for targeting tumor regions.
The acidity ~pH) of tumor tissues appears to be lower ~han ~hat of normal tissues. Studie~; conducted more than half a century ago showed that m~lignant tumors metabolize carbohydrates mainly by anaerobic glycolysis, even under aerobic conditions (Warburg et al., ~iochem~ A., 152, p. 309 (1924)). The oxidation of glucose stops at the stage of glucose oxidation to pyruvic acid, followed by reduction to lactic acid (30xer and Devlin, Science, 134, p. lg95 (1961)). Most of this lactic acid is either removed or buffered by surrounding extracellular fluid, but some of it accumulates extracellularly. ~his results in a lower ?H
within the tumor than in normal tissues. Elevation of the blood-sugar by intravenous infusion of glucose should accel-erate anaerobic metabolism resulting in even more lactic acid in the tumor, and this should further increase the pH
difference between tumors and normal tissues.
Following Warburg's studies, there have been several reports of lower pH in tumors of both experimental animals, (Voegtlin et al., Nat'1. Inst. Hlth. 3ull., 164, p. l (1935); Xahler and Robertson, J Nat. Cancer Inst., 3, p. 495 (1943); and human patients, ~aeslund, Acta Soc. Med.
U~sal., 60, p. 150 (1955); Pampus, Acta ~eurochir., 11, p. 305 (1963)).
Meyer et al., in Cancer ~es., 8, p. 513 (19~8) reported that the pH of malignant human tumors is lower Ihan in nor-mal tissues. In twelve out of fQurteen cases, where both normal and neoplastic tissues ~rom the same pa~ients could be studied ln vivo, there was a difference in pH which aver-aged 0.~9 and ranged from 0.17 to 1.15.
Ashby, (Lancet, August 6, p. 312 (1966)), found that the mean pH of malignant tumors from nine patients was 6.~
(ranging between 6.6 and 6.9). Raising of the blood sugar by intraveneous infusion of dextrose further decreased the tumor pH to a mean of 6.5 (range 6.3 - 6.8).
Van Den seerg et al., Eur. J. Cc ~ , 18, p~ 457 (1982), showed that the pH of twenty-two human mammary carcinomas was 7.29 (~0.05, SEM), as compared to 7.63 (~0.03, SEM) in human subcutis, and observed similar differences in rat tumors. The differences between pH in tumors and normal tissues were highly statistically si~nifi-cant, although they were lower than those reported in the studies discussed above.
Thistlethwaite et al., Int. J. Radiation Oncoloqy 3iol.
~y~, 11, p. 1647 (1985), showed, likewise, that the pH of human tumors as measured by readings on fourteen tumors was below the physiological level with an average of 6.81'0.09 (SEM). They speculated that the reported therapeutic effec-tiveness of hyperthermia depends on the lower extracellular p~ of tumors as compared to normal tissues.
Trouet et al., U.S. Patent No. 4,376,765, describe drug compounds composed of a protein macromolecule ~carrier) linked via a peptide chain ("spacer arm") to an amino func-tion of a drug. The carrier facilitates endocytic take-up by target cells so that the spacer arm may be cleaved ~ithin the cell. Recently, attention has been directed to developing antibody drug conjugates ~hicn release a drug within a tumor cell once the conjugate has crossed the cell membrane and encountered acidic pH (3.5-5.5~ within the -5~
cell. U.5. Patent No. 4,569,789 by Blattler et al., describes chemical formation of conjugates using cross-linking structures which can link amino-group substances such as chemotherapeutic drugs to the sulfhydryl portion of a compound such as an antibody reactive with tumor cell sur-face antigens capable of crossing the tumor cell membrane.
One limitation of such a method of forming conjugates is that the antibody must contain a sulfhydryl group. This reduces the number of possible drug-antibody conjugates which may be formed using such procedures.
In spite of the published evidence that tumors have lower pH than normal tissues, and that acid-cleavable com-plexes may be formed between antibodies and drugs, this evi-dence has not yet resulted in the development of immunoconjugates which are composed of antibodies reactive with tumor associated antigens and chemotherapeutic agents, and which could be targeted to tumor tissues and are capable of selectively releasing the chemotherapeutic agents in the presence o the lower pH of cancer tissues for uptake by the tumor cells, but not at the pH of normal tissue.
Summary of the Invention In the present invention, pH sensitive immunoconjugates are provided for treating tumors in mammals by delivering a chemotherapeutic agent to tumor tissue. The immunocon-jugates comprise an antibody reactive with a tumor-associated antigen coupled to a chemotherapeutic agent by a link which renders the conjugate unstable at low pH. In particular, the immunoconjugates comprise a monoclonal anti-body which does not have to be internalized by tumor cells, and the chemotherapeutic agent is a compound such as an anthracycline compound effective in the treatment of tumors and possessing at least one free amino residue. A species of --b-- 3L3~ ~3 immunoconjugate showing particularly desirable ~ropertieS
for pH sensitivity in the range of pH of human tumor tissue, is that comprised of the L6 monoclonal antibody coupled by a poly-L-Lysine spacer to ~he drug Daunomycin.
Brief Descri~tion of the Drawinqs ; The present invention will be described in connection ~ with the accompanying drat~ings in which:
- : FIGURE 1 is a graph depicting gel chromatographs of the ; antibody-Daunomycin immunoconjugate reaction mixture FIGURE 2 is a photograph of an electrophoretic (5DS) gel of modified and unmodified antibody;
FIGURE 3 depicts the absorption spectra of the free and conjugated forms of Daunomycin;
FIGURE 4 illustrates the effects of changes in pH on the kinetics of release of Daunomycin conjugated to hu~nan : Ig~;
FIGURE 5 is a graph of the toxicity to melanoma cells of various doses of free Daunomycin as measured by 3[H¦
thymidine uptake by cells over time;
: `
FIGURE 6 is a graph of the toxicity to lung carcinoma cells of various doses of Daunomycin as measured by 3[H]
thymidine uptake by cells over time;
FIGURE 7 is a graph of the binding of the L6 antibody : to lung carcinoma cells, and cf the 96.5 antibody to melanoma cells at different pH;
FIGURE 8 is a graph of the competition binding assay of :~ the L6-Poly-L-Lysine-Daunomycin (L6-PLS~ADM) conjugate to :~ the fixed cell line 33~7 (M~85 designates the conjuga~e);
; ' : ` .
~ t33 FIGURE 9 is a graph of thymidine inhibition of the L6 - PLS -ADM con~ugate;
FrGuRE 10 is a graph of colony inhibition showing toxicity of the L6-PLS-ADM conjugate at pX 6 and pH 7 (M214 designates the conjugate);
FIGURE 11 is a graph showing the blood clearance of the L6-PLS-ADM conjugate in nude mice. Thi.s graph compares the blood clearance of the conjugate to nat:ive L6 antibody and to the non-specific antibody lF5;
FIGURE 12 is a graph of the in vivo tumor uptake of L6-PLS-ADM conjugate, L6 antibody alone and the non-specific IF5 antibody;
FIGURE 13 is a graph of the localization index (LoI~) as a function of time for the L6-P~S-ADM conjugate and the native L6 anti~ody;
FIGURE 14 is a graph of the 1n vlvo kidney uptake of the L6-PLS-ADM conjugate, L6 antibody alone and the I~5 non-specific antibody; and FIGURE 15 is a graph of the ln vivo liver uptake of the L6-PLS-ADM conjugate, L6 antibody alone and the IF5 non-specific antibody.
Accordingly, the present in~ention provides novel immunoconjugates composed of antibodies selectively reactive with tumor-associated antigens to target tumor tissues linked to chemotherapeutic agents. The immunoconjugates are unstable in low pH tumor tissues. The conjugates have a low toxicity at the pH of normal tissue, but when the conjugates localize in low pH tumor tissue as a result of recognition by the antibodies of ~he antigens associated with tumor ~L3~ 3 cells because of the chemical inst~bility of the conjugates, the chemotherapeutic agent is released and can be taken up by the tumor cells. Therefore, it is unnecessary for the entire conjugate to be internalized within the tumor cell, i.e., for the antibody to cross the cell membrane, for cell death to occur. In addition, those tumor cells which lack the target antigen can still be killed by the chemothera-p~utic agent, provided a sufficient number of cells within the tumor express the antigen recognized by the antibody of the immunoconjugate. In addition, the invention includes methods for using these pH-sensitive immunoconjugates in chemotherapy, by introducing the conjugates into a patient to localize in low pH tumor tissue, where the chemotherapeutic is released and allowed to diffuse into the tumor cells. Thus, the expres~ion of tumor-associated anti-gens in only a minimal number of the targeted tumor cells or tumor-associated tissue is required for tumor therapy, using the present invention. The examples set forth below demon-strate the ability of immunoconjugates prepared according to the invention, to localize in tumor tissue in an animal model.
To form the i~munoconjugates of this invention, suit-able antibodies must be selected or developed. The antibodies used for the conjugates are preferably monoclonal antibodies of either mouse or human origin, which are reactive with antigens that are expressed most strongly at the surface of tumor cells and/or in the close vicinity (i.e. outside the cell membrane~ of tumor cells. Monoclonal antibodies may be ?roduced using procedures such as those described by Kohler and Milstein in Nature, 256, p. 495, (1975). An example of one such monoclonal antibody, and the antibody preferred for use in this invention, is the L6 antibody (American Type Culture Collection "ATCC," No.
`-" ~ g ~iL3Q~.~9~
HB8677), an IgG2a mouse immunoglobulin which is specific for a gaglioside antigen and which reacts with most human carci-nomas. The ganglioside antigen (referred to as the "L6 antigen"), is expressed at the surface of cells of most human carcinomas, including non-small lung carcinomas, breast carcinomas, colon carcinomas and ovarian carcinomas~
The L6 antibody and the L6 antlgen are described in copending Canadian application Serial No. 497,251 filed 10 December, 1985 and assigned to the same assignee as the present invention.
.
The L6 antigen does not modulate in the presence of L6 antibody (i.e., the antigen antibody complex is not internalized), indicating that the L6 antibody remains at the cell surface and is not taken up by tumor cells.
Additional monoclonal antibodies of mouse, rat, human or other origin can be generated to the L6 antigen, or other tumor-associated antigens. Chimeric antibodies, obtained by splicing together genes for the variable region of the anti-body molecule (of mouse origin) and genes for the constant region (of human origin~ as are exemplified by the work of Morrison et al., Proc. Natl. Acad. Sci., 81, p. 6a51 (1984), and Takeda et al., Nature, 314, p. ~52 (1985), may also be used. The immunoconjugates can also be made by using polyclonal sera which are prepared in various species, including rabbits and monkeys. Various fragments which are, for example, obtained by proteolytic digestion of antibody molecules, and include Fab, (Fab')2, and Fc fragments can also be used. The present invention can equally well be carried out by using antibodies and fragments which are spe-cific for antigens other than the L6 antigen, as long as the A
10- ;~3~9 ~ r33 antibodies and fragments have a high affinity constant (103 M or better) and the antigen is either expressed in high levels at the tumor cell surface (at least 50,000 molecules per cell) or is present at relatively high levels in the immediate vicinity of the tumor cells~
Suitable chemotherapeutic agents for use in the present invention are those which have-a cytotoxic and/or grow~h inhi~itory effect on cancer cells. These include therapeutic agents of the type commonly used in the treat-ment of human cancer, including antineoplastic drugs such as the anthracycline compounds Daunomycin, Mitomycin C, Adriamycin, and antimetabolites such as the folic acid antagonist, for example, Methotrexate.
In the present invention, the i~munoconjugates must be unstable at lo~ p.~ to release the chemotherapeutic agent.
This may be ac-cmplished using several methods of chemical synthesis. In one approach, a pH-sensitive link such as aconitic anhydride, is attached to a chemotherapeutic agent and the carboxyl group (-COOH) of the agent is then coupled to the lysine group of the antibody. This approach is simi-lar to the chemistry described by Shen and ~yser, 3iochem.
3ioDhys. Res. Comm., 102, p. 10~8 (1981), . Stable immunoconjugates between toxins and anti~odies to certain lymphocyte populations for carry-ing the conjugate into the target cells, have been developed using such procedures; these immunotoxins have been found to be immuno-suppressive. Diener et al., Science, 231 p. 118 (1985).
The pH unstable immunoconjugates of the present inven-tion may also be formed using an aconitic anhydride link to couple the chemotherapeutic agent to the antibody. These reactions are depicted below. In Step I of such a .
11 ~
procedure, the labile gamma-carboxyl group of aconitic anhydride is reacted with a suitab].e chemotherapeutic agent, such as Daunomycin, containing at least one free amino group forming an intermediate compound (1). In the next step (II), this inte~nediate is reacted with an available antibody containing at least one lysine group, in the presence of carbodiimide reagent to form an immunoconjugate consisting of Daunomycin and antibody coupled by the link. This immunoconjugate ~2j will dissociate in low pH medium such as tumor tissue as #2 shown in Step III.
0 01~
I. ~ COR ~H-~ ~C/
CH30 0 Y ~ ~0 l ~ COOH
HO ~
Daunomycin ~ N-Cis Aconitic Anhydride (DM) ~
C~3 ~ ~ COR
CH3 OH O ~
~ ~ W (ADM) Hl 8 NH C -,CIH
~ C-COOH
COOH
~3~L2~3 - lla -IgG--(~)--[Lysine Group~ --Antibody I I 7M } Drug NH
~~C=O
LINK
~ \COOH ) (~) O=C--NH--IgG } Antibody I I I At Low pH
(~) ~ IgG-NH2 ~ DM + LINK
.
~' '' .
:
:: :
,, .
' .'~
-i2 ~3~
When the above chemistry is used to conjugate a monoclonal antibody such as L6 to a chemotherapeutic agent, for example, the anthracycline Daunomycin, relatively low yields of reaction may be obtained so that the amount of drug associated with antibody, which will be released, may be too low for optimum therapeutic effectiveness. In addi-tion, the reactivity of the antibody may be affected by a polymerization reaction induced by thle carbodilmide reagent used in the above reaction. Therefore, although the reac-tion may be used to form the immunoconjugates of this inven-tion, it is preferable to improve the above reaction, for example, by using activating reagents, or by the use of spacer molecules.
Thus, to improve the reactions, a succinated intermedi-ate of the anhydride modified chemotherapeutic agent and N-hydroxysuccinimide may be prepared using a carbodiimide reagent such as l-ethyl-3-(3-dimethylaminopropyl) carbo-diimide hydrochloride (EDC) to promote ~he activation of the carboxylic groups of the aconitic anhydride. This interme-diate is then reacted with the amino group of an available lysine of the antibody to form an immunoconjugate ~ith an amide bond. Such immunoconjugates are described, and the reactions shown, in Example II below.
Particularly useful immunoconjugates may be prepared which incorporate spacer molecuLes, preferably polyamino acids containing at least three amino acids such as poly-L-Lysine and poly-L-Glutamic acid and includins protein molecules, for e~ample, albumin. In a preferred conjugation process, the amino group of a lysine in a lysine-containing antibody is modiCied by thiolation, for example using S-acetylmercaptosuccinicanhydride (SACA) to provide free sulfhydryl groups (-SH). A spacer molecule, such as poly-L-Lysine is complexed ~ith the anhydride ~odified 2~`~3 chemotherapeutic agent prepared as described above, and ~he lysine group of the spacer molecule of the complex is then modified with a reagent such as maleiimide reagent for exam ple, sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate. The thiolated antibody is then conjugated ~ith the maleiimide-modified spacer molecule-chemotherapeutic agent complex to form an immunoconjugate capable of dissociation at low pH.
Alternatively, in a series of reactions mediated by a reagent such as N-succinimidyl 3-(2~pyridyldithio) propionate (SPDP), lysine groups in 2 spacer molecule such as albumin are attached to the carboxyl group of the anhydride-modified chPmotherapeutic agent (obtained as described above), and to the amino group of a lysine in the antibody. Immunoconjugates containing spacer molecules are set forth in Examples I'~ and V below.
Immunoconjugates having a spacer !ink may thus be pre-pared with several molecules of chemotherapeutic agent per antibody molecule (up to 50 molecules of agent per antibody molecule) which, in turn, enhances drug delivery to the tumor tissue, ~ithout significantly altering the reactivity of the antibody.
The level of conjugation using the above-described pro-cedures may be further improved by modifying the pH of the reactions so that the pH is in the range of from 6.5 to 8.5, or by increasing the temperature during the reactions in the range of from 4C to 37C. Additionally, the time of incu-bation may be modified to increase the amount of drug cou-pled to antibody from 3 up to 24 hours. Further, the ratio of chemotherapeutic agent introduced to the antibody in Step II of the reaction between antibody and the chemotherapeutic agent may be changed; final ratios of agent to antibody from lO to 50 are preferred.
Z~3 ~ or the above approach of making the low pH unstaole immunoconjugates usin~ an aconitic anhydride link, the chemotherapeutic agent should possess at least one free amino group. Since the amino group is believed to be neces-sary for biological activity, the spacer is preferably com-pletely hydrolyzed from the chemotherapeutic agen~ to free the amino group. Suitable chemotherapeutic agents which meet these requirements are the anthracycline compounds Daunomycin, Mitomycin C, Adriamycin, and methotrexate.
These compounds also contain a quinone structure and an acyl (-COR) moiety, both of which are believed to be important for tumor cell destruction. ~ pH stable conjugate can be made, as a control, by using another spacer, maleic anhydride, in place of the aconitic anhydride.
A second approach for linking a chemotherapeutic agent to an antibody ro form pH uns~able imm~noconjugates is based on chemical reac ons using cyanosen bromide, similar to those described by Axen et al,, in Nature, 214, p. 1302 (1967)~ Axen et al.
describes coupling proteins to polysaccharide resins such as Sephadex. To carry out these reactions, the chemo-therapeutic agen~, for example, Daunomycin, is activated using cyanogen bromide ~CNBR) at an alkaline pH (e.g., pH
11.0). The activated Daunomycin is then added to a solution of an appropriate antibody and a buffer solution, such as a sodium bicarbonate solution, to maintain an alkaline pH.
The resulting con,usate is purified, for example, by column chromatography. ~he immunoreac.ivity of the conjugated antibody is ~es,ed by procedures such as immunohistology using the PAP ~echnique (Garriques et a1., Inter. S. Cancer, 29 p. 511 (1982)), or by radioactive binding assays.
Immunoconjugates formed in this manner may then be tested in the pH range of tumor tissues, ?referably in the range of .~`' ~, .
3~4Z~
.
from pH 5.6 to pH 6.7. These reactions may be summarized as follows. 3riefly, the carbon of the cyanoqen bromide is reacted with hydroxyl ~-OH) groups of the chemotherapeutic agent to form a mixture of intermediates (l). The interme-diates ar-e reacted with the amino group of a lysine amino acid of the antibody to form a mixture of immunoconjugates (2), having an amino group of a lysine amino acid of the antibody coupled via a carboxyl link to hydroxyl groups of the chemotherapeutic agent.
These reactions are:
,OH ~0\
I DRUG~+ CNBr >DRUG C=NH + DRUG-0-C-N
OH ~0 ~ ~ O OH
~ IgG-NH-C-0-DRUG~OH
II W + IgG-NH2 ANTIBODY IgG-N=C~ ~DRUG
Since the i~munoconjugates (2) are not stable at !ow pH, the conjugate dissociates in low pH medium into free drug and antibody as shown below.
Acidic Medium ANTIBODY
III ~ > IgG-NH2+DRUG
The chemotherapeutic agent should contain at least two hydroxyl groups, and the antibody should contain at least one lysine amino acid for these reactions.
A third approach for forming acid-cleavable bonds uses diazotization, following a method described by Cuatrecasas for forming conjugates, J. Biol. Chem., 245, p. 3059 (1970), and consists of the fol-lowing steps. The chemotherapeutic agent, for example, Daunomycin, is activated using a reagent such as P-nitro benzyl chloride in solution. After incubation, the nitro group is reduced, using a stannous chloride solution. The ~3 product of this reduction is then diazotized by adding HCl while cooling on ice. Sodium nitrite is added to induce dia~otization. The activated Daunomycin is then conjugated with an available tyrosine amino acid of a suitable antibody using sodium bicarbonate to form a nitro-benzoyl link between the antibody and the drug. The pH is then adjusted, preferably to a pH of approximately ~Ø The conjugated antibody is then purified using column chromatography, and the immunoreactivity of the conjugated antibody is tested.
These immunoconjusates are tested for release of the Daunomycin at pH in the range of 5.6 to 6.7. These reac-tions are represented as follows:
I DRUG-OH ~ Cl-CH2- ~ No2 > DRUG- ~CH2 ~ No2 ~ Sn/HC' Reduc~ion, DRUG-0-CH2- ~ NH2 ~3~;~g~
_7--I I I (~) + NaN02/HCl , DRUG-O-CH2 ~; N+--N Cl Dlazotlzat1on Antibody IV ~ ~ IgG~ ~ ~IgG- ~ N= ~ 0-DRUG
ANTI~OD ~U OH OH
Tyrosine Since the immunoconjuyate is unstable at low pH, the drug will be released into the tumor tis~ue by dissocia-tion (Step V).
Low pH
V ~ ~ Ig5 ~ DRUG
The chemotherapeutic agent employed in the above reac tions (the third approach), should contain at least one hydroxyl (-OH) group, and preferably the antibody should contain at least one tyrosine amino acld in its structure.
The following examples are presented to illustrate the present invention and to assist one of ordinary skill in the art in making and using the same. The examples are not intended in any way to otherwise limit the scope of the dis-closure or the protection granted by Letters Pa~ent hereonO
XAMPLE I
pH of Tumor Tissue in Humans To investigate the in VLVO pH of several types of tumor tissues in humans, the following study was performed at the Virginia Mason Hospital in Seattle, Washington, in March of lg~6 .
Ten patients (8 females and 2 males; mean age 67.3 years) with different types of tumors were entered in an acute study during surgery. A flexible pH probe, diameter 1.2 mm (Microelectrode 20142, Microelectrodes, Inc., New Hampshire, U.S.A.) connected to a digital pH meter (Bechman Model 3500) was inserted into normal tissue and tumor tissue through a 14-gauge needle ~ith the patient's index finger connected to a reference electrode (NMI-401, Micro~
electrodes, Inc.). The probe was calibrated before and after the procedure for each patient by use of commercially available buffers, pH 7, (3eckman) and pH 2 (Ricca Chemi-cal , Arlington, TX). The probe was sterilized with Turgicos solution (Johnson & Johnson, Arlington, TX~. The pH values were recorded after stabilizataon, usually within S-10 minutes in normal tissue, and the same procedure was repeated in t~mor tissue. Two of the patients received 50 ml of a 50% glucose solution intraveneously (ni.v."). The glucose was given over a 30 min. period beginning one hour before surgery. The findings of this study are sumrnarized in Table 1, which demonstrates a consistent, highly signifi-cant difference (0.8 pH units) between various tumors and normal tissue.
9- ;~3~
Table 1 pH Measurements in ~.umors and Normal Tissues from Ten Patien~s A
5iven pH of pH of Difference i.v. nor~al tumor (A-a) Age Sex Diagnos1s ~lucose 1 e tissue 1) 76 F Cancer of the Yes 7.2 Subc. ;.9 1.3 colon with mats 2) 57 M Undif. mesenchymal Yes 7.4 5ubc. 6~6 0.8 tumor 3~ 80 F Rectal cancer No 6.9 Para- 6.4 0.5 rectal 4) 46 F Mammary cancer No 7.4 Subc. 6.7 0.7 5) 68 F Malignant melanoma No 6.9 Subc. 6.0 0.3 6) 48 M Lymphoma with No 7.4 Subc. 6.7 0.7 axillary me~s 7) 78 F Cancer of the No o.3 Subc. 6.0 0.9 cardia adenocarcinoma 8) 77 F Mammary cancer No 7.1 Subc. 6.5 0.6 mets 9~ 76 F Hypernephroma No 7.3 Subc. 6.6 0.7 10) 67 F Cancer of the No 7.3 Subc. 6.2 1.1 esophagus -Mean + SEM = 7.2 ~ 0.1 normal tissue.
Mean + SEM = 6.4 1 0.1 tumor tissue.
mets= metastasis Subc. = subcutaneous P value = 1.3 -06 -2~-EXA~PLE II
Daunomycin-Antibody Immunoconjugate Preparation of Anhydride-Modified Daun y~ DM~
12 mg of Daunomycin ("DM") (Signta Chemical Co., St.
Louis, MO) were dissolYed in ice-colcl water, and a solution of 3 ml dioxane containing 12 mg of c:is-aconitic anhydride was added drop-wise. The p~l was adjusted to 9.0 by the addition of 0.5 N NaOH. The mixture was stirred for fifteen minutes, after ~hich the pH was decreased to 7 by addinq 0.5 M HCI. The solution ("ADM solution") was ~irred for an additional hour. This derivative was designated "ADM".
The proportion of free (unmodified) to modified DM
("ADM") was estimated using thin-layer chromatography on a mixture of acetone:chloroform:acetic acid (17:3:1). The "~f" of free drug ~as approximately 0.1 and that of the spacer-DM (hereafter ADM) was approximately 0.5.
Spectroscopy showed that both DM and ADM had absorbance peaks at 475 nm and at 28~ nm. (Figure 3~.
Preparation of Antibody-Daunomycin Immunoconiuaate L6 antibody (ATCC No. H38677), was dissolved in phos-phate buffered saline (PBS), pH 7, and 0.6 ml of the ADM
solution prepared as described above was added drop-wise to 10 mg of the L6 antibody in 0.8 ml of PaS. Subsequently, 10 mg of (l-ethyl-3)3-dimethylamino-propyl) carbodiimide hydro chloride (EDC) was added, and the mixture was kept at ~C
and at a pH of ~.0 for 3 hours. The mixture was then loaded onto a Sephadex G-50 column (38 X 1.8 cm~, and l ml frac-tions were collected. The antibody-drug conjugate exhibiting the yellow color of the drug, was eluted in .
~, ~3~ 3~
.
fractions 16 and 17, and the free DM was eluted in fractions 35-42, as shown in Figure 1. The yield of the conjugation reaction was 7-10~, and a ratio of 3:1 DM molecules per antibody molecule was obtained.
Tests by i~munohistology, following the PAP procedures of Garrigues et al., supra, were performed to study the ability of the conjugate to bind to tumors expressing the L6 antigen. The tests showed that the immunoreactivity of the conjugate was pre-served, although it ~as weaker than that of the native anti-body. These tests were followed by cell binding assays using techniques desc~ibed by ~eaumier et al., J. Nuclear Med., 27 p.824 (1986). Approximately 80% of the original immunoreactivity was ~reserved. Gel-electrophersis (7% SDS) showed only one band of the conjugated protein. This band was identical ~o hat of unmodified TsG (MW, 150k), Figure 2, indicating that most of the conjugate remained in a monomeric s.ate and did not polymerize.
An absorption spectrum of the purified ?roduct showed a ne-~ 2eak at 370 nm. (Figure 3). This peak indicates that a - convaient bond ~as formed in the conjugation between the DM
and the L6 antibody.
Release of Daunomycin ~rom the I~munoconiuqate at Low ~ in a Cell-Free Mediu~
The purified immunoconjugate was mixed with citrate-phosphate buffers of .our different ?Hs: pH 4, 5, 6 or 7, after which the ."ixtures ~ere incubated a~ 37~C and 1 ml aliquots removed a~ cifferent time intervals. In order ~o separate DM which was released from the conjugate, conju-gates were fil~ered .hrough a Centricon-10 Fil~er (Amicon, Danvers, MA) which has a filtration cut-off at lO;OC0 ~ Trademark ,iJ ~
~9 J
~q?4~
daltons molecular weight, after which the absorbance of the supernatant was checked for presence of free DM (which absorbs at ~75 nm~. Figure 4 depicts data obtained with a conjugate prepared by coupling 3M to h~an IgG which serves as a readily available model for conjugation, rather than to the L6 antibody. Figure 4 shows that after 24 hours of incubation at pH 4 or 5, between 30-40% of the DM has been released from the conjugate. At pH 6 approximately 15% of the DM was released. No significant release was noticed at a neutral pH.
_ cin on Cultured Cell Lines The ability of DM to inhibit 3[H] thymidine uptake by cells from an explanated human lung carcinoma, 2981 (Oncogen, Seattle, wA), which can bind the L6 antibody, and by cells from melanoma M-2669 (Oncogen, Seattle, WA), which cannot, was measured. As shown in Figu.e 5, free DM ~as very effective even at a low dose, less than 0.5 ug/ml.
Cytotoxicity was observed after only 16 hours incubation with the drug, as illustrated in Figure 6.
3indinq of AntibodY to Tumor Cells The antibody used to form the immunoconjugate herein, L6, as well as another antibody 96.5, demonstrate the abil-ity to bind to eumor cells (lung carcinoma and melanoma) in the range of pH from 5 to 7. (Figure 7). Thus, antibody binding is not likely to be inhibited by the pH found in tumor eissue. (~able l).
23~
EXAMPLE III
Amide-Linked Daunomycin-Antibody Immunoconjugate Pre~aration of Succinated ADM
To maximize the amount of chemotherapeutic agent asso-ciated with the antibody of the i~munoconjugates of this invention, ADM solution was prepared as described above in ~ample II. To 4 ml of ADM solution, 10 mg of N-hydroxysuccinimide (Fluka, Basel, Switzerland) and 5 mg of EDC was added. This mi~ture was stirred at room temperature for 24 hours (pH 5) to make the succinated product (~ADM-SUC").
Coniuqation ~o Ant.bodv 1.0 ml of .~3~-SUC was added to 1 ~1 of L6 antibody (5 mg/ml in PBS buffer). The pH was adjus~ed to 8.5 with 1 M
NaO~. The mixt~ure ~as lncubated for 24 ~ours at ~C, then purified using a G-50 sephadex column. ~he in~unoconjugate was isolated as described in ~xample II, and c~ntains an amide link between the antibody and the Daunomycin. The reactions may be depicted as foLlows:
ADM - COOH + 1{0 - ~-O Cnrbodilmide, ADM - C - O
DI~UG O (~D~I-SUC) ~Anhydride- (,`I-I{ydroxysuccinimide) Daunomycin) O~
ADM - C - O ~ 1 + H ~
2~ ~ Ab ~ AD~'~i - C - NH - Ab (AD~_suc) (-' ntibody) *Trademark ., .. , . . , , , . ~ ~ ... . . .. . .
-24~
The conjugation yield was higher for the reactions in this example, and a DM to antibody ratio of 10:1 was obtained.
EXAMPLE _IV
Daunomycin-Antibody Immunoconjugate Using Albumin Spacer Modification of Ant _odY
- To l-ml of the antibody L6 (5 mg~m}) was added 63~1 of a solution of SPDP ~7 mg/5ml ethanol~, and the mixture was incubated for 30 minutes at room temperature to modify a lysine amino acid of the antibody. The SPDP-modified anti-body was then purified on a PD-10 (Pharmacia, Sweden) chromatography column, prewashed with a 0.1 M sodium acetate solution (pH 4.5). The eluted peak was then reduced with 0.24 ml of dithiothreitol (DTT) (O.SM) for 10 ~inutes.
Attachment of Albumin to Daunom~
.
1 ml of human serum albumin (HSA) was added to 0.65 ml of anhydride-modified Daunomycin (ADM) solution (prepared as described in Example II). 20 mg of ~DC were added to the mixture to form a DM-HSA complex and incubated for 20 hours at 4C. The comple~ed ~DM-HSA was then purified on a G-50 sephadex column. The molar ratio of ADM to HSA was 7:1.
Modification of ADM-HSA
.
The ADM-~SA solution was incubated with 21 ~1 of SPDP
solution (7 mg/5 ml of ethanol) for 30 minutes at room tem-perature to form SPDP modified (ADM-HS~) which was then purif ied on a PD-10 column.
:
: ' .
', ,' , ~4.3Q'~2~3 Coniuqation The reduced, SPDP modified L6 antibody and the S2DP
modified ADM-HSA were then mixed together to form an immunoconjugate of Daunomycin coupled to albumin by an albumin spacer. The ratio of DM to albumin was approxi-mately 7:1. The reactions were:
L6 + SPDP - > L6-SPDP DTT~ L6-SPDP-S~
ANTIBODY
ADM t HSA - > ADM - HSA ~ SPDP - > ADM - HSA - SPDP
ADM - HSA - SPDP + L6 - SPDP - SH- - -> ADM - HSA - L6 IMMUNOCONJU~ATF
EXAMPLE V
Daunomycin-Antibody Immunoconjugate Using Poly-~-Lysine Spacer Prepara~ion of Anhydride-Modified Daunomycin (ADM) Sixteen (16) mg of Daunomycin ("DM") (Sigma Chemical Co., St. Louis, MO) were dissolved in 1.5 ml of ice-cold water. 16 mg cis-aconitic anhydride was slowly added to the dissolved Daunomycin. The pH was adjusted to 9.0 by the addition of 0.5 N NaO~. The mixture was stirred for 15 min, and the pH was then decreased to 3 by adding HCl. The solu-tion was stirred in the cold (4C) for 15 min. The pellet wa~ then isolated by centrifugation for 15 min at 4C at 3000 rpm. The pellet was resuspended in 1 ml of PBS and the pH adjusted to approximately a. This derivative was desig-nated "ADM".
~93 Attachment of Poly-L-Lvsine to Dauno~ycin To lO mg of poly L-lysine (PLS) (Sigma Che~ical Co., St. Louis, MO), MW 53,000, 0.67 ml of ADM solution, pH 7, was added. Then 20 mg of EDC were added to the reaction mixture. The mixture was stirred for 20 hours at 4C, then the modified PLS (ADM-PLS) was purified on a G-50 sephadex column, as described in Example III. More than 70% of the ADM became associated with the eluted PLS.
Prevaration of DaunomYcin Poly-L-Lvsine-AntibodY
Immunocon~uqate Thiolation of_L6_Ant _ody (L6-SH) lO mg of L6 antibody were dissolved in l ml of PBS.
The pH was then adjusted to 6.5 with l N HCl. 40 ~l of S-acetylmercaptosuccinicanhydride solution ~SACA) (stock:
12.6 mg reagent in 0.1 ml dried dimethyl formamide~ was added to the antibody solution. The dimethyl formamide was freshly dried over molecular sieves (Aldrich Company, Milwaukee, WI). Thiolation was conducted for 30 min a~
25C. The following reagents were then added: 0.1 ml 0.1 M
Tris-HCl, pH 7, lO ~l 0.1 M EDTA pH 7, and 0.1 ml lM
hydroxlamine, pH 7. The mixture was incubated for 5 min at 30C and was then loaded on a G-25 Sephadex column (25 x 1.8 cm). The column was prewashed with phosphate buffer O.lM, pH 6, which contained 5 mM EDTA. The fractions of the modified antibody were collected, pooled and concentrated to a volume of 0.3 ml.
Maleiimide reaction of PLS-ADM
To 1 ml of PLS-ADM complex ~pH-7.2) 25 ~l of sulfosuccinimidyl-~-(N-maleimidomethyl) cyclohexane-1-carboxylate solution (maleiimide reagent, "ME") (17 mg -27- ~ ~Q~3 maleiimide reagent per 50 ~1 dried dimethylformamide) was added dropwise. The mixture was incubated for 30 min at 30C. It was then loaded on a Sephadex G-25 column (12 x 1.8 cm~ and eluted with PBS. Fractions containing modified (PLS-ADM-ME) were pooled and concentrated to a volume of 0.6 ml, Linkin~ of ADM-PLS tc, L6 antibodv and purlfication of the To 0.3 ml of modified L6 (L6-SH), 0.6 ml of modified PLS-ADM-ME were added dropwise. The pH was adjusted to 6.2.
Nitrogen was then purged into the mixture for 3 min. The mixture was incubated for one hour at 30C in a sealed tube.
2 mg of 2-ethyl maleiimide were then added to block excess -SH groups on the antibody. The reaction was continued for 20 min at 30C. The L6-PLS-ADM conjugate was purif;ed by precipitating ~ith saturated (55%) ammonium sulfate solution (30 min at 4C). The sample was then spun down at 90C0 xg for 10 min at 4C and the pellet which formed contained the conjugate. The pellet was resuspended in 0.5 ml of PBS
buffer. The pH was adjusted to 7.5. The molar ratio achieved by using this approach was between 18 and 25 mole-cules of Daunomycin for each antibody molecule. The purified conjugate was subsecuently tested for binding to tumor cells and for cytotoxicity.
The reactions were:
L6 + SACA > L6-SH
ANTIBODY
PLS + ADM > PLS-ADM > PLS-ADM-ME
(ME) PLS-ADM-ME + L6-SH > L6-PLS-ADM
IMMUNOCONJUGATE
;~3t~ '33 Bindinq of the L6-PLS-ADM immunoconiuqate to a carcinoma cell line The binding of the immunoconjugate L6-PLS-ADM prepared as described above to tumor cells was tested. The binding was done using a competition assay in which different amounts of both native and conjugated L6 antibody were incu-bated with the tumor (metastatic colon carcinoma) cell line 3347 (Oncogen, Seattle, WA) and displaced using 10 ng of a fluorescent derivative (fluorescein isothiocyanate (FITC)) of L6 (FITC-L6). The level of inhibition of binding was compared as a function of the amount of introduced ~cold"
antibody. The immunoconjugate and the native L6 antibody produced the same binding curve. A conjugate to a non-specific antibody (Ig2a), Pl.17, (ATCC No. TIB10) showed no binding. These data are shown in Figure 8.
Cytotoxicity of the L6-PLS-ADM immunoconjuqate The L6-PLS-ADM immunoconjugate was tested for its potential to inhibit growth of tumor cells. Two inhibition assays were used: 1) 3[H3Thymidine, and 2) colony assay.
soth assays correlated well. These results are summarized in Figures 9 and 10. The immunoconjugate was toxic even at pH 7-7.5, with an inhibition constant of 5 ~g/ml (based on drug concentration). The conjugate was less toxic, however, than free drugs: DM (at 0.2 ~g/ml) and ADM ( at 2 ~g/ml).
When the conjugate was exposed to low pH ~pH 6) the toxicity was 15-25% higher than in neutral pH and was similar to the free ADM toxicity. L6 antibody alone did not inhibit under these conditions.
13~ 2~?3 EXAMPLE VI
Localization of the L6-PLS-ADM Immunoconjugate in vivo The ability of ~6-PLS-ADM conjugate prepared as described in Example V to localize in tumors, compared to unconjugated L6 monoclonal antibody, was examined in nude mice bearing human tumor xenografts. Two randomized groups of 19 mice each were used. Each mouse bore 2 L6 antigen-positive bilateral subcutaneous human metastatic lung carci-noma tumors (H2981) (Oncogen, Seattle, WA) of appro~imately 7 x 7 mm at the start of the experiment. The viability of the tumors was deter~ined by observing enlargement of each tumor for two weeks following implantation.
125I was used to label the specific L6 antibody by the chloramine T method described by Beaumier et al, J. Nuc.
Med., 27, P. 824 (1986). Each mouse received approximately 5 ~Ci of L6 antibody (specific activity approximately 10 ~Ci/~g) along with either 50 ~9 L6 or 50 ~g unlabelled L6-PLS-A~M conjugate. In addition, each mouse also received a comparable 131I-labelled non-specific monoclonal antibody (IF5) (Oncogen, Seattle WA) of the same subclass (IgG2a) described by Clark et al, PNAS, 83 p. 4~94 ~1986), along with 50 ~9 of unlabelled IF5 antibody, coadministered i.v.
At selected time points, (6, 24, ~a, 72, and 120 hours), 4 animals from each group were anesthesized, exsanguinated through the orbital plexus and sacrificed.
Selected tissues, tumor, blood, liver, spleen and kidney were removed, weighed and counted in a gamma counter capable of differentiating between 125I and 131I.
Blood clearance was slower with the L6-PLS-ADM conju-gate compared to either L6 or IFS antibody, as seen in ~3~ 3~3 Figure ll. This was probably caused by the increased size of the L6-PLS-ADM conjugate.
Most significantly, the tumor uptake was similar for both the L6 antibody and the L6-PLS-ADM conjugate, both of which were much higher than the uptake of non-specific IF5 antibody as shown in Figure 12. The peak uptake was between 48 and 72 hrs. The localization index (L.I.) for the L6 antibody and the L~-PLS-ADM conjugate peaked at 72 hrs.!
with values of 3.3 and 20~ respectiveLy (Figure 13), Uptake by normal tissue, kidneys and liver was comparable for all the preparations, as illustrated in Figures 14 and 15.
The examples presented above demonstrate that pH sensi-tive immunoconjugates for joining antibodies reactive wi~h tumor associated antigens with chemotherapeutic agents may be formed according to the present invention and used to target tumor cells, without requiring ~hat the immunoconjugates cross the tumor cell membrane. As shown by these examples, the immunoconjugates will be unstable within the range of pH of human tumor tissue (p~ 4-6). While the examples demons~rate that up to 25 molecules of chemotherapeutic agent were bound per antibody molecule, higher ratios of drugs to antibody may be achieved using the procedures described herein, or by varying aspects of these procedures to maximize the association of chemotherapeutic agent with the antibody.
The immunoconjugates described herein have relatively little toxicity until they reach tumor tissue where, due to the low pH, the conjugates release Ihe active chemotherapeutic agent which can then diffuse into tumor cells.
.
~3~ 3~
-3i-From the pH values for tumor tissue obtained in the above study, set forth in Table 1, the efficacy of the immunoconjugates described in this invention may depend on the level of chemotherapeutic agent releasable in the range of pH 6.0 to pH 6.7. In addition, since only a small per-centage of an injected dose of immunoconjugate will reach the tumor vicinity (depending on the ability of the antibody to act as a carrier, i.e., to target the tumor site, which is a function of the antibody's affirlity for the tumor-associated antigen with which it reacts) it is necessary to obtain conjugates with as many molecules of chemotherapeutic agent bound to an antibody molecule as possible, preferably 10 to 50 molecules of agent per molecule of antibody.
In addition, during nonequilibrium pH conditions ln vivo in the tumor tissue region, portions of the agent may dissociate from the conjugate and be ~a;cen up by the tumor cells, so that higher amounts of chemotherapeutic agent will ultimately be released from the antibody leading to higher effective therapeutic levels of the drug at the tumor for a given dose of immunoconjugate. The ability of the immunoconjuqates of the present invention to take advantage of the low pH occurring in tumor tissues in chemotherapy may be enhanced by further lo~ering the pH of such tissue, for example by intravenous infusion of large doses of glucose.
Ashby, Lancet, Aug. 6, pp. 312-313 (1966).
The above esults demonstrate that the immunoconjugates of the present invention can localize in tumors in an animal model and may be useful for directing chemotherapeutic agents to tumors in humans for treatment.
The chemotherapeutic effectiveness of the immunoconjugates of the present invention may bP determined experimentally, for example, by administering a range of ~ -32- ~3~
doses of the immunoconjugateS into tumor-bearing animal models. Effectiveness of the immunoconjugates may then be assessed by determining the extent of destruction of tumor cells. In addition, where tumors consist of mixed popula-tions of antigen positive and antigen negative tumor ceLls, observations on the number of nonantigen bearing tumor cells (i.e., "antigen negative" cells) destroyed out of the total mixed population of tumor cells, can provide information on the number of an~.igen positive cells required to achieve chemotherapeutically effective levels of drug in tumor tis-sues using the immunoconjugates of the present invention.
In addition, the immunoconjugates may be radiolabeled using standard procedures for administration to humans, for exam-ple, to determine the dose per gram of immunoconjugate required to achieve a therapeutic effect.
While the present invention has been described in con-junction with preferred embodiments, one of the ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equiva-lents, and alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent thereon be limited only by the appended claims and equivalents thereof.
Claims (42)
1. A pH-sensitive immunoconjugate for delivering a chemotherapeutic agent to tumor tissues comprising:
an antibody reactive with tumor-associated anti-gens, a chemotherapeutic agent, and a link between the antibody and chemotherapeutic agent, said link unstable at low pH, whereby said immunoconjugate dissociates in low pH
tumor tissue releasing said chemotherapeutic agent in said tumor tissue.
an antibody reactive with tumor-associated anti-gens, a chemotherapeutic agent, and a link between the antibody and chemotherapeutic agent, said link unstable at low pH, whereby said immunoconjugate dissociates in low pH
tumor tissue releasing said chemotherapeutic agent in said tumor tissue.
2. The immunoconjugate according to Claim 1, wherein said antibody is isolated from polyclonal sera.
3. The immunoconjugate according to Claim 1, wherein said antibody is a monoclonal antibody.
4. The immunoconjugate according to Claim 3, wherein said monoclonal antibody is L6 and said tumor-associated antigen is L6 antigen.
5. The immunoconjugate according to Claim 3, wherein said antibody is not internalized by tumor cells.
6. The immunoconjugate according to Claim 1, wherein said chemotherapeutic agent is selected from the group con-sisting of Daunomycin, Mitomycin C, Adriamycin and Methotrexate.
7. The immunoconjugate according to Claim 6, wherein said chemotherapeutic agent is Daunomycin.
8. The immunoconjugate according to Claim 1, wherein said immunoconjugate dissociates, releasing said chemo-therapeutic agent in said tumor tissue, in the range of pH 4 to 7.
9. The immunoconjugate according to Claim 1, wherein said immunoconjugate dissociates, releasing said chemo-therapeutic agent in said tumor tissue, in the range of pH
5.6 to 6.7.
5.6 to 6.7.
10. The immunoconjugate according to Claim 1, wherein said pH unstable link is an amide bond and said immunoconjugate comprises the following structure:
where X is an amino group of said chemotherapeutic agent, and Y is an amino group of a lysine amino acid of said antibody.
where X is an amino group of said chemotherapeutic agent, and Y is an amino group of a lysine amino acid of said antibody.
11. An immunoconjugate a-cording to Claim 1, where said pH unstable link is an amide bond, and said immunoconjugate comprises the following structure:
where X is an amino group of said chemotherapeutic agent, Z is a spacer containing at least three amino acids and containing at least two lysine amino acids, and Y is an amino group of a lysine amino acid of the antibody.
where X is an amino group of said chemotherapeutic agent, Z is a spacer containing at least three amino acids and containing at least two lysine amino acids, and Y is an amino group of a lysine amino acid of the antibody.
12. The immunoconjugate according to Claim 11, wherein said spacer is a polyamino acid.
13. The immunoconjugate according to Claim 12, wherein said spacer is poly-L-Lysine.
14. The immunoconjugate according to Claim 11, wherein said spacer is human serum albumin.
15. A pH-sensitive immunoconjugate, unstable in low pH
tumor tissue, comprising the following structure:
where X is a chemotherapeutic agent, L is Lysine, Z is a spacer comprising a polyamino acid containing at least three amino acids, and Y is an antibody containing a lysine amino acid.
tumor tissue, comprising the following structure:
where X is a chemotherapeutic agent, L is Lysine, Z is a spacer comprising a polyamino acid containing at least three amino acids, and Y is an antibody containing a lysine amino acid.
16. The immunoconjugate according to Claim 15, wherein said antibody is not internalized by tumor cells.
17. The immunoconjugate according to Claim 15 wherein said spacer is poly-L-Lysine.
18. A pH-sensitive immunoconjugate unstable in low pH
tumor tissue, comprising the following structure where X is a chemotherapeutic agent, L is Lysine, Z-M is a maleiimide-modified spacer containing at least three amino acids and containing at least two lysine amino acids, and R-NH-Y is a thiolated antibody.
tumor tissue, comprising the following structure where X is a chemotherapeutic agent, L is Lysine, Z-M is a maleiimide-modified spacer containing at least three amino acids and containing at least two lysine amino acids, and R-NH-Y is a thiolated antibody.
19. The immunoconjugate according to Claim 18 wherein said antibody is not internalized by tumor cells.
20. An immunoconjugate according to Claim 1, wherein said pH unstable link is a diazo-benzyl bond and said immunoconjugate comprises the following structure:
where X is a chemotherapeutic agent, and Y is an antibody.
where X is a chemotherapeutic agent, and Y is an antibody.
21. A pH-sensitive immunoconjugate, unstable in low pH
tumor tissue, comprising the following structure:
where X is a chemotherapeutic agent and Y is an antibody.
tumor tissue, comprising the following structure:
where X is a chemotherapeutic agent and Y is an antibody.
22. The immunoconjugate according to Claim 21 wherein said antibody is not internalized by tumor cells.
23. A pH-sensitive immunoconjugate unstable in low pH
tumor tissue, comprising the following structure:
where X is a chemotherapeutic agent and Y is an antibody.
tumor tissue, comprising the following structure:
where X is a chemotherapeutic agent and Y is an antibody.
24. The immunconjugate according to Claim 23 wherein said antibody is not internalized by tumor cells.
25. A process for producing a pH-sensitive immunoconjugate between an antibody reactive with a tumor-associated antigen and a chemotherapeutic agent comprising:
(a) attaching aconitic anhydride to an available carboxyl group of a chemotherapeutic agent, to form a first intermediate with a free carboxyl group;
(b) reacting said free carboxyl group of said first intermediate with N-hydroxysuccinimide and carbodiimide reagent to form a second intermediate; and (c) coupling an antibody via the amino group of an available lysine of said antibody to the carboxyl group of said anhydride of said second intermediate to form an amide link, said link unstable at low pH, to form a pH sensitive immunoconjugate, whereby said immunoconjugate dissociates in low pH
tumor tissue, releasing said chemotherapeutic agent in said tumor tissue.
(a) attaching aconitic anhydride to an available carboxyl group of a chemotherapeutic agent, to form a first intermediate with a free carboxyl group;
(b) reacting said free carboxyl group of said first intermediate with N-hydroxysuccinimide and carbodiimide reagent to form a second intermediate; and (c) coupling an antibody via the amino group of an available lysine of said antibody to the carboxyl group of said anhydride of said second intermediate to form an amide link, said link unstable at low pH, to form a pH sensitive immunoconjugate, whereby said immunoconjugate dissociates in low pH
tumor tissue, releasing said chemotherapeutic agent in said tumor tissue.
26. The immunoconjugate prepared by the process according to Claim 25.
27. A process for producing a pH-sensitive immunoconjugate between an antibody reactive with a tumor-associated antigen and a chemotherapeutic agent comprising:
(a) attaching aconitic anhydride to an available carboxyl group of a chemotherapeutic agent, to form a first intermediate with a free carboxyl group;
(b) reacting said free carboxyl group of said first intermediate with the amino group of a lysine-containing amino acid spacer molecule using a first activating reagent to form a chemotherapeutic agent-spacer-reagent compound:
(c) reacting the amino group of a lysine amino acid of an antibody with a second activating reagent to form modified antibody;
(d) reducing said modified antibody; and (e) reacting said chemotherapeutic agent-spacer reagent compound with said reduced, modified antibody to form an immunoconjugate comprising a chemotherapeutic agent coupled by a spacer molecule to an antibody, whereby said immunoconjugate dissociates in low pH
tumor tissue, releasing said chemotherapeutic agent in said tumor tissue.
(a) attaching aconitic anhydride to an available carboxyl group of a chemotherapeutic agent, to form a first intermediate with a free carboxyl group;
(b) reacting said free carboxyl group of said first intermediate with the amino group of a lysine-containing amino acid spacer molecule using a first activating reagent to form a chemotherapeutic agent-spacer-reagent compound:
(c) reacting the amino group of a lysine amino acid of an antibody with a second activating reagent to form modified antibody;
(d) reducing said modified antibody; and (e) reacting said chemotherapeutic agent-spacer reagent compound with said reduced, modified antibody to form an immunoconjugate comprising a chemotherapeutic agent coupled by a spacer molecule to an antibody, whereby said immunoconjugate dissociates in low pH
tumor tissue, releasing said chemotherapeutic agent in said tumor tissue.
28. The process according to Claim 27 wherein said first activating reagent is carbodiimide reagent.
29. The process according to Claim 28 wherein said carbodiimide reagent is (1-ethyl-3)3-dimethylamino-propyl)-carbodiimide hydrochloride.
30. The process according to Claim 27 wherein said second activating reagent is N-succinimidyl 3-(2-pyridyldithio) proprionate.
31. The immunoconjugate prepared by the process according to Claim 27.
32. A process for producing a pH-sensitive immunoconjugate between an antibody reactive with a tumor-associated antigen and a chemotherapeutic agent comprising:
a) attaching aconitic anhydride to an available carboxyl group of a chemotherapeutic agent to form a first intermediate with a free carboxyl group;
b) reacting said free carboxyl group of said first intermediate with the amino group of a lysine con-taining amino acid spacer molecule using a first activating reagent to form a chemotherapeutic agent-spacer-reagent com-pound;
c) reacting said chemotherapeutic agent-spacer reagent compound with a second activating reagent to form modified chemotherapeutic agent-spacer-reagent compound;
d) reacting the lysine group of an antibody using a thiolating reagent to form modified antibody; and e) reacting said modified chemotherapeutic agent-spacer reagent compound with said modified antibody to form an immunoconjugate comprising a chemotherapeutic agent coupled by a spacer molecule to an antibody, whereby said immunoconjugate dissociates in low pH
tumor tissue, releasing said chemotherapeutic agent in said tumor tissue.
a) attaching aconitic anhydride to an available carboxyl group of a chemotherapeutic agent to form a first intermediate with a free carboxyl group;
b) reacting said free carboxyl group of said first intermediate with the amino group of a lysine con-taining amino acid spacer molecule using a first activating reagent to form a chemotherapeutic agent-spacer-reagent com-pound;
c) reacting said chemotherapeutic agent-spacer reagent compound with a second activating reagent to form modified chemotherapeutic agent-spacer-reagent compound;
d) reacting the lysine group of an antibody using a thiolating reagent to form modified antibody; and e) reacting said modified chemotherapeutic agent-spacer reagent compound with said modified antibody to form an immunoconjugate comprising a chemotherapeutic agent coupled by a spacer molecule to an antibody, whereby said immunoconjugate dissociates in low pH
tumor tissue, releasing said chemotherapeutic agent in said tumor tissue.
33. The process according to Claim 32 wherein said spacer molecule is poly-L-Lysine.
34. The process according to Claim 35 wherein said first activating agent used to form the chemotherapeutic agent-spacer-reagent compound is (1-ethyl-3,3-dimethylamino-propyl) carbodiimide hydrochloride.
35. The process according to Claim 33 wherein said second activating reagent is maleiimide reagent.
36. The process according to Claim 35 wherein said maleiimide reagent is sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1- car-boxylate.
37. The process according to Claim 33 wherein said thiolating reagent is S-acetylmercaptosuccinicanhydride.
38. The immunoconjugate prepared by the process according to Claim 32 or 33.
39. The process according to Claim 32 wherein said chemotherapeutic agent is selected from the group consisting of Daunomycin, Mitomycin C, Adriamycin and Methotrexate.
40. The process according to Claim 39 wherein said chemotherapeutic agent is Daunomycin.
41. The process according to Claim 39 wherein said antibody is L6 monoclonal antibody.
42. The process according to Claim 41 wherein said antibody is an antibody which is not internalized by tumor cells.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US880,674 | 1978-02-23 | ||
| US88067486A | 1986-06-30 | 1986-06-30 | |
| US07/047,161 US4997913A (en) | 1986-06-30 | 1987-05-12 | pH-sensitive immunoconjugates and methods for their use in tumor therapy |
| US047,171 | 1998-03-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1304293C true CA1304293C (en) | 1992-06-30 |
Family
ID=26724681
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000541057A Expired - Fee Related CA1304293C (en) | 1986-06-30 | 1987-06-30 | Immunoconjugates and methods for their use in tumor therapy |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4997913A (en) |
| EP (2) | EP0253202A3 (en) |
| JP (1) | JPS63115827A (en) |
| AU (2) | AU7466387A (en) |
| CA (1) | CA1304293C (en) |
| IL (1) | IL83012A0 (en) |
Families Citing this family (79)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5252317A (en) * | 1986-11-10 | 1993-10-12 | The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Amplifier molecules for diagnosis and therapy derived from 3,5-bis[1-(3-amino-2,2-bis (aminomethyl)-propyl) oxymethyl] benzoic acid |
| US5567411A (en) * | 1986-11-10 | 1996-10-22 | State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Dendritic amplifier molecules having multiple terminal active groups stemming from a benzyl core group |
| US5135737A (en) * | 1986-11-10 | 1992-08-04 | The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Amplifier molecules for enhancement of diagnosis and therapy |
| US5144012A (en) * | 1988-08-08 | 1992-09-01 | Eli Lilly And Company | Cytotoxic drug conjugates |
| US5006652A (en) * | 1988-08-08 | 1991-04-09 | Eli Lilly And Company | Intermediates for antibody-vinca drug conjugates |
| US5094849A (en) * | 1988-08-08 | 1992-03-10 | Eli Lilly And Company | Cytotoxic antibody conjugates of hydrazide derivatized vinca analogs via simple organic linkers |
| US5028697A (en) * | 1988-08-08 | 1991-07-02 | Eli Lilly And Company | Cytotoxic antibody conjugates of hydrazide derivatized methotrexate analogs via simple organic linkers |
| US5171563A (en) * | 1988-09-30 | 1992-12-15 | Neorx Corporation | Cleavable linkers for the reduction of non-target organ retention of immunoconjugates |
| US5010176A (en) * | 1988-11-10 | 1991-04-23 | Eli Lilly And Company | Antibody-drug conjugates |
| US5977307A (en) * | 1989-09-07 | 1999-11-02 | Alkermes, Inc. | Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins |
| US5672683A (en) * | 1989-09-07 | 1997-09-30 | Alkermes, Inc. | Transferrin neuropharmaceutical agent fusion protein |
| US5154924A (en) * | 1989-09-07 | 1992-10-13 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical agent conjugates |
| US5182107A (en) * | 1989-09-07 | 1993-01-26 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates |
| US5527527A (en) * | 1989-09-07 | 1996-06-18 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical agent conjugates |
| US6329508B1 (en) | 1989-09-07 | 2001-12-11 | Alkermes, Inc. | Transferrin receptor reactive chimeric antibodies |
| US5140013A (en) * | 1989-11-28 | 1992-08-18 | Universite Laval | Maleic anhydride derivatives used as conjugation agents of anti-tumor agents on desired carriers |
| US5314995A (en) * | 1990-01-22 | 1994-05-24 | Oncogen | Therapeutic interleukin-2-antibody based fusion proteins |
| CA2075927A1 (en) * | 1990-02-16 | 1991-08-17 | Victor A. Raso | Hybrid reagents capable of selectively releasing molecules into cells |
| DE69126124T2 (en) * | 1990-09-28 | 1997-08-28 | Neorx Corp | POLYMERS CARRIER FOR RELEASING COVALENT-RELATED ACTIVE SUBSTANCES |
| FI101678B (en) * | 1990-12-31 | 1998-08-14 | Akzo Nv | Acid labile molecules |
| US5505931A (en) * | 1993-03-04 | 1996-04-09 | The Dow Chemical Company | Acid cleavable compounds, their preparation and use as bifunctional acid-labile crosslinking agents |
| US5382582A (en) * | 1993-03-26 | 1995-01-17 | Chan; Carcy L. | Methotrexate analogs and methods of using same |
| US6232295B1 (en) | 1994-10-12 | 2001-05-15 | Jon Faiz Kayyem | Cell-specific contrast agent and gene delivery vehicles |
| US6962686B2 (en) | 1994-10-12 | 2005-11-08 | California Institute Of Technology | Cell-specific gene delivery vehicles |
| US6015555A (en) * | 1995-05-19 | 2000-01-18 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates |
| US5698556A (en) * | 1995-06-07 | 1997-12-16 | Chan; Carcy L. | Methotrexate analogs and methods of using same |
| US5830478A (en) * | 1995-06-07 | 1998-11-03 | Boston Biomedical Research Institute | Method for delivering functional domains of diphtheria toxin to a cellular target |
| US7635687B2 (en) * | 1997-06-04 | 2009-12-22 | Oxford Biomedica (Uk) Limited | Vector system |
| WO2001036486A2 (en) * | 1999-11-18 | 2001-05-25 | Oxford Biomedica (Uk) Limited | Scfv antibodies against disease associated molecules |
| DE69841204D1 (en) * | 1997-06-04 | 2009-11-12 | Oxford Biomedica Ltd | Tumor targeted vector |
| US7276488B2 (en) * | 1997-06-04 | 2007-10-02 | Oxford Biomedica (Uk) Limited | Vector system |
| CA2248592A1 (en) | 1998-08-31 | 2000-02-29 | Christopher D. Batich | Microspheres for use in the treatment of cancer |
| TWI242000B (en) | 1998-12-10 | 2005-10-21 | Univ Southern California | Reversible aqueous pH sensitive lipidizing reagents, compositions and methods of use |
| BRPI0003386B8 (en) * | 2000-08-08 | 2021-05-25 | Cristalia Produtos Quim Farmaceuticos Ltda | homo- or heterodimeric prodrugs useful in treating diseases or disorders mediated by phosphodiesterases; pharmaceutical compositions containing the prodrug or its pharmaceutical acceptable salts; process of obtaining these prodrugs |
| EP1333811A4 (en) * | 2000-10-16 | 2004-03-03 | Neopharm Inc | Liposomal formulation of mitoxantrone |
| WO2002049672A2 (en) * | 2000-12-21 | 2002-06-27 | Mcgill University | Conjugates of antibodies and anticancer drugs |
| EP2335728A1 (en) | 2001-05-11 | 2011-06-22 | Ludwig Institute for Cancer Research Ltd. | Specific binding proteins and uses thereof |
| US20100056762A1 (en) | 2001-05-11 | 2010-03-04 | Old Lloyd J | Specific binding proteins and uses thereof |
| GB0126378D0 (en) * | 2001-11-02 | 2002-01-02 | Oxford Biomedica Ltd | Antigen |
| US7585491B2 (en) | 2002-12-13 | 2009-09-08 | Immunomedics, Inc. | Immunoconjugates with an intracellularly-cleavable linkage |
| US8435529B2 (en) | 2002-06-14 | 2013-05-07 | Immunomedics, Inc. | Combining radioimmunotherapy and antibody-drug conjugates for improved cancer therapy |
| US7591994B2 (en) | 2002-12-13 | 2009-09-22 | Immunomedics, Inc. | Camptothecin-binding moiety conjugates |
| US9770517B2 (en) | 2002-03-01 | 2017-09-26 | Immunomedics, Inc. | Anti-Trop-2 antibody-drug conjugates and uses thereof |
| US8420086B2 (en) | 2002-12-13 | 2013-04-16 | Immunomedics, Inc. | Camptothecin conjugates of anti-CD22 antibodies for treatment of B cell diseases |
| US20040151766A1 (en) * | 2003-01-30 | 2004-08-05 | Monahan Sean D. | Protein and peptide delivery to mammalian cells in vitro |
| US20050112786A1 (en) * | 2003-11-25 | 2005-05-26 | Qing Wang | Method of immobilizing a substance of interest to a solid phase |
| US20080312201A1 (en) * | 2004-09-10 | 2008-12-18 | Patrick Fogarty | Reduced Toxicity Methotrexate Formulations and Methods for Using the Same |
| US10058621B2 (en) | 2015-06-25 | 2018-08-28 | Immunomedics, Inc. | Combination therapy with anti-HLA-DR antibodies and kinase inhibitors in hematopoietic cancers |
| US9707302B2 (en) | 2013-07-23 | 2017-07-18 | Immunomedics, Inc. | Combining anti-HLA-DR or anti-Trop-2 antibodies with microtubule inhibitors, PARP inhibitors, bruton kinase inhibitors or phosphoinositide 3-kinase inhibitors significantly improves therapeutic outcome in cancer |
| NZ568288A (en) | 2005-11-28 | 2012-02-24 | Verrow Pharmaceuticals Inc | Compositions comprising a a cyclodextrin and a nephrotoxic anticancer drug |
| US7572625B2 (en) * | 2006-05-18 | 2009-08-11 | Boston Scientific Scimed, Inc. | Medical devices coated with drug carrier macromolecules |
| CN104043123B (en) * | 2007-01-25 | 2019-08-13 | 达娜-法勃肿瘤研究所公司 | Purposes of the anti-egfr antibodies in the mutant mediated disease for the treatment of EGFR |
| WO2008112192A2 (en) * | 2007-03-08 | 2008-09-18 | Kalobios Pharmaceuticals, Inc. | Epha3 antibodies for the treatment of solid tumors |
| AU2008227123B2 (en) * | 2007-03-15 | 2014-03-27 | Ludwig Institute For Cancer Research Ltd. | Treatment method using EGFR antibodies and src inhibitors and related formulations |
| MX2010001757A (en) | 2007-08-14 | 2010-09-14 | Ludwig Inst Cancer Res | Monoclonal antibody 175 targeting the egf receptor and derivatives and uses thereof. |
| WO2009114325A2 (en) | 2008-03-03 | 2009-09-17 | Tosk, Incorporated | Methotrexate adjuvants to reduce toxicity and methods for using the same |
| WO2010011684A2 (en) * | 2008-07-21 | 2010-01-28 | The Regents Of The University Of California | Prodrug and fluoregenic compositions and methods for using the same |
| LT3912643T (en) | 2009-02-13 | 2023-02-10 | Immunomedics Inc. | Immunoconjugates with an intracellularly-cleavable linkage |
| US20110076232A1 (en) * | 2009-09-29 | 2011-03-31 | Ludwig Institute For Cancer Research | Specific binding proteins and uses thereof |
| US9382329B2 (en) | 2012-08-14 | 2016-07-05 | Ibc Pharmaceuticals, Inc. | Disease therapy by inducing immune response to Trop-2 expressing cells |
| CN107753954A (en) | 2012-12-13 | 2018-03-06 | 免疫医疗公司 | The dosage of the antibody that effect is improved and toxicity reduces and SN 38 immunoconjugates |
| US10744129B2 (en) | 2012-12-13 | 2020-08-18 | Immunomedics, Inc. | Therapy of small-cell lung cancer (SCLC) with a topoisomerase-I inhibiting antibody-drug conjugate (ADC) targeting Trop-2 |
| US10206918B2 (en) | 2012-12-13 | 2019-02-19 | Immunomedics, Inc. | Efficacy of anti-HLA-DR antiboddy drug conjugate IMMU-140 (hL243-CL2A-SN-38) in HLA-DR positive cancers |
| US9931417B2 (en) | 2012-12-13 | 2018-04-03 | Immunomedics, Inc. | Antibody-SN-38 immunoconjugates with a CL2A linker |
| US9492566B2 (en) | 2012-12-13 | 2016-11-15 | Immunomedics, Inc. | Antibody-drug conjugates and uses thereof |
| US10137196B2 (en) | 2012-12-13 | 2018-11-27 | Immunomedics, Inc. | Dosages of immunoconjugates of antibodies and SN-38 for improved efficacy and decreased toxicity |
| WO2015012904A2 (en) | 2012-12-13 | 2015-01-29 | Immunomedics, Inc. | Antibody-sn-38 immunoconjugates with a cl2a linker |
| US10413539B2 (en) | 2012-12-13 | 2019-09-17 | Immunomedics, Inc. | Therapy for metastatic urothelial cancer with the antibody-drug conjugate, sacituzumab govitecan (IMMU-132) |
| US11253606B2 (en) | 2013-07-23 | 2022-02-22 | Immunomedics, Inc. | Combining anti-HLA-DR or anti-Trop-2 antibodies with microtubule inhibitors, PARP inhibitors, Bruton kinase inhibitors or phosphoinositide 3-kinase inhibitors significantly improves therapeutic outcome in cancer |
| CA2926087C (en) * | 2013-10-10 | 2023-03-14 | Beth Israel Deaconess Medical Center, Inc. | Tm4sf1 binding proteins and methods of using same |
| WO2015123595A1 (en) | 2014-02-14 | 2015-08-20 | The Regents Of The University Of California | Cyclic peroxides as prodrugs for selective delivery of agents |
| CN107428837A (en) | 2015-04-22 | 2017-12-01 | 免疫医疗公司 | Circulate separation, detection, diagnosis and/or the identification of the positive cancer cells of TROP 2 |
| US10195175B2 (en) | 2015-06-25 | 2019-02-05 | Immunomedics, Inc. | Synergistic effect of anti-Trop-2 antibody-drug conjugate in combination therapy for triple-negative breast cancer when used with microtubule inhibitors or PARP inhibitors |
| CN108601841A (en) | 2016-02-10 | 2018-09-28 | 免疫医疗公司 | The combination of ABCG2 inhibitor and SACITUZUMAB GOVITECAN (IMMU-132) overcome the resistance to SN-38 in the cancer for expressing TROP-2 |
| WO2017189279A1 (en) | 2016-04-27 | 2017-11-02 | Immunomedics, Inc. | Efficacy of anti-trop-2-sn-38 antibody drug conjugates for therapy of tumors relapsed/refractory to checkpoint inhibitors |
| EP3568159A4 (en) | 2017-01-11 | 2020-08-05 | Bristol-Myers Squibb Company | Psgl-1 antagonists and uses thereof |
| KR20190128198A (en) | 2017-03-14 | 2019-11-15 | 파이브 프라임 테라퓨틱스, 인크. | Antibodies that bind VISTA at acidic pH |
| CN110392570A (en) | 2017-03-27 | 2019-10-29 | 免疫医疗公司 | With husky trastuzumab lattice dimension for the triple negative breast cancer of health and RAD51 inhibitor for treating expression TROP-2 |
| EP3606964A4 (en) | 2017-04-03 | 2020-12-09 | Immunomedics, Inc. | Subcutaneous administration of antibody-drug conjugates for cancer therapy |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4376765A (en) * | 1980-03-31 | 1983-03-15 | Institut International De Pathologie Cellulaire Et Moleculaire | Medicaments, their preparation and compositions containing same |
| US4631190A (en) * | 1981-06-26 | 1986-12-23 | Shen Wei C | Acidity-sensitive spacer molecule to control the release of pharmaceuticals from molecular carriers |
| JPS5843926A (en) * | 1981-09-08 | 1983-03-14 | Suntory Ltd | Selective carcinostatic agent |
| US4625014A (en) * | 1984-07-10 | 1986-11-25 | Dana-Farber Cancer Institute, Inc. | Cell-delivery agent |
| US4542225A (en) * | 1984-08-29 | 1985-09-17 | Dana-Farber Cancer Institute, Inc. | Acid-cleavable compound |
-
1987
- 1987-05-12 US US07/047,161 patent/US4997913A/en not_active Expired - Fee Related
- 1987-06-24 AU AU74663/87A patent/AU7466387A/en not_active Abandoned
- 1987-06-28 IL IL83012A patent/IL83012A0/en unknown
- 1987-06-29 JP JP62162201A patent/JPS63115827A/en active Granted
- 1987-06-30 CA CA000541057A patent/CA1304293C/en not_active Expired - Fee Related
- 1987-06-30 EP EP87109414A patent/EP0253202A3/en not_active Ceased
- 1987-06-30 EP EP19900125137 patent/EP0428188A3/en not_active Withdrawn
-
1991
- 1991-06-04 AU AU78131/91A patent/AU636493B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| EP0428188A2 (en) | 1991-05-22 |
| AU7466387A (en) | 1988-01-07 |
| EP0428188A3 (en) | 1992-07-01 |
| JPH0588686B2 (en) | 1993-12-24 |
| US4997913A (en) | 1991-03-05 |
| AU7813191A (en) | 1991-08-29 |
| EP0253202A2 (en) | 1988-01-20 |
| AU636493B2 (en) | 1993-04-29 |
| JPS63115827A (en) | 1988-05-20 |
| EP0253202A3 (en) | 1988-10-05 |
| IL83012A0 (en) | 1987-12-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1304293C (en) | Immunoconjugates and methods for their use in tumor therapy | |
| AU610062B2 (en) | Diagnostic and therapeutic antibody conjugates | |
| EP0550663B1 (en) | Modified antibodies with controlled clearance time | |
| AU627785B2 (en) | Vasopermeability-enhancing conjugates | |
| US5990286A (en) | Antibodies with reduced net positive charge | |
| Shih et al. | Anthracycline immunoconjugates prepared by a site-specific linkage via an amino-dextran intermediate carrier | |
| WO1989007269A1 (en) | Method for the preparation of antibody-fragment conjugates | |
| JP2012131808A (en) | Antibody having reduced net positive charge | |
| US5084560A (en) | Immunoconjugates and methods for their use in tumor therapy | |
| Yang et al. | Pharmacokinetics and mechanism of action of a doxorubicin-monoclonal antibody 9.2. 27 conjugate directed to a human melanoma proteoglycan | |
| Lavie et al. | Monoclonal antibody L6-daunomycin conjugates constructed to release free drug at the lower pH of tumor tissue | |
| US6524823B2 (en) | Vasopermeability-enhancing conjugates | |
| Griffin et al. | Selective cytotoxicity of a ricin A-chain-anti-carcinoembryonic antigen antibody conjugate for a human colon adenocarcinoma cell line | |
| MATSUI et al. | Suppression of human melanoma growth in nude mice injected with anti high-molecular-weight melanoma-associated antigen monoclonal antibody 225.28 S conjugated to purothionin | |
| WO1997025069A9 (en) | Antibodies with reduced net positive charge | |
| Kojima et al. | Anti‐tumor activity of T‐2 toxin‐conjugated A7 monoclonal antibody (T‐2‐A7 MoAb) against human colon carcinoma | |
| CN1255378A (en) | Monoclonal antibody Fab'-Pingyangmycin conjugate and its anticancer action | |
| CA2000479C (en) | Vasopermeability-enhancing conjugates | |
| WO1991000108A1 (en) | Antibody-oxidase conjugates with non-systemic substrates | |
| EP0213811A2 (en) | Bateriocin-Targeted compounds for cancer therapy | |
| CA2005162A1 (en) | Method for antibody targeting of diagnostic or therapeutic agents | |
| Harapanhalli et al. | Lysine-directed conjugation of ethidium homodimer to B72. 3 mAb: retention of immunoreactivity and altered tumor targeting | |
| MXPA98005565A (en) | Antibodies with positive reduced load |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |