DD283634A5 - PROCESS FOR PRODUCING NEW COMPOUNDS - Google Patents

Abstract

The present invention relates to a process for the preparation of novel compounds. The prodrug is less cytotoxic than the corresponding parent drug and is converted into the parent drug in the area of the tumor in the presence of the antibody bound enzyme. The prodrugs, antibody-antibody conjugates, and combinations of this invention provide better selective ablation of tumor cells and are thus useful in the field of medicine for the treatment of cancer and other tumors. manufacture; prodrug; Parent Drug; cytotoxicity; Immunoconjugate / Prodrugkombination; Antitumor agents; therapeutic use}

Description

M / 29 283

Field of application of the invention

The present invention relates to a process for the preparation of prodrugs. These are converted into an active cytotoxic agent by antibody-enzyme conjugates which bind to tumor cells in the region of the tumor by the antibody-bound enzyme. With the aid of the prodrugs prepared according to the invention, it is possible to circumvent a large number of the disadvantages currently associated with the treatment of cancer and other tumors by antibody-mediated release of medicaments.

Characteristic of the known state of the art

The use of interferons to selectively transfer cytotoxic agents to tumor cells in cancer treatment is known in the art. The release of cytotoxic agents in the area of the tumor cells is

p 1/1, 1

of great advantage / because of a systemic administration

this remedy often besides the elimination of tumor cells 5

a killing of normal body cells also results. In the currently used transmission systems for anti-

Tumor agent is therefore a cytotoxic agent conjugated to a tumor-specific antibody / whereby an immune

, conjugate is formed, which binds to the tumor cells and thereby releases the cytotoxic agent in the region of the tumor. The immunoconjugates used in this targeting system include antibody-drug conjugates (see, eg, RW Baldin et al., "Monoclonal Antibodies For Cancer Treatment," Lancet, pp. 603-05 (Mar. 15, 1986), and antibody-toxin conjugates (see, eg, PE Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review" in Monoclonal Antibodies '84: Biological And Clinical Applications: A. Pinchera et al. (Ed.), Pp. 475-506 (1985)).

Both polyclonal and monoclonal antibodies are used for these immunoconjugates (see, for example, K. Ohkawa et al.). Selective In Vitro And In Vivo Growth Inhibition Against Human Yolk Sac Tumor Cell Lines By Purified Antibody Against Human Oc-Fetoprotein Conjugated With Mitomycin C Via Human Serum Albumin "/ Cancer Immunol Immunothera. / 23 / pp. 81-66 (1986) and GF Rowland et al." Drug Localization And Growth Inhibition Studies Of Vindesine Monoclonal Anti-CEA Conjugates In A Human Tumor Xenograft. "Cancer Immunol. Immunother. 21, pp. 183-87 (1986)). These immunoconjugates include drugs such as daunomycin (e.g., J. Gallego et al., Preparation of Four Daunoraycin-Monoclonal Antibody 791T / 36

* Conjugates With Anti-Tumor Activity "/ Int. J. Cancer, 33, pp. 737-44 (1984) and R. Arnon et al." In Vitro And In Vivo

»Efficacy of Conjugates of Daunomycin with Anti-tumor

Antibodies "/ Immunological Rev. 62 / p. 5-27 (1982),

Methotrexate (N.Endo et al., "In Vitro Cytotoxicity Of A Human Serum Albumin Mediated Conjugate Of Methotrexate With Anti-MM46 Monoclonal Antibody", Cancer Research, 47, pp. 1076-80 (1987), Mitomycin C (K. Ohkawa et al., Supra) and vindesine (GF Rowland et al / supra) Antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, as well as plant toxins such as ricin (see * eg, FL Moolten et al.) Antibodies Conjugated To Potent Cytotoxins As Specific Antitumor Agents, Immunol, Rev., pp. 47-73 (1982)).

Despite the high research effort required to apply immunoconjugates for therapeutic purposes, several limitations have emerged for the aforementioned delivery systems (see, eg, MJ Embleton, "Targeting Anti-Cancer Therapeutic Agents By Monoclonal Antibodies," Biochemical Society Transactions, 14, pp. 393-395 (615th meeting, Belfast 1986)).

First, the drug concentration required to kill the particular tumor cell is often not adjustable / because the transfer rate of the drug is limited by the number of tumor-associated cell surface antigens as well as the number of drug molecules bound per antibody molecule. This limitation has led to the use of more cytotoxic agents *, e.g. of plant toxins, to form the conjugates and to develop polymer-bound antibody-drug conjugates with a high number of drug molecules bound (see, eg PE Thorpe, supra, pp. 475-506 and RW Baldwin et al., Design And Therapeutic Evaluation Of Monoclonal Antibody 791T / 36 - Methotrexate Conjugates "in Monoclonal. *** Antibodies And Cancer Therapy, p. 215.31 (Alan R. Liss, Inc. 1985)). Despite the load with a large number of drug molecules or despite the use of more effective

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Toxins / many immunoconjugates still have no optimal cytotoxic activity and, despite complete saturation of the antigen binding sites can not cause complete k killing of the tumor cells.

Second, it has been recognized / that the cytotoxic activity of an immunoconjugate often depends on the uptake into the tumor cell by the antibody component of the conjugate - (see, eg, JW Larabert et al / Purified Immunotoxins That Are Reactive With Human Lymphoid Cells / J. Biol. Chem. 260 (No. 22), p. 12035-12041 (1985)). This internalization step is the use of an antibody-drug conjugate / wherein the drug attacks an intracellular site of action, or using an antibody toxin-o

Conjugates crucial. However, the vast majority of tumor-associated antigens and thus the antibody-drug or antibody-toxin conjugates bound thereto are not internalized. Those conjugates / which

are internalized / often become the lysosomes of the 20

Transported cell / where then the drug or toxin is degraded (see E.S. Vitetta et al., Science / 238 / p 1098-1104 (1987)). Accordingly, an antibody drug or antibody-toxin conjugate, despite

Dye binding properties are only a very limited

have limited cytotoxic activity / because the site of action within the cell is not reached.

It is also well known / that tumor cell populations

are often very heterogeneous in their antigen expression 30

See, e.g., A.P. Albino et al., Heterogeneity In Surface Antigen And Glycoprotein Expression Of Cell Lines Derived From Different Melanoma Metastases Of The Same Patient J. Exp. Med., 154 / p. 1764-78 (1981)).

5 2:36

Furthermore, it was possible to show that antigen-negative cells can form antigen-negative daughter cells (see, for example, M. Yen et al., "Clonal Variation For Expression of A Human Melanoma Antigen Defined By A Monoclonal Antibody" / J. Immunol. No. 4) / p. 1312-17 (1981)). Thus, in each tumor cell population a certain number of cells will be found / which have no antigen specific for the particular immunoconjugate. The immunoconjugates therefore can not bind to these cells and cause their killing.

Due to these disadvantages, in vivo treatment with the currently available transmission systems for antitumor drugs or toxins has been particularly unsuccessful

connected.

In addition to the immunoconjugates discussed above, antibody-enzyme conjugates in combination with a second unbound enzyme were assayed in vitro by converting the unbound enzyme iodide or arsphenamine into the corresponding toxic form to enhance antibody-mediated cytotoxicity (see, eg, CW Parker et al. * "Enzymatic Activiation and Trapping of Luminol-Substituted Peptides and Proteins." "A Possible Means of Amplifying the Cytotoxicity of Anti-Tumor Antibodies" Proc. Natl. Acad. Sei. USA, 72 (No I) / p. 42 (1975) and GW Philpott et al., "Affinity Cytotoxicity Of Tumor Cells With Antibody-Clucose Oxidase Conjugates / Peroxidase /

And Arsphenamine "/ Cancer Research / 34, pp. 2159-64 (1974))

In these in vitro studies, the enzyme glucose oxidase was bound to an antibody and used with iodide, together with the non-bound enzyme peroxidase

or arsphenamine into the cytotoxic iodine or arsenic to 35

convict. This method thus sets a steering of

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Glucose oxidase with the help of the antibodies to the tumor cells as well as the presence of two non-guided agents in the area of the tumor The probability * that all three compounds are found in vivo in the area of the tumor at the same time / is rather small and therefore it seems unlikely Method of therapeutic importance.

Canadian Patent 1 216 791 describes the conjugation of an antibody with an enzyme / which catalyzes the release of ammonium ions from substrates. The ammonium ions are said to enhance the cytotoxic effect of certain tumor-targeted immunotoxins.

Finally, European Patent Application 84302218.7 describes a method of treating diseased cell populations / e.g. a tumor / wherein an antibody is used to transport a non-metabolizable antigen to the tumor cells. In this case, the antigen accumulates at least in a certain proportion of the tumor cells / which subsequently lyse / wherein the antigen is released in a ubiquitous fibronectin matrix formed in the region of the tumor. Then, an iodine-containing ligand is administered / which specifically attaches to the matrix-bound antigen. The cytotoxic iodine then destroys the tumor cells in this area. This application also describes a variety of alternative embodiments / wherein in one of these variants an antibody-enzyme conjugate is used to bring an enzyme into the region of a tumor / there to convert a administered non-lethal substrate into a cytotoxic compound (see EP-Anm. Pages 34-35). In this application, however, no disclosure for carrying out this embodiment is included. Similarly, Hellstrom et al. Describes "Antibodies For Drug Delivery" in Controlled Drug Delivery

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(2nd ed.) * Robinson and Lee (eds.) / P. 639 (1987)) suggest that "non-toxic drugs at the tumor site until their activation by one agent (eg, an enzyme) are considered as another approach can be

Object of the invention 10

The aim of the invention is to provide prodrugs which, when used together with antibody-enzyme conjugates, effectively kill the treated tumor tissue

allow

 15

Explanation of the essence of the invention

T _he invention is the object of / a process for the preparation of new prodrugs to provide for disposal.

The present invention contributes to the solution of the above problems by providing a novel method of transferring cytotoxic agents to tumor cells using antibody-enzyme conjugates together with prodrugs. According to the invention, therefore, an enzyme which catalyzes the reaction of a slightly or non-toxic prodrug into an active cytotoxic drug is conjugated to a tumor-specific antibody. This antibody

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Enzyme conjugate is administered to the tumor patient and binds to the surface of such tumor cells due to the antibody specificity / which tumor antigens are / for which the antibody is specific. Subsequently, the prodrug is administered to the patient and transferred in the region of the tumor due to the action of the antibody-bound enzyme in the cytotoxic more active drug.

The present invention further encompasses a method for transferring cytotoxic drugs to tumor cells wherein a plurality of prodrugs are activated by a single antibody-bound enzyme. In addition, according to the invention, a number of different immunoconjugates may be used. Tumor specific antibodies / to which various enzymes are bound can be used to convert a number of different prodrugs into their more cytotoxic effective form for treating the tumor. Alternatively, a variety of immunoconjugates may be used to convert a prodrug or a series of prodrugs into their more cytotoxically active form wherein the specificity of the antibody component of each conjugate varies / ie Each immunoconjugate contains an antibody to another antigenic determinant on the tumor cell.

A preferred embodiment of the present invention relates to antibody-enzyme conjugates / containing the enzyme alkaline phosphatase ("AP") / and together with a new prodrug etoposide-4'-phosphate or 7- (2-aminoethylphosphate) mitomycin or a combination of both Destruction of the tumor cells are used. A further embodiment of the present invention relates to an antibody-enzyme conjugate / which contains the enzyme penicillin V amidase (PVA) and is used together with a new prodrug N- (p-hydroxyphenoxyacetyl) adriamycin for the destruction of tumor cells.

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A further embodiment of the present invention relates to the use of an antibody-enzyme conjugate which contains the enzyme cytosine deaminase (CD) / together with the prodrug 5-fluorocytosine for the destruction of 5

Tumor cells.

The immunoconjugates and prodrugs of the invention can be used in antitumor agents / which contain a pharmaceutically effective amount of at least one immunoconjugate or prodrug of the invention in a pharmaceutically acceptable carrier. In addition, the immunoconjugates and prodrugs may be used in combinations as well as in methods for the treatment of tumors in mammals / wherein the patient is treated with a pharmaceutically effective amount of the inventive agent ¹ ^.

The methods / immunoconjugates / prodrugs / pharmaceutical agents and combinations of the invention provide a relatively simple and direct method of transfer

cytotoxic drugs on tumor cells, which is characterized by more selective cytotoxicity, and at the same time the problems associated with heterogeneous antigen expression known from conventional immunotherapeutic methods using antibodies.

* Antigen / antibody internalization and inadequate efficacy of the drug should be avoided.

Brief description of the drawings:

FIG. 1 shows the principle used for activating FIG. 30

Prodrugs on tumor cells that bind antibody-enzyme conjugates.

Figure 2 shows an SDS polyacrylamide gel analysis (5-12 / 5%

Gradient gel / non-reducing) of (A): 96.5-AP Immuno-35

conjugate; (B) L6-AP immunoconjugate (C) AP; (D) monoclonal antibody 96.5; and (E) monoclonal antibody L6.

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Figure 3 shows preparation and hydrolysis of etoposide phosphate and etoposide thiophosphate prodrugs of the present invention.

Figure 4 shows the high pressure liquid chromatography (HPLC) (taken at 254 nm) of: (A) Etoposide 4'-phosphate alone # i. in the absence of AP or L6-AP conjugate; (B) etoposide alone; (C) the product after a 5 minute reaction of Etoposide 4'-phosphate prodrug with AP; and (D) the product after a 5 minute reaction of etoposide 4'-phosphate prodrug with the L6-AP conjugate of the invention.

FIG. 5 shows a comparative graph of FIG. 15

Percent of etoposide released as a function of time / when reacting etoposide-4 ^1- phosphate or etoposide-4'-thiophosphate with alkaline phosphatase.

FIG. 6 shows the percentage binding of monoclonal antibodies L6 and 96.5 as well as conjugates according to the invention L6-AP and 96.5-AP to H3347 tumor cells.

FIG. 7 shows the percentage binding of monoclonal antibodies L6 and 1F5 as well as conjugates according to the invention L6-AP and 1F5-AP to H3347 tumor cells.

FIG. 8 is a comparative graph of the percentage of killed tumor cells as a function of

the concentration of etoposide or etoposide phosphate prodrug. 30

The figure shows an increased percent kill as a result of a reaction of the relatively non-toxic prodrug with either the L6-AP conjugate of the invention or the investigational 96. 5-AP conjugate.

FIG. 9 shows a comparative graph of the percent inhibition of H-thymidine incorporation in the DNA of H3347 tumor cells when reacted with 9 : Etoposide / O: EP / D: L6-AP + EP or: 1P5-AP + EP. The graph shows the increase in cytotoxic activity / obtained by treatment of the tumor cells with L6-AP and EP / compared to activity with EP alone treatment.

FIG. 10 shows analyzes of the phosphatase activities in untreated tumors or treated with conjugates according to the invention.

FIG. 10a shows the total phosphatase activity of H3347 tumors as a function of time in untreated mice or in mice / which had been treated 24 hours previously with the L6-AP conjugate according to the invention.

Figure 10b shows tumor cross-sections of untreated or L6-AP or IF5-AP pretreated mice stained with either hematoxylin and eosin or with an AP substrate. Dark areas indicate a high phosphatase

Activity

 25

Figure 11 is a comparative graph of tumor voluhnia versus time in mice / ♦: untreated; or treated with 0: etoposide: S: EP; o: 1F5-AP + EP or 4: L6-AP + EP. Arrows indicate the beginning of drug treatment τ the conjugates were administered 18 to 24 hours earlier. The graph shows the pronounced antitunior effect observed in treatment with L6-AP and EP.

2 3 3 6 3

FIG. 12 shows the chemical structures of the mitomycin derivatives used according to the invention / including the novel prodrug 7- (2'-aminoethyl phosphate) mitomycin (MOP).

Figure 13 shows the reaction of MOP with the enzyme alkaline phosphatase as a function of time. The course of the reaction was recorded by means of HPLC via the released MOH (the mitomycin alcohol derivative of MOP).

FIG. 14 shows the percentage binding of monoclonal antibodies L6 and 1F5 as well as conjugates according to the invention L6-AP and 1F5-AP to H2981 tumor cells.

Figure 15 is a comparative graph of the percent inhibition of H-thymidine incorporation into the DNA of H2981 tumor cells / which contain A rEtoposide,: EP, α: AP + EP, •: L6-AP + EP, or O: 1F5-AP + EP were treated. The graph shows the increase in cytotoxic activity as compared to EP / in the case that the tumor cells were pretreated with the L6-AP conjugate according to the invention.

Figure 16 is a comparative graph of the percent inhibition of H-thymidine incorporation into the DNA of H2981 tumor cells / which are with α: mitomycin C (MMC); A: MOH; a: MOP; Δ: MOP + AP, @: L6-AP + MOP or O: 1F5-AP + M0P. The graph shows an increase in cytotoxic activity in pretreatment of the tumor cells with the L6-AP conjugate of the present invention followed by treatment with MOP compared to activity following MOP treatment alone.

Figure 17 is a comparative graph of percent inhibition of H-thytnidine incorporation into the DNA of CEM cells labeled A: MMC, π: MOH / m : MOP, •: L6-AP + MOP, or O: 1F5-AP + M0P were treated. The graph demonstrates the specificity of the enhanced cyto-

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toxicity according to Figure 16, as a significant increase in CEM cells lacking the L6 antigen can not be observed.

Figure 18 is a comparative graph of tumor volume versus time in Figure 9 : untreated (control) or 9: MOH / O: MOP, A: 1F5-AP + MOP or D: L6-AP + M0P treated mice. Arrows indicate the respective medication treatment; Conjugates were administered 18 to 24 hours before each drug treatment, the graph shows the enhanced antitumor effect after treatment of tumors with L6-AP + M0P.

Figure 19 shows a comparative representation of the tumor volume as a function of time at £ 8: untreated (control) or with CJ: MOP / EP, 0: 1F5-AP + M0P / EP or 9: L6-AP + M0P / EP treated mice. Arrows indicate the respective drug treatment, conjugates were administered 18 to 24 hours before each drug treatment. 2, CL The graph shows the enhanced antitumor effect after treatment of the tumor with the L6-AP conjugate according to the invention and a combination of prodrugs, namely MOP and EP.

FIG. 20 shows the chemical structure of an adriamycin prodrug according to the invention ("APO") and its production from adriamycin.

FIG. 21 shows a comparative graph of the percentage of released adriamycin as a function of time 3Φ on reacting APO with A: free penicillin V-amidase or O and α: with the L6-PVA conjugate according to the invention at 10 and 100 μg, respectively total protein / ml. The course of the reaction was determined by means of HPLC.

Figure 22 shows the percent binding of monoclonal antibody L6 and the conjugates according to the invention L6-PVA and

1F5-PVA on H? 981 tumor cells.

Figure 23 shows a comparative plot of the percent inhibition of H-thymidine incorporation in the DNA of H2981 tumor cells treated with 0: adriamycin (ADM) / ·: APO, Λ: L6-PVA + APO or Δ: 1P5-PVA + APO. The graph shows the increase in cytotoxic activity upon pretreatment of the tumor cells with L6-PVA conjugate / followed by treatment with APO versus antitumour activity with exclusive treatment with APO.

Figure 24 shows the percent binding of antibodies L6 and 1F5 as well as conjugates according to the invention L6-PVA and 1F5-PVA to Daudilymphomazellen.

Figure 25 shows a comparative plot of percent inhibition of H-thymidine incorporation into the DNA of daudi lymphoma cells treated with 9 : ADM / O: APO / ffl: L6-PVA + APO or D: 1F5-PVA + APO. The graph shows an increase in cytotoxic activity following pretreatment of tumor cells with 1F5-PVA conjugates followed by treatment with APO / versus cytotoxic activity following treatment with APO alone.

Figure 26 describes the chemical structure of 5-fluorocytosine ("5-FC") and 5-fluorouracil ("5-FU"). 5-FC represents a prodrug which is converted to 5-FU via the methods of the present invention.

FIG. 27 shows a comparative graphical representation of the amount of product formed as a function of time when cytosine (cyto) is reacted with *: CD / 0: L6-CD or 3: 1F5-CD or when 5-FC is reacted with α: CD / A: L6-CD or Δ: 1F5-CD. When using cytosine as a substrate

2 8 3 0 3

üracil is formed and when 5-FC is used as a substrate 5-PU is formed. The course of the reaction was recorded spectrophotoroetrically.

FIG. 28 shows the percentage binding of the monoclonal antibody L6 and the conjugates according to the invention L6-CD and 1F5-CD to H2981 tumor cells.

_Figure 29 is a comparative graphical representation

the percent inhibition of H-leucine incorporation into the protein of H2981 tumor cells after treatment with: 5-FU / O: 5-FC, 0: L6-CD + 5-FC, or B: 1F5-CD + 5-FC. The graph shows the increase in cytotoxic activity following pretreatment of the tumor cells with L6-CD conjugate followed by treatment with 5-FC compared to activity following treatment with only 5-FC.

The present invention relates to a novel method for the transfer of cytotoxic agents to tumor cells and

causes more selective elimination of tumor cells in the treatment of cancer / e.g. of carcinomas / heianomas and other tumors.

According to the method of the invention, an antibody-enzyme conjugate is administered to the tumor-affected patient (mammals including human). This antibody-enzyme conjugate consists of a tumor-specific antibody / which is linked to an enzyme. The enzyme becomes a prodrug, which has a less cytotoxic effect on tumor cells than their parent drug converts to the more active parent drug. After transfer to the patient, the conjugate is directed to the site of the tumor by its antibody component, which recognizes an antigen on the tumor cells, and binds to the tumor cells there. The antibody transports

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thus the enzyme to the place of the tumor. Subsequently, as a substrate for the enzyme, a prodrug is introduced into the patient and transferred in the region of the tumor by the enzyme into the active cytotoxic drug. The drug is thus activated extracellularly and can diffuse into all tumor cells in this area / ie both into those cells which carry the antigen specifically recognized by the antibody of the conjugate as well as those cells which lack this antigen but are nevertheless present in the tumor (see Fig. 1). The method of the invention thus obviates the present problem of tumor antigen heterogeneity and the need for antibody-conjugate internalization in the context of conventional immunoconjugate drug delivery methods.

As in the case of the present method, a direct binding of the drug to the antibody is not required and thus the amount of the drug that can be transferred is not limited, there are also no conventional problems regarding the effectiveness of the drug in the region of the tumor. By the present method, the number of drug molecules at the site of the tumor is amplified / because the antibody-bound enzyme of the conjugate can each turn several substrate molecules into the active compound.

In addition, the active drug can be released by the method according to the invention specifically in the area of the tumor / not in other tissues / on the other hand. This is made possible by the fact that the enzyme concentration in the area of the tumor is higher than in other tissues / because antibody-enzyme conjugates are bound to the tumor cells.

The antibody component of the immunoconjugate of the invention includes any antibody which can specifically bind to a tumor-associated antigen. Non-limiting gg examples of such antibodies are antibody specific for

Carcinomas / melanomas / lymphomas as well as bone and soft tissue sarcomas and other tumors. Preferred are those antibodies which persist for a long time on the cell surface.

c remain bound flat or be internalized very slowly. These antibodies may be polyclonal or preferably monoclonal / consisting of intact antibody molecules or fragments / containing the active binding sites of an antibody / eg Fab or F (ab ') ₂ > ^{un (} 3 may be prepared by conventional methods (see eg RA DeWeger et al / Eradication Of Murine Lymphoma And Melanoma Cells By Chlorambucil-Antibody Complexes / Immunological Rev. 62 / p. 29-45 (1982) (tumor specific polyclonal antibodies produced and used in

conjugates); M. Yeh et al. "Cell Surface Antigens Of Io

Human Melanoma Identified By Monoclonal Antibodies "/" Proc. Natl. Acad. Be. 76 / p. 2927 (1979): J.P. Brown et al. "Structural Characterization of Human Melanoma Associated Antigen p97 with Monoclonal Antibodies" / J.

Immunol. 127 (No. 2), pp. 539-546 (1981) (tumor-specific 20

monoclonal antibodies produced); and J.P. Mach et al., "Improvement Of Colon Carcinoma Imaging: From Polyclonal Anti-CEA Antibodies And Static Photoscanning To Monoclonal Fab Fragments And ECT" / in Monoclonal Antibodies For

Cancer Detection And Therapy / R.W. Baldwin et al. (Ed.) / 25

Pp. 53-64 (Academic Press 1985 (antibody fragments produced and used to localize to tumor cells)). Monoclonal antibodies may be mouse or human antibodies as well as chimeric antibodies (see, e.g., V.T.Oi / "Chimeric Antibodies" / BioTechniques / 4 (No. 3) / p. 214-221 (1986)).

The enzyme component of the immunoconjugate according to the invention comprises all enzymes / which convert a prodrug into their more active ones

cytotoxic form can pass. The in connection with 35

The term "prodrug" used in the invention describes one

Precursor or a derivative of a pharmaceutically active substance / which in comparison to their parent drug (parent compound) acts less cytotoxic to tumor cells and can be enzymatically activated or converted into a more active starting shape (see, eg, DEV Wilman / "Prodrugs in Cancer Chemotherapy" / Biochemical Society Transactions / 14, pp. 375-382 (615th Meeting, Belfast 1986) and VJ Stella et al., "Directed Drug Delivery" / R. Borchardt et al. # (Eds) / pp. 247-267 (Humana Press 1985) " ).

Nonlimiting examples of enzymes which can be used according to the invention are the alkaline phosphatase for converting phosphate-containing prodrugs into their free parent compounds / arylsulfatase for converting sulfate-containing prodrugs into their free parent compounds / cytosine deaminase for converting 5-fluoro-cytosine into the anticancer agent 5-fluorouracil / proteases / such as serratiaprotease / thermolysin, subtilisme, carboxypeptidases and cathepsins (such as cathepsins B and L) which are useful for converting peptide-containing prodrugs into their free parent compounds / D-alanyl carboxypeptidases, useful for converting prodrugs with D-amino acid substituents, carbohydrate cleaving enzymes / such as ß- galactosidase and neuraminidase, suitable for the conversion of glycosylated prodrugs into their free parent compounds, / ö-lactamase / suitable for converting p-lactam-derivatized drugs into their free starting compounds / and penicillin amidases / as the peni cillin V amidase or penicillin G amidase, suitable for the conversion of drugs which have been derivatized on the amine nitrogen with phenoxyacetyl or phenylacetyl groups. In addition, antibodies having enzymatic activity (also known as "abzymes") can be used to convert the prodrugs of the invention into their free parent compounds (see RJ Massey / Nature, 328, pp. 457-458 (1987)).

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Antibody-abzyme conjugates can be prepared as described herein and used to transfer the abzyme to a tumor cell population.

Nonlimiting examples of the above-mentioned prodrugs are phosphate-containing, thiophosphate-containing / sulphate-containing / peptide-containing / D-amino acid-modified, glycosylated, β-lactam-containing, optionally substituted phenoxyacetamide-containing or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine -Prodrugs / which can be converted by the conjugate enzyme into a more active / cytotoxic free drug. Non-limiting examples of cytotoxic agents ι which can be converted into a prodrug according to the invention is / are etoposide / teniposide, adriamycin /

Daunomycin / carminomycin / aminopterin / dactinomycin / mitomycin / cis-platinum and cis-platinum analogs / bleomycins / esperamicins (see U.S. Patent 4,675,187) / 5-fluorouracil / melphalan and other related nitrogen mustard compounds.

The prodrug is preferably selected from etoposide phosphates etoposide thiophosphates / etoposide sulfates / teniposide phosphates / teniposide phosphates, teniposide sulfates / adriamycin phosphates / adriamycin sulfates / N-phenoxyacetyl derivatives of adriamycin / N-phenylacetyl derivatives of adriamycin, mitomycin phosphates / mitomycin sulfates / 5-fluorouridine mono-

phosphates and 5-fluorocytosine

 25

The enzymes used according to the invention can be covalently bound to the antibodies according to the invention by known methods. For this purpose, the heterobifunctional crosslinking reagents SPDP (N-succinimidyl

3- (2-pyridyldithio) propionate) or SMCC (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (see, for example, PE Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates / Immunological Rev./). 62, p.119-58 (1982): JM Lambert et al., Supra, p. 12038; GF Rowland et al., Supra, pp. 183-84 and J. Med.

Gallego et al., Supra, pp. 737-38). In addition, it is possible to use fusion proteins which at least comprise the antigen-binding region of an antibody used according to the invention.

body, which is at least connected to a functionally active part of an enzyme used in the invention. These fusion proteins are produced by known recombinant DNA techniques (see e.g.

M.S. Neuberger et al., Nature 312, pp. 604-608 (1984)). These fusion proteins act in exactly the same way as the antibody-enzyme conjugates described herein.

In a preferred embodiment of this invention, an antibody specific for a human cancer antigen has been conjugated to the enzyme alkaline phosphatase and used in the present invention to convert a 4'-phosphate derivative of the epipodophyllotoxin glucoside into an active anticancer agent. These derivatives include etoposide-4 ^1- phosphate, etoposide-4'-thiophosphate and teniposide-4 ^1- phosphate (see Figure 3 for the structures of these derivatives: the teniposide derivative has a 2-thienyl group instead of the methyl group in the sugar residue shown). Other embodiments of this invention include phosphate derivatives of these glucosides / wherein the phosphate moiety is attached to other hydroxy groups of the glucoside. In a particularly preferred embodiment of this invention, etoposide-4'-phosphate or etoposide-4'-thiophosphate are used as prodrugs.

According to the invention, alkaline phosphatase / AP was covalently linked to the monoclonal antibody L6 / IgG2a antibody, which recognizes a glycoprotein antigen on human lung carcinoma cells (I. Hellstrom, et al., "Antitumor Effects Of L6, AN IgG2A Antibody That Reacts With Most Human Carcinomas Proc. Natl. Acad. Sci. USA, 83, pp. 7059-63 (1986)). The immunoconjugate thus obtained shows no decrease in enzymatic activity compared to the unconjugated enzyme. In addition, the majority of the binding activity of the immunoconjugate remained

LO 21

L6 antibody obtained.

By means of an in vitro cytotoxicity assay, it has been shown that treatment of cells of a human carcinoma cell line with the Le-AP immunoconjugate / followed by addition of an etoposide phosphate prodrug to the cells results in a similar cytotoxicity / cytotoxicity of etoposide itself on these cancer cells. , "On the other hand, etoposide phosphate itself showed only low cytotoxicity.

In addition, in vivo studies on nude mice have shown that the L6-AP conjugates accumulate in L6 positive tumor xenografts. Histological studies of these tumors revealed that the AP enzyme was distributed throughout the Turoormasse.

In addition, the L6-AP conjugates showed strong in vivo antitumor activity in therapy experiments. This was the

2Q conjugate was administered to nude mice / which had subcutaneous L6-positive tumors »followed by treatment with an etoposide phosphate prodrug. The antitumor effect of this treatment caused complete regression of some tumors and was superior to the effect of exclusive treatment with the prodrug or its parent compound.

In another preferred embodiment of this invention, the L6-AP immunoconjugate was used to convert the novel mitomycin phosphate prodrug into the active mitomycin drug, as in the case of the etoposide phosphate prodrug, by the AP enzyme of the conjugate Phosophatgruppe removed from the prodrug / whereby the active tumor drug is released. In the mitomycin phosphate prodrug may be, according to the invention to produce an N-C, _ _Q alkyl

phosphate derivative of mitomycin C or porfiromycin or

η 7

1.Ii 111 ·

the pharmaceutically acceptable salts thereof. N denotes the nitrogen atom / which is bonded in the 7-position to the mito anchors of the starting compound. In a particularly preferred embodiment, the derivative used is 7- (2'-aminoethyl phosphate) raitomycin (MOP) (see FIG. 12 for the structure of mitomycin C and MOP in the form of the disodium salt: the porfiromycin derivative corresponding to the MOP has a methyl group on the aziridin nitrogen of mitomycin C). The MOP compound is also referred to as 9a-methoxy-7-II (phosphonoxy-methyl-amino-mitosan disodium salt Other embodiments of this invention include N-alkyl-midomycin phosphorothioates as prodrugs.

In vitro studies in which human lung tumor cell lines were treated with L6-AP immunoconjugates followed by addition of MOP / gave a cytotoxicity effect on these tumor cells comparable to that of the recognized antitumor agent mitomycin alone. Exclusive application of the micromycin phosphate prodrug to the tumor cells revealed low cytotoxicity. Similarly, the L6-AP immunoconjugates showed a pronounced in vivo antitumor effect in therapy experiments. For this purpose, nude mice / which human lung tumors carried the conjugate were administered and then treated with the mitomycin phosphate prodrug. The antitumour effect was more pronounced here than with exclusive treatment with prodrug or with the parent compound or after treatment with a non-binding antibody-AP conjugate followed by a prodrug dose.

In a further embodiment of the present invention, a penicillin amidase is covalently linked as an enzyme with the monoclonal antibody L6. That here

9 "- M t 23 · ι ο ο - ο -»

The obtained immunoconjugate is used to convert a novel adriamycin prodrug into the active antitumor compound adriamycin. The amidase "penicillin V amidase (PVA) used here was isolated from Fusarium oxysporum / hydrolyzes phenoxyacetylamide bonds. Thus, in particular, N- (p-hydroxyphenoxyacetyl) adriamycin (APO) is used as a prodrug / which is hydrolyzed by the amidase to form the active antitumor agent adriamycin. The L6-PVA immunoconjugate shows no loss of enzymatic activity compared to the unconjugated qazym and the binding activity of the L6 antibody is largely retained in the conjugate.

In vitro studies revealed that treatment of human lung cancer cells with the L6-PVA conjugate followed by addition of the APO prodrug to the cells resulted in cytotoxicity values comparable to the values of adriamycin alone. In addition, the APO prodrug exhibited much lower cytotoxicity to the tumor cells.

Similar in vitro studies were performed with the 1F5-PVA conjugate / wherein the PVA enzyme is conjugated to the monoclonal antibody 1F5 / which reacts with an antigen of lymphoma cells. Treatment of Daudi lymphoma cells with the 1F5-PVA conjugate / followed by addition of APO to the cells / resulted in toxicity levels / comparable to treatment of adriamycin alone / only low cytotoxicity on treatment of the cells with APO alone was watching.

Although only the synthesis of the novel adriamycin prodrug / N- (p-hydroxyphenoxyacetyl) adriamycin is described herein, the present invention encompasses the same

27 .;

Synthesis and Use of Other Related Adriamycin Prodrugs / Which Can Be Derivatized Using Substantially Equal Procedures. The invention includes, e.g. also the prodrug N- {PhenoxyacetyDadriamycin which can be prepared by the process described herein / wherein p-hydroxyphenoxyacetic acid is replaced by phenoxyacetic acid (see Example 4 / below). In addition, this invention encompasses adriamycin prodrugs / which contain additional N-hydroxyphenoxyacetyl derivatives of adriamycin / so e.g. substituted at various positions of the phenyl ring derivatives and N-phenoxyacetyl derivatives / which contain other substituents on their phenyl ring than the hydroxy group described herein.

The present embodiment also includes other amidases / e.g. the penicillin G amidase as an enzyme component of the immunoconjugate and prodrugs / which are derivatized to match the respective amidase and can be hydrolyzed by this to the active antitumor form.

If e.g. a penicillin G amidase is used as an enzyme, the prodrug should contain a phenylacetylamide group (as opposed to the phenoxyacetyl group of APO) / as penicillin G amidases hydrolyze this type of amide bonds (see, e.g., A.L. Margolin et al / Biochim.

Biophys. Acta 616 / p. 283-89 (1980)). The prodrugs of the invention thus comprise N- (p-hydroxyphenylacetyl) adriamycin / N- (phenylacetyl) adriamycin as well as other optionally substituted N-phenylacetyl derivatives of adriamycin.

The present invention includes all prodrugs which are obtained by reacting the amine group of the starting compound with the carboxy group of phenoxyacetic acid / phenylacetic acid or other similar acids. The present invention thus includes anthracycline prodrugs other than adriamycin / which can be derivatized and are substantially similar to those described herein

~ I.

_w - * ^ J s.

Adriamycin prodrugs. Examples of other prodrugs which can be prepared and used according to the invention include hydroxyphenoxyacetylamide derivatives, hydroxyphenylacetylamide derivatives, phenoxyacetylamide derivatives, and phenylacetylaraid derivatives of anthracyclines, such as daunomycin and carminomycin. Further amine-containing medicaments / such as melphalan / mitomycin / aminopterin / bleomycin and dactinomycin can be modified according to the information contained herein to form prodrugs according to the invention.

The present invention thus also encompasses compounds of the general formulas I and II:

o 9 ^H o

(I)

worm

R ¹ for R ¹ for R ² for and

Hydrogen and R is OH or OCH-; or

3 OH and R stand for OCH ₃ ? and is hydrogen or OH;

0 OH

(II)

, ο 6 3

worm

R ^{1 is} hydrogen and R ^{3 is} OH or OCH ₃ ?

R ^{1 is} OH and R ^{3 is} OCH-;

2 ^y

R is H or OH.

Another preferred embodiment of the present invention involves the conjugation of the enzyme cytosine dearoinase (CD) to L6 monoclonal antibodies. The deaminase catalyzes the conversion of 5-fluorocytosine (5-FC) / a compound lacking antineoplastic activity / to the active antitumor compound 5-pluoruracil. (5-FU) (see Fig. 24). The L6-CD immunoconjugate according to the invention was used to convert the prodrug 5-FC to 5-FU / whereby a significant cytotoxic effect on tumor cells in vitro could be observed.

In a similar way as for the previously described Iromunkonjugate / was no significant in L6-CD Kon3ugat

* Q decrease in enzymatic or binding activity observed as a result of conjugation. In vitro studies also showed that treatment of human lung tumor cells with L6-CD conjugate # followed by addition of the prodrug 5-FC to the cells, has a cytotoxic effect.

This effect was comparable to the cytotoxic effect of the antagonist 5-FU alone on the cells. Exclusive treatment of the tumor cells with the prodrug did not produce a significant cytotoxic effect.

it is evident from the extensive data contained herein that the immunoconjugate / prodrug combination of the invention is a selective mechanism for elimination of tumor cells / wherein a prodrug with reduced cytotoxic activity is administered / which prodrug is in the presence of the antibody-directed enzyme

: je j

in the vicinity of the tumor cells in a highly cytotoxic state is transferred. In addition, the cytotoxicity achieved by this method is improved compared to conventional antibody targeting techniques. In contrast to antibody drug conjugates, as already described there are / no physical limitations / since the release of the active drug in the region of the tumor is not impaired. It will thus be seen that the method of the invention is one way of increasing selective toxicity to tumor cells in the treatment of cancer and other tumors.

Another embodiment of the invention includes a method of combination chemotherapy using

Γ5 of several prodrugs and a single antibody enzyme conjugate. Accordingly, various prodrugs are used / wherein all substrates represent the same immunoconjugated enzyme. Thus, a variety of different prodrugs are converted to their cytotoxic form by a specific antibody-enzyme conjugate, thereby producing increased antitumor activity in the region of the tumor. For example, an enhanced antitumor effect was observed in in vivo studies / wherein the L6-AP immunoconjugate of the invention was administered to naked / human lung tumor infected mice / which was then subsequently treated with a combination of new prodrugs / i. treated with a mixture of etoposide phosphate and mitomycin phosphate prodrug. Similarly, administration of an etoposide phosphate, adriamycin phosphate (see U.S. Patent 4,185,111) and 5-fluoro uridine monophosphates (see, for example, C. Heidelberger et al / Fluorinated Pyrimidines And Their Nucleosides / Adv. Enzymol. Relate Mol. Biol. 54, p. 57-119 (1983)) after pretreatment with an antitumor antibody-AP conjugate a combination of effective antitumor agents, ie Etoposide, adriamycin and 5-fluorouridine in the area of the tumor.

I ₍

In another embodiment, a variety of immunoconjugates are used , wherein the enzyme component of the conjugate varies. In this case, one or more corresponding prodrugs in the region of the tumor can be converted into the cytotoxic form by each immunoconjugate. For example, an antitumor antibody can be linked to AP and cytosine deaminase, respectively, to a conjugate. Both immunoconjugates are then administered to the tumor patient and will bind to the tumor antigen due to their antibody specificity in the region of the tumor. Following administration of the prodrugs etoposide phosphate and 5-fluorocytosine, etoposide and 5-fluorouracil / both form effective anti-tumor agents in the area of the tumor.

Another embodiment of this invention involves the use of various immunoconjugates / wherein the specificity of the antibody component of the conjugate varies / i. Immunoconjugates are used / wherein each antibody specifically binds to another tumor antigen. The enzyme component may be the same or vary.

In particular, this embodiment is very useful / if the number of different antigens on the tumor surface is unknown and it is to be ensured that sufficient enzyme is delivered to the tumor. The simultaneous use of conjugates / specific for different antigens of a tumor increase the likelihood / that enough enzyme is brought to the tumor / so that a reaction of one or more prodrugs occurs. In addition, this results in a high degree of specificity / because the probability is low / that normal cells have all the tumor-associated antigens (see I. Hellstrom et al./ "Monoclonal Antibodies To Two Determinants Of Melanoma-Dependent p97 Act Synergistically In Complement Dependent Cytotoxicity "/ J. Immunol. / 127 (No. 1), pp. 157-160 (1981)).

The present invention also encompasses pharmaceutical agents / combinations and methods for the treatment of cancer and other tumors. In particular, the invention includes combinations / comprising an antibody-enzyme conjugate according to the invention and the corresponding prodrug or prodrugs for use in a method of tumor treatment, wherein a patient in a pharmaceutically acceptable manner with a pharmaceutically effective amount of an antibody-enzyme conjugate or conjugates and a

LO is treated pharmaceutically effective amount of a prodrug or prodrugs. The combinations and methods according to the invention are suitable for the treatment of all mammals / including humans / dogs / cats and horses. In a preferred embodiment, the antibody-enzyme conjugate is administered to the patient prior to introduction of the prodrug. The time between administration of the conjugate and the prodrug should be such / that the antibody of the conjugate can direct the enzyme to the tumor and fix it there. Depending on the administered conjugate are for this 12 h to

2 "D sufficient for a week.

The conjugates and prodrugs of the invention can be administered by conventional methods. Non-limiting examples of these are intravenous / intraperitoneal / oral, intralymphatic administration or administration directly into the tumor. Intravenous administration is preferably used.

The compositions of the invention / which comprise immunoconjugates or prodrugs may be in a variety of forms. Nonlimiting examples of these are liquid solutions or suspensions / tablets, pills, powders, suppository polymeric microcapsules or microvesicle liposomes as well as injectable or infusible solutions. The particular preferred form is dependent on

the mode of administration as well as the therapeutic use. For example, oral administration of antibody-enzyme conjugates is inconvenient * since the conjugate protein in case of oral administration e.g. can be broken down in the stomach in tablet form.

The conjugate- or prodrug-containing agents preferably also include common pharmaceutically acceptable carriers and adjuvants such as human serum albumin / ion exchanger AlumimsiDXid, lecithin, buffer substances / such as phosphates / glycine / sorbic acid / potassium sorbate and salts or electrolytes / such as protamine sulfate.

The most effective mode of administration and dosage form of the agent according to the invention depends on the severity and course of the disease / the state of health of the patient / its response to the treatment as well as on the judgment of the respective treating physician. Accordingly, the particular dose of immunoconjugate and prodrug should be adapted to the individual patient.

The effective dose of an antibody according to the invention

2 Enzyme conjugate can range from 1/0 to about 100 mg / m. The effective amount of a prodrug according to the invention depends on the prodrug used and on its parent drug. Since the prodrug is less cytotoxic than the parent drug, a higher dosage can be made / than is described for the starting compound in the prior art. An effective dose for etoposide

2 prodrugs are e.g. in the range of about 75 to 500 mg / m.

An effective dose of mitomycin phosphate prodrugs may range from 50 to 1000 mg / m. An effective dose for Adriamycm prodrugs may range from 15 to 150 mg / m. An effective dose of 5-fluorocytosine and other 5-fluorouridine prodrugs may range from

0 7

600 to 200 mg / m are. For ease of understanding of the present invention, the following non-limiting examples are given.

embodiments example 1

The following example illustrates the use of the iramun conjugates of the present invention and methods for the reaction of an etoposide phosphate prodrug into etoposide using antibody-bound alkaline phosphatase and those derived therefrom

resulting in vitro cytotoxicity to tumor cells 15

and in vivo antitumor effects obtained using the methods of the invention.

Preparation of the antibody-alkaline phosphatase conjugate according to the invention:

In this example, three immunoconjugates are prepared and tested # which comprise the monoclonal antibodies L6 # 96.5 or 1F5 / conjugated to the enzyme alkaline phosphatase (AP). L6 is a monoclonal antibody of the IgG2a subclass / and specifically binds to a glycoprotein antigen on human lung carcinoma cells (see I. Hellstrom et al / (1986) / as above). 96.5 is an IgG2a monoclonal antibody specific for p97 / a melanoma-associated antigen (see J.P. Brovn et al.

3Q "Structural Characterization of Human Melanoma Associated Antigen p97 With Monoclonal Antibodies" / J. Immunol. 127 (No. 2), pp. 539-46 (1981)). 1P5 is an IgG2a monoclonal antibody specific for the CD-20 antigen on normal and neoplastic B cells (see E. A. Clark et al., Role Of The Bp35 Cell Surface Polypeptides In Human

B-Cell Activation "/ Proc Natl., Acad. S / USA / 82 / p. 1766-70 (1985)). The L6 hybridoma which produces the raonoclonal antibody L6 is available from the American Type Culture Collection ( ATCC) filed under accession number HB8677 in connection with EP patent application 207. The 1F5 hybridoma / producing the monoclonal antibody 1F5 is deposited under ATCC No. HB9645 The monoclonal antibodies 96.5 are commercially available.

The antibody-enzyme conjugates are linked by covalent attachment of AP to the monoclonal antibodies L6, 96.5 or 1F5 via a thioether bridge. The methods used for this purpose are similar to those of J.M. Lambert et al / in "Purified Immunotoxins That Are Reactive With Human Lymphoid Cells" / J-Biol. Chein. 260 (No. 22), p. 12035-12041 (1985)). According to an experimental protocol, the conjugates L6-AP and 96.5-AP are prepared as follows:

2-Iminothiolane (50 mM in 0.5 M triethanolamine hydrochloride

with 10 mM EDTA at pH 8.0) are added to a solution (8.0 mg / ml) of the antibody L6 or 96.5 (in 50 mM triethanolamine hydrochloride and 1 mM EDTA at pH 8/0) / so that the final concentration of 2 -Iiminothiolane 1/3 mM Be '25 carries. After 90 min. At 0 ^° C, the reaction is stopped by gel filtration over Sephadex G-25 using phosphate buffered saline (PBS) as eluant at pH 7.2. The reaction of the antibodies with 2-iminothiolane introduces sulfhydryl groups, the number of which is 1.9 to 3 _f 5 as determined by Ellman's assay (see PW Riddles et al., "Ellman's Reagent: 5,5'-dithiobis (2 -nitrobenzoic Acid) -A Reexamination ", Analytical Biochemistry, 94, pp. 75-81 (1979)).

Alkaline phosphatase (calf intestine, Boehringer Mannheim, 10 mg / ml) in 100 mM phosphate buffer at pH 7.0 is with

Sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (Pierce Chemical Co./20 mM in dimethylformamide (DMF)) / so that the final concentration of sulfo-SMCC is 2/4 mM. After 30 min at 30 ^° C., the modified enzyme is purified by gel filtration over Sephadex g-25 and elution with PBS.

The modified AP is then added in a molar ratio of 2: 1 with the thiolated antibody. Reaction of AP with sulfo-SMCC introduces maleimido groups into the enzyme / which, when reacted with sulfhydryl groups of the modified antibodies, form a thioether linkage between antibody and AP. Iodoacetamide (final concentration 1 mM) is added to the protein solution after 1 h reaction time / to block unreacted thiols. Subsequently, the conjugates are purified on Sephacryl S-300 with PBS as eluent. The fractions are registered at 280 nm and the AP activity of each fraction (64,000-fold diluted) is tested at pH 9/5 with p-nitrophenyl phosphate as substrate (P. Tijssen / Laboratory Techniques In Biochemistry And Molecular Biology / p. 67 / (Elsevier Press 1985)). Conjugate-containing fractions with a suitable AP antibody ratio are assayed by SDS-PAGE on a 5-12 / 5% gradient gel (see Pig. 2) and then pooled. The protein concentration is determined at 280 nm / whereby a 0/1% solution of the antibody or of AP absorbs 1/4 or 0/76 ODs. An analysis of the conjugates on the gel gives predominantly a 1: 1 ratio of antibody to enzyme. Under the denaturing conditions in the gel, AP / migrates a homodimer with a molecular weight of 140 kd in the native state / as a single band of 70 kd. This 70 kd protein band is also obtained in the gel on those traces / which show the high molecular conjugate bands / because one subunit of the enzyme dissociates from the covalently cross-linked antibody-enzyme conjugate (see Fig. 2 / lanes A and B).

Gel filtration over a Sephacryl S-300 column shows / that there is no free enzyme in the conjugate preparation.

A second very similar protocol is used to prepare the conjugates-L.6-AP and 1F5-AP of the invention / wherein the antibody is modified with iminothiolane {0/5 mM) to introduce a single free thiol group. AP is modified with succinimidyl 4- (N-maleimidomethylcyclohexane-1-carboxylate (SMCC) (Pierce Chemical Co./ Rockford / IL) at a final concentration of SMCC of 1/0 mM The modified proteins are pooled and the resulting conjugates Purified by gel filtration over Sephacryl S-300 Subsequent SDS-PAGE analysis shows that these conjugate preparations contain neither unconjugated proteins nor aggregates As described above, the protein concentration of the preparations is determined by their absorbance at 280 nm / solutions of 1 mg / ml of the antibody (mol. wt. 160 kd) and AP (molar wt. 140 kd) absorbing 1.4 and 0/76 OD units, respectively.

Preparation of the prodrugs etoposide phosphate and etoposide thiophosphate '.

Accordingly, in the next step of the present invention, each antibody-enzyme conjugate is reacted with a new etoposide phosphate or etoposide thiophosphate prodrug, more particularly as prodrugs of the 4'-disodium phosphate ester of etoposide and the 4'-disodium thiophosphate ester of etoposide with the Formulas used in accordance with FIG. 3.

Etoposide phosphate and etoposide thiophosphate are prepared by reacting etoposide with phosphoryl chloride and thiophosphoryl chloride, respectively, to give either a dichlorophosphate or dichlorothiophosphate as an intermediate. The phosphorylation reaction is carried out in a

suitable anhydrous organic solvents such as e.g. Acetonitrile and preferably in the presence of a tertiary amine base / e.g. N / N-diisopropylethylamine performed. The course of the reaction is monitored by means of thin-layer chromatography (TLC) * whereby the optimum reaction time can be determined on the basis of the product formation and / or the consumption of the starting material. Experience has shown that the reaction can take 4 to 72 hours, depending on the quality of the phosphorus compound used. Hydrolysis of the dichlorophosphate or dichlorothiophosphate intermediates to the corresponding disodium phosphate or disodium thiophosphate prodrugs occurs by adding an aqueous sodium bicarbonate solution (20 to 50 fold excess) to the reaction mixture and then stirring the mixture for a period of 1/5 to 3 hours room temperature. After partitioning between ethyl acetate and water followed by reverse phase chromatography of the aqueous phase using a water-methanol mixture and lyophilization or evaporation of the aqueous medium in vacuo gives the desired prodrugs.

A more detailed description of the preparation of the 4'-disodium phosphate dexvate of etoposide / a prodrug used in the invention is as follows:

A magnetically stirred suspension of Etopos®cl (Bristol-Myers Co./ 2/30 g / 3/91 mmol) in dry acetonitrile (210 ml) is heated / the compound is almost completely dissolved / cooled to room temperature and treated with N, Treated N-diisopropylethylamine (2/36 mli 13/5 mmol).

The mixture is cooled to 0 ⁰ C and treated via syringe over a period of 30 sec. With phosphoryl chloride / (POCl-, 666 mg, 4.34 mmol). The mixture is allowed to warm slowly to room temperature over 2 to 3 h and stirred at room temperature for 63 h. The reaction mixture is then treated with a solution of sodium bicarbonate.

36 'ό <3

carbonate (6/0 g, 71/4 mmol) in deionized water (110 ml). The mixture is stirred at room temperature for 80 min and then partitioned with aqueous sodium bicarbonate solution (20 ml), deionized water (125 ml) and ethyl acetate (350 ml). The organic phase is extracted again with deionized water (1 × 50 ml) and the combined aqueous phases are washed with ethyl acetate (250 ml) and then evacuated at room temperature for 1 h to remove the solvent (0.5 ramHg). The aqueous portion is then applied to an octadecylsilane (C-18) silica gel column (0 = 4 cm, height = 15 cm / packed in methanol and equilibrated with H ₃ O). After applying the entire aqueous phase, the column is washed with H ₃ O (175 ml) to remove inorganic salts. The product is then eluted with 20% methanol in water. Concentration of the solvent at 0.5 torr yields 744 mg (36%) of the pure etoposide phosphate compound as a colorless solid. In contrast, lyophilization gives the pure compound in the form of a very flaky, low density solid. In another embodiment, the etoposide phosphate prodrug of the invention is prepared as follows:

A magnetically stirred suspension of etoposide (10/50 g, 17.84 mmol / dried over P ₃ O ₅ at 80 ° C / 0.5 torr) in dry acetonitrile (450 mL) is treated with diisopropylethylamine (4.20 mL, 24 / 1 mmol). Diphenylchlorophosphate (2/00 ml, 9.65 mmol) is then added via syringe. The mixture is stirred under nitrogen for 2 h at 50 ⁰ C, whereby the etoposide dissolves. There is a further addition of diphenylchlorophosphate (1.80 ml, 8.68 mmol) and the reaction mixture is maintained at 45 ° C for 72 h. After a further addition of amine base (0.75 ml) and diphenylchlorophosphate (0.80 ml / 3.86 mmol), the geraisch is stirred at 40 to 45 ° C for 27 h, again with diphenylchlorophosphate

37 «: 'w 4 3

(0.40 ml) and kept at 40 to 45 ° C for 22 h. Add isopropanol (20 mL) / evaporate the solvent in vacuo and dissolve the solid residue in CH ₂ Cl ₂ (500 mL) and partition with H ₃ O (400 mL). The aqueous phase is extracted again with CH ₂ Cl ₂ (100 ml) and the combined organic extracts are washed with brine (250 ml) and dried (Na ₂ SO ₄ / MgSO ₄ ). Concentration in a rotary evaporator / followed by flash chromatography over silica gel using 2 to 3% CH ₃ OH in CH ₂ Cl ₂ gives 12.5 g (85%) of Etoposide 4'-diphenyl phosphate as a colorless solid.

In the next step, platinum oxide (0/198 q, 0.87 mmol) is added from a freshly opened bottle (Aldrich Chemical Co.) to the solution of etoposide 4'-diphenyl phosphate (0/79 g, 0.962 mmol) in 95 ml of absolute ethanol added. The solution is hydrogenated in a Parr apparatus under 45 to 50 PSI for 4 hours at room temperature. The reaction mixture is filtered through a pad of Celite using ethanol as eluent. After concentration in vacuo, and 14 hours of drying under vacuum over P ₂ O 5 _to ~ ^he keeps one etoposide-4'-phosphate as a white solid (0.627 g / 94%):

FAB MS m / e 669 (M + H) ⁺

-1

IR (KBr) 3440, 2930, 1778, 1604, 1498 cm *.

¹ H NMR _{DMS0-d6) ί 6.93 (s, lH), 6:46 (s, lH), 6.12 (s, 2H), 5.94 (m, 2H), 5.17 (bs, lH), 4.86 (d, J = 3.93Hz, lH), _en 4.64 (q, J = 7.5.5.8Hz, lH). 4.51-4.42 (m, 2H), 4.20

(d, J = IO.7Hz, IH), 4.01 (dd, J = 12.1.5.3Hz, IH), 3.51 (s, 6H), 3.51-2.75 (m, 7H), 2.83 (m.lH), 1.16 (d, J = 5.IHz, 3H).

¹³ C NMR (DMSO-d ₆ ) δ 174.5, 151.2, 151.1, 147.7, 146.2, 126.1, 132.3, 128.8, 109.8, 109.7, 107.9, 101.5, 101.2,

98.5, 80.0, 74.3, 72.7, 71.7, 67.6, 67.2, 65.7, 55.8, 43.0, 37.1, 20.2. 18.5.

Elemental analysis for

calculated: found:

50.95% 51.42%

5.11% 4.97%

Etoposide 4'-phosphate is then added to 2.90 g (4.34 mmol) of etoposide 4'-phosphate by addition of deionized water (50 ml) and solid sodium bicarbonate (3.00 g, 35.7 mmol). converted into its disodium salt. The mixture is stirred at room temperature for 0.5 h, during which the formation of CO ₂ subsides. The mixture is applied directly to a C-18 column according to the embodiment described above. The column is first washed with 300 ml of deionized water to remove excess salts and then eluted with 4: 1 H ₂ O / CH ₃ OH to give 1.90 g (61%) of the pure etoposide 4'-phosphate disodium salt in the form of a fluffy white solid after lyophilization.

The 4'-dinatriuiq phosphate or thiophosphate derivatives of etoposide are etoposide prodrugs with high water solubility and reduced cytotoxic activity. By reacting these compounds with alkaline phosphatase, the phosphate or thiophosphate group is cleaved off, releasing the effective anticancer agent etoposide (see FIG. 3).

An experiment in which etoposide-4'-phosphate and etoposide-4'-thiophosphate are reacted with alkaline phosphatase shows _; that both prodrugs are a substrate of the enzyme. As shown in Figure 5, the etoposide phosphate is hydrolyzed faster by the enzyme than the etoposide thiophosphate prodrug. However, etoposide thiophosphate can be

, ο 5 3

may be of particular benefit due to its increased hydrolytic stability.

Reaction of the antibody-alkaline phosphatase conjugate with an etoposide phosphate prodrug:

The conjugates of the invention show no significant decrease in enzymatic activity as a result of the binding of the enzyme to the antibody. This is evident from the fact that both conjugates and the free enzyme with p-nitrophenyl phosphate (see P. Tijssen, supra) or etoposide phosphate substrates have the same activities.

So you give, for example either AP alone or the antibody-enzyme conjugate L6-AP prepared as described above (final AP concentration 5 μg / ml) to a solution of etoposide 4'-phosphate (0.1 mM) in Tris buffer (100 mM) with MgCl- ( 1 pm) and ZnCl (0/1 mM) at pH 7/0. The reaction is monitored by HPLC using an IBM C-18 column (3μ / 4 / 5χ 100mm) using 50% aqueous methanol as eluent (0/5 ml / min / detection at 254nm). tracked. This shows that AP causes hydrolysis of at least 85% of the etoposide 4'-phosphate to etoposide within 5 min. After the start of the reaction either in free form or as part of the Lö antibody-enzyme conjugate (see Fig. 4C and 4D). It can be seen from the figures that no AP enzyme activity is lost by the binding to the antibody in the conjugate. In the absence of the enzyme, no phosphate hydrolysis occurs (see FIG. 4A). An aqueous solution

of etoposide phosphate or etoposide thiophosphate is stable for at least 8 h at room temperature or for several days at 4 ° C.

Binding of the antibody-alkaline phosphatase conjugate to H3347 tumor cells:

Fig. 6 shows the results of a conjugate binding assay _for checking the binding ability of the -L6-AP and 96.5-AP conjugates / as well as the free antibodies L6 and 96.5 to tumor cells of the inetastatic human colon cancer cell line H3347.

The binding assay is carried out as follows: The immunoconjugates or the free antibodies are diluted in incompletely modified Delbecco's medium (IMDM / Gibco) and 100 .mu.l aliquots each are incubated at 4.degree. C. with 10 cells for 30 min. The cells are washed and incubated with 50μl FITC goat anti-mouse antibodies (Tago, 1:12/5 diluted) at 4 ° C for a further 30 min. The cells are washed and analyzed on a Coulter Epics C fluorescence cell analyzer. Dead cells are rejected and the mean logarithmic green fluorescence intensity of each sample is determined. This value is transferred to linear values and the ratios between negative control (cells + FITC-goat anti-mouse antibody) and all samples are determined.

Fig. 6 shows that most of the binding ability of the antibodies is retained in the conjugate, that is, that the binding ability of the antibody is not affected by the conjugation. In addition, this figure shows the binding specificity of the antibodies / that is, both the free antibody 96.5 and the 96.5-AP conjugate bind much less to the tumor cells compared to antibody L6 and L6-AP conjugate, respectively. This result is to be expected / considering that H3347 tumor cells are derived from a human carcinoma and antibody L6 is specific for a carcinoma antigen.

41 -. w -. J -,

while antibody 96/5 reacts specifically with a melanoma antigen.

Similar binding experiments using L6 / L6-AP / 1F5 and 1P5-AP also revealed that L6 and L6-AP are bound to the H3347 carcinoma cell line (saturation at 10 μ9 / πι1 antibody) / while only a very slight or undetectable Binding of 1P5 or 1P5-AP is observed (see Fig. 7). This result again demonstrates the binding specificity of the conjugate / where the L6-AP conjugate binds to L6 positive tumor cell lines and does not bind the B lymphoma cell specific 1F5 conjugate.

In vitro cytotoxicity of a conjugate / prodrug combination according to the invention:

Next, the cytotoxic effect of conjugate / prodrug combinations of the invention will be demonstrated in vitro using either a clonogenic cytotoxicity assay or an H-thymidine incorporation assay.

The clonogenic cytotoxicity test corresponds to the colony inhibition assay of I. Hellstrom et al., "Colony Inhibition And Cytotoxicity Assays" in In Vitro Methods In Cell-Mediated Immunity / Bloom and Glade (ed.) / P. 409-14 (1971)). The cells used to determine cytotoxicity are the H3347 tumor cells described above. The conjugates L6-AP and 96.5-AP were tested for their ability to convert the prodrug into the free drug.

The H3347 cells (10 / ml) are suspended in IMDM growth medium (each containing 10 μg / ml of Iiranun conjugate / based on the antibody concentration) and incubated for 30 min. At 37 ° C. The cells are washed 2 χ / resuspended in IMDM and treated with medium dissolved drug or prodrug.

The mixture is then incubated at 37 ° C for 15 h. After twice

Washing, the cells are plated and the number of colonies (> 8 cells per colony) are counted 7 to 10 days later g.

The test result is shown in FIG. 8 (the percent inhibition corresponds to an average of 6 samples). As the figure shows, etoposide (ICc ₀ = 0/20 μΜ) is much more cytotoxic than etoposide-4 ^1- phosphate. EP) (IC ₅₀ = 5/8 μΜ). The prodrug alone shows a very low cytotoxic activity. Treatment of the H3347 cells with the L6-AP conjugate / followed by addition of etoposide phosphate gives a very large increase in cytotoxic activity compared to the prodrug alone / wherein the increased cytotoxic activity is comparable to the cytotoxic activity determined for etoposide alone. Treatment of cells with 96.5-AP conjugate and etoposide phosphate results in a much lower increase in cytotoxic activity compared to the result for the prodrug. As already discussed above, this result is due to the small amount of 96.5-AP conjugate / which binds to the H3347 cells (see Figure 6). The conjugates themselves are not cytotoxic / because exclusive treatment of the cells with the conjugate does not lead to cell death.

The cytotoxic effect of the conjugates according to the invention is also determined via the H-tymidine incorporation test. In this test, a suspension of 10 H3 347 tumor cells in

0/1 ml IMDM with 10% fetal calf serum for 1 h at 4 ° C 30

in the presence of 5 pg / ml conjugate. The cells are washed twice with a medium of 10% fetal calf serum, resuspended (1 ml) and placed in microtiter plates (96 wells / 10,000 cells / well).

depression). Subsequently, the drug 35

or the prodrug in IMDM

⁴³ I , 3 u 3

added and incubated at 37 ° C for 6 h. The cells are washed twice and then incubated for an additional 12 hours followed by a 6 hour pulse with H-thyraidine (1/10 c well). The plates are frozen at -20 C to detach the cells and the cells are placed on glass fiber discs isolated. The filters are then counted on a Beckman 3801 scintillation counter.

, ₀ This test determines the H-thymidine incorporation rate into the DNA of the tumor cells and thus the cytotoxic effect of etoposide or the prodrug EP on the cells in the absence or presence of the L6-AP or IF5-AP conjugates, respectively. As shown in Fig. 9, etoposide (IC ₅₀ = 1 μΜ) is more than

100 times more toxic than EP (35% inhibition at 100 μΜ). An io

Pretreatment of cells with 1F5-AP prior to EP addition does not increase toxicity. However, a substantial increase in cytotoxic activity is obtained / when the cells are first treated with L6-AP followed by EP. Thus, in both experiments for determining the in vitro cytotoxicity, it can be seen that the cytotoxic effect of the conjugate / prodrug combination according to the invention is comparable to the effect of etoposide alone and that this effect can be observed specifically for antigens.

since the cytotoxicity of EP by treatment of H3347-25

Cells with the control conjugates 96.5-AP and 1F5-AP are not raised in a similar manner.

Localization of Conjugates in Tumor Xenografts in Mice:

Next, one attempts to find out by in vivo localization how fast and how high the conjugates of the invention accumulate in a tumor. This information proves useful in determining

44: 3 3

a suitable time interval between administration of the antibody-enzyme conjugate and the prodrug in tumor therapy studies.

First, female BALB / C-nu / nu mice (4 to 6 weeks old / Life Sciences / St. Petersburg / Florida) are injected 10 H3347 Turaor cells subcutaneously (s.c.) into the left and right posterior planks. The tumor cells are obtained from in vitro cultures / which are suspended by treatment with trypsin (0/5 g / l) and EDTA (0/2 g / l) for two minutes. The cells are washed twice with IMDM and incubated for 1 h at 37 ° C in IMDM with 10% fetal calf serum. The cells are washed / suspended in PBS and stored at 4 ° C until injection. With the localization and therapy studies described here, one starts / if the Turaore have.

Turaore reached an average size of 225 mm

125 Localization studies include L6 and L6-AP with J and 1F5 and 1F5-AP with J using the iodogen method (see PJ Fraker et al / Biochem Biophys Res Coraraun. 80 / pp. 849-857 (1978)). Two days before the localization experiments, the animals are switched to 0/5% (v / v) Lugol ¹ S-JOd solution. Each mouse is injected intraperitoneally (lp) with 100 μg of 25 (based on each monoclonal antibody) of one of the following solutions: L6-AP (5 μίί) and 1F5-AP (2.5 μίί) in 0/2 ml PBS at pH 7 , 2 or a combination of L6 (5 μ <2ί) and 1F5 (2/5 μΰΐ) in 0/2 ml PBS. At periodic intervals, the mice are anaesthetized / withdraws the blood via the orbital plexus and subsequently kills the mice. The tissues are weighed and measured in a gamma counter. Localization is accomplished by comparing ¹²⁵ J-L6 and ¹³¹ J-1F5 / dh

125 Determination of the ratio of specific (J) to nonspecific (J) uptake in different

4. /,, 1.1 .. IW J

Tissues determined. The results for uptake rates into tumor and liver are summarized in Table I.

Table 1 % injected dose of administered proteins per g / tissue

L6

Tumor Liver 1.6 (8.0) 4.9 (2.0)

Time

2 h

24 h 3.6 (12.0) 2.3 (1.4)

48h 4.0 (8.0) 2.5 (1.3)

L6 AEP

Tumor 1.5 (7.5) 1.0 (10.0) 0.5 (5.0)

liver

5.2 (0.7)

1.3 (1.3) 0.8 (1.0)

The numbers in parentheses show the ratio of L6 / 1F5 and L6-AP / 1F5-AP, respectively

The table shows that L6 unconjugated antibodies are effectively localized to the tumor within 24 hours and remain there for at least 48 hours. During this time, the ratio of L6 to 1F5 in the tumor remains at 8 to 12 / while the ratio in the liver is relatively low (1/3 - 1/4). The maximum value for specific uptake of L6-AP into the tumor is approximately 24 h, with the ratio of L6-AP to 1F5-AP reaching 10/0. These results show that the L6-AP conjugate accumulated in the tumor much better than 1F5-AP / but not as well as unmodified antibody L6.

Next, the natural phosphatase activity in the tumor and its increase by tumor-directed AP targeting with

IW -> ·

Help of a! Conjugates invention determined. For this purpose, tumors are removed from mice which had previously been treated for 24 h with 100 μg L6-AP (based on L6). The total phosphatase activity is determined using p-nitrophenyl phosphate as substrate according to the following procedure: The removed tumor is washed and then washed at 23 C in p-nitrophenyl phosphate (1 mg / ml) in pH 9/5 Tris (100 mM) with NaCl ( 100 mM) and MgCl ₂ (5 mM). The course of the reaction is recorded by determining liberated p-nitrophenol at 410 nm, taking into account the tumor weight. It has been shown that tumors from mice / which were administered the L6-AP conjugate / give more than 10χ higher phosphatase activity than tumors from untreated mice (see Pig 10A).

A more detailed histological analysis of the phosphate activity of the tumor is obtained by Tu-morquerschnitte

from untreated or previously 24 h with 300 ug (relative

on the antibody) L6-AP and 1F5-AP treated mice, respectively. The phosphatase activity is determined immunohistochemically by using a phosphatase substrate / which forms a black precipitate in the range of the enzyme activity: Rapid freezing of distant tumors at -28 ° C and producing with a Reichert-Jung microtome sequential 8 μΐη cross sections. Phosphatase activity is determined with an AP substrate kit from Vector Laboratories (Burlingame / CA) and the results compared to hematoxylin and eosin stained sections (H. and E. / see Figure 10B).

Fig. 10B shows that only a low enzyme activity could be detected in tumors from untreated or IFF-treated mice. In L6-AP-treated mice, phosphatase activity is greatly increased and distributed throughout the tumor. A microscopic examination shows that the majority of the tumor cells from L6-AP-treated mice have a strong

47 shows positive staining due to phosphatase activity.

In vivo antitumor effect of a conjugate / etoposide phosphate prodrug combination according to the invention; 5

Therapy experiments are performed on nude mice with subcutaneous tumors of about 225 mm in volume. The conjugates L6-AP and 1F5-AP are administered 18 to 24 h before an EP treatment (ip). Tumor growth LO- 'is associated with growth in untreated mice or with growth at the maximum tolerated dose of etoposide or Compared to EP-treated mice.

Specifically, 8 nude mice with bilateral H3347-LS 'tumors are treated either exclusively with etoposide (0/2 ml, containing 1/2 mg etoposide in 2: 3 DMSOrH ₂ O) or EP (0.2 ml, containing 2 mg of EP in H ₃ O) or with L6-AP (0/1 ml containing 300 pq of antibody in PBS) or 1F5-AP (0/1 ml / containing 300 ug of antibody in PBS) followed by EP. Each experiment contains a control group of untreated mice. The tumor volume is estimated several days after the Turoorimplantation using the following formula:

[(vertical height / 2) Tj χ largest length

l:

These experimental results are shown in FIG. 11. Etoposide has a very low influence on tumor growth

at the dose used, with a higher dose remaining unchanged

is tragic. The prodrug EP is less toxic to the animals and the higher dose that can be administered therefore causes a stronger antitumor effect compared to etoposide. Similar antitumor activity is observed in mice / which controls the control conjugate 1F5-AP before treatment.

Ί L.

received action with EP. However, when mice are treated with L6-AP and subsequently with EP, a more pronounced antitumor effect is observed. L6-AP all have no influence on tumor growth (results not shown).

A summary of the results of tumor therapy is shown in Table II below. Of the 16 tumors in eight mice treated with L6-AP and EP, six tumors show complete regression and two more tumors are smaller in size than at the beginning of treatment. The other treatment trials show no complete or partial reaction.

Table II 15

Influence of different treatment methods on tumor growth

20 medium increase REACTION partial decrease complete decrease "none 18 0 0 E-fcoposid · 12 stable 0 0 25 EP 6 0 0 IFS-AP + EP 9 0 0 0 L6-AP + EP 3 4 2 6 10 30 7 5

The data show the response of 16 tumors in each experimental group, each 23 days after tumor implantation.

Response: Increase - continued tumor growth τ oo

stable - no further tumor growth;

partial decrease - decrease in size * complete decrease - complete regression of the tumor.

The present example clearly demonstrates the applicability of the method according to the invention for the transfer of a cytotoxic antitumor agent to tumor cells with the aid of a tumor-specific antibody-enzyme conjugate and a prodrug _f which is converted by the enzyme from a relatively unwontitive form into a highly toxic form.

Example 2

This example illustrates the use of immunoconjugates of the invention and methods for converting a relatively non-toxic mitoin mycine phosphate prodrug into an active mitomycin compound which is cytotoxic to tumor cells in vitro. Like Example 1, the following example demonstrates the applicability of the immunoconjugates according to the invention ι prodrugs and methods for the in vivo transfer of a cytotoxic antitumor agent to tumor cells.

In this example, the imraine conjugates L6-AP and 1F5-AP prepared in Example 1 are used. In this embodiment of the invention, each of these antibody-enzyme conjugates is reacted with a novel mitoinycin phosphate prodrug. In particular, the Dinatriurosalz an N-C, _ _g -Alkylphosphates of mitomycin C is used here as a prodrug. The antitumor agent released in the reaction is a mitomycin alcohol derivative. The L6-AP / mitomycin phosphate-prodrug combination according to the invention causes a cytotoxic effect in vitro against tumor cells and in vivo a pronounced antitumor effect in mice.

Preparation of a new mitomycin phosphate prodrug:

The new mitomycin phosphate prodrug 7- (2'-aminoethyl phosphate) 35

mitomycin (hereinafter referred to as MOP) is the 2-amino

ethyl phosphate derivative of mitomycin C (MMC) and is prepared as follows:

A solution of 2-aminoethyl dihydrogen phosphate (56 mg / 5

0/4 mmol) in water (0/35 ml) and triethylamine (0.3 ml /

2 mmol) is reacted with mitomycin A (hereinafter referred to as MMA) (140 mg / 0/4 mmol) in methanol (6 ml) overnight at room temperature. Then 1/4 ml of saturated aqueous sodium bicarbonate solution is added and the reaction mixture is partitioned with water and methylene chloride. The aqueous phase is concentrated to dryness and treated with several portions of methanol and concentrated again. The residue is taken up in methanol / filtered and applied to a 2 × 10 cm C-18 (reverse phase) silica gel, the product is eluted with water and all volatiles are removed, methanol is added and evaporated as described above and the product is evaporated The residue thus obtained was dried over phosphorus pentoxide under high vacuum in a desiccator for 24 h The mitomycin- Q phosphate derivative MOP is obtained as a fine blue powder (190 mg, 97%).

360 MHz ¹ H-NMR (D ₂ O) 6 1.94 (6, 3H, CH ₃ ), 2.9-3.1 (sa, 4H), 3.20 (s, OCH-), 3.28 (s, IH), 3.36 (s, IH), 3.5-3.65 (m, 4H), 4.1-4.25 (m, 2H), 4.50-4.57 (dd, IH, 10-H).

MOP is thus prepared by replacing the 7-methoxy group of MMA with 2-aminoethyl phosphoric acid (see FIG. 12). The product is converted to the water-soluble disodium salt by treatment with sodium bicarbonate.

The corresponding known mitomycin alcohol derivative

51 - Ö O ν

7- (2-hydroxyethyl) amino-9a-methoxymitosane (hereinafter referred to as MOH) is prepared by reacting MMA (100 mg, 0/286 mmol) with ethanolamine (26 mg / 0/429 mmol) according to the procedure described in U.S. Pat

Method of B.S. Iyengar et al. "Mitomycin C and 5

Porfiromycin Analogues With Substituted Ethylamines At Position 7 ", J. Med. Chem 26, 16-20 (1983)) to give a finely powdered blue product (850 mg / 54%).

Reactivity and stability of the mitomycin phosphate prodrug:

MOP is then tested for its reactivity with AP. To a solution of MOP (1 mM) in 200 mM Tris buffer / pH 7/2 is added at room temperature either intestinal

** Calf AP or placental human AP (final concentration 1 μg / ml). The course of the reaction is monitored by HPLC using a C-18 column (4/6 x 150 mm) and following conditions: detection at 280 nm; 30-95% methanol in acetate buffer (100 mM, pH 5/2) within 8 min. / Re-equilibration after 15 min .; Flow rate 0/8 ml / min. Under these conditions MMC is eluted at 7/0 min / MOH at 8/5 min and MOP at 4/0 min. As shown in Figure 13, the phosphate group of MOP is rapidly cleaved by AP. With the help of HPLC it is confirmed that the

2 <-> speaking alcohol MOH is formed. At the reaction conditions used, the half-life for the hydrolysis of MOP is about 10 minutes and the reaction is completed within 40 minutes.

The stability of MOP and EP in human serum is determined by HPLC / determining the rate of decrease of the prodrugs as well as the rates of formation of MOH and etoposide. For example, A solution of MOP (1 mM in 100 mM Tris / pH 7/2) is added to fresh human serum and the final concentration of the agent is adjusted to 0.1 mM.

Prepare 0/25 to of which each is diluted with methanol (0.25 ml) and EDTA (50 μΐ at 100 mM), precipitating the serum proteins and stopping the reaction. The samples are centrifuged and analyzed by HPLC as indicated above. It appears that 50% hydrolyzes within 1 h, but only 25% hydrolyzes MOP within 4 h Rapid rapid hydrolysis is achieved by addition of AP to the serum.

Binding of the Antibody-Alkaline Phosphatase Conjugate to H2981 Tumor Cells:

Subsequently, the binding ability of the inventive antibody-enzyme conjugates L6-AP and 1F5-AP is determined on H2981 tumor cells. The H2981 cell line is derived from a lung primary human adenocarcinoma (see I. Hellstrom et al., "Monoclonal Mouse Antibodies Raised Against Human Lung Carcinomas," Cancer Res. 46, (No. 8) / p. 3917). 23 (1986)). It is known that the antibody strongly binds L6 to H2981 cells (saturation at 10 pg / ml), whereas 1F5 binds only very weakly to these cells.

The binding test is carried out according to Example 1. A FACS analysis reveals that L6 and L6-AP are strongly bound to the cells, while a much lower binding of 1F5 and 1F5-AP occurs (see Figure 14).

In Vitro Toxicity of the Conjugate / Prodrug Combination of the Invention on H2981 Tumor Cells:

The cytotoxic effect of the conjugate according to the invention

Prodrug combinations is in vitro according to Example 1 on the

H-thymidine incorporation test shown: in this case one uses

H2981 tumor cells for in vitro toxicity and CEM cells as control. The T cell ALL cell line

 'Π ^

»U ν-

CEM (obtained from the ATCC) does not bind to the monoclonal antibodies L6 or 1F5. The cytotoxic effect of the prodrugs EP and MOP on tumor cells in the presence or absence of L6-AP or IF5-AP-iromun conjugates is examined. The cytotoxic effect of these combinations is further compared with the cytotoxic effect of the respective stamane compound (parent drug).

A suspension of 10 H2981 or CEM cells in 0.1 ml IMDM containing 10% fetal calf serum is incubated for 1 h at 4 C in the presence of 10 pg / ml conjugate. The cells are washed twice with media containing 10% fetal calf serum / resuspended in 1 ml of phosphate buffered saline * pH 7 # 2 (PBS) and plated in microtiter plates (96 wells / 10,000 cells / well). Subsequently, the PBS solution is added to the prodrug and incubated at 37 ° C for 1 h (for MOP) or 5 h (for EP). The cells are then washed twice and incubated a further total of 24 hours (including a 6-hour pulse with H-thymidine / 1/0 μCi / well). For detaching the cells are frozen, the plates stored at -70 ⁰ C, and transmits the cells after thawing onto glass fiber filters. , The filters are measured in a Beckman 3801 scintillantixin counter and the cytotoxic effects of 3 conjugate / prodrug combinations are determined in comparison to the cytotoxicity of exclusive treatment of the cells with the prodrug or parent compound. FIGS. 15 to 17 show the test results.

As can be seen from FIG. 15, Etoposide (IC-q = 2 μΜ) on H2981 cells is much more toxic than EP (20% killing at 30 μΜ). Pre-treatment of the cells with 1F5-AP prior to addition of the prodrug causes a very small increase in cytotoxicity. A substantial increase in cytotoxic activity, however, results in a treatment

54 2 3 3-:

of the cells with L6-AP before treatment with EP. This cytotoxic effect is comparable to the effect of etoposide.

A similar result is obtained using the mitomycin derivatives. Fig. 16 shows / MMC and MOH that act equally toxic to cells and H29ßl IC _S0 have values of about 1 μΜ. The phosphate prodrug MOP is far less cytotoxic (5% cell death at 10 μΜ) / possibly due to its inability to enter the cell. However, the activity of MOP is then to be compared with the activity of MOH and MMC / if the tumor cells are previously incubated with the L6-AP conjugate according to the invention. This increase is antigen-specific / as the non-binding conjugate 1F5-AP does not show a significant effect on the cytotoxic activity of the prodrug. Neither L6-AP nor 1F5-AP can significantly enhance the cytotoxic effect of MOP on CEM cells / because the said conjugates are incapable of binding to this cell line (see Figure 17). These results show that the phosphate group of each prodrug examined inactivates the active ingredient and that the active cytotoxic agent (EP or MOP) is formed by hydrolysis of the phosphate group by an antibody-enzyme conjugate bound to the surface of the tumor cell.

25 In vivo Antitumor Effect of a Conjugate / Mitomycin Prodrug Combination According to the Invention:

Before determining the in vivo antitumor activity of MOP in combination with the L6-AP conjugate according to the invention, the respective cytotoxicity of the prodrug or the released active derivative MOH / in BALB C nu / nu mice is first determined. When administering (ip) the active substances in two equal doses 4 days _{apart, LD50} values of 45 and 90 mg active substance / kg body weight are determined for MOH and MOP. It also shows / that one

- δ 3 ο

considerably larger amount of the active ingredient may be administered in the form of smaller doses over a longer period of time. Total amounts up to 40 mg / kg MOH and 100 mg / kg HOP are tolerated in the form of four equal doses over a period of 25 days. These studies also show that significantly more mitomycin prodrug is tolerated due to decreased toxicity.

Therapy studies are performed on naked BaIb C nu / nu female mice (4 to 6 weeks old / 6 mice per treatment group) (Life Sciences / St. Petersburg / Fla.) / Who previously had an H2981 tumor implanted from an in vivo source (so, right back flank). The experiments are carried out / as soon as the tumor has reached a volume of about 100 mm. The L6-AP and LC5-AP conjugates (0/1 ml / 250 ug antibody in PBS containing) 18 to 24 hours prior to treatment with MOP (0/2 ml / 0/6 mg MOP in H ₂ O containing ) (ip). Tumor growth is compared with growth in untreated mice or in mice fed with the maximum tolerated dose of MOP (0/2 ml / 0/6 mg MOP in H ₃ O) or MOH (0.2 ml / 0 / Containing 2 mg of MOH in H 2 O).

From Figure 18 it can be seen that both MOH and MOP have significant in vivo antitumor activity. To reach an average tumor size of 750 mm, MOH-treated mice require 45 days / MOP prodrug-treated mice 63 days and the control group 27 days. As mentioned above, the MOP prodrug has a less toxic effect on the animals / therefore a higher dose can be administered / which in turn results in the greater antitumor effect compared to the MOH derivative. The activity of MOP is also slightly enhanced by the non-binding conjugate 1F5-AP, but a much more definite effect is found in the group

56 "2 8 :::.

who received L6-AP before MOP treatment. As shown, the tumors pretreated with L6-AP conjugate (after ¹ MOP treatment) show on day 70 about 1/3 the size of tumors previously treated with 1F5-AP conjugate Table III below / that up to € 3. Day after implantation, three of the six tumors in the L6-AP + MOP-treated mice completely regressed and the remaining three

Tumors no longer enlarged since the beginning of treatment. The 10

In contrast, three of the five tumors in the 1F5-AP + MOP-treated group show an increase in size / two of the five tumors are stable and no partial or complete regression is observed.

TABLE III

Tumor response (up to 63rd day) to treatment with antibody-AP conjugates and mitomycin derivatives

Tumor responses

Group 'increase stable partial full f _ ₅ regression regression

Control 6/6 - -

MOH 6/6 - -

MOP 4/5 1/5

1P5-AP + MOP 3/5 2/5

L6-AP + MOP - 3/6 - 3/6

These experiments clearly show the in vivo-observed specificity and the enhanced antitumor effect of the

according to the invention antibody-enzyme / MOP combination. 35

₅₇ k 3 3 ο _v

example 3

This example illustrates the applicability of the immunoconjugates / prodrugs of the present invention and methods for transferring a number of different drugs to tumor cells, using the antibody-alkaline phosphatase conjugate L6-AP in combination with the prodrugs EP and MOP, increased antitumor activity in vivo can be demonstrated. The present invention thus enables the use of a single antibody-directed enzyme with a variety of prodrugs for combination chemotherapy against tumors.

Prodrugs EP and MOP are prepared as described in Examples 1 and 2. The preparation of the immunoconjugates L6-AP and 1P5-AP is described in Example 1. The in vivo studies on nude mice are carried out according to Examples 1 and 2. Naked / H2981 tumor implant-bearing mice are treated with L6-AP or IF5-AP 18-24 hours prior to treatment with a combination of MOP / EP (containing 2 ml, 1 mg EP and 0.3 mg MOP in HO) Pretreated conjugate. Tumor growth is compared to growth in untreated mice as well as growth in mice treated with MOP / EP alone.

Fig. 19 shows that the antitumor activities of exclusive MOP / EP combination treatment, and MOP / EP combination treatment / after 1F5-AP pretreatment are approximately the same. In addition, it can be seen from the following Table IV that all tumors of these two groups as well as the tumors of an untreated control group increase in size. Pretreatment of the tumor / bearing mice with L6-AP conjugate / followed by combination treatment with MOP / EP, however, leads to a marked antitumor effect. As is apparent from Fig. 19 /

9 λ η

show tumors pretreated with L6-AP (followed by a combined MOP / EP treatment) on day 70 only 1/3 the size of a tumor »pretreated with 1F5-AP conjugate. In addition, Table IV shows that "63 days after implantation, one of six tumors in the L6-AP pretreated group is completely regressed" that three of the six tumors cease their growth and that only two of the six tumors increase in size.

TABLE IV

Tumor response (after 63 days) to treatment with antibody-AP conjugates and mitomycin / etoposide combinations

tumor responses

Group increase stable partially complete

Rückbildg. Rückbildg.

Control 6/6 -

MOP / EP 5/5 -

1P5-AP + MOP / EP 6/6 -

L6-AP + MOP / EP 2/6 3/6 - 1/6

These in vivo studies thus suggest the utility of the conjugates, prodrugs and methods of the invention for combination therapy against tumors.

In addition, the conjugates of the invention / such as L6-AP may be used with other combinations of prodrugs such as EP, adriamycin 14-phosphate, and 5-fluorouridine monophosphate for the delivery of a number of different cytotoxic agents to tumor cells.

59 * - ° - ° * *

The preparation of the antibody-enzyme conjugate L6-AP, as well as the prodrug etoposide-4 ¹ -phosphate again according to Example 1. Adriamycin 14-phosphate is prepared according to US Patent 4,185,111. 5-fluorouridine monophosphate is prepared according to MJ Robins et al. / Can. J. Chem. 53, p. 1302-1306 (1975)).

The reaction of L6-AP with the three prodrugs mentioned above is carried out as follows: either AP or the L6-AP conjugate (final EP concentration 5 μg / ml) is added to a solution of etoposide 4'-phosphate or adriamycin Added 14-phosphate (0/1 mM) in Tris buffer (100 mM) with MgCl ₂ (1 mM) and ZnCl ₂ (0.1 mM) at pH 7/0. To convert the 5-fluorouridine prodrug, a solution of 5-fluoro-uridine (3 μΜ) in phosphate buffer (100 mM) at pH 8.0 is required. The reaction of L6-AP with etoposide or 5-fluorouridine prodrug is recorded according to Example 1. "The reaction of L6-AP with adriamycin 14-phosphate is monitored by HPLC / using an IBM C-18 column (3 μ / 4 / 5χ 100 mm) and 65% methanol in water (3% ammonium acetate containing) as eluent (0.5 ml / min, detection at 495 nm).

The reaction of the antibody AP conjugate with the respective prodrug effects the hydrolytic cleavage (s) thereby liberating the free drug (see, e.g., R. B. McComb et al / Alkaline Phosphatase / Plenum Press (New York 1979)).

The cytotoxicity of each of the three prodrugs to tumor cells in the presence of the L6-AP conjugate of the present invention can be demonstrated by the colony inhibition assay described in Example 1. After removal of the phosphate group from the prodrugs by the conjugate, etoposide, adriamycin and 5-fluorouridine are released. Each of these agents is known to be an effective antitumor agent (see, e.g., P.J. O'Dwyer et al / Etoposide: Current Status of

60 2 - 3 ο "

To Active Anticancer Drug, 312: 692-700 (1985): MJ Embleton et al. Antibody Targeting Of Anti-Cancer Agents and VS

5 Byers (eds) / pp. 321-22 (Academic Press 1985); U.S. Patent 4,185,111 / supra: S.T. Crooke and S.D. Reich (ed.) / Anthracyclines: Current Status And New Developments, Academic Press (New York 1980); and C. Heidelberger et al. "Fluorinated Pyrimidines And Their Nucleosides" / in Adv.

Enzymol. Relat. Areas MoI. Biol., 54 / pp. 57-119 (1983)).

The prodrugs of etoposide / adriamycin and 5-fluorouridine may therefore be used together or in succession to release the corresponding known antitumour compound in the region of the tumor with the aid of the antibody-alkaline phosphatase conjugates according to the invention. For example, anti-tumor agents administered in combination have been shown to work together synergistically (see, for example, S. Monfardini et al / Manual of Cancer Chemotherapy / UICC Technical Report Series / 56/1981)). This embodiment of the present invention thus describes a method for combined tumor chemotherapy.

Example 4

The following example describes the use of further imraunconjugates according to the invention and prodrugs for the reaction of a relatively weakly toxic prodrug into an active antitumor compound / which has a cytotoxic effect on tumor cells in vitro. In this example, an L6-penicillin V-amidase (hereinafter referred to as PVA) immunoconjugate is used to convert an N-phenoxyacetyl derivative of adriamycin into the known antitumor compound adriamycin.

2 B 3 ο 3

Preparation of the antibody-penicillin-V-amidase conjugate according to the invention:

In this example, an L6-PVA immunoconjugate and a 1F5-PVA conjugate are prepared. The antibodies L6 and 1F5 as well as their descent are already described above. The amidase penicillin V-amidase used is obtained from a fungal culture of Fusarium oxysporum as described in DA Lowe et al., "Enzymatic Hydrolysis Of Penicillin-V to 6-Aininopenicillic Acid By Fusarium Oxysporum" Biotechnology Letters, 8 (3), p 151-56 1986). Fusarium oxysporum strains / »from which this enzyme can be isolated * are deposited with the ATCC. PVA is thus a commonly available enzyme / which converts penicillin V ^1S into penicillanic acid. , The phenoxyacetamide-reactive enzyme can therefore be used to excite prodrugs of known cytotoxic agents, such as phenoxyacetic acid or p-hydroxyphenoxyacetic acid

were derivatized

 20

The antibody-PVA conjugates of this embodiment of the invention are prepared according to the method of preparing ".A, AP conjugates according to Example 1. The antibodies L6 and IF5 are reacted with iminothiolane as described and the number of sulfhydryl groups introduced per antibody is from 1 to 2 ,

The enzyme PVA is dissolved in PBS at a concentration of 9 mg / ml and incubated with SMCC (Pierce Chemical Co./100 mM in DMF).

^ treated / so that the final concentration is 5 mM. By treating with SMCC, maleimide groups are introduced into the enzyme. After 30 min. At 30 ^° C., the modified enzyme is purified by gel filtration on G-25 PD-10 Sephadex (Pharmacia / üpsalla / Sweden) and elution with PBS.

modified PVA then becomes a solution of

62 2 8 ο ί 3

thiolated antibody added in a molar ratio 3: 1. Each reaction mixture is saturated with nitrogen and allowed to stand at room temperature for 3 h. It is then incubated at 4 C for a further 18 h. After that

δ is added to 2-aminoethanol (1 mM final concentration) to each solution / to block unreacted maleimides.

Each reaction mixture is then passed through a gel filtration column (G-25) using 20 mM Tris buffer / pH 7/2 with 50 mM NaCl as eluent. The resulting mixtures are purified over D EAE Sephadex columns (2/5 x 10 cm). The fractions are measured at 280 nm. Unreacted antibodies of each mixture do not bind to the column / while conjugate and unreacted PVA are eluted with 20 mM Tris buffer pH 7/2 with 0/5 M NaCl. The PVA and conjugate-containing fractions are concentrated with an Amicon S-300 ultrafiltration filter and purified on a Sephacryl S-300 column (2/5 χ 95 cm) with PBS as eluent. The fractions are measured at 280 nm and the conjugate-containing fractions (determined by SDS-PAGE / 4 to 12% gradient gel) are pooled.

Preparation of a new adriamycin prodrug:

With each of the antibody-PVA conjugates prepared above, a novel adriamycin prodrug is reacted. The prodrug used for this purpose is N- (p-hydroxyphenoxyacetyl) adriamycin (hereinafter referred to as APO) / wherein adriamycin is acylated at the amino sugar position with p-hydtoxyphenoxyacetic acid (see Fig. 20). This adriamycin prodrug is prepared as follows:

In 10 ml of tetrahydrofuran is added 84 mg (0/5 mmol) of p-hydroxyphenoxyacetic acid / 57 mg (0/5 mmol) of N-hydroxysuccinimide and 100 mg (0/49 mmol) of dicyclohexylcarbodiimide.

3, 034

This mixture is stirred for 2 h * then filtered and the filtrate with 200 mg (0/35 mmol) Adriamycinhydrochlorid added. To Reaktionsgeraisch 0/1 ml of triethanolamine is added and stirred for a further 4 h. The reaction mixture is filtered through glass wool and evaporated under high vacuum to a residue. The resulting mixture is eluted through a silica gel 60 column (2/5 χ 20 cm) with 95: 5 dichloromethane: methanol. The pooled fractions are again purified with the same column to give 70 mg (0/1 mmol / 30% yield) of pure N- (p-hydroxyphenoxyacetyl) adriamycin.

FAB MS m / e 694.2125 (M + H) ⁺ . calculated ^C 35 ^H 3 & ^No i4 '

· 360 MHz · ¹ H NMR (CDCl ₃ ) δ 1.06 (d, 3H, chipper CH-), 15

1.5-2.2 (m, 6H, H sugar), 3.0 (q, 2H) 4.0 (s, 3H, OCH ₃ ), 4.35 (s, 2H, COCH ₂ O), 4.8-5.0 (m, 3H), 5.2 and 5.4 (s, IH), 6.6-6.8 (dd, 4H, p-phenoxy ArH), 7.4-7.9 (m, 3H, 2.3,4-H), 9.0 (s, IH, Ar ¹ OH), 11.61 and 12.39 (s, IH, ArOH).

It is understood that other phenoxyacetylamide derivatives of adriamycin can be prepared by substantially the same procedure as described above. For example, N- (phenoxyacetyl) adriamycin can be prepared as described herein wherein the p-hydroxyphenoxyacetic acid is replaced by 0.5 mmol (76 mg) of phenoxyacetic acid. Similarly, N- (p-hydroxyphenoxy)

acetyl) melphalan or Daunomcyin-Prodrgus or N- (phenoxy-30

acetyl) melphalan or daunomycin prodrugs are prepared according to this protocol / using 100 mg of Welphalan or 200 mg of Daunomycin (Oi35 mmol).

2 ^

Reaction of an Antibody-Penicillin V Araidase Conjugate with an AdrLamycin Prodrug:

The ability of the antibody-PVA conjugate / L6-PVA to convert the new prodrug APO to adriamycin is determined as follows: To a solution of APO (0/1 mM) in PBS is added either (a) PVA alone (final concentration 50 μg / ml), (b) 100 pg / ml of the L6-PVA conjugate (final PVA concentration: 25 μg / ml) or (c) 10 μg / ml of L6-PVA (final PVA concentration: 2/5 pg / ml). Each reaction is determined by HPLC using a column of Phenominex C-18 (3 pm, 4.5 χ 100 nm) and an elution gradient of 20-60% tetrahydrofuran in water with 0/1% H ₃ PO ₄ (1/0 ml /Hin./detection at 495 nra).

Under these conditions, adriamycin is eluted after 8/9 min. And APO after 12/2 min. The results are shown in FIG.

As shown, the amide group of APO is hydrolyzed by PVA / where adriamycin is formed, under the conditions used the half-life of the hydrolysis of APO by PVA is approximately 20 min. In addition, within 40 min Reaction both PVA and the antibody-PVA conjugate effect at least 80% hydrolysis of APO to adriamycin. At a conjugate concentration of 10 μg / tnl (2/5 μg / ml PVA) this rate of hydrolysis is reached in 120 min. In addition, it is apparent from these studies that the antibody-PVA conjugate of the present invention shows no appreciable loss of enzymatic activity due to the binding of the enzyme to the antibody. This is evident from the fact that both conjugate and free enzyme show similar hydrolysis activities of APO to adriamycin.

2 336 3

Serum stability of the novel adriamycin prodrug according to the invention:

The stability of APO int human serum is determined by HPLC / decreasing rate of

APO and the rate of formation of adriamycin is measured. A solution of APO (10 roM in dimethylformamide) is added here to fresh human serum so that the final concentration is 0/1 mM. Samples of 50 μΐ are diluted with LO methanol (50 μΐ) / where the serum protein precipitates. These samples are then centrifuged and analyzed by HPLC as described above. No hydrolysis of APO to adriamycin can be detected within * 2 h.

Binding of antibody-PVA conjugates to H2981 tumor cells:

The binding ability of the L6-PVA and 1F5-PVA conjugates according to the invention to H2981 Turaor cells is determined according to Examples 1 ^and 3. The results of the binding assay are shown in Pig.

A FACS analysis shows that both L6 and the L6-PVA conjugate bind tightly to the tumor cells / while the 1F5-PVA conjugate shows no significant binding. This binding test shows firstly that the conjugation with an enzyme does not significantly affect the binding ability of the antibody component of the immunoconjugate of the present invention. Second, this test again shows the binding specificity of the conjugate; the L6-PVA conjugate binds to the L6-positive H2981 tumor cells / while the 1F5-PVA conjugate shows no binding due to the lack of specificity of the 1F5 antibodies for these tumor cells.

2 3 3 5 3

OO

In Vitro Toxicity of the Antibody-PVA Conjugate / Adriamycin Prodrug Accordance to H2981 Tumor Cell of the Invention:

The in vitro cytotoxic effect of the antibody-PVA / adriamycin prodrug combination according to the invention on H2981 tumor cells is carried out by means of the H-thymidine incorporation test according to Examples 1 and 2. The H2981 tumor cells are plated on microtiter plates (96 wells) in IMDM (10,000 cells / well) and incubated for 18 h at 37 ° C

Antibody PVA conjugates L6-PVA or 1F5-PVA are added ₍ closing at a concentration of 10 pg / ml (based on the antibody) and the plates are incubated for 30 min at 4 ° C, followed by washing the plates 4 χ with IMDM and add different concentrations of APO in IMDM After 2 h, wash again / add IMDM and incubate again for 18 h at 37 ° C. Then add H-thymidine (1 pci / well) and after 6 h the plates are frozen at -70 ° C to detach the cells.

Thawing the cells are transferred to glass fiber filters. 3 H-thymidine incorporation is monitored in a Beckman 3801 scintillation

measured and compared with cells / which were treated exclusively with APO or Adriamycin (ADH).

C_ 25 This assay determines the inhibition of ³ H-thyraidine incorporation into DNA of the tumor cells and thus the cytotoxic effect of prodrugs APO on cells with or without pre-treatment of cells with L6-PVA or 1F5-PVA conjugate. The cytotoxic effects of these combinations are compared with the cytotoxicity values which are obtained after treatment of the cells exclusively with the parent compound adriamycin. As shown in FIG. 23, adriamycin is significantly more toxic to conjugate-untreated tumor cells (ICt ₀ = 38 nM) than APO with an ICjQ value of 2 μΜ. This result is due to earlier

2-32034

Ό /

Reports / in which a lower toxicity of adriamycinatnide to adriamycin has been demonstrated (as for example Y. Levin and BA SeIa / FEBS Letters "98 / p. 119 (1979) and R. Baurain et al / J.Med.Chem , P. 1171 (1980)). Pre-treatment of the cells with L6-PVA increases the cytotoxicity of APO 20-fold compared to adriamycin alone. Pretreating the cells with 1F5-PVA will not affect the toxicity of APO. These results indicate that the L6-PVA conjugate is capable of hydrolyzing the relatively weakly toxic prodrug APO and thereby killing tumor cells to the same extent as adriamycin alone. In addition, this cytotoxicity is antigen-specific / because the 1F5-PVA conjugate that does not bind to this specific tumor cell line does not cause cytotoxicity.

Binding of the antibody-PVA conjugate to Daudi lymphoma cell

Furthermore, the binding ability of the L6-PVA * and 1F5-PVA conjugates of the invention was determined on Daudi cell lines. This cell line represents a Burkitt's lymphoma cell line / which is deposited in the ATCC (ATCC * CCL 213) and expresses the CD antigen to which the 1F5 antibody binds.

The binding assay is performed according to Example 1 / using Daudl cells. The results are shown in FIG.

In this case, monoclonal antibodies 1F5 and 3g bind the 1F5-PVA conjugate tightly to the lymphoma cells.

This study again demonstrates that the binding ability of the conjugate is not significantly affected by the conjugation procedure.

In addition, it can be seen from the figure that the antibodies L6 and the conjugate L6-PVA do not bind significantly

⁶ * 2 3 3 0

Daudi cells show. This is to be expected / because Daudi tumor cells have no Lö antibody-specific antigen. Together with the binding assays previously described, this study clearly demonstrates the binding specificity of the conjugates of the invention / i. L6-containing conjugates bind specifically to L6-positive cells and IF5-containing conjugates bind specifically to CD-20-positive cells.

In Vitro Toxicity of the Antibody-PVA Conjugate / Adriamycin Prodrug Combination Against Daudi Cells According to the Invention:

In the next step, the in vitro cytotoxicity of the L6-PVA or 1F5-PVA conjugates in combination with the prodrug APO is investigated in comparison to Daudi cells.

The H-thymidine test is carried out according to the examples described above with minor modifications due to the non-adherent Daudi cells. About 250,000 Daudi cells in IMDM are placed in each well of a 96-well microtiter plate and spiked with the antibody-enzyme conjugate. The reaction mixture is incubated at 4 ° C for 30 min. Unbound antibody-enzyme conjugate is removed by centrifugation at 500 g for 5 minutes and supernatant. The cells are resuspended in IMDM and the wash described is retrieved for complete removal of unbound conjugate 3χ. Add APO in IMDM for 2 h and wash 1 χ as described above. The remaining steps of the test are performed according to the previously described examples.

This test is used to inhibit H-thymidine incorporation into the DNA of Daudi cells and thus the cytotoxic effect of the prodrug APO on these cells, with or without

2 ο υ £ 3

Treatment of cells with L6-PVA or 1F5-PVA conjugate determined. The cytotoxic effects of these combinations are compared with the cytotoxicity of a cell treatment exclusively with adriamycin. As shown in Figure 25, adriamycin on conjugate-untreated Daudi cells is significantly more toxic than APO. Pre-treatment of the cells with 1F5-PVA conjugate leads to a substantial enhancement of the cytotoxicity of the prodrug APO and is comparable to the cytotoxicity of adriamycin alone. Pretreatment of cells with L6-PVA conjugate will not produce comparable enhancement.

It should be noted that these binding and cytotoxicity studies resulted in opposite results from previously obtained results from studies with the same conjugates and H2981 tumor cells. Here, the combination L6-PVA + APO showed an increased cytotoxic effect / while 1F5-PVA + APO caused no comparable effect. This is to be expected on account of the different specificities of the conjugate antibodies L6 and 1F5 and once again clearly demonstrates the specificity of the cytotoxic effects achieved with the conjugate / prodrug combinations according to the invention.

^AyJ This study also demonstrates that the 1F5-PVA conjugate in combination with APO is useful for generating a cytotoxic effect in vitro on tumor cells. The in vitro cytotoxicity studies described in this example thus demonstrate that any conjugate /

® which contains an antibody reactive with a tumor-associated antigen / can be used according to the invention for the treatment of a tumor / which reacts with this antibody.

₇₀ 2 8 3 5

Example 5

This example relates to the use of the immunoconjugates of the invention and to methods of converting the prodrug 5-pluorocytosine (hereinafter referred to as 5-FC ") into the antitumor agent 5-fluorouracil (hereinafter referred to as 5-FU) using the antibody-linked enzyme cytosine -Deäminase (CD) (see Figure 26) .The antibody-CD conjugate / S-FC prodrug combination of the present invention exhibits a marked cytotoxic effect on tumor cells in vitro.

t producing the antibody cytosine Deaminasekonjugates invention;

L6-CD and 1F5-CD immunoconjugates are prepared based on the earlier examples from the monoclonal antibodies L6 or 1F5 and the enzyme cytosine deaminase. Although CD enzymes have been found in and isolated from various microorganisms (see, for example, West et al., Biochem. Biophys. Acta., 719 / pp. 251-58 (1982)), the compressed baker's yeast compact used herein is specifically described in U.S. Pat Description of PL Ipata et al., "Baker's Yeast Cytosine Deaminase. Some Enzymatic Properties and Allosteric Inhibition By _ Nucleosides And Nucleotides "/ Biochemistry / 10 / p. 4270-76 ^{( 25} (1971)).

For this purpose, yeast cells (Saccharomyces cerevisiae) (2.0 kg) are plasmolysed with ethyl acetate. To prepare a crude enzyme preparation, an ammonium sulphate precipitate (50-73%) SQ is carried out twice. The ammonium sulfate precipitate is dialyzed against 10 mM Tris-Cl buffer, pH 8/0, applied to a Q-Sepharose anion exchange column (Pharmacia) and eluted with a KCl gradient (0-0 / 3M).

₇₁ 2 8 3 0

The resulting fractions are assayed for CD activity by using 3 roM cytosine (or 5-PC) as a substrate in PBS at 27 ° C according to the protocol of T. Nishiyama et al., Antineoplastic Effects in Rats of 5-Fluorocytosine In Combination With Cytosine Deaminase Capsules "/ Cancer Research / 45 / p. 1753-61 (1985)). A small amount of the enzyme preparation is added to the substrate cytosine (or 5-FC). The course of the reaction is determined by means of UV spectrophotometry / in which, after stopping the samples with 0/1 N HCl, the üracil. (or 5-FU). The ratios

250/280 (for cytosine) and 255/290 (for 5-FC) are used to determine the formed üracil resp. 5-FU amount used. This method for determining the CD activity is used after each purification step of the enzyme and of the inventive CD-containing conjugate (see below).

The active fractions of the KCl gradient are pooled / concentrated and purified on a Sephadex G-75 column. Subsequent SDS-PAGE (14% / non-reducing) shows that the CD-containing fractions consist of one major protein

a molecular weight of 18 kd and smaller amounts of protein at 20 and 30 kd. The CD activity of these fractions is 10 U / mg protein (using cytosine as substrate). Other preparations show an activity of 17 U / mg protein. All protein determinations are made using the BCA Protein Detection Reagent (Pierce / Rockford / IL).

The purified CD is subsequently attached to the monoclonal ₃ lbs. Antibody L6 or 1F5 bound by means of the method described for the AP conjugates in Example 1. The crude conjugates (untreated with iodoacetamide) are purified by gel filtration over S-200 Sepharose with PBS as eluent. The fractions are measured at 280 nm and

_o The CD activity of the fractions is as described above ob

certainly. Conjugate-containing fractions with a suitable

72 2 8 3 6

CD-antibody ratio is pooled and analyzed by SDS-PAGE on a 4 to 12% non-reducing gradient gel to give purified L6-CD and 1F5-CD conjugate preparations.

Reaction of the antibody-cytosine deaminase conjugate with the prodrug 5-fluorocytosine:

The ability of the inventive conjugates L6-CD and 1F5-CD / the prodrug 5-FC to 5-FÜ or the substrate cytosine to uracil * is determined as follows:

Either give free CD (final concentration 5 μg / ml) / the L6-CD conjugate (final CD concentration 5 μg / Inl) or the 1F5-CD conjugate {final CD concentration 5 μg / ml) to a 3 mM solution from (a) cytosine or (b) 5-FC in PBS at 27 ° C, and determines the amount of resulting product as a function of time spectrophotometrically (as above

described). The results are shown in FIG. 20

The figure shows that 5-FU is released from the prodrug 5-FC both by free CD and by the antibody CD conjugates according to the invention. It can also be seen from the figure that as a result of the binding of the enzyme to the antibody of both conjugates, there is no substantial decrease in the CD enzyme activity since the conjugates have essentially the same activities as free CD. The specific activity of free enzyme and conjugate is approximate

4 U / mg enzyme

 30

For comparison, the reactivity of the conjugate is determined instead of 5-FC with cytosine as the substrate. It can be seen from the figure that the conjugates also have the same CD activities with cytosine as substrate _/ compared to free CD. The specific activity of the conjugate - 10 U / mg bound

Enzyme - also indicates that the original enzyme activity of CD is retained in the conjugate. This activity value is maintained for several weeks / when the conjugate is stored in PBS at 4 ° C.

Binding of the antibody-CD conjugate to H2981 tumor cells;

The binding ability of the L6-CD- and .-. 1F5-CD conjugates to H2981 tumor cells are determined according to Examples 1 and 2. The results of the binding assay are shown in FIG. FACS analysis shows that both L6 and L6 CD strongly bind to the tumor cells * while the 1F5 immunoconjugate does not bind to the cells. As with previous binding studies, this assay shows that both the binding activity of these conjugates is maintained despite conjugation of the antibody to the enzyme, as well as the binding specificity of these conjugates.

In vitro cytotoxicity of the antibody-CD conjugate / 5-FC prodrug combination according to the invention against H2981 tumor cells!

The in vitro cytotoxicity of the antibody CD / 5-FC prodrug combination according to the invention against H2981 tumor cells becomes 3

with the help of an H-Leucineinbauversuches / comparable to the. H-thymidine incorporation experiment according to Examples 1 and 2, determined.

In this experiment, a suspension of 10 H2981 cells *

in 0.1 ml IMDM with 10% (vol / vol) fetal calf serum in microtiter plates (96 wells) and incubated overnight at 37 C for binding. The plates are

and washed with L6-CD or 1F5-CD conjugate (10 μg total protein / ml, 10 U / mg bound protein CD-

Containing activity / using cytosine as substrate) in 0.1 ml IMDM. After 30 min. At 4 ° C, the plates are washed 4 χ. Next, add 0/15 ml of leucine-free RPMI media / containing different concentrations of compounds 5-FC or 5-FU to each chamber. The cells are incubated for 18 h at 37 ° C and pulsed for 6 h with H-leucine (1 pCi / well) in 0.05 ml leucine-free RPMI. The plates are then according to

the details in Examples 1 and 2 for the H-thymidine incorporation test further.

This assay is used to determine the inhibition of H-leucine incorporation into the protein of the tumor cells and thus the cytotoxic effect of the prodrug 5-FC on the cells with or without pre-treatment of the cells with L6-CD or 1F5-CD conjugates. The cytotoxic effects of these combinations are compared with the cytotoxic effects after treatment of the cells exclusively with the parent compound 5-FU As shown in Figure 29, 5-FC on conjugate-untreated tumor cells has very little cytotoxic effect, while 5-FU cell growth is inhibited with an IC ₅₀ of 20 μΜ, however, pretreatment of the cells with L6-CD conjugate raises the cytotoxicity of 5-FC to a value which is associated with the cytotoxicity.

² ^ toxicity of 5-FU alone is comparable. This is because antigen-linked L6 CD converts the non-toxic prodrug 5-FC to 5-FU. The non-binding conjugate 1F5-CD causes no comparable increase / which in turn demonstrates the antigen specificity of the increased cytotoxicity.

From the above described embodiments of the invention it is evident that will describe by changing the basic construction of other embodiments of $ 3 to which the inventive method, Imraun-

use conjugates and prodrugs. The scope of the present invention will therefore be determined not by the examples described above, but by the appended claims.

Claims (2)

M / 29 283 Patent claims 1. Process for preparing the compounds of the general formula (I) a) 0 OH (D wherein R is hydrogen and R ^{3 is} OH or OCH-; or R ^{1 is} OH and R ^{3 is} OCH ₃ ; and R ^{2 is} H or OH. b) or the compounds of the formula (II) 0 OH Z υ ο. · Ο where A R is hydrogen and R is OH or OCH ₃ ; or R is OH and R is OCH ₃ ; and R ^{2 is} H or OH, characterized in that a compound of the general formula 20 25 30 35 a) with p-HydrcKyphenoxyessigsäure or with phenoxyacetic acid or a preferably with N-hydroxysuccinunide or dicyclohexylcarbodiimide activated derivative thereof to give a compound of general formula (I), or b) with phenylacetic acid or hydroxyphenylacetic acid or a preferably with N-hydroxysuccinimide or dicyclohexylcarbodiimide activated derivative thereof to give a compound of general formula (II). 2: - 3 6 3 Process according to claim 1, characterized in that roan N- (p-hydroxyphenoxyacetyl) adriamycin or N- (phenoxyacetyl) adriamycin is obtained. Ηι € ί? UJ / J pins? Cicb MUHO 10 "^